Standardized bioflavonoid composition for regulating the homeostasis of host defense mechanisms

Abstract

A bioflavonoid composition for establishing and regulating homeostasis of host defense mechanisms is disclosed, comprising at least one standardized bioflavonoid extract enriched in at least one free-B-ring flavonoid and at least one standardized bioflavonoid extract enriched in at least one flavan. The intended composition is effective against respiratory diseases and conditions.

Description

Background Art

[0001] Aging is a natural phenomenon and a complex degenerative process that affects the functions of both the mind and body over time. Poor host defense response is one of the most common changes seen in aging. Understanding the underlying mechanism of the decreased host defense response in the elderly is the first step towards its alleviation. Chemically induced accelerated aging models, such as the D-galactose-induced thymic injury and immune aging mouse model, are one of the preferred options for studying the effects of aging on the immune system. In chemically induced animal aging models, animals exhibit immune aging similar to the decreased host defense response frequently observed in the elderly (Azman 2019). The D-galactose-induced aging model is one of the animal models widely used and well-verified in anti-aging research. D-galactose is converted to glucose at normal concentrations in the body, but at high concentrations, it is easily converted to aldose and hydroperoxide, leading to the generation of free radicals derived from oxygen. It may also react with the free amines of proteins and peptides to produce advanced glycation end products (AGEs) by non-enzymatic glycation. When these reactive oxygen species (ROS) accumulate and AGEs increase in this model, the normal organs and host defense homeostasis become imbalanced, resulting in oxidative stress, systemic inflammation, decreased immune response, mitochondrial dysfunction, and apoptosis (e.g., of thymocytes), ultimately accelerating the aging process. These changes are counted among the natural pathological features of aging and senescence.

[0002] Sepsis is a severe organ dysfunction caused by the dysregulation of the host defense response to infection and may lead to organ failure. Sepsis is mainly a state mediated by macrophages / monocytes and is caused by the overproduction of several early cytokines such as TNF-α, IL-1, IL-6, and interferon-γ, and late mediators such as HMGB1. High-mobility group box 1 protein (HMGB1) is an endogenous damage-associated molecular pattern (DAMP) protein that can be released or secreted from cells by injury stimuli or cytokines and is located in the nucleus or cytoplasm. Nuclear HMGB1 is a structural chromatin-binding factor that plays a role in maintaining genomic integrity, while extracellular HMGB1 released from activated or damaged cells is a mediator that causes inflammation and immune dysfunction in response to various stresses such as oxidative damage and pathogen infection. Since HMGB1 is released from macrophages and monocytes activated in response to endogenous and exogenous inflammatory signals, it is an important mediator of sepsis (Wang et al., 1999), increasing the imbalance of the host defense mechanism, leading to multiple organ failure, and ultimately potentially resulting in death. Since the late sustained release of HMGB1 can sufficiently trigger an inflammatory response, patients who survive may also continue to have an inflammatory response (Gentile and Moldawer, 2014).

[0003] When actively released from stimulated monocytes or passively released from necrotic cells, HMGB1 acts as an alarmin (danger signal) that functions to counteract the reduction of intracellular homeostasis with neighboring cells and activate the host immune response. HMGB1 functions as a chemokine that aids the migration of immune cells to the site of infection and, as a DAMP that activates other immune cells to secrete inflammatory cytokines, plays an important role in activating the innate immune response (Yang et al., 2001). When inflammatory cytokines are produced at low (optimal) concentrations, they exert a defensive function against viral or microbial invasion, but when overproduced, as in the case of a "cytokine storm," they can be harmful to the host by mediating a harmful inflammatory response. In most cases, it is thought that in hosts with underlying diseases such as immunodeficiency or immunosuppression, or in the elderly, these cytokine storms cause acute systemic inflammatory syndrome. Even if the patient survives, there is a possibility of delayed-onset inflammatory mediation, leading to persistent inflammatory responses, immunosuppressive responses, and catabolic responses. In addition to functioning as a chemoattractant for a number of cell types, including all inflammatory cells, HMGB1 is thought to be involved in the "cytokine storm" through activation of the NFκB signaling pathway because it causes the secretion of TNF-α, IL-1β, IL-6, IL-8, and macrophage inflammatory protein (MIP) into inflammatory cells (Bianchi and Manfredi, 2007). Extracellular HMGB1 has the potential to induce a devastating inflammatory response and has been reported in many studies to promote the progression of sepsis and acute lung injury (Entezari et al., 2014). In contrast to TNF-α and IL-1β, which are secreted within minutes of endotoxin stimulation, HMGB1 is secreted hours later, both in vitro and in vivo, and is judged to mediate late-stage inflammation. Indeed, administration of an HMGB1 neutralizing antibody 24 hours after the onset of sepsis provides protection against lethal endotoxemia, indicating that HMGB1 plays a major role as a late mediator of lethal sepsis (Wang et al., 1999).Clinically, it has also been demonstrated that there is a strong association between persistent high concentrations of HMGB1 and subjects in the late stage of sepsis or those who died from sepsis (Angus et al., 2007). Recently, several clinical trials have shown that chloroquine and its analog (hydroxychloroquine) are beneficial for the clinical efficacy against COVID-19 and viral clearance (Andersson et al., 2020, Gao et al., 2020; Gautret et al., 2020). In a mouse sepsis model, the anti-malaria drug chloroquine tested prevented lethality, and its protective effect was mediated by inhibiting the release of HMGB1 from macrophages, monocytes and endothelial cells, preventing HMGB1 cytokine-like activity, and inhibiting NF-κB activation (Yang et al., 2013). Dietary antioxidants have been reported to significantly reduce hyperoxia-induced acute inflammatory lung injury by enhancing macrophage function through suppressing the accumulation of HMGB1 in the airway (Patel et al, 2020). In view of the above, the natural bioflavonoid composition containing free B-ring flavonoids and flavans described in the text of the protected subject of the present application inhibits HMGB1 and NF-κB, prevents sepsis lethality, inhibits AGE formation, induces endogenous antioxidant enzymes, promotes macrophage phagocytosis, increases bacterial clearance, prevents acute lung injury, can apply the historical usage safe for maintaining and protecting respiratory and lung health, and has been confirmed to prevent and treat pathological conditions such as lung injuries caused by viruses, microbial infections (e.g., COVID-19) and PM2.5 air pollutants, PM10 particles in the air, air pollutants, photochemical smog, tobacco, smoke from e-cigarettes, and smoke from recreational marijuana.

[0004] Acacia catechu Willd (Fabaceae) is commonly known as Cutch tree, Khair, and Khadira and is used as a traditional medicine in India and other Asian regions (Hazral et al., 2017). This plant is a deciduous tree that can grow up to 15 m tall. The bark is dark grayish-brown and peels off in long strips. The leaves are pinnate with a pair of spines at the base of the leaf axis. The flowers are pale yellow and arranged in cylindrical spikes. The legumes are hairless, flat, and elliptical. According to the Ayurvedic Pharmacopoeia of India, the heartwood of Acacia catechu is pale red and changes from reddish-brown to almost black with age, surrounded by a whitish sapwood, is crack-resistant, odorless, and astringent. Medium-sized trees with an age of about 8 years or more are harvested for the extraction of Acacia catechu extract. Since the appearance of Acacia catechu (timber), Uncaria gambir (a climbing plant), and cashew nut shells (pericarp) are very different, plant material supply and plant authentication are the main focus of the first supplier selection criteria. Acacia catechu is used in Ayurvedic medicine for the throat, mouth, and gums and is also used for coughs and diarrhea. Externally, it is used as an astringent and for cooling skin ulcers, swellings, and rashes. Powder is used for wound treatment. Acacia catechu has been found to increase the number of antibody-producing cells in the animal spleen, enhance the immune system, enhance the phagocytic activity of macrophages, and suppress the release of inflammatory cytokines (Sunil et al, 2019).

[0005] Skullcap (Scutellaria baicalensis Georgi) (Lamiaceae family) is a traditional Chinese medicine known by its common name Chinese skullcap (Huang Qin) and is used in several Asian countries as indicated in the Chinese Pharmacopoeia. This plant is a perennial herb, with stems that creep horizontally and then become erect, tinged with purple. The leaf stalks are short, lanceolate, hairy, and bear medium green leaves. From early summer to early autumn, it bears hairy flowers with a dense blue upper lip and a pale blue lower lip in a raceme. During spring or summer, the biennial roots are collected and air-dried for commercial purposes. According to the Chinese Pharmacopoeia, the roots are about 8 - 25 cm in length and 1 - 3 cm in diameter. The roots are yellowish-brown or dark yellow, with scattered traces of where they were dug on the outside. The upper part is flaky, with twisted longitudinal wrinkles or irregular networks, and the lower part has longitudinal veins and fine wrinkles. The texture is hard and brittle, easily broken, with a yellow fracture, a reddish-brown core, and the center of old root bark is dark brown or blackish-brown, withering or hollowed out. It has almost no smell and a bitter taste. The dried roots usually contain less than 10% of bioflavonoids such as baicalin. The roots used for skullcap extract are tested by TLC and HPLC methods based on the identification and quantification methods of the Chinese Pharmacopoeia.

[0006] Skullcap has been recorded since the Eastern Han Dynasty (around 200 AD or about 2,200 years ago) in the "Shen Nong Ben Cao Jing", a classic Chinese medical book. According to the latest list of the top 30 herbs for the treatment of respiratory infections in Traditional Chinese Medicine (TCM) based on the analysis of two TCM databases (World Traditional Medicine Patent Database (WTM) and Saphron TCM database), Huang Qin (Radix Scutellaria) is ranked as the second most frequently used herb and is included in 38% of all TCM compositions for the treatment of respiratory infections (Ge et al. 2010).

[0007] Huanggon was included in the TCM composition recommended by the Chinese government in 2003 during the SARS epidemic. The use of baicalin (Yuan et al, 2009) and flavonoids derived from plants of the genus Scutellaria (Zhong, et al., 2006) has subsequently been patented for the treatment of SARS and COVID-19 (Song et sl. 2020). According to recent scientific research on Huanggon, bioactive components of this herb with biological functions related to antioxidant, anti-inflammatory, suppression of allergic reactions and antibacterial activity (Bejar et al., 2004), bioflavonoids, especially baicalin and baicalein, have been identified (Shen et al, 2021). For infection, it is essential for the virus to bind to and germinate from host cells, but baicalin and baicalein also showed strong antiviral activity by inhibiting the proteins required for the virus to bind and germinate (Yu et al, 2011). In mice infected with the influenza A H1N1 virus (swine influenza), the extract from Huanggon regulated its inflammatory response to reduce disease severity, reduced lung tissue damage, and ultimately increased its survival rate (Zhi et al, 2019).

[0008] Flavonoids are a group of natural products that are widely distributed and sold. It has been demonstrated that taking flavonoids is inversely related to the risk of non-vascular dementia. The mechanism of action is unknown, but it is presumed to be due to the antioxidant effect of flavonoids (Commenges et al. 2000). Polyphenol flavone acts at the mRNA level on genes including cox-2, nuclear factor κBB (NFκB) and bcl-X(L) to induce programmed cell death, differentiation and growth inhibition in transformed colon cells (Wenzel et al. 2000). It has been reported that the number of hydroxyl groups on the B ring is important for the suppression of cox-2 transcriptional activity (Mutoh et al. 2000).

[0009] Free B-ring flavonoids are relatively few. Among the 9,396 total flavonoids isolated from synthetic or natural sources, only 231 free B-ring flavonoids are known (The Combined Chemical Dictionary, Chapman and Hall / CRC, Version 5: 1 June 2001). Free B-ring flavonoids have been reported to have various biological activities. For example, galangin (3,5,7-trihydroxyflavone) acts as an antioxidant and free radical scavenger and is considered a promising candidate for anti-genotoxicity and cancer chemoprevention (Heo et al. 2001). This compound is an inhibitor of tyrosinase monophenolase (Kubo et al. 2000), an inhibitor of rabbit heart carbonyl reductase (Imamura et al. 2000), and has antimicrobial activity (Afolayan and Meyer 1997) and antiviral activity (Meyer et al. 1997). Baicalein and two other free B-ring flavonoids have anti-proliferative activity against human breast cancer cells (So et al. 1997).

[0010] Generally, flavonoids are tested for activity randomly based on their availability. Claims that substitution on the B-ring is necessary for specific biological activities are scattered, for example, high-affinity binding to p-glycoprotein (Boumendjel et al. 2001), cardiotonic action (Itoigawa et al. 1999), endothelial cell protective action against toxicity induced by linoleic acid hydroperoxide (Kaneko and Baba 1999), COX-1 inhibitory activity (Wang, 2000), and prostaglandin endoperoxide synthase (Kalkbrenner et al. 1992) are said to require B-ring substitution. Very few literatures mention the significance of the unsubstituted B-ring of free B-ring flavonoids. One example is the use of 2-phenylflavone as a potential anticoagulant that inhibits NADPH quinone acceptor oxidoreductase (Chen et al. 2001).

[0011] Regarding the mechanisms of action reported in relation to the anti-inflammatory activities of various free B-ring flavonoids, various theories have been proposed. The main bioactive free B-ring flavonoids of Celosia argentea L. have been reported to suppress inflammatory cytokines (Liao, et al, 2021). The anti-inflammatory activities of free B-ring flavonoids such as chrysin (Liang et al.2001), wogonin (Chi et al.2001) and galangin (Raso et al.2001) are related to the inhibition of inducible cyclooxygenase and nitric oxide synthase by the activation of peroxisome proliferator-activated receptor γ (PPARγ), and are said to affect degranulation and AA release (Tordera et al.1994). Oroxylin, baicalein and wogonin have been reported to inhibit 12-lipoxygenase activity without affecting cyclooxygenase (You et al.1999). More recently, the anti-inflammatory activities of wogonin, baicalin and baicalein have been reported to occur through the inhibition of inducible nitric oxide synthase and cox-2 gene expression induced by nitric oxide inhibitors and lipopolysaccharides (Chen et al.2001). It has also been reported that oroxylin acts by suppressing NFκB activation (Chen et al.2001). Finally, wogonin has been reported to inhibit inducible PGE2 production in macrophages (Wakabayashi and Yasui 2000).

[0012] Catechin is one of the bioactive flavonoids well-documented in the literature (Bae et al.2020). Catechin and its isomer epicatechin have an IC 50It inhibits prostaglandin endoperoxide synthase at a value of 40 μmol / L (Kalkbrenner et al. 1992). Five flavan-3-ol derivatives containing (+)-catechin and gallocatechin isolated from four plant species, Atuna racemosa, Syzygium carynocarpum, Syzygium malaccense (Malay rose apple), and Vantanea peruviana, have an inhibitory activity against COX-2 that is equal to or lower than that against COX-1, and the IC 50 values range from 3.3 μM to 138 μM (Noreen et al. 1998). (+)-Catechin isolated from the bark of Ceiba pentandra (kapok) inhibits COX-1 at an IC 50 value of 80 μM (Noreen et al. 1998). Commercially available pure (+)-catechin inhibits COX-1 at an IC 50 value of approximately 183 - 279 μM depending on the experimental conditions, but it has no selectivity for COX-2 (Noreen et al. 1998).

[0013] To date, approximately 330 compounds have been isolated from various Acacia species. Flavan, a type of water-soluble plant pigment, is the largest class of compounds isolated from Acacia. Approximately 180 different flavonoids have been identified, 111 of which are flavans. Terpenoids are the second largest class of compounds isolated from Acacia species, and 48 compounds have been identified. Other classes of compounds isolated from Acacia include alkaloids (28 species), amino acids / peptides (20 species), tannins (16 species), carbohydrates (15 species), oxygen-containing heterocycles (15 species), and aliphatic compounds (10 species). (Buckingham, The Combined Chemical Dictionary, Chapman and Hall CRC, version 5:2, Dec. 2001).

[0014] When green tea catechins are supplemented to the diet of Sprague Dawley male rats, the activity level of platelet phospholipase A2 decreases, and the cyclooxygenase concentration in platelets significantly decreases (Yang et al. 1999). Catechin and epicatechin have been reported to slightly suppress cox-2 gene transcription in human colorectal cancer DLD-1 cells (IC 50 = 415.3 μM) (Mutoh et al. 2000). The neuroprotective effect of (+)-catechin derived from red wine is rather due to the antioxidant property of catechin than the inhibitory effect on intracellular enzymes such as cyclooxygenase, lipoxygenase or nitric oxide synthase (Bastianetto et al. 2000). Catechin derivatives such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and theaflavin, purified from green tea and black tea, show inhibition of cyclooxygenase and lipoxygenase-dependent metabolism of arachidonic acid in human colorectal mucosa and colorectal tumor tissues (Hong et al. 2001), and induce COX-2 expression and PGE2 production (Park et al. 2001).

[0015] Regarding the effect of suppressing the LPS-induced inflammatory response via the NF-κB, MAPK, and PI3K-Akt signaling pathways in type II alveolar epithelial cells, studies on extracts of Machilus thomsonii and Celosia cristata have recently been published (Feng et al., 2019). U.S. Patent Nos. 9,061,039, 8,535,735, 7,972,632, and 7,192,611, with the invention title "Identification of Free-B-Ring Flavonoids as Potent COX-2 Inhibitors," and U.S. Patent Nos. 9,168,242, 8,568,799, 8,124,134, 7,108,868, with the invention title "Isolation of a Dual COX-2 and 5-Lipoxygenase Inhibitor from Acacia," respectively describe methods for isolating, purifying, and using compositions containing free-B-ring flavonoids or flavans. Regarding the combined composition of free-B-ring flavonoids and flavans based on COX / LOX dual inhibitors and their use in joint care, mental acuity, oral care, skin care, etc., U.S. Patent Nos. 9,849,152, 9,655,940, 9,061,039, 8,535,735, 7,674,830, 7,514,469, with the invention title "Formulation of a mixture of Free-B-Ring flavonoids and flavans as a therapeutic agent," U.S. Patent Nos. 8,652,535, 8,034,387, 7,695,No. 743, Invention Title: "Formulation of a mixture of Free-B-Ring flavonoids and flavans for use in the prevention and treatment of cognitive decline and age-related memory impairments", U.S. Patent No. 9,622,964, 8,790,724, Invention Title: "Formulation of dual cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors for skin care", U.S. Patent No. 8,945,518, Invention Title: "Formulation of Dual Eicosanoid System and Cytokine System Inhibitors for the Use in the Prevention and Treatment of Oral Diseases", and U.S. Patent No. 7,531,521, Invention Title: "Formulation for prevention and treatment of carbohydrate induced diseases and conditions", the entire disclosure of which is incorporated herein by reference.,

Prior Art Documents

Patent Documents

[0016]

Patent Document 1

Patent Document 2

Patent Document 3

[0017] [Non-Patent Document 1] Azman 2019 [Non-Patent Document 2] Wang et al., 1999 [Non-Patent Document 3] Gentile and Moldawer, 2014 [Non-Patent Document 4] Yang et al., 2001 [Non-Patent Document 5] Bianchi and Manfredi, 2007 [Non-Patent Document 6] Entezari et al., 2014 [Non-Patent Document 7] Angus et al., 2007 [Non-Patent Document 8] Andersson et al. 2020 [Non-Patent Document 9] Gao et al., 2020 [Non-Patent Document 10] Gautret et al., 2020 [Non-Patent Document 11] Yang et al., 2013 [Non-Patent Document 12] Patel et al, 2020 [Non-Patent Document 13] Hazral et al., 2017 [Non-Patent Document 14] Sunil et al, 2019 [Non-Patent Document 15] World Traditional Medicine Patent Database (WTM) [Non-Patent Document 16] Saphron TCM Database [Non-Patent Document 17] Ge et al. 2010 [Non-Patent Document 18] Yuan et al, 2009 [Non-Patent Document 19] Zhong, et al., 2006 Non-Patent Document 20 Song et al., 2020 Non-Patent Document 21 Bejar et al., 2004 Non-Patent Document 22 Shen et al., 2021 Non-Patent Document 23 Yu et al., 2011 Non-Patent Document 24 Zhi et al., 2019 Non-Patent Document 25 Commenges et al., 2000 Non-Patent Document 26 Wenzel et al., 2000 Non-Patent Document 27 Mutoh et al., 2000 Non-Patent Document 28 The Combined Chemical Dictionary, Chapman and Hall / CRC, Version 5: 1 June 2001 Non-Patent Document 29 Heo et al., 2001 Non-Patent Document 30 Kubo et al., 2000 Non-Patent Document 31 Imamura et al., 2000 Non-Patent Document 32 Afolayan and Meyer, 1997 Non-Patent Document 33 Meyer et al., 1997 Non-Patent Document 34 So et al., 1997 Non-Patent Document 35 Boumendjel et al., 2001 Non-Patent Document 36 Itoigawa et al., 1999 Non-Patent Document 37 Kaneko and Baba, 1999 Non-Patent Document 38 Wang, 2000 Non-Patent Document 39 Kalkbrenner et al., 1992 Non-Patent Document 40 Chen et al., 2001 Non-Patent Document 41 Liao, et al., 2021 Non-Patent Document 42 Liang et al., 2001 Non-Patent Document 43 Chi et al., 2001 Non-Patent Document 44 Raso et al., 2001 Non-Patent Document 45 Tordera et al., 1994 Non-Patent Document 46 You et al., 1999 Non-Patent Document 47 Wakabayashi and Yasui, 2000 Non-Patent Document 48 Bae et al., 2020 Non-Patent Document 49 Noreen et al., 1998 Non-Patent Document 50 Buckingham, The Combined Chemical Dictionary, Chapman and Hall CRC, version 5:2, Dec. 2001 Non-Patent Document 51 Yang et al., 1999 Non-Patent Document 52 Bastianetto et al., 2000 Non-Patent Document 53 Hong et al., 2001 Non-Patent Document 54 Park et al., 2001 Non-Patent Document 55 Feng et al., 2019 Summary of the Invention

[0018] Disclosed is a bioflavonoid composition for establishing and regulating the homeostasis of the host defense mechanism, comprising at least one standardized bioflavonoid extract with a high concentration of at least one free B-ring flavonoid and at least one standardized bioflavonoid extract with a high concentration of at least one flavan. The intended composition is effective against respiratory diseases and conditions.

Brief Description of the Drawings

[0019]

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Modes for Carrying Out the Invention

[0020] Detailed Description Disclosed are compositions and methods for regulating the homeostasis of host defense mechanisms, comprising a combination of one or more free B-ring flavonoids derived from Scutellaria baicalensis and one or more flavans derived from Acacia catechu. Also disclosed are compositions for maintaining the homeostasis of host defense mechanisms by modulating HMGB1, suppressing oxidative stress, and inducing the production of immunoglobulins and T cells in mucosal immunity, particularly in the immune system and the respiratory system. As a method for treating, managing, promoting, protecting the phagocytic activity of macrophages as the front line of innate immune defense cells in mammals, and providing an important host defense mechanism for populations with increased exposure to pathogenic stress and oxidative stress caused by air pollution, viruses such as SARS-CoV-2, and microbial infections, particularly for hosts with chronic inflammatory disorders including aging and chronic inflammatory disorders of the respiratory system, also disclosed is a method comprising administering an effective amount of the composition at 0.01 mg to 500 mg per kg of body weight of the mammal.

[0021] The subject of protection of the present application relates to the synergistic regulation of host defense homeostasis, and uses a standardized bioflavonoid composition containing free B-ring flavonoids and flavans to regulate the extracellular protein HMGB1, suppress oxidative stress, and induce mucosal immunity, particularly the production of immunoglobulins and T cells, thereby improving the host's immune function, respiratory health, and lung function. IgA is the second most abundant antibody in serum and is at the forefront of defense against lung and systemic infections by inhibiting the adhesion of microorganisms and viruses to epithelial cells and neutralizing bacteria, air pollutants, and viruses. It should be noted that the expected composition does not act or function by directly inhibiting microbial infections or viruses to achieve the expected effects. The expected embodiments regulate the homeostasis of the host's self-defense mechanism so as to suppress microbial or viral infections by the host's defense function.

[0022] The subject of protection of the present application designates the homeostasis of the host defense mechanism to the lungs and the whole body. Although it is expected that the subject of protection of the present application maintains systemic mucosal homeostasis in the gastrointestinal tract and urogenital tract, according to the data shown in the main text of the subject of protection of the present application, its main function is mainly to regulate HMGB1 and to induce the forefront of respiratory defense mucosal immunity such as immunoglobulin A (IgA), and it was confirmed that it lies in protecting the structural integrity and function of the respiratory system. The lung protection effect of the subject of protection of the present application was evaluated in vivo in live hosts using a lipopolysaccharide (LPS)-induced acute lung injury model and a microbial infection model under hyperoxia, and also evaluated in vitro using macrophages with reduced function under hyperoxia. When a bioflavonoid composition containing free B-ring flavonoids and flavans was tested in macrophages with reduced function under hyperoxia, the phagocytic activity (innate immune defense) of macrophages was enhanced by inhibiting the release of HMGB1. As in vivo verification of these results, the bioflavonoid composition showed an increase in bacterial clearance in the airways and lungs, significantly suppressed the accumulation of HMGB1 in the airways, reduced the total protein in the lungs of mice exposed to hyperoxia and microbial infection, and was judged to be available for protecting the respiratory tract and lungs. The same respiratory and lung protection effects of the subject of protection of the present application were also observed in the LPS-induced acute lung injury model, and when the bioflavonoid composition was supplemented, the main symptoms of inflammation were reduced and biomarkers and lung injury were decreased. The systemic host defense homeostasis effect of the subject of protection of the present application was also evaluated in a lipopolysaccharide (LPS)-induced sepsis model and a D-galactose-induced accelerated aging model under and without influenza vaccine immunization. In all the tested models, the subject of protection of the present application containing free B-ring flavonoids and flavans showed a statistically significant improvement in the host defense mechanism, and it was verified that it can be used to restore host defense homeostasis locally or systemically.

[0023] Disclosed is a bioflavonoid composition for establishing and regulating the homeostasis of the host defense mechanism, comprising at least one standardized bioflavonoid extract with a high concentration of at least one free B-ring flavonoid and at least one standardized bioflavonoid extract with a high concentration of at least one flavan. The intended composition is effective against respiratory diseases and conditions. As described below, the at least one standardized bioflavonoid extract with a high concentration of at least one free B-ring flavonoid and the at least one standardized bioflavonoid extract with a high concentration of at least one flavan in the composition are in the range of 1% to 98% based on the weight of each extract, and the optimal weight ratio is 80:20. The intended embodiments further include embodiments in which the at least one standardized bioflavonoid extract with a high concentration of at least one free B-ring flavonoid is highly concentrated and standardized from the roots of Sedum aizoon, and the at least one standardized bioflavonoid extract with a high concentration of at least one flavan is highly concentrated and standardized from the heartwood of Machilus thomsonii.

[0024] The object of protection includes a bioflavonoid composition that combines free B-ring flavonoids and flavans. The composition has been shown to inhibit extracellular HMGB1 secretion as a local effect in bronchoalveolar lavage fluid and as a systemic effect in spleen homogenates in hosts exposed to hyperoxia and microbial infection respectively, and in a D-galactose-induced accelerated aging model. Based on major immune or inflammatory response biomarkers such as HMGB1 and NFκB, and in vivo changes related to immunosenescence, an objective evaluation of the composition of the present invention was carried out. The bioflavonoid composition containing free B-ring flavonoids and flavans can increase the survival rate in vivo by regulating HMGB1 and NFκB, significantly enhance macrophage phagocytosis in vitro, and reduce inflammatory cytokines such as TNF-α, IL-1β, IL-6, CRP and CINC3, and is determined to be available for restoring, regulating and maintaining the homeostasis of the host defense mechanism. Similarly, the bioflavonoid composition containing free B-ring flavonoids and flavans disclosed in the present application has been shown to reverse immunosenescence, as evidenced by stimulation of innate and adaptive immune responses (increase in complement C3, CD3+ T cells, CD8+ cytotoxic T cells, CD3-CD49b+ natural killer cells, NKp46+ natural killer cells and CD4+ TCRγδ+ gamma delta T cells), enhancement of antioxidant capacity (decrease in advanced glycation end products, increase in glutathione peroxidase), and protection from age-related functional decline and structural damage of major immune organs such as the thymus.

[0025] The desired composition maintains the immune homeostasis of mammals by optimizing or balancing the immune response; improves immune deficiency due to aging and immune organ senescence; prevents immune deficiency due to chronic inflammation and inflammation; helps maintain a healthy immune response to influenza vaccination and COVID-19 vaccination; helps maintain a healthy immune function against viral and bacterial infections; or protects the immune system from oxidative stress damage induced by air pollution. Furthermore, the desired embodiment includes a composition that modulates HMGB1 as an endogenous or exogenous response attack trigger and shifts the host defense response to restore homeostasis, wherein the HMGB1 is released by immune cells, viruses or microorganisms, immune cells contaminated with air pollutants, host respiratory cells, or cardiovascular cells deteriorated by immune aging, inflammation, or oxidative stress.

[0026] Most importantly, it has been proven in human clinical trials that supplementation with the novel bioflavonoid composition containing free B-ring flavonoids and flavans disclosed in the present application induces IgA, a major mucosal defense-related immunoglobulin. IgA is the most important antibody class present on the mucosal surface of the respiratory tract and plays a role in protecting the mucosal surface from the invasion of microorganisms and foreign antigens. The bioflavonoid composition to be protected in the present application was found to significantly increase the immunoglobulin IgA as a result of supplementation with the bioflavonoid composition disclosed for the protection in the present application in a randomized double-blind placebo-controlled comparative human clinical trial. In subjects supplemented daily with UP446, a standardized bioflavonoid composition containing free B-ring flavonoids and flavans shown for the protection in the present application for 56 days, and in subjects immunized with influenza vaccine on the 28th day and supplemented for a total of 56 days, IgA increased. The increase in IgA indicates enhanced mucosal protection at the inlets of the gastrointestinal tract, respiratory tract, and urogenital tract.

[0027] Regarding the advantages of the combined use of these standardized bioflavonoid extracts derived from two medicinal plants, safflower and Magnolia officinalis Rehd. et Wils., which are the objects of protection of this application, as described in the main text of the objects of protection of this application, an unexpected synergistic effect was observed in the in vivo test using the LPS-induced sepsis model. Generally, considering the host defense mechanism as a lever and the bioflavonoid composition containing free B-ring flavonoids and flavans as a pivot point, the catabolic HMGB1 on one side of the lever was downregulated, and at the same time, the host defense homeostasis or lung protection was achieved by promoting the induction of mucosal immunity on the opposite side, particularly the production of (IgA).

[0028] In a desired embodiment, the standardized bioflavonoid extract in the composition is extracted with any suitable solvent including supercritical fluid of CO2, water, acidic water, basic water, acetone, methanol, ethanol, propenol, butanol, alcohol-water mixture, mixed organic solvents, or a combination thereof.

[0029] Free B-ring flavones and flavonols have the following general structure:

[0030]

Chemical formula

[0031] In a desired embodiment, the at least one standardized bioflavonoid extract with a high concentration of at least one free B-ring flavonoid contains 0.5% to 99.5% of one or more free B-ring flavonoids. In other embodiments, the at least one standardized bioflavonoid extract with a high concentration of at least one flavan contains 0.5% to 99.5% catechin.

[0032] In a desired embodiment, the free B-ring flavonoid includes at least one of baicalin, baicalein, baicalein glycoside, wogonin, wogonin glucuronide, wogonin glycoside, oroxylin, oroxylin glycoside, oroxylin glucuronide, chrysin, chrysin glycoside, chrysin glucuronide, scutellarin and scutellarin glycoside, norwogonin, norwogonin glycoside, galangin, or a combination thereof.

[0033] As demonstrated in Example 1, free B-ring flavonoids were extracted from plants using organic solvents or aqueous solvents. The extraction yield varies depending on the specific plant species and plant part to be extracted, ranging from the first digit before the decimal point of the total biomass to approximately 25%. The free B-ring flavonoids in the extract can be isolated, identified, and quantified by an analytical method combining a UV spectrophotometer and a PDA detector with high-performance liquid chromatography (HPLC). The content of free B-ring flavonoids in the solvent extract ranged from a low level of less than 1% to a high level of >35% (Table 2 of Example 1). Furthermore, in Example 2, the concentration and standardization of free B-ring flavonoids were demonstrated. After optimizing the extraction solvent and extraction conditions, neutralizing, precipitating, and filtering the extract solution from the organic solvent extract of the roots of *Scutellaria baicalensis*, the content of the target free B-ring flavonoids was increased from approximately 35% to 60 - 90%. In Example 2, RM405 containing more than 75% baicalin was produced as the main free B-ring flavonoid derived from the roots of *Scutellaria baicalensis*. A standardized bioflavonoid extract derived from the roots, stems, or whole plants of the genus *Scutellaria* can be obtained by a method of precipitating a basic aqueous extract solution after neutralization with an acidic solution, or by recrystallization in water, or by column chromatography using various resins as carriers, and the bioflavonoids can be concentrated 2 - 3 times to a free B-ring flavonoid purity of 20% - 99%.

[0034] Flavans have the following general structure:

[0035]

Chemical formula

[0036] In certain contemplated embodiments, the at least one standardized bioflavonoid extract having at least one flavan concentrated is selected from catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epiteafolin, epicatechin gallate, gallocatechin gallate, theaflavin, theaflavin gallate, or combinations thereof.

[0037] Catechin is a flavan mainly present in Acacia catechu, Uncaria gambir, cashew nut shells, and green tea, and has the following structure.

[0038] [Chemical formula]

[0039] As demonstrated in Example 3, flavan extracts were produced from various plants by extraction with organic solvents, aqueous solvents, and alcohol solvents. The contents of catechin and epicatechin as total flavans in these plant extracts were quantified by HPLC, and the results are shown in Table 4. After a standardized flavan extract (RM406) derived from the heartwood of Acen yaknok was produced by aqueous extraction, it was concentrated, precipitated, and recrystallized to increase and standardize the flavan content from about 10% to 65%. A standardized bioflavonoid extract derived from the heartwood or bark or whole herb of Acen yaknok or Gambier nut or the shell of cashew nuts can be obtained by a method of precipitation after concentration of the plant extract solution, or a method of recrystallization in an ethanol / water solvent, or column chromatography using various resins as carriers, and the bioflavonoid can be concentrated 2 to 8 times to a flavan purity of 10% to 99%.

[0040] Example 4 demonstrated a method for producing a bioflavonoid composition called UP446 by combining an Acacia extract (RM406 of Example 3) with a total flavan content of >65% as catechin and epicatechin, and a Scutellaria extract (RM405 of Example 2) with a free B-ring flavonoid content of >75% as baicalin, baicalein, etc., and maltodextrin as an excipient. The main and minor component bioflavonoid contents as individual free B-ring flavonoids and flavans were quantified and shown in Table 5, and the total bioflavonoid content was 86%. Table 6 showed four different bioflavonoid compositions derived from various free B-ring flavonoid sources such as the roots (UP446) or stems (UP223) of Calendula officinalis, and various flavan sources such as the heartwood of Machilus thomsonii (UP894-II) or the whole herb of Gambierdiscus toxicus (UG0408). The blend ratios of these compositions varied depending on the bioflavonoid content in each standardized extract and were adjusted according to the intended use and biological functionality. In the subject matter of the present application, the unexpected synergistic effect of using UP446 and UP894-II and combining two different bioflavonoid species, and the unexpected functionality in the regulation of host defense homeostasis were revealed, leading to an improvement in immune function, respiratory health, and protection of lung function.

[0041] Maintaining strict host defense homeostasis is essential for human physiological functions to defend against invading microorganisms, viruses, fungi, pollutants from the outside, remove dead cells, and initiate reconstruction and regeneration functions. When the immune function is overly stimulated, allergic reactions and destructive autoimmune diseases may occur. In addition to aging, oxidative stress, psychological stress, and systemic inflammation, many chronic diseases such as diabetes, obesity, and metabolic syndrome may shift the tipping point of host defense homeostasis and lead to a decline in host defense function. In addition to daily balanced nutrition, exercise, and stress management, well-known healthy lifestyle habits such as supplementation with natural compounds with antioxidant, anti-inflammatory, and immunomodulatory (immunosuppressive or immunostimulatory depending on the state of imbalance of host defense function), antiviral agents, antibiotics, steroids, and prescription drugs of DTHE can exert beneficial balance effects to return the host defense mechanism in a favorable direction. Many polyphenols including bioflavonoids have been classified as immunosuppressive agents because they have been reported to suppress cytokine production essential for the initiation of host defense responses against infection or vaccination. Therefore, the use of polyphenols in the real world to support the host defense mechanism has not been proven in clinical trials.

[0042] Unfortunately, there is very little information regarding what the essential tipping points are for maintaining the homeostasis of the host defense mechanism, and whether there are major biological, physiological, and pathological pathways and biomarkers that can act as tipping point factors accelerating the shift towards the downward spiral process of the host defense mechanism response to pathogens, and the elucidation of this has also not received much attention. Identifying such tipping points is important. Whether active compounds can be found to manufacture compositions that can keep away from the destructive direction of the tipping point and restore the homeostasis of the host defense mechanism is even more important. HMGB1 is such a biomarker that acts as an alarmin related to the decline of intracellular homeostasis balance and can assist in very strong biological responses under viruses such as coronavirus, SARS-CoV-2, microbial infections, and PM2.5 pollutants that lead to destructive decline of host defense function, and I am convinced of this.

[0043] In the airways of animals and humans exposed to long-term oxidative stress, the concentration of the nuclear protein HMGB1 is overwhelmingly high (100-fold compared to healthy controls). HMGB1 was initially identified as a nuclear protein that regulates transcription by stabilizing the structure of nucleosomes and mediating conformational changes in DNA. In contrast to its nuclear role, extracellular HMGB1 induces a marked inflammatory response. Interestingly, evidence has been accumulating in several animal models of lung infection that when high concentrations of extracellular HMGB1 accumulate in the airways, the host defense mechanism against bacterial and viral infections can be directly reduced due to the decline in macrophage function.

[0044] Therefore, the bioflavonoid composition UP894-II (Table 6), which contains 70 - 80% free B-ring flavonoids and 15 - 20% flavans, was utilized to evaluate its effect on macrophages under high oxygen stress. As shown in Example 5, at 8 - 128 μg / mL of UP894-II, the survival rate of macrophages under 24-hour high oxygen exposure did not change (Figure 4). As demonstrated in Figure 5 of Example 6, UP894-II significantly enhanced the phagocytic activity of macrophages in a dose-dependent and statistically significant manner at a low concentration of 3.7 μg / mL. Surprisingly, such protection of the phagocytic activity of macrophages under oxygen stress by UP894-II was closely correlated with the reduction of high oxygen-induced HMGB1 release in macrophages under the administration of UP894-II, and it had exactly the same dose-dependence (Figure 6 of Example 7).

[0045] That is, as a result of the reduction of HMGB1 concentration or the inhibition of its activity in the airways by UP894-II, the bioflavonoid composition disclosed in the present application, the phagocytic activity of macrophages, which are at the forefront of innate immune defense cells, is protected, providing an important host defense mechanism for populations with increased exposure to pathogenic stress and oxidative stress caused by air pollution, viruses such as SARS-CoV-2, and bacterial infections, especially for hosts with chronic inflammatory disorders.

[0046] As described in the text of the protected subject matter of the present application, in multiple in vivo tests (such as the LPS-induced sepsis model of Examples 9 to 12, the LPS-induced acute lung injury model of Examples 13 to 21, and the hyperoxia-exposed microbial infection acute lung injury model of Examples 35 to 39), the objective administration-response effect of the bioflavonoid composition containing the free B-ring flavonoid and flavan disclosed in the present application was evaluated. According to the data shown in these examples of the protected subject matter of the present application, when the standardized composition was orally administered to sepsis or acute lung injury test subjects, a remarkable host defense homeostasis effect was obtained.

[0047] Using the Colby's conventional formula with the data obtained from the LPS-induced survival rate tests demonstrated in Examples 10 and 11, the significant value of combining the free B-ring flavonoids derived from the extract of the genus *Tatsunamisoa* and the flavans derived from the extract of the genus *Acacia* was evaluated and confirmed. According to the Colby method, a standardized preparation containing two or more materials is presumed to have an unexpected synergistic effect when the measured value is higher than the predicted value. In the subject matter of the present application, it was intended to confirm that the bioflavonoid composition has an unexpected synergistic effect on the reduction of mortality and the increase of survival rate. As shown in Example 12, by combining the free B-ring flavonoid extract and the flavan extract, an unexpected synergistic effect was observed on the reduction of mortality or the increase of survival rate. The beneficial effect observed when the said composition was administered exceeded the predicted effect obtained by simply summing up the effects obtained by each of its components at the specified ratio (Table 13). 144 hours after LPS sensitization, the survival rate (SR%) was statistically significantly increased compared to the normal control only in the bioflavonoid composition containing the free B-ring flavonoids and flavans (Table 10). In fact, 24 hours after administration, no death of animals was observed in the bioflavonoid composition (survival rate 100%), but in the groups administered with the genus *Tatsunamisoa* (RM405) and the genus *Acacia* (RM406) alone, mortality rates of 15.4% and 30.8% were observed respectively (Table 10 in Example 11). Although the beneficial uses of these medicinal plants have been reported, to the best of the inventors' knowledge, this is the first time that the administration of a combination of standardized extracts derived from these medicinal plants has resulted in an unexpected outcome of reducing mortality and increasing survival rate in LPS-induced sepsis. In addition to these unexpected outcomes, due to other favorable innate immune responses and adaptive immune responses, particularly the increase in IgA observed in human clinical trials, and the decrease in extracellular HMGB1 described in the subject matter of the present application, the bioflavonoid composition containing the free B-ring flavonoids and flavans has a unique uniqueness in that it guides the direction of the host immune response to a balanced activity and brings about a comprehensive host defense homeostasis.

[0048] Example 13 demonstrated the effect of a standardized bioflavonoid composition containing free B-ring flavonoids and flavans on reducing lipopolysaccharide (LPS)-induced acute inflammatory lung injury in rats. As a result of improving host defense homeostasis by balancing HMGB1, the biomarkers serum TNF-α (Example 14) and IL-1β (Example 15), IL-6 (Example 16), CRP (Example 19), IL-10 (Example 20) and total protein (Example 18) in bronchoalveolar lavage fluid (BAL), and the concentration of CINC-3 (Example 17) in lung homogenate changed significantly, and these changes were subsequently confirmed by histological examination of lung tissue. In Example 21, a statistically significant decrease in the overall severity of lung injury was observed in animals administered the composition disclosed in the present application. In Examples 11 and 12, the advantages of formulating free B-ring flavonoids derived from an extract of the genus Cirsium and flavans derived from an extract of the genus Acacia were evaluated in an LPS-induced sepsis model, and an unexpected synergistic effect was also observed. Data from the protected subject matter of the present application suggest that a bioflavonoid composition containing free B-ring flavonoids and flavans helps maintain the homeostasis of the host defense mechanism by balancing and breaking the vicious cycle including extracellular HMGB1 upstream and subsequent NFκB signaling and cytokine storm. Therefore, these major features of the composition lead to new uses that require a balanced host defense mechanism to protect respiratory function from sepsis or acute or chronic injuries such as, but not limited to, air pollution, seasonal influenza or viruses (e.g., COVID-19) and bacterial infections.

[0049] When LPS is directly infused into the lungs, through partial activation of NFκB, alveolar macrophages release a significant amount of HMGB1, thereby activating the innate immune response, which is known to lead to an increase in the production of primary cytokines such as TNF-α, IL-1β, and IL-6, and the inflammatory protein CRP. These cytokines, either alone or in cooperation, can cause significant lung pathologies and may induce the activation of cytokine and chemokine cascades essential for disease pathology. For example, during an acute inflammatory response, chemotactic cytokines induce neutrophil chemoattractant (CINC-3), which plays an important role in neutrophil recruitment in LPS-induced acute lung injury. The suppression of HMGB1 is a major checkpoint for immune homeostasis to control these major cytokines and chemotactic factors involved in the acute inflammatory response of the lungs. The balance of HMGB1 is a major phenomenon in lung pathologies and has important clinical relevance for cytokine storm intervention and the reduction of the severity of acute respiratory distress syndrome (ARDS).

[0050] Protein or fibrin leakage into the interstitial space is an important factor in pulmonary edema, and an increase in exudate serves as an indicator of disease severity. When the composition is administered, the total protein in the bronchoalveolar lavage fluid decreases in both LPS-induced acute lung injury and acute lung injury in hyperoxia-exposed / PA-infected mice, and the composition is judged to be significant for the alleviation of lung pathologies. From these significant changes in biomarkers in serum, BAL, and homogenates, it is demonstrated that the administration strategy of the composition leads to a statistically significant decrease in the overall severity of lung injury, and such a decrease was subsequently confirmed by histopathological evaluation. Based on the decrease in HMGB1 concentration and NFκB, the increase in airway and lung bacterial clearance, the decrease in total lung protein, the decrease in cytokines, the improvement of histopathological data, and the induction of IgA shown in this application, it is clear that the bioflavonoid composition actually regulates the checkpoint of immune homeostasis and is effective in cytokine storm suppression and the reduction of the severity of acute inflammatory lung injury.

[0051] Therefore, in the protection scope of this application, a bioflavonoid composition containing the free B-ring flavonoid and flavan disclosed in this application was evaluated in hyperoxia-sensitized and Pseudomonas aeruginosa (PA)-infected mice by comparing it with resveratrol as a positive control (Example 35). In this model, first, UP446 (Table 6), a bioflavonoid composition containing more than 60% free B-ring flavonoid and more than 10% flavan, was tested for its ability to increase the survival rate of mice after 7 days of administration. The mortality rate of mice maintained in room air (RA) was 9%, while a mortality rate of 64.29% was observed in mice exposed to hyperoxia for 2 days before PA inoculation (Table 36). On the other hand, after prophylactically administering resveratrol (RES) and UP446 for 7 days before exposing to hyperoxia for 2 days and then inoculating with PA, the mortality rates of the mice were 27.27% and 28.57%, respectively (Table 36). Next, the bioflavonoid composition was tested using a mouse model of oxidative stress / lung infection-induced acute lung injury with both PA-induced lung infection and hyperoxia-induced oxidative stress, and the effect of UP446 on the suppression of acute lung injury induced by lung infection and exacerbated by oxidative stress was investigated (Example 36). The bioflavonoid composition containing the free B-ring flavonoid and flavan produced statistically significant results in mice exposed to hyperoxia and PA infection in terms of a) a decrease in the accumulation of HMGB1 in the airway (Table 40 in Example 39); b) an increase in airway and lung bacterial clearance (Tables 38 and 39 in Examples 37 and 38); and c) an improvement in lung injury reflected by a decrease in BAL total protein (Table 37 in Example 36). This correlates with the fact that UP446 has a significantly high ability in improving host defense against microbial infections that affect the lung. Furthermore, UP446 improved host defense against bacterial infections in the lung and airway. These effects play an important role in the prevention of septic shock and systemic inflammatory response. The population with reduced host defense function due to oxidative stress and viral or microbial infection is increasing, but the data from this study clearly show that UP446, a composition of free B-ring flavonoid and flavan, is beneficial to such a population.

[0052] As demonstrated in the accelerated aging model in Example 22, D-galactose was administered to mice to induce an aging phenotype. Four weeks after the induction of D-galactose, UP446, a composition of free B-ring flavonoids and flavans disclosed in the present application, was administered to mice at two concentrations for 4 weeks. Then, as an immunization, influenza vaccine was inoculated, and host defense mechanisms were measured in multiple assays to examine whether UP446 contributed to a balanced host defense phenotype similar to that of control mice. The significant outcomes are highlighted below. A) In Example 23 and Table 23, the thymus indices of the normal control group and the UP446+D-Gal administration groups at both concentrations were significantly higher than those of the D-Gal group. It was determined that UP446 contributed to the reversal of thymic involution, i.e., the decrease in thymus size associated with aging, which seems to affect the ability of the living body to generate an immune response. B) In Example 24 and Table 24, it was found that there were significant changes in humoral immunity among the immunized groups. In the D-Gal+UP446 (200 mg / kg) group, complement C3 was significantly increased compared to the D-Gal alone group, and in the UP446 administration group, the humoral immune response after immunization was prolonged compared to the D-Gal group. C) In Example 28, where white blood cells in whole blood from various groups were measured, it was found that there were significant differences among the immunized mouse groups. In the immunized UP446+D-Gal group, CD49b+ cells (Table 28) and NKp46+ natural killer cells (Table 29) increased compared to the immunized D-Gal alone group. From these data, it was determined that UP446 promoted the increase in the natural killer cell population and the percentage of innate immune cells increased. D) It was found that there were also significant differences among non-immunized mouse groups. The D-Gal+UP446 group showed a strong tendency towards an increase in CD3+ T cells (P = 0.055 in Table 25), and CD8+ cytotoxic T cells (Table 27), NKp46+ natural killer cells (Table 28), CD4+TCRγδ+ gamma delta T cells (Table 30), and IL12p70 (Table 31) were significantly increased compared to the D-gal alone group. According to these data demonstrated in Examples 25 to 30, UP446, which is the bioflavonoid composition disclosed in the present application, appears to prime the inactivated immune system in non-immunized mice, increase the immune cell population, and enhance immune "readiness". E) To monitor the antioxidant pathway, antioxidant enzymes and biomarkers were tested. The aging phenotype induced by the D-Gal model is based on an increase in advanced glycation end products (AGE), resulting in oxidative stress and damage similar to the levels seen in aged animals (Azman KF, 2019). It is thought that by enhancing the antioxidant pathway, the effect of oxidative stress can be suppressed. First, in Example 31, the concentration of AGE in serum samples of immunized and non-immunized mice was measured. It was found that AGE in mouse serum from the non-immunized D-Gal+UP446 group (both concentrations) was decreased compared to the D-gal alone group (Table 32). Therefore, it was determined that animals administered with UP446 had a low free radical concentration, specifically the free radical concentration contributing to the aging phenotype of the D-Gal model. Next, in Example 32, attention was paid to the activity of glutathione peroxidase (GSH-Px) in mouse serum from immunized animals. It was found that the immunized UP446+D-Gal group at both concentrations had significantly higher GSH-Px activity compared to the immunized D-Gal group (Table 33), and it was determined that animals administered with UP446 had an enhanced ability to neutralize free radicals. F) The protein concentrations in the spleens of animals in the immunized group were also analyzed. The spleen is one of the major organs of the immune system. The spleen contains a high concentration of white blood cells and controls the concentration of immune cell types in the blood. In Example 33, where NFκB, an inflammatory transcription factor activated in response to inflammation, was measured, it was found that NFκB was decreased in the high-dose D-Gal+UP446 administration group (Table 34). From this, it was determined that the decrease in the concentration of NFκB is one of the mechanisms of UP446 for regulating the inflammatory response during host defense homeostasis. HMGB1 is an alarmin protein that is a transcription factor and is a nuclear protein under non-inflammatory conditions, but when it exits the nucleus, it is secreted into the extracellular space and further amplifies the inflammatory signal. As demonstrated in Example 34, in the non-immunized high-dose D-Gal+UP446 group, it was found that the HMGB1 concentration was significantly decreased compared to the D-Gal group (P = 0.053 in Table 35). All of these results indicated that administration of UP446 suppressed oxidative stress and inflammation in the spleens of non-immunized mice.

[0053] Progressive deterioration of tissues and organs, which is partially reflected as antioxidant defense system dysfunction and immune system disorders, is a characteristic of aging. According to the free radical theory of aging, the main contributing factor to aging and age-related degenerative structure and dysfunction of tissues and organs is oxidative damage (imbalance between free radicals and antioxidants) (Azman and Zakaria 2019). An increase in advanced glycation end products (AGE) is known to accelerate the aging process and is considered the main pathway of the aging mechanism in the D-galactose-induced accelerated aging model characterized by poor immune response and antioxidant defense system disorders. In an experiment using the D-galactose-induced animal model to reproduce these natural phenomena, which are the subject of protection of this application, in mice administered D-Gal + solvent, increased oxidative stress, decreased antioxidant enzyme activity, and decreased immune response were observed. In contrast, supplementation with a bioflavonoid composition containing free B-ring flavonoids and flavans reversed age-related structural and functional changes. Supplementation with the bioflavonoid composition UP446 resulted in a statistically significant dose-correlated decrease in serum AGE, and the maximum decrease rate was 58% in the high-dose group (Table 32 in Example 31). Furthermore, the most effective defense mechanism of cells against oxidative damage mainly involves the action of endogenous enzymatic antioxidants such as glutathione peroxidase (GSH-Px). In fact, the bioflavonoid composition exerted a strong antioxidant boosting effect, and GSH-Px increased significantly statistically at all doses (Example 33 in Example 31). Considering the induction of mucosal immunity, preservation of immune organs, decrease in AGE, and increase in endogenous antioxidant enzymes, the bioflavonoid composition containing free B-ring flavonoids and flavans prevents age-related immunoregulatory deficiency and antioxidant defense system dysfunction.

[0054] Supplementation of a bioflavonoid composition to chemically aged mice enhanced innate immunity. Activation and augmentation of natural killer cells are major immunomodulatory methods for maintaining host defense homeostasis. Natural killer cells are an important component of the innate immune system known to respond rapidly to a wide range of pathologic sensitizations; air pollutants; viral, microbial and fungal infections; and cellular oxidative and hormonal disorders without priming or pre-activation. Natural killer cells monitor cell integrity to detect changes in cell surface molecules and deploy their cytotoxic effector mechanisms. Natural killer (NK) cells function as cytotoxic lymphocytes and producers of immunomodulatory cytokines. After stimulation, NK cells produce large amounts of cytokines, mainly interferon-γ (IFN-γ) and tumor necrosis factor (TNF). These cytokines and the like produced by NK cells act directly during the early immune response and are important modulators of the late adaptive immune response mediated by T cells and B cells. As a result of oral administration of the bioflavonoid composition, the marked increase in NK cells, which are the intended protected subjects of the present application, clearly shows that the protected subjects have an important influence on innate immune regulation, and suggests that its immediate and effective immune induction activity is involved in building the basis of immune homeostasis. This activation of innate immunity as natural killer cells is another aspect in which the bioflavonoid composition induces a response to protect the respiratory tract and maintain mucosal homeostasis.

[0055] The induced levels observed in CD4+ TCRγδ+ gamma-delta T cells, which are known to be responsible for immune regulation, promotion of immune surveillance, and immune homeostasis, confirmed the mucosal immune regulation and host defense homeostasis activities of the subject matter of the present application. Gamma-delta T cells are a unique subset of T cells that mainly exist at many inlets in the living body, including the lungs and intestines. They move to such locations at the early stage of development and survive as resident cells. Due to their strategic anatomical positions (the mucosal layers of the respiratory and gastrointestinal systems), gamma-delta T cells provide the front line of defense based on their innate immune-like responses by directly killing infected cells, mobilizing other immune cells, activating phagocytosis, and restricting the migration of pathogens or contaminants to the systemic compartment. These cells are known to rapidly expand their population upon secondary sensitization and provide pathogen-specific defense. Their ideal positions in the respiratory and intestinal tracts also help maintain respiratory and intestinal epithelial integrity. Generally, the physiological roles of gamma-delta T cells include defensive immunity against extracellular and intracellular pathogens or contaminants, surveillance, regulation, tissue healing, and epithelial cell maintenance of innate and adaptive immune responses, as well as regulation of physiological organ functions. Gamma-delta T cells share characteristics with natural killer (NK) cells. Both are usually regarded as components of innate immunity, recognize transformed / damaged cells, play a prominent role in antiviral defense, promote downstream adaptive immune responses, and are powerful cytolytic lymphocytes. Furthermore, gamma-delta T cells also act as antigen-presenting cells (Ribot et al., 2021; Bonneville et al., 2010). The bioflavonoid composition UP446, which is the subject matter of the present application, induced these rapidly responding immune cells (gamma-delta T cells and NK cells), resulting in mucosal immune regulation and host defense homeostasis.

[0056] In summary, in D-Gal mice administered with UP446, which is a composition of free B-ring flavonoids and flavans, significant changes were observed compared to the D-Gal alone group. The host defense mechanism of the aging animals was reversed so as to approach the phenotype of normal control mice, or at least the priming and activation of the host defense system were enhanced. The thymus index, serum complement, natural killer cells, and glutathione peroxidase activity in the immunized D-Gal+UP446 group were higher than those in the D-gal alone group, and it was determined that the host defense system of the UP446-administered group could respond better to vaccination than the D-Gal-induced aging alone group. CD8+ cytotoxic T cells, natural killer cells, and CD4+ TCRγδ+ gamma delta T cells in the non-immunized D-Gal+UP446 group were higher than those in the D-gal alone group, and the AGE concentration and NFκB concentration were decreased compared to the D-gal group. It was determined that both the innate immune response and the adaptive immune response were primed and oxidative stress and inflammation were reduced. From these results, it is clear that UP446, which is a composition of free B-ring flavonoids and flavans, is useful for helping to activate the host defense system in both vaccination or during the activity of infection and preventive measures so as to prime the host defense system against infection.

[0057] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged RNA virus and is the cause of the coronavirus disease 2019 (COVID-19) pandemic, which results in various clinical outcomes ranging from asymptomatic infection to lung injury, inflammation, respiratory distress, multiple organ failure, and death. Extracellular HMGB1 secreted in the lungs infected with SARS-CoV-2 is considered a therapeutic target in severe pulmonary inflammation of COVID-19 (Andersson et al 2020). Herbal medicines are being investigated for the treatment of SARS-CoV-2 virus adhesion, acute respiratory failure, and sepsis by inhibiting HMGB1 release (Wyganowska-Swiatkowska et al.2020). In view of the binding of human angiotensin I-converting enzyme 2 (hACE2) as the main entry point of the virus to the SARS-CoV-2 spike protein, a transgenic mouse model expressing human ACE2 was sensitized with SARS-CoV-2 to enhance the effects of modeling and intervention. As shown in Example 40, transgenic mice infected with the SARS-CoV-2 virus and administered a solvent showed a statistically significant two-fold increase in lung HMGB1 protein expression compared to uninfected normal transgenic control mice. In contrast, when transgenic mice infected with the SARS-CoV-2 virus were supplemented with UP894-II, a bioflavonoid composition containing 70-80% free B-ring flavonoids and 15-20% flavans, the expression of HMGB1 protein in lung tissue decreased to the level of uninfected normal control transgenic mice (Figure 8). This decrease in the lung HMGB1 expression level as a result of the administration of the bioflavonoid composition was statistically significant compared to transgenic mice infected with SARS-CoV-2 and administered a solvent. HMGB1 is a major late alarmin known to activate a complex sequence of host immune responses, and if not suppressed, it leads to cytokine storm, disruption of host defense homeostasis, and ultimately harmful clinical symptoms as seen in hospitalized COVID-19 patients.Since the expression of HMGB1 in the lung tissue of this transgenic mouse infected with SRS-Cov2 was significantly and statistically significantly reduced, it was determined that the host defense mechanism was improved, homeostasis was induced, and cytokine storm lethality caused by SARS-CoV-2 coronavirus infection and related lung and other organ damage were reduced by the bioflavonoid composition containing free B-ring flavonoids and flavans.

[0058] Presumably, the most prominent primary outcome of the regulation of the host defense mechanism from UP446, a unique bioflavonoid composition containing more than 60% free B-ring flavonoids and more than 10% flavans, was the change in serum IgA in healthy volunteers demonstrated from a human clinical trial in Example 41. In a double-blind placebo-controlled comparative clinical trial, healthy middle-aged subjects (Table 42) were supplemented with 250 mg of UP446 twice a day or placebo daily for 28 days before sensitizing their immune system with the influenza vaccine (Table 41). The subjects continued to take UP446 or placebo for another 28 days, and blood samples were taken at baseline, after 28 days of intake, and after 56 days of intake (28 days after vaccination) to perform host defense biomarker measurements. After 8 weeks of intake, it was found that in the subjects who took UP446, a bioflavonoid composition, mucosal immune indices such as immunoglobulin A before and after influenza vaccination were significantly increased compared to the placebo group. In the UP446 intake group, the changes in IgA from day 0 to 56 days and from day 28 to 56 days were significantly higher in the within-group comparison. Throughout the supplementation period, the subjects who took UP446, a bioflavonoid composition, showed a significant increase in IgA concentration after influenza vaccination compared to the placebo group. These data clearly show that IgA, which is the main immunoglobulin of a healthy respiratory system and is considered the most important immunoglobulin for mucosal defense, is one of the main indicators for improving the homeostasis of the host defense mechanism in humans.

[0059] The respiratory system (i.e., the lungs and upper airway) is frequently exposed to various pathogens and contaminants inhaled during breathing, and has a large mucosal surface area (400 - 500 m 2 ) at the preferred sites that serve as its inlets. Continuously attacked by numerous airborne microorganisms, particles, contaminants, and environmental antigens, the mucosal surface of the respiratory tract must engage in robust non-specific and specific defense mechanisms to protect against respiratory infections and injuries. In addition to physiological defenses (coughing, sneezing, and mucociliary clearance) and the removal of particles and microorganisms by alveolar macrophages, the mucosal humoral immune response, more specifically, the induction of IgA production in the respiratory tract, is the most important aspect of respiratory system protection. IgA is thought to function as an effective front line of respiratory / lung defense against foreign substances in cooperation with non-specific natural immune factors without inducing potentially harmful inflammatory responses. In fact, bioflavonoid compositions containing free B-ring flavonoids and flavans correspond to the natural immune response by enhancing macrophage phagocytic activity, while promoting the adaptive immune response by stimulating mucosal immunity, particularly the production of IgA. IgA, the major class of immunoglobulins in the respiratory mucosa, is the most important immunoglobulin for respiratory and lung defense, and it is known to a) protect the mucosal surface from invasion by microorganisms and foreign antigens, b) neutralize bacterial products, c) eliminate pathogens or antigens that have invaded the mucosal surface via the secretory pathway mediated by IgA, d) agglutinate microorganisms and interfere with bacterial motility, and e) neutralize viruses intracellularly by interacting with viral antigens during transcytosis and interfering with virus synthesis or assembly (Pilette et al., 2001). As described in the main text of the present application and demonstrated in particular in the human clinical trial shown in Example 41, supplementation with a bioflavonoid composition containing free B-ring flavonoids and flavans induces mucosal immunity, particularly increasing the production of IgA in human clinical trials and enhancing the phagocytic activity of hyperoxic macrophages, suggesting that the main roles of the present application's protection target are lung protection and maintenance of mucosal immune homeostasis.

[0060] In summary, using both cell culture and animal models, it is shown that prolonged exposure to oxidative stress during oxygen therapy, which is routinely used to treat COVID-19 patients, dramatically releases HMGB1, shifts the balance of the immune response, induces a decline in innate immunity, reduces macrophage function, and consequently impairs host defense against pathogens invading the respiratory tract and lungs, leading to acute inflammation of the respiratory tract and lung injury, and ultimately death. Using these model systems, it is demonstrated that HMGB1 is a novel cellular and molecular mechanism underlying the cause of susceptibility to lung infections induced by oxidative stress, and that a bioflavonoid composition containing free B-ring flavonoids and flavans, as shown in FIGS. 1 and 2, improves innate immunity and alleviates respiratory function decline by shifting HMGB1 in these hosts. In an example of administration of a bioflavonoid composition containing free B-ring flavonoids and flavans, the accumulation of extracellular HMGB1 was suppressed, respiratory function was improved, innate immunity was enhanced against bacterial and viral infections, and the inflammatory response was suppressed by improving the homeostasis of the host defense mechanism.

[0061] The subject of protection of the present application, which regulates HMGB1 by free B-ring flavonoids and flavans, although not limited, as shown in Figure 3, a) targets the active or passive release of HMGB1 by preventing cytoplasmic translocation or preventing vesicle-mediated release, or inhibits HMGB1 release or interferes with its action by inhibiting the formation of intramolecular disulfide bonds in the nucleus; b) directly targets HMGB1 at the time of release and neutralizes its action; c) can be expressed as a result of blocking HMGB1 pattern recognition receptors such as Toll-like receptor (TLR)-2 / 4 / 7 / 9 and receptor for advanced glycation end products (RAGE), or inhibiting their signal transduction. Inhibition of oxidative stress-mediated HMGB1 release in infection, inflammation, and cell death can target 1) the nuclear export of HMGB1 by CRM1 in activated immune cells, 2) HMGB1 release by PARP1 in necrosis, 3) HMGB1 release by caspases 3 / 7 in apoptosis, 4) HMGB1 release by ATG5 in autophagy, 5) HMGB1 release by PKR in pyroptosis, and 6) HMGB1 release by PAD4 in NETosis. The action of the bioflavonoid composition containing free B-ring flavonoids and flavans can also be expressed by preventing the clustering or self-association of HMGB1, and can be achieved by targeting specific physicochemical factors such as ionic strength (the concentration of HMGB1 tetramers decreases with increasing ionic strength), pH (the degree of self-association is highest at pH 4.8), metal ions, especially zinc (the addition of low-dose Zn2+ promotes the formation of HMGB1 tetramers), and redox environment (under oxidative conditions similar to the extracellular environment, HMGB1 mainly exists as tetramers, and under reducing conditions similar to the intracellular environment, more dimer species exist). The bioflavonoid composition prevents the formation of HMGB1 tetramers and interferes with the binding affinity of HMGB1 for TLR and RAGE by changing the physicochemical microenvironment.

[0062] In the descriptions above and below, certain specific matters are described in order to enable a full understanding of various embodiments of the present disclosure. However, as is obvious to those skilled in the art, the subject matter of the present application can be implemented without these matters without limitation.

[0063] In the description of the present application, unless otherwise specified, all concentration ranges, percentage ranges, ratio ranges, or integer ranges should be understood to include all integer values within the specified range and, where appropriate, fractions thereof (for example, one tenth or one hundredth of an integer). Also, all numerical ranges specified in the present application regarding all physical characteristics such as polymer subunits, sizes, or thicknesses should be understood to include all integers within the specified range unless otherwise specified. The terms "about", "comprising", "consisting of", and "essentially consisting of" used in the present application, unless otherwise specified, mean the average of the specified range, numerical value, or structure ±20%. The indefinite article used in the present application should be understood to mean "one or more" of the recited elements. The use of alternatives (e.g., "or" or "and / or") should be understood to mean any one, both, or any combination of those alternatives. Unless the context is inconsistent, throughout this specification and the claims, the terms "comprising" and its variations such as "(singular) comprising" and "comprises", and synonyms such as "including" and "having" and their variations should be interpreted in a broad inclusive sense, that is, in the sense of "including but not limited to".

[0064] Whenever the term "one embodiment" or "an embodiment" is used anywhere in this specification, it means that the specific features, structures, compositions, or characteristics described in connection with this embodiment are included in at least one embodiment of the subject matter of the present application. Therefore, when the expression "in one embodiment" or "in an embodiment" is used at various places throughout this specification, it does not necessarily mean the same embodiment in all cases.

[0065] Also, the term "prodrug" means a compound in which the active compound of the present disclosure is covalently bonded to any carrier and is released in vivo when such a prodrug is administered to a mammalian subject. The prodrugs of the compounds of the present disclosure can be prepared by modifying the functional groups present in the compounds of the present disclosure so as to cleave either in routine operations or in vivo to return to the parent compounds of the present disclosure. Examples of prodrugs include those in which a hydroxy group, an amino group or a mercapto group is bonded to any group of the compounds of the present disclosure and which cleave to form a free hydroxy group, a free amino group or a free mercapto group, respectively, when the prodrug of the compound of the present disclosure is administered to a mammalian subject. Examples of prodrugs include acetic acid, formic acid and benzoic acid derivatives of the alcohol moiety in the compounds of the present disclosure or amide derivatives of amine functional groups, etc.

[0066] The terms "stable compound" and "stable structure" mean a compound that is isolated from a reaction mixture to a useful purity and is sufficiently robust to be formulated into an effective therapeutic agent having a reasonable shelf life.

[0067] A "biomarker" or "marker" component or compound is one or more chemical components or compounds unique to the plants, plant extracts or combined compositions of two or three plant extracts disclosed in the present application, and is used to control the quality, consistency, integrity, safety or biological function of the compositions of the present invention.

[0068] "Mammal" includes, in addition to humans, domestic animals such as laboratory animals or family pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals such as wild animals.

[0069] "Optional" or "optionally" means that the element, component, event or circumstance modified by this term may or may not be present, and that there are cases where these elements, components, events or circumstances are present and cases where they are not present. For example, "optionally substituted aryl" means that this aryl group may or may not be substituted, and includes both substituted aryl groups and unsubstituted aryl groups.

[0070] "Pharmaceutically or nutritionally acceptable carriers, diluents or excipients" includes any adjuvant, carrier, excipient, lubricant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that has been approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. In a desired embodiment, the composition further contains a pharmaceutically or nutritionally acceptable active ingredient, adjuvant, carrier, diluent or excipient, and the pharmaceutical or nutraceutical formulation contains from about 0.1 weight percent (wt%) to about 99.9 wt% of an active compound in the at least one standardized bioflavonoid extract.

[0071] "Pharmaceutically or nutritionally acceptable salts" include both acid addition salts and base addition salts. "Pharmaceutically or nutritionally acceptable acid addition salts" refer to salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc., which maintain the biological effectiveness and properties of the free base and are not inappropriate biologically or otherwise.

[0072] "Pharmaceutically or nutritionally acceptable base addition salts" means base addition salts that maintain the biological effectiveness and properties of the free acid and are not inappropriate biologically or otherwise. These salts are produced by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In certain embodiments, the inorganic salt is an ammonium salt, a sodium salt, a potassium salt, a calcium salt, or a magnesium salt. Salts derived from organic bases include primary amines, secondary amines, tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and salts of basic ion exchange resins, for example, salts of ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly useful organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0073] Solvates of the compounds of the present disclosure are often formed by crystallization. The term "solvate" as used in this application means an aggregate containing one or more molecules of the compound of the present disclosure and one or more molecules of a solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Accordingly, the compounds of the present application can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvates. The compounds of the present disclosure can be true solvates, but the compounds of the present disclosure may simply hold incidental water or may be a mixture of water and some incidental solvent.

[0074] The "pharmaceutical composition" or "nutraceutical composition" means a formulation consisting of the compound of the present disclosure and a medium generally acceptable in the art for delivering this bioactive compound to a mammal (e.g., a human). For example, the pharmaceutical composition of the present disclosure may be formulated or used as a single composition, or may be formulated or used as a component or an active pharmaceutical ingredient (API) in a prescription drug, an over-the-counter (OTC) drug, a botanical drug, a crude drug, a natural drug, a homeopathic agent, or any other form of health care product that has been reviewed and approved by a government agency. A typical nutraceutical composition of the present disclosure may be formulated or used as a single composition, or may be formulated or used as a nutritional or bioactive ingredient in a food, a functional food, a beverage, a bar, a food flavor, a medical food, a dietary supplement, or a crude drug product. The medium generally acceptable in the art includes all carriers, diluents, or excipients that are pharmaceutically or nutraceutically acceptable for this purpose.

[0075] As used herein, "concentration increase" means a plant extract or other formulation in which the amount of one or more active compounds is increased by at least 2-fold to about 1000-fold compared to the amount of one or more active compounds present in the weight of the plant material or other raw material before extraction or other manufacturing processes. In certain embodiments, the weight of the plant material or other raw material before extraction or other manufacturing processes can be the dry weight, wet weight, or a combination thereof. In a desired embodiment, the standardized bioflavonoid extract is concentrated by solvent precipitation, neutralization, solvent partitioning, ultrafiltration, enzymatic digestion, silica gel, XAD, HP20, LH20, C-18, alumina oxide, polyamide, ion exchange resin, column chromatography using CG161 resin as a carrier, or a combination thereof, either individually or in combination.

[0076] As used herein, the "main active ingredient component" or "main active ingredient" refers to one or more active compounds present in a plant extract or other formulation, or concentrated to a high concentration in a plant extract or other formulation, and capable of exerting at least one biological activity. In certain embodiments, the main active ingredient component of the highly concentrated extract will be one or more active bioflavonoid compounds concentrated to a high concentration in said extract. Generally, one or more main active ingredients in said bioflavonoid composition will directly or indirectly provide the majority (i.e., more than 60%, or more than 50%, or more than 20% or more than 10%) of one or more measurable biological activities or effects as compared to other extract components. In certain embodiments, the main active bioflavonoid may be a minor component present in a weight percentage less than half of the extract (e.g., less than 50%, less than 25%, or less than 10% or less than 5% or less than 1% of the bioflavonoid contained in the extract), but can provide the majority of the desired biological activity. All bioflavonoid compositions of the present disclosure containing a main active ingredient such as baicalein as one of the free B-ring flavonoids may further contain epicatechin as a flavan which is a minor active ingredient, and said minor active ingredient may or may not contribute to the pharmaceutical or nutraceutical activity of the highly concentrated composition, but does not contribute to the concentration of the main active ingredient, and the minor active ingredient may not be effective alone in the absence of the main active ingredient component.

[0077] "Effective amount" or "therapeutically effective amount" means an amount of the bioflavonoid compound or composition of the present disclosure sufficient to shift the set point of host defense mechanisms to effect an improvement in immune function including any one or more of: (1) stimulation of innate immunity; (2) enhancement of adaptive immunity, particularly increases in CD4+ and CD8+, complement C3, CD3+ T cells, CD8+ cytotoxic T cells, CD3-CD49b+ natural killer cells, NKp46+ natural killer cells, and CD4+ TCRγδ+ gamma delta T cells; (3) suppression of chronic systemic inflammation and oxidative stress; (4) protection of immune cells, respiratory cells, and lung cells against HMGB1-induced cytokine storm damage; (5) functioning as a potent antioxidant to suppress oxidative stress, reduce NF-kb, reduce advanced glycation end products, increase glutathione peroxidase, neutralize reactive oxygen species, and prevent destruction of the structural integrity and decline in function of the respiratory, lung, and immune systems due to oxidative stress; (6) maintenance of the homeostasis of innate and adaptive immune responses; (7) increase in the phagocytic index of macrophages in humoral and cellular immune responses; (8) inhibition of the activation of transcription factors such as NF-kB, NFAT, and STAT3; (9) inhibition of lymphocyte activation and inflammatory cytokine gene expression (IL-2, iNOS, TNF-α, COX-2, and IFN-γ); (10) decrease in the concentrations of inflammatory cytokines such as IL-1β, IL-6, and TNF-α; (11) downregulation of the expression of COX-2, NOS-2, and NF-κB; (12) inhibition of eicosanoid production by inhibiting phospholipase A2 and TXA2 synthase activities; (13) suppression of Th1 and Th17 cell responses; (14) decrease in neutrophil chemotaxis due to decreased expression of ICAM and VCAM; (15) inhibition of MAPK phosphorylation, adhesion molecule expression, and signal transducer and activator of transcription 3 (STAT-3); and (16) activation of the transcription factor NRF2 and induction of heme oxygenase-1 when administered to mammals such as humans.

[0078] Compositions for modulating the homeostasis of the host defense mechanism by variously combining two or three plant extracts (e.g., UP446 or UP894-2 containing free B-ring flavonoids and flavans as disclosed herein, without limitation). "Biomarkers" related to host defense functions, lung structure integrity, and function modulated thereby include, without limitation, hemagglutinin inhibition (HI) titer against specific virus strains, IgA, IgG, IgM, CD3+, CD4+, CD8+, CD45+, TCRγδ+, CD3-CD16+56+, GM-CSF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, TNF-α, TNF-β / LTA150, G-CSF, CCL2 / 3 / 5, IP-10, CXCL10, CRP, HMGB1, Nrf-2, INF-α / β / γ, NF-κB, PDGF-BB, MIP-1α, D-dimer, angiotensin II, cardiac troponin, VEGF, PDGF, albumin, SOD, MDA, 8-isoprostaglandin F2α, catalase (CAT), advanced glycation end products (AGEP), glutathione peroxidase, iNOS, COX1, COX2, LO5, LO12, LO13.

[0079] "Viruses" used in this application include, without limitation, highly pathogenic avian influenza (A type H5N1 virus strain), A type influenza (H1N1, H3N2, H5N1), B type influenza / Washington / 02 / 2019-like virus, B type influenza / Puert / 3073 / 2013-like virus, hepatitis A, B, C, and D viruses, coronavirus SARS-CoV, SARS-CoV-2 (COVID-19), MERS-CoV (MERS), respiratory syncytial virus (RSV), enterovirus A71 (EV71), parainfluenza, and adenovirus.

[0080] The "microorganisms" used in the present application include, but are not limited to, pathogenic bacteria that infect the respiratory system. The most common bacterial pathogens include Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, and Moraxella catarrhalis. The main pulmonary fungal pathogens in upper and lower respiratory tract infections include Aspergillus, Cryptococcus, Pneumocystis, Histoplasma capsulatum, Blastomyces, Cryptococcus neoformans, Pneumocystis jiroveci, Candida spp., and endemic fungi. The main bacterial pathogen of pharyngitis and tonsillitis is Streptococcus pyogenes. Bacterial infections may also occur after contracting viral diseases such as colds and influenza.

[0081] As used herein, the "respiratory tract and lungs" include, but are not limited to, the airways that deliver air to the lungs and oxygen from the lungs to all other organs of the host, the mouth and nose which are openings for taking in air from outside the host into the host respiratory system, the sinuses which are cavities between the bones of the host head and help regulate the temperature and humidity of the air inhaled by the host, the pharynx (throat) which is a tube that delivers air from the host's mouth and nose to the trachea (windpipe), the trachea which is a passage connecting the host's larynx and lungs, the bronchi which are tubes leading from the bottom of the host's windpipe to each lung, the lungs which are organs composed of two parts that extract oxygen from the air and deliver it into the host's blood, the bloodstream that delivers carbon dioxide to the lungs and oxygen from the lungs to all organs and other tissues of the host, and the muscles and bones that help move the air inhaled by the host in and out of the host's lungs.

[0082] "Respiratory infections" include the symptoms of the common cold, such as nasal congestion, runny nose, sneezing, mild fever, headache, sore throat, chest tightness, wheezing, dry cough and coughing, fatigue, shortness of breath, congestion, hoarseness, pain and difficulty in swallowing, swollen lymph nodes, facial tenderness (especially under the eyes or the bridge of the nose). Some warning signs that a common cold is progressing from a viral infection to a bacterial infection include symptoms lasting for 10 to 14 days or more, fever exceeding 100.4 degrees, fever that worsens without improvement for several days, spots with white pus on the tonsils, sinusitis with postnasal drip, nasal congestion / stuffy nose, toothache, cough, greenish nasal discharge, facial tenderness (especially under the eyes or the bridge of the nose), bad breath, fatigue, fever, but are not limited thereto.

[0083] "Pulmonary infection" or "pneumonia" is the most common bacterial or viral lower respiratory tract infection. It may also be caused by exposure to air pollutants, tobacco, e-cigarettes, or recreational marijuana smoking. It is an infection that causes inflammation in the air sacs of one or both lungs, and mucus or pus accumulates in these air sacs. Symptoms of pneumonia include, but are not limited to, cough with sputum or pus, fever, chills, difficulty breathing, severe chest pain, dehydration, fatigue, loss of appetite, skin sweating and stickiness, rapid breathing, shallow breathing, shortness of breath, wheezing, high heart rate, and decreased blood oxygen saturation. "Pulmonary infection" or "pneumonia" can be diagnosed by chest X-ray, CT scan, blood tests, and sputum culture. Resident macrophages function to protect the lungs from foreign pathogens, are triggered by the inflammatory response of pathogens, and are the cause of the histopathological and clinical findings seen in pneumonia. Macrophages engulf these pathogens, trigger signal molecules or cytokines such as TNF-a, IL-6, and IL-1, and recruit inflammatory cells such as neutrophils to the site of infection. Macrophages further present these antigens to T cells, trigger both cellular and humoral defense mechanisms, activate complement, and function to form antibodies against these organisms. As a result, inflammation of the lung parenchyma occurs, the alveolar wall capillaries become more "leaky", causing exudative hemorrhage, leading to the onset of pneumonia.

[0084] The amount of the compounds, extracts or compositions of the present disclosure that constitute a "therapeutically effective amount" or "nutritional effect amount" varies depending on the bioactive compound, or nutritional component, or biomarker of the disease state to be treated and its severity, the mode of administration, the administration period or the dietary supplement period, or the age of the subject to be treated, but can be routinely determined by a person having ordinary knowledge in the art based on their own knowledge and in light of the present disclosure. In certain embodiments, the "effective amount" or "therapeutically effective amount" or "nutritional effect amount" can be expressed as an amount per mammalian body weight (i.e., 0.005 mg / kg, 0.01 mg / kg, or 0.1 mg / kg, or 1 mg / kg, or 5 mg / kg, or 10 mg / kg, or 20 mg / kg, or 50 mg / kg, or 100 mg / kg, or 200 mg / kg or 500 mg / kg). By utilizing the FDA guidelines taking into account the differences in total body surface area and body weight between animals and humans, the human equivalent daily dose can be estimated from the "effective amount" or "therapeutically effective amount" or "nutritional effect amount" in animal studies.

[0085] The "dietary supplement" used in the present application is a product that improves, promotes, enhances, manages, controls, maintains, optimizes, modifies, suppresses, inhibits, establishes, or prevents an imbalance, decrease, suppression, or overstimulation of a specific pathological condition, biological function, phenotypic state, or defense mechanism related to homeostasis, balance, natural state, or biological processes, or structural and functional integrity (i.e., it is not used for diagnosing, treating, alleviating, curing, or preventing diseases). For example, regarding the host defense mechanism, it can enhance the efficacy of vaccines, strengthen the phagocytic activity of macrophages, improve the natural killing activity of NK cells, regulate the production levels of inflammatory cytokines, reduce inflammation and tissue damage, induce antibody responses and production, strengthen antibody-dependent cell cytotoxicity, stimulate T cell proliferation, promote the generation of regulatory T cells which are immunosuppressive cells, protect immune cells and lung cells from HMGB1-induced cytokine storm damage, suppress the uncontrolled activation of NFκB, and protect organs or tissues from oxidative stress. As an immunoadjuvant specific to immunostimulants, the "dietary supplement" can be used to regulate, maintain, manage, balance, suppress, or stimulate any component of adaptive immunity or innate immunity. In certain embodiments, the dietary supplement is a dietary supplement of a special classification, natural nutrition, food, functional food, or medical food, and is not a pharmaceutical product.

[0086] As used in the present application, "treating" or "treatment" means treating the corresponding disease or pathological condition in mammals such as humans having the said corresponding disease or pathological condition, and (i) in particular, when a mammal has a predisposition to the said disease or pathological condition but has not yet been diagnosed as having the said disease or the like, preventing the occurrence of the said disease or pathological condition in the mammal; (ii) suppressing the said disease or pathological condition, i.e., stopping its occurrence; (iii) alleviating or modifying the said disease or pathological condition, i.e., causing regression of the said disease or pathological condition; or (iv) alleviating the symptoms caused by the said disease or pathological condition without addressing the underlying disease or pathological condition (for example, alleviating cough and fever, alleviating pain, suppressing inflammation, suppressing pulmonary edema, alleviating pneumonia); (v) includes balancing the regulation of immune homeostasis or changing the phenotype of the said disease or pathological condition.

[0087] The terms "disease" and "pathological condition" used in this application may be used synonymously, or may not be synonymous in the sense that a specific pathological condition or the causative agent of a pathological condition is unknown (therefore, the cause has not yet been determined), and thus it has not yet been recognized as a disease, but some specific symptom groups have been confirmed by clinicians and it is only recognized as an undesirable pathological condition or syndrome. A disease or pathological condition may be acute, such as a viral infection (SARS, COVID-19, MERS, hepatitis, influenza) or a microbial infection, or may be chronic, such as lung damage caused by air pollution or exposure to smoke. As a result of a persistent imbalance leading to a weakened immune function, it may cause a disease or pathological condition, make a mammal more susceptible to infectious diseases, or lead to secondary organ and tissue damage directly or indirectly related to viral or bacterial infections or air pollutants.

[0088] The term "statistical significance" used in this application means that when calculated using Student's t-test, the p-value is 0.050 or less, indicating that it is unlikely that a particular event or measurement result occurred by chance.

[0089] For the purpose of administration, the compound to be protected in this application may be administered as a crude compound or formulated as a pharmaceutical or nutraceutical or food composition. The pharmaceutical or nutraceutical composition to be protected in this application contains a compound having the structure described in the object of protection of this application and a carrier, diluent or excipient acceptable in pharmaceuticals or nutraceuticals or conventional foods. The compound having the structure described in this application is in an effective amount for treating a specific corresponding disease or pathological condition or for supplementing natural nutrients, i.e., sufficient to establish the homeostasis of the host defense mechanism or to promote any of innate immunity or adaptive immunity or immune homeostasis in general or other related effects described in this application, and is generally present in the composition in an amount that is not toxic to the host or is acceptable.

[0090] Administration of the compounds or compositions of the present disclosure, or pharmaceutically or nutraceutically acceptable salts thereof, in pure form or as suitable pharmaceutical or nutraceutical compositions, can be effected by any of the modes of administration acceptable for agents of similar use. The pharmaceutical or nutraceutical compositions of the present disclosure can be manufactured by adding a pharmaceutically or nutraceutically acceptable carrier, diluent or excipient appropriate for the compounds of the present disclosure, and can be formulated into solid, semi-solid, liquid or gaseous preparations, for example, tablets, capsules, powders, granules, ointments, solutions, beverages, suppositories, injections, inhalants, gels, creams, lotions, tinctures, sachets, instant beverages, masks, microspheres, and aerosols. The bioflavonoid compositions disclosed in the present application can also be formulated as conventional foods, functional foods, nutritional foods, and medical foods together with other food ingredients. Typical routes of administration of such pharmaceutical or nutraceutical compositions include oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, or intranasal. The term parenteral as used in the present application includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0091] The pharmaceutical or nutraceutical compositions of the present disclosure are formulated such that when the composition is administered to a patient, the active ingredient contained in the composition becomes bioavailable. The composition administered to a subject or patient or mammal will be in the form of one or more dosage units. For example, one tablet can be taken as one dosage unit, and a container filled with an aerosol of the compound or extract of the present disclosure or a composition of two to three plant extracts can hold multiple dosage units. The actual manufacturing methods of such dosage forms are known to those skilled in the art or will be readily understood. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any case, the composition to be administered will contain a therapeutically effective amount of the compound of the present disclosure or a pharmaceutically or nutraceutically acceptable salt thereof for treating the relevant disease or condition in accordance with the teachings of the subject matter protected by the present application.

[0092] The pharmaceutical or nutraceutical composition of the present disclosure may be solid or liquid. In one embodiment, the carrier is granular, and thus the composition is in the form of, for example, tablets or powders. The carrier may be liquid, and the composition is, for example, an oral syrup, an injection solution, or an aerosol useful for inhalation administration.

[0093] For oral administration, the pharmaceutical or nutraceutical composition is solid or liquid, and semi-solid, semi-liquid, suspension, and gel forms are included in the dosage forms regarded as solid or liquid in the present application.

[0094] As a solid composition for oral administration, the pharmaceutical or nutraceutical composition can be formulated into dosage forms such as powders, granules, compressed tablets, pills, capsules, chewing gums, sachets, wafers, bars, etc. Such solid compositions generally contain one or more inert diluents or edible carriers. Further, binders such as carboxymethyl cellulose, ethyl cellulose, cyclodextrin, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate and Sterotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose and saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and one or more coloring agents may be added.

[0095] When the pharmaceutical or nutraceutical composition is in the form of a capsule (for example, a gelatin capsule), in addition to the above types of materials, it can contain a liquid carrier such as polyethylene glycol and oils.

[0096] The pharmaceutical or nutraceutical composition may be in the form of a liquid preparation, for example, it may be an elixir, tincture, syrup, solution, emulsion or suspension. The liquid preparation, to give two examples, may be for oral administration or for delivery by injection. In the case of oral administration, useful compositions contain, in addition to the compounds of the present application, one or more of a sweetening agent, a preservative, a dye / colorant and a flavor enhancer. In the composition for injection administration, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent can be added.

[0097] The liquid pharmaceutical or nutraceutical composition of the present disclosure, regardless of the dosage form such as solution or suspension, can contain one or more of the following adjuvants, namely, water for injection, saline such as physiological saline, Ringer's solution, isotonic sodium chloride, synthetic mono- or diglycerides and other non-volatile oils that can be used as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol or other solvents as a sterile diluent; antibacterial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and tonicity regulators such as sodium chloride and dextrose. The parenteral preparation can be enclosed in an ampoule made of glass or plastic, a disposable syringe or a multi-dose vial. Physiological saline is a generally useful adjuvant. The pharmaceutical or nutraceutical composition for injection is sterile.

[0098] The liquid pharmaceutical or nutraceutical composition for parenteral or oral administration of the present disclosure should contain an amount of the compound of the present disclosure such that an appropriate dosage can be obtained.

[0099] The pharmaceutical or nutraceutical composition of the present disclosure may be for topical administration. In that case, the carrier may appropriately contain a solution, an emulsion, a cream, a lotion, an ointment, or a gel base. The carrier may contain, for example, one or more of diluents, emulsifiers, and stabilizers such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol. A thickening agent may be added to the pharmaceutical or nutraceutical composition for topical administration. In the case of transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0100] The pharmaceutical or nutraceutical composition of the present disclosure can also be for rectal administration. For example, it can be in the form of a suppository that melts in the rectum to release the drug. The composition for rectal administration can contain an oily carrier as an appropriate non-irritating excipient. Examples of such carriers include lanolin, cocoa butter, and polyethylene glycol.

[0101] The pharmaceutical or nutraceutical composition of the present disclosure can contain various materials that change the physical form of solid or liquid dosage units. For example, the composition can contain a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and can be selected, for example, from sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0102] The solid or liquid pharmaceutical or nutraceutical composition of the present disclosure can contain a substance that binds to the compound of the present disclosure and aids in the delivery of the compound. Suitable substances capable of performing this function include monoclonal or polyclonal antibodies, proteins, or liposomes.

[0103] The solid or liquid pharmaceutical or nutraceutical compositions of the present disclosure can, for example, have their particle size reduced to improve bioavailability. The size of powders, granules, particles, microspheres, etc. in the composition, with or without excipients, can be macro (e.g., visible or at least 100 μm in size), micro (e.g., sized in the range of about 100 μm to about 100 nm), nano (e.g., sized 100 nm or less), and any size in between or any combination thereof, to improve size and bulk density.

[0104] The pharmaceutical or nutraceutical compositions of the present disclosure can also be composed of dosage units for administration as an aerosol. The term aerosol is used to represent various systems ranging from colloidal systems to systems composed of pressurized packages. Delivery can be effected by a liquefied gas or compressed gas or a suitable pump system for dispensing the active ingredient. The aerosol formulations of the compounds of the present disclosure can be delivered in a single-phase, two-phase or three-phase system for delivering the active ingredient. Delivery of the aerosol formulation includes the necessary container, actuator, valve, inner container, etc., which can be integrally formed into a kit. One skilled in the art can determine the optimal aerosol formulation without undue experimentation.

[0105] The pharmaceutical or nutraceutical compositions of the present disclosure can be manufactured by methods well known in the pharmaceutical or nutraceutical arts. For example, a pharmaceutical or nutraceutical composition for parenteral administration can be manufactured by adding sterile distilled deionized water to the compounds of the present disclosure to form a solution. A surfactant may be added to assist in forming a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the compounds of the present disclosure and aids in the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0106] The compounds of the present disclosure or pharmaceutically or nutritionally acceptable salts thereof are administered in a therapeutically effective amount, which will vary depending on various factors such as the activity of the particular compound utilized, the metabolic stability and duration of action of the compound, the age, weight, general health, gender and diet of the patient, the mode and time of administration, the rate of excretion, drug combinations, the severity of the particular disorder or condition, and the subject being treated, etc.

[0107] The compounds of the present disclosure or pharmaceutically or nutritionally acceptable derivatives thereof may further be administered simultaneously with, before, or after food, water, and one or more other therapeutic agents. Such combination therapies include, in addition to methods of administering a single pharmaceutical or nutraceutical formulation containing the compounds or extracts of the present disclosure or a composition consisting of two to three plant extracts and one or more other active agents, methods of administering the free B-ring flavonoid and flavan composition consisting of the compounds or extracts of the present disclosure or two to three plant extracts and each active agent as separate pharmaceutical or nutraceutical formulations. For example, the composition consisting of the compounds or extracts of the present disclosure or two to three plant extracts and another active agent can be administered to a patient together as a single oral dosage composition such as a tablet or capsule, or each active agent can be administered as a separate oral dosage formulation. When separate dosage formulations are used, the compounds of the present disclosure and one or more other active agents can be administered essentially at the same time, i.e., simultaneously, or at staggered times, i.e., sequentially, and it should be understood that combination therapies include all of these regimens.

[0108] Of course, in the description of the present application, such combinations are permitted only when stable compounds can be obtained by combining the substituents or variables of the represented formula.

[0109] Also, as will be apparent to those skilled in the art, in the methods described in the present application, it may be necessary to protect the functional groups of the intermediate compounds with appropriate protecting groups. Such functional groups include hydroxy groups, amino groups, mercapto groups, and carboxylic acid groups. Appropriate protecting groups for hydroxy groups include trialkylsilyl groups or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl group, t-butyldiphenylsilyl group, or trimethylsilyl group), tetrahydropyranyl group, benzyl group, etc. Appropriate protecting groups for amino groups, amidino groups, and guanidino groups include t-butoxycarbonyl group, benzyloxycarbonyl group, etc. Appropriate protecting groups for mercapto groups include C(O)-R” (wherein R” is alkyl, aryl, or arylalkyl), p-methoxybenzyl group, trityl group, etc. Appropriate protecting groups for carboxylic acid groups include alkyl groups, aryl groups, or arylalkyl ester groups. The protecting groups can be added or removed according to standard techniques, and such techniques are known to those skilled in the art and are as described in the present application. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As will be apparent to those skilled in the art, the protecting groups may also be polymer resins such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin.

[0110] Similarly, as will be apparent to those skilled in the art, such protected derivatives of the compounds to be protected in the present application may not have pharmacological activity in their original state, but after administration to mammals, they can be metabolized in the body to form the pharmacologically active compounds of the present disclosure. Therefore, such derivatives can be referred to as “prodrugs”. All prodrugs of the compounds to be protected in the present application are included within the scope of the present disclosure.

[0111] Furthermore, all compounds or extracts of the present disclosure in the free base or free acid form can be converted into their pharmaceutically or nutritionally acceptable salts by treating them with appropriate inorganic bases, organic bases, inorganic acids or organic acids by methods known to those skilled in the art. The salts of the compounds of the present disclosure can be converted into their free base or free acid form by standard techniques.

[0112] In any of the above embodiments, the composition containing the extract or the mixture of compounds can be mixed in a specific weight ratio. For example, but not limited to, the extract of the genus Tatsunamisou containing bioflavonoids each containing baicalin and catechin and the extract of the genus Acacia can be blended at a weight ratio of 4:1 respectively. In a given embodiment, the (weight) ratio of two extracts or compounds of the present disclosure ranges from about 0.5:5 to about 5:0.5. The same range applies when using three or more (e.g., 3, 4, 5) extracts or compounds. Typical ratios include 0.5:1, 0.5:2, 0.5:3, 0.5:4, 0.5:5, 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:2, 2:3, 2:4, 2:5, 3:1, 3:2, 3:3, 3:4, 3:5, 4:1, 4:2, 4:3, 4:4, 4:5, 5:1, 5:2, 5:3, 5:4, 5:5, 1:0.5, 2:0.5, 3:0.5, 4:0.5, or 5:0.5. In other embodiments, the individual free B-ring flavonoid extract of the genus Tatsunamisou extract disclosed in the present application and the flavan extract of the genus Acacia are blended at a blend ratio of 4:1 as a non-limiting example to form a composition called UP446.

[0113] In other embodiments, individual extracts of the genus Potentilla and the genus Acacia are variously combined to form such formulations (including, but not limited to, for example, UP446, or UP223, or UP894-II, or UG0408), and evaluated in in vitro, ex vivo or in vivo models for the recognized biological functional advantages / disadvantages and unexpected synergistic / antagonistic effects, and the effective regulation of the homeostasis of the host defense mechanism, and the reduction of cytokine storm, oxidative stress and organ damage caused by sepsis. Based on the unexpected synergy measured in in vitro, ex vivo or in vivo models, due to the diversity of chemical components in each extract, the different mechanisms of action from different types of bioactive flavonoid compounds in each extract, and the possibility of improving the ADME of bioflavonoid compounds in the composition to maximize the biological and nutritional output, the best composition containing individual extracts of flavans or free B-ring flavonoids in a specific blend ratio was selected.

[0114] In any of the above embodiments, a composition containing a mixture of extracts standardized for free B-ring flavonoids and flavans as bioflavonoid compounds can be present at a predetermined percentage level or ratio. In a given embodiment, a composition containing Potentilla root extract powder or Acacia heartwood extract can contain 0.1% - 99.9% or about 10% - about 40% or about 60% - about 80% free B-ring flavonoids, 0.1% - 99.9% or about 1% - about 10% or about 5% - about 50% flavans, or combinations thereof. In a given embodiment, a composition containing a free B-ring flavonoid extract powder of the genus Potentilla or a flavan extract of the genus Acacia can contain about 0.01% - about 99.9% baicalin or catechin, or alternatively at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%, 95% baicalin or catechin.

[0115] In certain examples, the compositions of the present disclosure can be formulated to further include a pharmaceutically or nutritionally acceptable carrier, diluent, or excipient, and the pharmaceutical or nutraceutical formulation contains from about 0.05 weight percent (wt%), or 0.5 weight percent (wt%), or 5%, or 25%, or 50% or 80% to about 99 wt% of the active ingredient or the main active ingredient of the extract mixture. In other embodiments, the pharmaceutical or nutraceutical formulation contains from about 0.05 weight percent (wt%) to about 90 wt% of bioflavonoids, from about 0.5 wt% to about 80 wt% of baicalin, from about 0.5 wt% to about 86 wt% of total bioflavonoids, from about 0.5 wt% to about 90 wt%, from about 0.5 wt% to about 70 wt%, from about 1.0 wt% to about 60 wt%, from about 1.0 wt% to about 20 wt%, from about 1.0 wt% to about 10 wt%, from about 3.0 wt% to about 9.0 wt%, from about 5.0 wt% to about 10 wt%, from about 3.0 wt% to about 6 wt% of the main active ingredient of the extract mixture, etc. In any of the above formulations, the compositions of the present disclosure are formulated as tablets, hard capsules, soft gel capsules, powders, or granules.

[0116] In the present application, conversions of the compounds disclosed in the present application are also envisioned. Such products are obtained, for example, mainly by enzymatic processes such as oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound. Accordingly, the envisioned compounds are compounds produced by a method that includes administering the compound or composition to a mammal for a sufficient time to produce metabolites of the intended compound or composition. Such products are generally identified by radiolabeling or not radiolabeling the compounds of the present disclosure and administering them to animals such as rats, mice, guinea pigs, dogs, cats, pigs, sheep, horses, monkeys, or humans at a detectable dose, and then isolating the conversions from urine, blood, or other biological samples after sufficient time has elapsed for metabolism to occur.

[0117] The expected compound, pharmaceutical composition and composition can further comprise, or further include, or consist of at least one pharmaceutically or nutritionally or cosmetically acceptable carrier, diluent or excipient. The term "pharmaceutically or nutritionally or cosmetically acceptable carrier, diluent or excipient" as used herein includes any adjuvant, carrier, excipient, lubricant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that has been approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. The expected compound, pharmaceutical composition and composition can comprise, or further include, or consist of at least one pharmaceutically or nutritionally or cosmetically acceptable salt. The term "pharmaceutically or nutritionally or cosmetically acceptable salt" as used herein includes acid addition salts and base addition salts.

[0118] The intended composition containing free B-ring flavonoids and flavans is Cannabis sativa full-spectrum extract, CBD oil or CBD / THC, turmeric extract or curcumin, Terminalia extract, willow bark extract, Aloe vera leaf gel powder, Poria cocos extract, rosemary extract, rosmarinic acid, devil's claw root extract, cayenne pepper extract or capsaicin, American ginseng bark extract, philodendron bark extract, hop extract, Boswellia extract, rose hip extract, Sophora extract, Withania somnifera (ashwagandha), Bupleurum falcatum (mitsima saiko), Radix Bupleuri (saiko), Radix Glycyrrhiza, Fructus Forsythiae (renkyo), Panax quinquefolium (American ginseng), Panax ginseng C.A.Meyer: Otaneninnjin), Lentinula edodes (Shiitake), Inonotus obliquus (Kabanotanake), Lentinula edodes (Shiitake), Lycium barbarum (Nagabakkou), Phellinus linteus (Meshimakobu) (fruiting body), Trametes versicolor (Kawaratake) (fruiting body), Cyamopsis tetragonolobus (Cluster bean), Trametes versicolor (Kawaratake), Cladosiphon okamuranus Tokida (Okinawamozuku), Undaria pinnatifida (Wakame), Mentha genus or peppermint extract, ginger or black ginger extract, green tea or grape seed polyphenols, omega 3 or omega 6 fatty acids, krill oil, gamma-linolenic acid, citrus bioflavonoids, acerola concentrate, astaxanthin, picnogenol, resveratrol, ascorbic acid, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B, vitamin A, L-lysine, calcium, manganese, zinc, amino acid chelate minerals, amino acids, boron and boron glycinate, silica, probiotics, camphor, menthol, calcium salts, silica, histidine, copper gluconate, CMC, beta-cyclodextrin, cellulose, dextrose, physiological saline, water, oils, UCII, shark and bovine cartilage, mushrooms, seaweeds, yeasts, brown algae, agarve nectar, brown seaweed, fermentable dietary fiber, grains, sea cucumbers, agave, artichokes, asparagus, leeks, garlic, onions, rye grains, wheat, pears, apples, guavas, quince, plums, celery, oranges and at least one other active ingredient selected from one or more of other citrus fruit trees, adjuvants, excipients or carriers, or can further contain, or can consist of them.

[0119] The intended composition containing free B-ring flavonoids and flavans can include, or further include, or be composed of at least one other natural phenolic active ingredient. In certain embodiments, at least one bioactive ingredient can include, or be composed of, plant powders or plant extracts, etc. Examples of plant species containing the above immunosuppressive natural phenolic compounds include, but are not limited to, Piper longum Linn (Piper longum Linn: Japanese pepper), Coptis chinensis Franch (Coptis chinensis Franch: Chinese goldthread), Angelica sinensis (Oliv.) Diels (Angelica sinensis (Oliv.) Diels: Chinese angelica), Toxicodendron vernicifluum (Toxicodendron vernicifluum: Japanese lacquer tree), Glycyrrhiza glabra (Glycyrrhiza glabra: Spanish licorice), Curcuma longa (Curcuma longa: Turmeric), Salvia Rosmarinus (Salvia Rosmarinus: Rosemary), Rosmarinus officinalis (Rosmarinus officinalis: Rosemary), Zingiber officinalis (Zingiber officinalis: Ginger), Polygala tenuifolia (Polygala tenuifolia: Milkwort), Morus alba (Morus alba: White mulberry), Humulus lupulus (Humulus lupulus: Hops), Lonicera Japonica (Lonicera Japonica: Japanese honeysuckle), Salvia officinalis L.: Sage), Centella asiatica (Gotu Kola), Boswellia carteri, Mentha longifolia (Long-leaved Mint), Picea crassifolia, Citrus nobilis Lour, Citrus aurantium L (Bitter Orange), Camellia sinensis L (Tea Plant), Pueraria mirifica, Pueraria lobata (Kudzu), Glycine max (Soybean), Capsicum species, Fallopia japonica (Japanese Knotweed). A large number of phenolic compounds can also be detected in various fruit trees and vegetables, such as tomatoes, cruciferous vegetables, grapes, blueberries, raspberries, mulberries, apples, chili peppers, etc.

[0120] The free B-ring flavonoids are composed of one or more of baicalin, baicalein, baicalein glucoside, wogonin, wogonin glucuronide, wogonin glycoside, oroxylin, oroxylin glycoside, oroxylin glucuronide, chrysin, chrysin glycoside, chrysin glucuronide, scutellarin and scutellarin glycoside, norwogonin and norwogonin glycoside, galangin or any combination thereof. Examples of free B-ring flavonoids that can be used according to the method of the present application for protection include compounds represented by the above general structure. The standardized free B-ring bioflavonoids in the composition are synthesized, metabolized, biodegradable, biotransformed, bioconverted, biosynthesized from small carbon units by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, and microbacteria.

[0121] One or more free B-ring flavonoids are highly concentrated and standardized, derived from at least one species of the higher plant genera including Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus, Alpinia, or a combination thereof.

[0122] One or more free B-ring flavonoids are Scutellaria baicalensis, Scutellaria barbata, Scutellaria orthocalyx, Scutellaria lateriflora, Scutellaria galericulata, Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria galericulata, Scutellaria indica, Scutellaria sessilifolia, Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria orientalis, Oroxylum indicum, Passiflora caerulea, Passiflora incarnata, Pleurotus ostreatus, Lactarius deliciosus, Suillus beriniiIt is highly concentrated and standardized, derived from a plant species containing at least one of bellinii), chamomile, carrot, mushroom, honey, propolis, passion flower, and Indian trumpet flower, or a combination thereof.

[0123] Flavan is composed of one or more of catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epiteafavin, epicatechin gallate, gallocatechin gallate, theaflavin, theaflavin gallate, or any combination thereof. Examples of flavans that can be used according to the method of the present application for protection include compounds represented by the above general structure. The standardized flavan bioflavonoid in the composition is synthesized, metabolized, biodegradable, biotransformed, bioconverted, biosynthesized from small carbon units by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, and microbacteria.

[0124] The flavans that are the subject of protection of this application are isolated from one or more plants selected from plants of the genus Acacia. In a preferred embodiment, the plant is Acacia catechu (Acacia catechu: black catechu), Senegalia catechu, Acacia concinna (Acacia concinna: Sompoy), Acacia farnesiana (Acacia farnesiana: mimosa), Acacia Senegal (Acacia Senegal: gum arabic), Acacia speciosa, Acacia arabica (Acacia arabica: false gum arabic), Acacia caesia, Acacia pennata (Acacia pennata: Chaom), Acacia sinuata, Acacia mearnsii (Acacia mearnsii: black wattle), Acacia picnantha (Acacia picnantha: golden wattle), Acacia dealbata (Acacia dealbata: silver wattle), Acacia auriculiformis (Acacia auriculiformis: earleaf acacia), Acacia holoserecia (Acacia holoserecia: strap wattle), Acacia mangium, Anacardium occidentale (Anacardium occidentale: cashew nut shell), Uncaria gambir (Uncaria gambir: white catechu), Uncaria rhynchophylla (Uncaria rhynchophylla: hook vine), Camellia sinensis (Camellia sinensis: tea plant), Camellia assamica (Camellia assamica: Assam tea), Euterpe oleracea (Euterpe oleracea: açaí), Caesalpinia decapetala (Caesalpinia decapetala: Chinese honeylocust), Delonix regia (Delonix regia: royal poinciana), Ginkgo biloba (Ginkgo biloba: ginkgo), Acer rubrum (Acerrubrum: (American dogwood), Cocos nucifera (coconut palm), Limonium Brasiliense, Acerola barbados, Vitellaria paradoxa (shea tree), Vitis vinifera (European grapevine), Lawsonia inermis (henna), Artocarpus heterophyllus (jackfruit), Medicago sativa (alfalfa), Lotus japonicus (lotus), Lotus uliginosus (bog lotus), Eisenia bicyclis (arame), Hedysarum sulfurescens, Robinia pseudoacacia (false acacia), apple, apricot, plum, cherry, grape leaves, strawberry, legumes, lemon, tea, black tea, green tea, rooibos tea, barley grains, green algae (Acetabularia ryukyuensis), red algae (Chondrococcus hornemannii), chocolate (cocoa), raw coffee beans, a group consisting of, or a combination thereof, and in some embodiments selected from these.

[0125] In certain embodiments, the free B-ring flavonoid or flavan compound or extract of the present disclosure can be isolated from plants or marine resources, such as the plant resources mentioned in the examples and elsewhere in this application. Suitable plant parts for the isolation of the compounds include leaves, bark, trunk, trunk bark, stem, stem bark, twig, tuber, root, rhizome, root bark, bark surface, shoot, seed, fruit, androecium, gynoecium, calyx, stamen, petal, sepal, carpel (gynoecium), flower, stem cell, or any combination thereof. In certain related embodiments, the compound or extract is isolated from a plant resource and synthetically modified to include any of the specified substituents. In this regard, the synthetic modification of compounds isolated from plants can be carried out using a number of techniques known in the art, including but not limited to transgenic microorganisms, P450 enzymes, glycosyltransferases, or enzyme combinations, and microbacteria for total organic synthesis, metabolism, biodegradation, in vivo conversion, in vivo transformation, biosynthesis from small carbon units. Such techniques are well within the knowledge of those having ordinary skill in the art.

[0126] Other embodiments of the subject matter of the present application relate to the use of a standardized bioflavonoid composition (such as, but not limited to, UP446 or UP894-II exemplified in the examples of the present disclosure) containing free B-ring flavonoids and flavans, in which two or three plant extracts are variously combined to regulate the homeostasis of the host defense mechanism, including, but not limited to, methods of optimizing or balancing the immune response; methods of assisting in maintaining a healthy immune function against viral and bacterial infections; methods of protecting the immune system from oxidative stress damage induced by air pollution; methods of protecting normal and healthy lung function from viral infection, bacterial infection, and air pollution; methods of assisting in a healthy inflammatory response; methods of maintaining healthy levels of cytokines and cytokine responses to infection; methods of increasing and maintaining anti-inflammatory cytokines such as TNF-α, IL-1β, IL-6, GM-CSF, IFN-α, IFN-γ, IL-1α, IL-1RA, IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, TNF-β / LTA, CRP, and CINC3; methods of controlling the oxidative response and alleviating oxidative stress; methods of enhancing antioxidant capacity by increasing SOD and NRf2; methods of reducing advanced glycation end products; methods of increasing glutathione peroxidase; methods of neutralizing reactive oxygen species and preventing damage to the structural integrity and functional decline of the respiratory system, lungs, and immune system caused by oxidative stress; methods of maintaining lung purification and detoxification capacity; methods of protecting lung structural integrity and oxygen exchange capacity; methods of maintaining respiratory passage and enhancing alveolar oxygen absorption capacity; methods of reducing lung damage caused by oxidative stress; methods of promoting pulmonary microcirculation and protecting normal coagulation function; methods of increasing the activity and number of white blood cells; methods of enhancing natural killer (NK) cell function; methods of increasing the number of T lymphocytes and B lymphocytes; methods of increasing the number of CD4+ and CD8+ cells; methods of increasing the number of CD3+, CD4+NKp46+ natural killer cells, TCRγδ+ gamma delta T cells, CD4+TCRγδ+ gamma delta T cells, and CD8+ cells; methods of protecting and promoting macrophage phagocytic activity; methods of assisting or promoting normal antibody production;Methods for maintaining a healthy lung microbiota or symbiotic system in a respirator; methods for alleviating or reducing cold / flu-like symptoms including, but not limited to, body pain, sore throat, cough, mild throat and bronchial irritation, nasal congestion, sinus congestion, sinus pressure, runny nose, sneezing, decreased sense of smell, decreased sense of taste, muscle pain, headache, fever and chills; methods for loosening phlegm (mucus), thinning bronchial secretions to make coughing easier; methods for reducing the severity of bronchial irritation; methods for reducing the severity of lung injury or edema or inflammatory cell infiltration caused by viral infection, microbial infection and air pollution; methods for supporting the bronchial system and comfortable breathing throughout the cold / flu or pollution season; methods for preventing or treating pulmonary fibrosis; methods for reducing the duration or severity of the common cold / flu; methods for reducing the severity or duration of viral and bacterial infections of the respiratory system; methods for preventing or treating or curatively treating respiratory infections caused by viruses, microorganisms and air pollutants; methods for managing or treating or preventing or reversing the progression of respiratory infections; methods for promoting and enhancing and rejuvenating the repair and regeneration functions of the lungs and the entire respiratory system, etc.

Example

[0127] [Example 1] Preparation and Quantification of Free B-Ring Flavonoids from Plants Plant materials derived from the roots of Scutellaria orthocalyx, or the roots of Scutellaria baicalensis (Skullcap), or the whole herb of Scutellaria lateriflora (Blue Skullcap) were ground to a particle size of 2 mm or less. The dried and ground plant material (60 g) was then transferred to an Erlenmeyer flask and methanol:dichloromethane (1:1) (600 mL) was added. The mixture was shaken for 1 hour, filtered, and the biomass was extracted again with methanol:dichloromethane (1:1) (600 mL). The organic extracts were combined and evaporated under reduced pressure to obtain an organic extract (see Table 1 below). After organic extraction, the biomass was air-dried and extracted once with ultrapure water (600 mL). The aqueous solution was filtered and freeze-dried to obtain an aqueous extract (see Table 1 below).

[0128]

Table 1

[0129] The presence and amount of free B-ring flavonoids in organic extracts and aqueous extracts derived from various plant species were confirmed and are shown in Table 5. A Luna C-18 column (250×4.5 mm, 5 μm) was used, and free B-ring flavonoids were quantitatively analyzed by HPLC using a 0.1% phosphoric acid / acetonitrile gradient of 80%→20% over 22 minutes. Free B-ring flavonoids were detected at 254 nm using a UV detector and identified by comparing the retention times with free B-ring flavonoid standards.

[0130]

Table 2

[0131] [Example 2] Generation of a standardized free B-ring flavonoid extract derived from plants The roots of Scutellaria baicalensis (Scutellaria baicalensis: goldenrod) were washed with water and thinly sliced into small pieces. The washed and thinly sliced roots were put into an extraction device and extracted twice with hot water at a temperature of 90 - 95°C. For every 1 kg of roots, about 8 L of water was added and extracted at 90 - 95°C for about 1 hour. After collecting the extract solution, the roots were extracted again with 6 L / kg of water at 90 - 95°C for another 1 hour. The extract solution was collected and combined with the first extract solution. After filtering the extract solution, the pH of the solution was adjusted to about 2 with hydrochloric acid water or sulfuric acid water. After allowing the acidic aqueous solution to stand for about 2 hours, the precipitate was filtered and washed with purified water. The precipitated extract was dried at 80 - 90°C. The dried powder was ground and blended. The extraction yield was 1 kilogram of high-concentration bioflavonoid extract from 10 - 15 kg of roots. When the content of bioflavonoids was quantified by the HPLC method in the same manner as in Example 1 above, a standardized extract called RM405 with a baicalin content of 75% or more and a drying loss of less than 5% was produced. The particle size of RM405 was controlled so that the passing rate through 80 mesh was 80%. The potential contamination of heavy metals such as lead, arsenic, Pb, Cd, and Hg was analyzed by ICP-MS. The potential contamination of coliforms, filamentous fungi, yeasts, and the total viable count of bacteria were also measured to meet the USP / AOAC / KFDA requirements.

[0132] A standardized bioflavonoid extract derived from the roots, or stems or whole plants of the genus Scutellaria can be obtained by a method of precipitating a basic aqueous extract solution after neutralization with an acidic solution, or a method of recrystallization in water, or column chromatography using various resins as carriers, and the bioflavonoids can be concentrated 2 - 10 times to a purity of 20% - 99%.

[0133] [Example 3] Production of a standardized bioflavonoid extract derived from Acacia catechu and cashew nut shells The bark of Acen yak no ki (500 mg of crushed bark) was extracted with the following solvent systems: (1) 100% water, (2) 80:20 water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5) 20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, and (8) 60:40 methanol:THF. The extracts were concentrated and dried under reduced pressure. The flavan content in the dried extracts was quantified by HPLC as follows, and the results are shown in Table 4.

[0134] Dried and ground cashew nut (Anacardium occidentale) shell powder (60 g) was placed in a 100 ml stainless steel tube, and the ASE350 automatic extraction device was used at 80 °C and a pressure of 1500 psi for two extractions with 70% ethanol DI aqueous solution as the solvent. The extract solution was automatically filtered and collected. The organic extract solutions were combined and evaporated under reduced pressure using a rotary evaporator to obtain a crude 70% ethanol extract (R00883-70E, 23.78 g, extraction yield 39.63%).

[0135] Using the following analytical method, the amount of free catechin in the bioflavonoid extract derived from the heartwood of Acen yak no ki or the cashew nut shell was quantified by a C18 reverse-phase column (Phenomenex, USA, Luna 5 μm, 250 mm × 4.6 mm) on a Hitachi HPLC / PDA system. Mobile phase A: 0.1% aqueous phosphoric acid solution, and mobile phase B: acetonitrile were used for elution at a flow rate of 1.0 ml / min (Table 3), and the UV absorbance at 275 nm was measured at a column temperature of 35 °C. A catechin reference standard was purchased from Sigma-Aldrich. The reference standard was dissolved in MeOH:0.1% H3PO4 (1:1) containing 0.5 mg / ml of catechin (C1251) and 0.1 mg / ml of epicatechin (E1753). The test sample was prepared at a concentration of 2 mg / ml in 50% methanol / 0.1% H3PO4 in a volumetric flask, sonicated until dissolved (for about 10 minutes), cooled to room temperature, mixed well, and filtered through a 0.45 μm nylon syringe filter. HPLC analysis was performed by injecting 20 μL of the sample into the HPLC.

[0136]

Table 3

[0137] Using catechin and epicatechin as standards, chemical components were quantified based on retention time and PDA data. The catechin quantification results from the extract of Acer mono Maxim are shown in Table 4. As shown in Table 4, the flavan extract produced by solvent extraction with 80% methanol / water had the best flavan component concentration. The bioflavonoid content in the 70% ethanol extract of cashew nut shell is 9.4% catechin and 6.1% epicatechin.

[0138]

Table 4

[0139] The bark was removed from the heartwood of Acer mono Maxim, washed with water, and thinly sliced into small pieces. The washed and thinly sliced heartwood was put into an extraction apparatus and extracted twice with hot water at a temperature of about 115 °C. For 1 kg of Acer mono Maxim, about 4 L of water was added and extracted at 105 - 115 °C for about 5 hours. After filtering the extraction solution, it was concentrated under reduced pressure at 50 - 60 °C. The concentrated solution was kept cold at a temperature of about 5 °C for 7 - 10 days, then the precipitate was filtered, the wet cake was frozen, and dried at about -20 °C for 1 day. The dried powder was pulverized, sieved, and blended after drying at 90 °C for 10 hours. The extraction ratio of the final extract with respect to the heartwood was about 1 kg of bioflavonoid extract from 20 kg of Acer mono Maxim heartwood. When the content of bioflavonoid was quantified by the HPLC method as follows, a standardized extract called RM406 was produced, in which the total content of catechin and epicatechin was 65% or more and the drying loss was less than 5%. The particle size of RM406 was controlled so that the 80-mesh passing rate was 80%. The potential contamination of heavy metals as lead, arsenic, Pb, Cd, and Hg was analyzed by ICP-MS. The potential contamination of coliforms, filamentous fungi, yeasts, and the total viable count were also measured to meet the USP / AOAC / KFDA requirements.

[0140] By a method of precipitation after concentration of the plant extract solution, or a method of recrystallization in an ethanol / water solvent, or column chromatography using various resins as carriers, a standardized bioflavonoid extract derived from the heartwood, bark, whole herb of Uncaria acida or Uncaria gambir, or the cashew nut shell can be obtained, and the bioflavonoid can be concentrated 2 to 10 times to a purity of 10% to 99%.

[0141] [Example 4] Formulation of the standardized bioflavonoid composition Two standardized extracts were used, an Acacia extract (RM406 of Example 3) with a total flavan content as catechin and epicatechin > 65%, and a Scutellaria extract (RM405 of Example 2) with a free B-ring flavonoid content as baicalin, baicalein, etc. > 75%. Three components including maltodextrin as an excipient were used to formulate a bioflavonoid composition called UP446. The ratio of flavan to free B-ring flavonoid can be adjusted based on the indication and functionality. The amount of the excipient is adjusted based on the actual active ingredient content in each component. It is necessary to create a blend sheet for each batch of the formulation based on the QC of the formulation specifications and each batch of the components. An excess amount of the active ingredient in the range of 2 to 5% is recommended according to the formulation specifications. A blend sheet of one batch of UP446 (lot number G1702) with a blend ratio of free B-ring flavonoid extract:flavan extract:maltodextrin of 80:17:3 is shown below.

[0142]

Table 5

[0143] A standardized extract derived from an Acacia heartwood extract with a total flavan content of >65% as catechin and epicatechin, and a standardized extract derived from a Tatsunamisou stem extract with a free B-ring flavonoid content of >75% as baicalin, baicalein, etc. were used to formulate a bioflavonoid composition called UP223. The blend ratio of the free B-ring flavonoid extract to the flavan extract was set at 90:10.

[0144] A standardized extract derived from an Acacia heartwood extract with a total flavan content of >90% as catechin and epicatechin, and a standardized extract derived from a Tatsunamisou root extract with a free B-ring flavonoid content of >90% as baicalin, baicalein, etc. were used to formulate a bioflavonoid composition called UP894-II. The blend ratio of the free B-ring flavonoid extract to the flavan extract was set at 4:1, the baicalin content was set at 70 - 80%, and the total catechin was set at 15 - 20% (Table 6).

[0145] [Table 6]

[0146] [Example 5] Using the MTT assay, the cell viability under hyperoxic exposure conditions for 24 hours in the presence of UP894-II was measured.

[0147] RAW264.7 cells were maintained in room air (21% oxygen O2) or exposed to 95% O2 for 24 hours in the presence of UP894-II (0 - 256 μg / ml), a standardized bioflavonoid composition shown in Example 4 and Table 6, or its solvent. Cell viability was measured by the MTT assay as described by the manufacturer.

[0148] When compared with the T0 control, which was the measured value at the time of seeding, significantly more viable cells were observed in the indoor air control group at T24. The cell viability of the O2 control group (95% O2) was significantly decreased compared with the indoor air control group. When the solvent DMSO was administered at concentrations of 0.16% and 0.32%, it had no effect on the cell viability under O2. To examine whether the formulation UP894-II could improve the macrophage function decreased by oxidative stress, first, the dose-response curve of this formulation on cell viability was created under normal culture conditions or high oxygen conditions. The attached graph (Figure 4) is a representative result of three independent experiments. At doses of UP894-II less than 128 μg / ml, the cell viability did not change significantly compared with the DMSO control group. Therefore, UP894-II was tested for its effect on enhancing macrophage function at doses less than 128 μg / ml.

[0149] [Example 6] UP894-II enhanced the phagocytic activity of macrophages.

[0150] RAW264.7 cells were maintained in indoor air (21% O2) or exposed to 95% O2 for 24 hours in the presence of UP894-II (0 - 100 μg / ml), which is the standardized bioflavonoid composition shown in Example 4 and Table 6. Next, the cells were incubated with FITC-labeled latex microbeads for 1 hour, stained with phalloidin and DAPI, and the actin cytoskeleton and nucleus were visualized, respectively. For the quantification of phagocytic activity, at least 200 cells in each group were counted, and the number of beads per cell was expressed as a percentage relative to the 21% O2 (0 μg / ml) control group. UP894-II was tested at 3.7 μg / ml, 11.1 μg / ml, 33.3 μg / ml, and 100 μg / ml. These doses were determined based on the cell viability assay.

[0151] As shown in Fig. 5, cultured macrophages were exposed to high oxygen for 24 hours in the presence of various concentrations of UP894-II or solvent alone. As is clear from the images, high oxygen exposure significantly reduced macrophage phagocytic activity. UP894-II significantly enhanced macrophage function even at a low dose of 3.7 μg / ml. These results suggest that UP894-II could be a good candidate for enhancing lung function under oxidative stress.

[0152] [Example 7] UP894-II suppresses high oxygen-induced HMGB1 release in macrophages.

[0153] RAW264.7 cells were maintained in room air (21% O2) or exposed to 95% O2 for 24 hours in the presence of UP894-II (0 - 33.3 μg / ml), a standardized bioflavonoid composition shown in Example 4 and Table 6. The HMGB1 concentration in the medium was analyzed by Western blot analysis. The blot is an image representing the HMGB1 concentration of each group and corresponds to the bar graph directly below it for every 2 lanes.

[0154] Compared to the room air control group (21% O2), HMGB1 release in the high oxygen control group (95% O2) was significantly increased. In DMSO, the solvent, there was no significant change in HMGB1 release compared to the high oxygen control group. In contrast, when UP894-II was administered, the HMGB1 concentration decreased dose-dependently and statistically significantly (75.9% - 89.7%) when measured at 3.7 μg / ml, 11.1 μg / ml, and 33.3 μg / ml (Fig. 6).

[0155] Particles generated from environmental air pollution can cause exogenous oxidative stress to the biological system through the generation of reactive oxygen species (ROS), leading to a decrease in host defense and inflammation, and resulting in lung injury. HMGB1 plays a major role in the pathology of lung injury in cooperation with ROS, causing apoptosis of alveolar macrophages, suppressing alveolar macrophage phagocytosis partially through the activation of NF-kB, leading to the upregulation of inflammatory cytokines and chemokines, and triggering cytokine storm. When these factors act in concert, they may cause pathological changes harmful to the lungs during pollution-induced lung injury, viral or bacterial infection. As an example of the interaction between these two factors, in fact, during oxygen therapy routinely used in the treatment of COVID-19 patients, long-term exposure to oxidative stress can reduce natural immunity, reduce macrophage function, and as a result, reduce the ability to remove pathogens invading the lungs, potentially causing acute inflammatory lung injury. Therefore, the population exposed to oxidative stress caused by cytokine storm, such as COVID-19 patients and patients with inflammatory disorders, is increasing, but reducing the HMGB1 concentration in the airway or blocking its activity can be an important treatment and prevention strategy for such populations. Therefore, according to the data shown in this example, UP894-II, a standardized bioflavonoid composition, can be used for such new applications through these clearly defined mechanisms in addition to the previously reported important uses. In the protection scope of this application, this concept was demonstrated, and the effects of the above-mentioned standardized composition were demonstrated in multiple disease models as described in the following examples.

[0156] [Example 8] Animals and Breeding CD-1 mice and Sprague Dawley rats were purchased from a vendor approved by the USDA. 8-week-old male CD-1 mice and SD rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). After arrival, the animals were acclimated and used in the tests. The animals were placed in a temperature-controlled room (71 - 72°F) and bred under a 12-hour light-dark cycle, and allowed free access to food and water.

[0157] Animals were housed in polypropylene mouse cages at 3 - 5 animals per cage and individually identified by attaching a unique number to the tail. Each cage was covered with a mouse or rat wire bar lid and a filter top (Allentown, NJ). The project number, test article name, dose level, group name, animal number, and sex were entered on a cage card and attached to each individual cage for identification. Harlan T7087 soft corn cob bedding was used and changed at least twice a week. The animals were allowed free access to fresh water and rodent diet #T2018 commercially available from Harlan (Harlan Teklad, 370W, Kent, WA).

[0158] [Example 9] Lipopolysaccharide (LPS)-induced sepsis model This model uses the survival rate of animals as an endpoint measurement (Wang et al., 1999). Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram-negative bacteria and a major contributing factor in the initiation of a systemic inflammatory process that can lead to endotoxin shock. Endotoxin shock is mainly a state mediated by macrophages / monocytes and is caused by the overproduction of several early cytokines such as TNF-α, IL-1, IL-6, and γ-interferon (IFN-γ), as well as the late mediator HMGB1. After dissolving a median lethal dose of LPS (25 mg / kg) in phosphate-buffered saline (PBS; Lifeline, lot number 07641) and administering it, the animals developed endotoxemia, HMGB1 was detected in the serum at 8 hours, and would reach peak plateau levels 16 - 32 hours after LPS administration. Without treatment, the mice would begin to die within 24 hours. In this study, the mice were monitored for 4 days after LPS injection. The survival rates were compared for LPS + sodium butyrate (SB; Aldrich, St. Louis, MO; lot number MKCG7272), LPS + vehicle (0.5% CMC; Spectrum, New Brunswick, NJ; lot number 1IJ0127), and LPS + UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6. The following groups were tested.

[0159] [Table 7]

[0160] In this model, before intraperitoneal injection of a lethal dose of LPS (from Escherichia coli 055:B5; Sigma, St. Louis, MO; lot number 081275) at 25 mg / kg together with 10 mL / kg of PBS, the mouse was pre-administered with UP446, a bioflavonoid composition shown in Example 4, for one week (7 days). The animals were observed hourly. Sodium butyrate was selected as the positive control for this test in view of the fact that it improved LPS-induced injury in mice by suppressing HMGB1 release (Li et al., 2018).

[0161] [Example 10] The standardized bioflavonoid composition improved animal survival rate under a lethal dose of endotoxin.

[0162] Three hours after the intraperitoneal injection of LPS, the mice began to show early signs of endotoxemia. The exploratory behavior of the mice decreased progressively, accompanied by piloerection (raising of the hair), reduced mobility, lethargy, and diarrhea. These signs and symptoms seemed to be present in all the dosing groups, but the severity was more pronounced in the vehicle control group.

[0163] Two mice in the solvent administration group and one mouse in the sodium butyrate (SB) group, which was the positive control, were found to have died 24 hours after LPS injection. When calculating the survival rates of these groups, it was found that they were 62.5% and 75% respectively (Table 8). The survival rate of the mice administered with UP446, which is the standardized bioflavonoid composition shown in Example 4 and Table 6, was 100% 24 hours after LPS injection. In the mice administered with UP446, SB, and solvent, survival rates of 87.5%, 62.5%, and 50% were observed respectively 34 hours after LPS injection. Approximately the most remarkable result was observed in the mice administered with UP446 48 hours after LPS injection. At this time point, the survival rate of the mice administered with the solvent was only 12.5%, while the mice administered with UP446 showed a survival rate of 75%. Even in the sodium butyrate group, which was the positive control, half of them had died at this time point. On the third day (72 hours after LPS injection), the survival rates of the groups of UP446, SB, and solvent were 62.5%, 50%, and 12.5% respectively. All the mice in the solvent control group had died 82 hours after LPS injection, and the survival rate of this group was 0%.

[0164] On the other hand, the mice administered with UP446 and SB showed a survival rate of 50%, and there was no change even 96 hours and 120 hours after LPS injection. These survival rates were statistically significant for both UP446 (p = 0.001) and SB (p = 0.01) compared to the animals administered with the solvent (Table 8). The surviving animals in these groups showed a gradual improvement in their good health condition. The mice seemed to be in good physical condition and gradually resumed normal behavior.

[0165]

Table 8

[0166] [Example 11] Comparison of the standardized bioflavonoid composition and its components in a LPS-induced sepsis model The advantages of combining free B-ring flavonoids derived from extracts of the genus *Tatsunamisoa* and flavans derived from extracts of the genus *Acacia* to form UP894-II at a specific ratio were demonstrated in Example 4. However, these advantages were evaluated in lipopolysaccharide (LPS)-induced endotoxemia. The *Tatsunamisoa* extract RM405 containing more than 60% baicalin shown in Example 3 and the *Acacia* extract RM406 containing more than 10% catechin shown in Example 4 were administered to male CD-1 mice (n = 13) at doses of 200 mg / kg and 50 mg / kg, respectively, for 7 days before LPS injection. On the 8th day, 25 mg / kg of LPS dissolved in 10 mL / kg of PBS was intraperitoneally (i.p.) injected into the mice. Mice in the UP894-II administration group were administered 250 mg / kg of UP894-II as a daily dose. During the test period, all mice were continuously administered their respective administered substances daily and the experiment ended on the 6th day after LPS injection. After intraperitoneal administration of a lethal dose of LPS (25 mg / kg), the animals were expected to develop sepsis within a few hours. Without treatment, the mice would begin to die within 24 hours. The animals were observed hourly. In this study, the mice were monitored for 6 days after LPS injection.

[0167]

Table 9

[0168] The survival rates of LPS + sodium butyrate (SB), LPS + solvent (0.5% CMC), LPS + UP894-II, LPS + *Tatsunamisoa* extract (RM405), and LPS + *Acacia* extract (RM406) were compared. PBS alone was intraperitoneally administered to normal control animals, and 0.5% CMC alone as the solvent was intragastrically administered to the solvent control. Sodium butyrate (SB) was selected as the positive control for this study in view of the fact that this compound improved LPS-induced injury in mice by suppressing HMGB1 release (Li et al., 2018).

[0169] The survival rate and mortality of the composition (UP894-II) were compared with the equivalent doses of the individual extracts contained in the same formulation, and the potential additive, antagonistic, or synergistic effects of the combination were investigated using Colby's formula (Colby, 1967). For the blend of these plant extracts to produce an unexpected synergistic effect, it is necessary for the measured inhibition value to exceed the calculated value.

[0170] Several hours after the intraperitoneal injection of LPS, the mice began to show early signs of sepsis. The exploratory behavior of the mice decreased progressively, accompanied by piloerection (raising of the hair), reduced mobility, lethargy, diarrhea, and tremors, and in some cases, eyelid closure. These signs and symptoms were present in all treatment groups, but were more pronounced in the vehicle-treated group and the Acacia extract (RM406)-treated group.

[0171] Twenty-four hours after LPS injection, it was found that 4 mice in the vehicle-treated group and the Acacia extract (RM406 shown in Example 4)-treated group, and 2 mice in the positive control group, the SB group, and the Tatsunamisou extract (RM405 shown in Example 3) group had died. When calculating the survival rate of these groups at this time point, it was found that the survival rate was 69.2% in the vehicle group and the Acacia extract (RM406) group, and 84.6% in the Tatsunamisou extract (RM405) group and the SB group (Table 10). The mice administered UP894-II had a survival rate of 100% 24 hours after LPS injection. Thirty-six hours after LPS injection, the survival rates of the mice administered UP894-II, the Tatsunamisou extract (RM405), the vehicle, SB, and the Acacia extract (RM406) were 84.6%, 61.5%, 53.9%, 53.9%, and 53.9% respectively. The most significant results were observed in the mice administered UP894-II 48 hours after LPS injection. At this time point, the survival rate of the mice administered the vehicle was only 15.4%, while the mice administered UP894-II showed a survival rate of 69.2%. The mice administered the Tatsunamisou extract (RM405), the Acacia extract (RM406), and SB showed survival rates of 46.2%, 38.5%, and 46.2% respectively 48 hours after LPS injection.

[0172] On the 3rd day (72 hours after LPS injection), the survival rates of the administration groups were 53.9%, 30.8%, 15.4%, and 46.2% for UP894-2, the extract of the genus Pulsatilla (RM405), the extract of the genus Acacia (RM406), and SB, respectively.

[0173]

Table 10

[0174]

Table 11

[0175]

Table 12

[0176] The survival rate of the mice administered with the solvent remained at 15.4% from the time point 48 hours after LPS injection until the end of the remaining test period. In contrast, the mice administered with the extract of the genus Acacia (RM406) continued to die until 96 hours after LPS injection. By the end of the 7-day observation period, the survival rate of the extract of the genus Acacia (RM406) group was only 7.7%. On the other hand, the mice administered with UP894-II and the extract of the genus Pulsatilla (RM405) maintained survival rates of 53.9% and 30.8%, respectively, from the time point on the 3rd day after LPS injection until the end of the remaining observation period. The positive control sodium butyrate (SB) group ended the test with a survival rate of 30.8%. The only group with a statistically significant survival rate compared to the solvent control was the UP894-II group (p = 0.01). The surviving animals in this group showed a progressive improvement in their good health status. The mice seemed to be in good physical condition and gradually resumed normal exploratory behavior.

[0177] [Example 12] In the standardized bioflavonoid composition, an unexpected synergistic effect was observed.

[0178] Using the LPS-induced survival rate test, the Corby method was used to evaluate whether there is a possible synergistic effect or unexpected effect when extracts derived from the genus Artemisia and the genus Acacia are used in combination at a specific ratio. When the standardized bioflavonoid composition UP894-II shown in Example 4 and Table 6 was administered to mice at a dose of 250 mg / kg, the survival rate was higher at each analysis time point than the predicted value calculated theoretically (Table 13). For example, the predicted survival rates 24 hours and 144 hours after LPS injection were 95.3% and 36.1% respectively, but the actual measured survival rates of UP894-II were 100% and 53.9% respectively. These results suggest that when two types of standardized extracts, the free B-ring flavonoid extract and the flavan extract derived from the genus Artemisia and the genus Acacia, are used in combination at a specific ratio, it is much more advantageous for extending the life of the test subjects during sepsis than using the Acacia extract or the Artemisia extract alone. Also using the Corby method, when examining the predicted mortality rates at these time points, it was found that the actual measured mortality rate of the mice administered with UP894-II was much lower than the estimated value, and it was confirmed that the survival prognosis of these test subjects was improved as a result of the combination therapy (Table 13).

[0179] For example, the predicted mortality rate of the mice administered with UP894-II was 41.4% 24 hours after LPS injection, but actually it was zero. Also, it was predicted that 97.6% of the test subjects would die at the end of the observation period, but the actual mortality rate of UP894-II only stopped at 46.2%. Thus, in this survival rate test, the advantages of using the Artemisia extract and the Acacia extract in combination were evaluated by the Corby formula. In this method, if the actual measured value of a predetermined endpoint measurement is greater than the predicted value calculated hypothetically, a formulation containing two or more bioflavonoid extracts is presumed to have an unexpected synergistic effect.

[0180]

Table 13

[0181] Using the survival and mortality values of the Tatsunamisou extract (RM405 shown in Example 3) (200 mg / kg) and the Acacia extract (RM406 shown in Example 4) (50 mg / kg) at 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 96 hours, 120 hours, and 144 hours after LPS injection, the calculated values of survival rate and mortality rate were obtained and compared with the actual measured survival rate of the composite UP894-II (250 mg / kg) at these specific time points. In this test, an unexpected synergistic effect was confirmed in the combined use of the Tatsunamisou extract (RM405) and the Acacia extract (RM406). The beneficial effect of UP894-II administration exceeded the sum of the effects of its components at all test time points. At the end of the observation period (i.e., 7 days after LPS injection and 14 days after oral administration of the above extracts and compositions), the survival rates of the UP894-II administration group, the Tatsunamisou extract (RM405) administration group, and the Acacia extract (RM406) administration group were 53.9%, 30.8%, and 7.7% respectively, suggesting that these plant extracts have an unexpected synergistic effect in protecting the host from cytokine storm and thus increasing the survival rate of patients during sepsis.

[0182] [Example 13] Effect of a standardized bioflavonoid composition (UP446) on reducing lipopolysaccharide (LPS)-induced acute inflammatory lung injury in rats - Test design This test was designed to evaluate the direct effect of alleviating LPS-induced acute lung injury when the bioflavonoid composition UP446, which contains free B-ring flavonoids and flavans shown in Example 4, was orally administered at 250 mg / kg (high dose) and 125 mg / kg (low dose). Acute lung injury is a clinical syndrome caused by damage to alveolar epithelial cells and capillary epithelial cells, leading to diffuse lung injury as seen in acute respiratory distress syndrome (ARDS). In this test, the test substance was orally administered to Sprague Dawley rats for 7 days before model induction by LPS. On the 8th day, 1 hour after oral administration, 10 mg / kg of LPS dissolved in 0.1 mL / 100 g of PBS was instilled intratracheally (i.t.) into each rat. The same amount of PBS alone was administered intratracheally to normal control rats.

[0183]

Table 14

[0184] LPS induces systemic and pulmonary responses, leading to the accumulation of inflammatory immune cells such as neutrophils and macrophages, and inflammatory cytokines such as IL-1, IL-8, IL-6, MIP-2 / CINC-3, and TNF-α. HMGB1 is actively secreted by macrophages and monocytes or passively released from necrotic cells, causing interstitial pulmonary edema, alveolar pulmonary edema, and epithelial cell damage.

[0185] The surviving animals were sacrificed 24 hours after intratracheal LPS administration. At autopsy, after injecting 1.5 mL of PBS into the right lung lobe via the trachea, bronchoalveolar lavage fluid (BAL) was collected by gently aspirating at least three times. The collected lavage fluid was pooled and centrifuged at 1500 rpm for 10 minutes at 4°C for use in measuring cytokines (e.g., IL-6) and lung protein concentration. This same right lung lobe was collected from each rat for tissue homogenization and used for MIP-2 / CINC-3 activity analysis. The left lung lobe was fixed in neutral buffered formalin and sent to Nationwide Histology for analysis by a board-certified pathologist for histopathological evaluation. Serum collected at autopsy was used to measure cytokines such as TNF-α and IL-1β. After intratracheal instillation of 10 mg / kg of LPS, all animals survived 24 hours after sensitization. The measurements of major cytokines and chemoattractants thought to be involved in the pathology of acute lung infection and the data from histopathological analysis were summarized in the following examples.

[0186] [Example 14] The bioflavonoid composition showed a dose-correlated and statistically significant decrease in serum TNF-α.

[0187] The amount of TNF-α in undiluted rat serum was measured as follows using a Rat TNF-α Quantikine ELISA Kit (Product No. RTA00) from R&D Systems. Undiluted serum was added to a microplate coated with TNF-α antibody. After 2 hours at room temperature, TNF-α in the serum was bound to the plate, and the plate was washed thoroughly. An enzyme-labeled TNF-α antibody was added to the plate and allowed to bind for 2 hours at room temperature. Washing was repeated, and an enzyme substrate was added to the plate. After developing for 30 minutes at room temperature, a stop solution was added, and the absorbance at 450 nm was read. The concentration of TNF-α was calculated based on the absorbance readings of the TNF-α standard curve.

[0188] As is clear from Table 15, a statistically significant increase in serum TNF-α was observed in rats that were sensitized with intratracheal LPS after administration of the solvent. When the standardized bioflavonoid composition UP446, shown in Example 4 and Table 6, was administered to rats, this increase was significantly reduced. A statistically significant dose-correlated decrease was observed in rats orally administered 250 mg / kg and 125 mg / kg of UP446. When these decreases in serum TNF-α concentration were calculated relative to the solvent control, it was found that they were 90.7% and 69.8% in the 250 mg / kg and 125 mg / kg UP446-administered groups, respectively. Sodium butyrate (SB), a positive control, showed a statistically significant (67.9%) decrease in serum TNF-α concentration.

[0189]

Table 15

[0190] [Example 15] The standardized bioflavonoid composition showed a dose-correlated and statistically significant decrease in serum IL-1β.

[0191] The amount of IL-1β in undiluted rat serum was measured as follows using a Rat IL-1β Quantikine ELISA Kit (Product No. RLB00) manufactured by R&D Systems. Undiluted serum was added to a microplate coated with an IL-1β antibody. After 2 hours at room temperature, IL-1β in the serum was bound to the plate, and the plate was washed thoroughly. An enzyme-labeled IL-1β antibody was added to the plate and allowed to bind for 2 hours at room temperature. Washing was repeated, and an enzyme substrate was added to the plate. After developing for 30 minutes at room temperature, a stop solution was added, and the absorbance at 450 nm was read. The concentration of IL-1β was calculated based on the absorbance readings of the IL-1β standard curve.

[0192] Also in this case, in rats administered with UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, a dose-correlated and statistically significant decrease in IL-1β was observed. In LPS-induced acute lung injury rats administered with a solvent, a statistically significant increase in serum IL-1β concentration was observed. Rats administered with UP446 showed a decrease of 81.2% and 61.8% in IL-1β concentration when orally administered at 250 mg / kg and 125 mg / kg, respectively (Table 16). The sodium butyrate (SB) group showed a 65.3% decrease in serum IL-1β concentration. These decreases were statistically significant in both the UP446 group and the sodium butyrate (SB) group.

[0193] [Table 16]

[0194] [Example 16] The standardized bioflavonoid composition showed a dose-correlated and statistically significant decrease in the IL-6 concentration in bronchoalveolar lavage fluid (BAL).

[0195] The amount of IL-6 in undiluted rat bronchoalveolar lavage fluid (BAL) was measured as follows using a Rat IL-6 Quantikine ELISA Kit (Product No. R6000B) from R&D Systems. Undiluted BAL was added to a microplate coated with an IL-6 antibody. After 2 hours at room temperature, IL-6 in the BAL was bound to the plate, and the plate was washed thoroughly. An enzyme-labeled IL-6 antibody was added to the plate and allowed to bind for 2 hours at room temperature. Washing was repeated, and an enzyme substrate was added to the plate. After developing for 30 minutes at room temperature, a stop solution was added, and the absorbance at 450 nm was read. The concentration of IL-6 was calculated based on the absorbance readings of the IL-6 standard curve.

[0196] Consistent with the data for TNF-α and IL-1β above, UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, showed a dose-correlated statistically significant decrease in the IL-6 concentration in BAL. At the high dose (250 mg / kg) of UP446, the IL-6 concentration in BAL decreased by 74.6%, and in the low-dose bioflavonoid composition, the decrease in the IL-6 concentration in BAL was 58.3% (Table 17). The decrease was statistically significant for both the high and low doses of UP446 compared to the acute lung injury rats administered the vehicle. The sodium butyrate (SB) group showed a statistically non-significant 37.7% decrease in IL-6 in BAL compared to the disease model administered the vehicle.

[0197] [Table 17]

[0198] [Example 17] Administration of the standardized bioflavonoid composition resulted in a statistically significant decrease in CINC-3.

[0199] CINC-3 / macrophage inflammatory protein 2 (MIP-2) belongs to a family of chemotactic cytokines called chemokines. MIP-2 belongs to the CXC chemokine family, is called CXCL2, and acts by binding to CXCR1 and CXCR2. It is mainly produced by macrophages, monocytes, and epithelial cells and is responsible for chemotaxis to inflammatory stimuli and neutrophil activation.

[0200] To the wells of a 96-well microplate coated with monoclonal CINC-3 antibody, 50 μL of each rat lung homogenate sample (10 samples per group in the solvent, sodium butyrate (SB), low-dose UP446, and high-dose UP446 groups, and 7 samples in the control group) and 50 μL of assay diluent buffer were added and allowed to bind for 2 hours. After washing the plate 5 times, enzyme-labeled polyclonal CINC-3 was added and allowed to bind for 2 hours. After further washing the wells 5 times, substrate solution was added to the wells to initiate the enzyme reaction, and the reaction was allowed to proceed at room temperature for 30 minutes in the dark. The enzyme reaction produced a blue pigment, which changed to yellow upon addition of the stop solution. The absorbance at 450 nm of each well (corrected at 580 nm) was read and compared to the standard curve of CINC-3 to estimate the amount of CINC-3 in each rat lung homogenate sample.

[0201] Oral administration of 250 mg / kg of UP446 daily for one week resulted in a statistically significant decrease in cytokine-induced neutrophil chemoattractant-3 (CINC-3) in LPS-induced acute lung injury (Table 18). The concentration of CINC-3 in normal control rats administered PBS alone by intratracheal injection was almost zero. In contrast, in intratracheal LPS-induced acute lung injury rats administered the vehicle, the average CINC-3 concentration in lung homogenates was 563.7 ± 172.9 pg / mL. In rats administered 250 mg / kg of UP446, this concentration decreased to an average value of 360.8 ± 110.7 pg / mL. This 36% decrease in CINC-3 concentration in rats administered 250 mg / kg of UP446 was statistically significant compared to the vehicle-administered disease model. The low-dose UP446 group and the sodium butyrate (SB) group had only a slight decrease in CINC-3 concentration in lung homogenates compared to vehicle-administered rats, stopping at 10.5% and 17.7%, respectively.

[0202]

Table 18

[0203] [Example 18] The standardized bioflavonoid composition decreased the total protein in bronchoalveolar lavage fluid (BAL).

[0204] The total protein amount in bronchoalveolar lavage fluid (BAL) was measured as follows using a Pierce BCA Protein Assay Kit (product number 23225) manufactured by ThermoFisher Scientific. BAL was diluted 5-fold, mixed with bicinchoninic acid (BCA) reagent in a microplate, and incubated at 37°C for 30 minutes. The absorbance at 580 nm was read, and the protein concentration in BAL was calculated based on the absorbance reading values of the bovine serum albumin standard curve.

[0205] In rats with LPS-induced acute lung injury administered with the solvent, it was found that the total protein concentration in the BAL increased by 3-fold compared to normal control rats. When UP446 at 250 mg / kg and 125 mg / kg was orally administered to rats daily for one week, the total protein content in the BAL decreased by 45.1% (p = 0.06 compared to the solvent) and 36.6% (p = 0.21), respectively, compared to rats with LPS-induced acute lung injury administered with the solvent (Table 19). In the sodium butyrate (SB) group, which is the positive control, the total protein concentration in the BAL decreased by 30.2% (p = 0.27) compared to rats with LPS-induced acute lung injury administered with the solvent.

[0206] [Table 19]

[0207] [Example 19] The standardized bioflavonoid composition showed a statistically significant decrease in CRP in the bronchoalveolar lavage fluid (BAL).

[0208] Using an Abcam C-reactive protein (PTX1) rat ELISA kit (product number ab108827), the abundance of CRP in a 1,000-fold diluted solution of rat BAL was measured as follows. A 1,000-fold diluted solution of BAL was added to a microplate coated with the CRP antibody. After placing it on a plate shaker and incubating at room temperature for 2 hours, CRP in the BAL was bound to the plate, and the plate was thoroughly washed. A biotinylated C-reactive protein antibody was added to the plate, and it was placed on a plate shaker and incubated at room temperature for 1 hour for binding. Washing was repeated, and streptavidin-peroxidase conjugate was added to the plate. After incubation at room temperature for 30 minutes, washing was repeated, and a chromogenic substrate was added. After developing at room temperature for 10 minutes, a stop solution was added, and the absorbance at 450 nm was read. The concentration of CRP was calculated based on the absorbance reading values of the CRP standard curve.

[0209] In rats with LPS-induced acute lung injury administered with a solvent, a statistically significant 5.6-fold increase in the CRP concentration in the BAL was observed compared to normal control rats. When the standardized bioflavonoid composition UP446 shown in Example 4 and Table 6 was orally administered to rats at a dose of 250 mg / kg for 1 week, the CRP concentration in the BAL decreased by 42.4% compared to the disease model administered with the solvent (Table 20). This decrease was statistically significant (p≤0.05). The positive control sodium butyrate (SB) group and the low-dose UP446 group showed a slightly statistically insignificant decrease in the CRP concentration compared to the disease rats administered with the solvent.

[0210]

Table 20

[0211] [Example 20] The standardized bioflavonoid composition showed a statistically significant decrease in IL-10 in the bronchoalveolar lavage fluid (BAL).

[0212] The abundance of IL-10 in the undiluted BAL was measured as follows using a rat IL-10 Quantikine ELISA kit (product number R1000) manufactured by RandD Systems. Undiluted BAL was added to a microplate coated with an IL-10 antibody. After 2 hours at room temperature, IL-10 in the serum was bound to the plate, and the plate was washed thoroughly. An enzyme-labeled IL-10 antibody was added to the plate and bound for 2 hours at room temperature. Washing was repeated, and an enzyme substrate was added to the plate. After developing for 30 minutes at room temperature, a stop solution was added, and the absorbance at 450 nm was read. The concentration of IL-10 was calculated based on the absorbance reading values of the IL-10 standard curve.

[0213] For 7 days before induction, after oral administration of UP446 daily at doses of 250 mg / kg and 125 mg / kg, the concentration of anti-inflammatory IL-10 in the BAL of diseased rats sacrificed 24 hours after intratracheal instillation of LPS was measured. IL-10 concentration often corresponds to the severity of the infection and inflammatory responses required by the host during infection or injury. As is clear from Table 21, in the rats administered the vehicle, it was found that the concentration of IL-10 was significantly increased 80-fold compared to normal control rats, and it was judged that the severity of acute lung injury was high. In contrast, the rats in the UP446 group showed a dose-correlated decrease in IL-10 in the BAL. When calculating these reduction rates, with UP446 at 250 mg / kg and 125 mg / kg respectively, they were 73.6% and 49.2%. With high-dose (250 mg / kg) UP446, the reduction rate was statistically significant, with p ≤ 0.05. At least in this specific model, due to the fact that reducing disease severity weakens the host's inflammatory response and thus may have the effect of weakening inflammation through upstream mechanisms possibly mediated by HMGB1 secretion, it can be explained that the anti-inflammatory cytokine decreased as a result of UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6. As evidence for this hypothesis, UP446 significantly decreased inflammatory cytokines such as IL-1β, IL-6, and TNF-α statistically significantly, leading to a significant decrease in the inflammatory response, and the need for anti-inflammatory cytokines such as IL-10 is less important for the host. In fact, the IL-10 concentration in the normal control group was almost zero, suggesting that the induction of anti-inflammatory cytokines is based on the presence or severity of acute lung injury. The significant decrease in IL-10 by the composition of free B-ring flavonoids and flavans demonstrated the establishment of the host defense mechanism.

[0214] [Table 21]

[0215] [Example 21] The standardized bioflavonoid composition reduced the overall severity of lung injury.

[0216] H&E-stained lung tissues were used to evaluate the severity of lung injury as a result of intratracheal LPS. The left lung lobe was used for histopathological analysis. As is clear from Table 22 and Figure 7, rats in the solvent administration group showed statistically significant increases in the severity of lung injury (3.5-fold increase), pulmonary edema (2.5-fold increase), and infiltration of polymorphonuclear (PMN) leukocytes (2.4-fold increase) due to intratracheal LPS. Oral administration of a high dose of UP446 (250 mg / kg) to rats daily for one week resulted in a statistically significant 20.8% decrease in the overall severity of lung injury compared to LPS-induced acute lung injury rats administered the solvent. Similarly, a strong tendency towards a decrease in pulmonary edema (23.3% decrease, p = 0.08) was observed in the high-dose UP446 group compared to rats administered the solvent. The sodium butyrate (SB) group, which was the positive control, and the low-dose UP446 group showed only slight changes in histopathological evaluation compared to diseased rats administered the solvent.

[0217]

Table 22

[0218] [Example 22] D-Galactose-Induced Immunosenescence Model as an Endogenous and Exogenous Attack Trigger Response Systemic administration of D-galactose induces accelerated immune cell aging and affects the immune response during sensitization, similar to aged mice. These phenomena are presumed to resemble the immune response profile of the elderly. The novel protected subject UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, was tested in this experimental aging mouse model, and its immune-stimulating effect was demonstrated. CD-1 mice (12 weeks old) for experiments were purchased and used in the accelerated aging test after 2 weeks of acclimation. The mice were randomly assigned to 4 immune groups and 4 non-immune groups. The immune groups were G1 = normal control + solvent (0.5% CMC), G2 = D-galactose + solvent, G3 = D-galactose + 200 mg / kg UP446, and G4 = D-galactose + 100 mg / kg UP446. The non-immune administration groups were G1 = normal control + solvent (0.5% CMC), G2 = D-galactose + solvent, G3 = D-galactose + 200 mg / kg UP446, and G4 = D-galactose + 100 mg / kg UP446. Ten mice were assigned to each administration group.

[0219] Mice were subcutaneously injected with 500 mg / kg of D-galactose daily for 10 weeks to induce aging. Four weeks after induction, oral administration of two doses (low dose 100 mg / kg and high dose 200 mg / kg) of UP446 suspended in 0.5% CMC was initiated in both the immune and non-immune groups. At week 8, each mouse except those in the non-immune group was injected with 3 μg of GSK's Fluarix quadrivalent IM (2020 - 2021 season influenza vaccine). This vaccine contained 60 μg of hemagglutinin (HA) per 0.5 mL human single-dose. This vaccine was formulated to contain 15 μg each of 4 influenza strains (such as H1N1, H3N2, B-Victoria lineage, and B-Yamagata lineage) for single immunization.

[0220] UP446 at two doses was administered orally via a gastric tube daily for 6 weeks from week 5 to week 10. At necropsy (i.e., 14 days after immunization), whole blood (1 mL) was collected, 110 μL was aliquoted for the flow cytometry immunopanel (kept cold on ice and transported to Flow Contract Site Laboratory, Bothell, WA), serum was separated from the remaining blood for antibody ELISA and enzyme assays (Unigen, Tacoma WA) (serum yield approximately 400 μL), 60 μL was placed into two tubes for cytokine analysis, and (transported by Fedex overnight to Sirona DX, Portland, OR). The weights of the thymus and spleen of each animal were measured, and the thymus index and spleen index were determined. Representative images of the thymus and spleen were taken from each group. At necropsy, the spleen was kept cold on dry ice and transferred to -80 °C for future use. The thymus fixed with paraformaldehyde and sucrose was sent to Nationwide histology for senescence-associated β-galactosidase staining and analysis.

[0221] [Example 23] UP446 produced a statistically significant increase in the thymus index.

[0222] Repeated subcutaneous administration of D-galactose to mice results in a poor immune response similar to the changes that occur during the normal aging process. The thymus is one of the most important immune organs affected by chronic exposure to D-gal. The thymus index is a good indicator of the strength of the body's immune function. The higher the thymus index, the more normal and stronger the non-specific immune response. In immunized mice, D-gal mice administered with the solvent showed a significant decrease (30.3%) in the thymus index compared to normal control mice. This decrease in the thymus index was reversed by administering the standardized bioflavonoid composition UP446, shown in Example 4 and Table 6, at two doses. Mice orally administered 200 mg / kg and 100 mg / kg of UP446 showed increases in the thymus index of 47.4% and 49.4% respectively compared to the D-gal group administered with the solvent. This reversal was statistically significant for both doses of UP446 compared to D-gal mice administered with the solvent. Similarly, non-immunized mice administered 200 mg / kg and 100 mg / kg of UP446 also showed statistically significant increases in the thymus index. These increases were found to be 27.4% and 31.6% respectively compared to D-gal mice administered with the solvent. In this study, regardless of the immune status, it was confirmed that UP446 supplementation protects mice from age-related thymic atrophy caused by D-galactose injection.

[0223]

Table 23

[0224] [Example 24] The bioflavonoid composition increased complement C3.

[0225] At the end of the test, serum was collected and evaluated for markers of humoral immunity such as the C3 component of the complement system. As is clear from Table 24, the complement C3 in the immunocompetent control group was significantly lower compared to the non-immunized control group. In both the immunized D-Gal+UP446 groups, the complement C3 was significantly higher than that in the immunized control group. In the non-immunized D-Gal+UP446 administration group, there was a tendency for the complement C3 to increase compared to the non-immunized D-Gal group, and in the immunized group administered with D-Gal+200 mg / kg UP446 (the standardized bioflavonoid composition shown in Example 4 and Table 6), the complement C3 was significantly increased compared to the immunized D-Gal group, demonstrating that humoral immunity was enhanced by UP446 in immunosenescent animals responsive to vaccination.

[0226]

Table 24

[0227] [Example 25] Effect of the bioflavonoid composition on CD3+ T cells (% of lymphocyte population) in whole blood CD3+CD45+ cells are a T cell population. When expressed as a percentage of total white blood cells (CD45+ cells), it was found that in non-immunized animals administered with 200 mg / kg UP446+D-Gal, the percentage of circulating T cells tended to be higher than that in the D-gal group, and UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, was judged to enhance CD3+ T cell expansion or differentiation in non-immunized animals.

[0228]

Table 25

[0229] [Example 26] Effect of the bioflavonoid composition on CD4+ helper T cells (% of lymphocyte population) in whole blood CD45+CD3+CD4+ cells are helper T cells, which recognize antigens on antigen-presenting cells and respond by cell division and cytokine secretion. When expressed as a percentage of total white blood cells (CD45+ cells), it was found that immunized animals administered D-Gal had a significantly lower percentage of circulating helper T cells two weeks after influenza vaccination compared to the control group. Both the immunized D-Gal group and the immunized D-Gal+UP446 (200 mg / kg) group also had significantly lower CD4+ helper T cells compared to the non-immunized group.

[0230]

Table 26

[0231] [Example 27] Effect of bioflavonoid composition on CD8+ cytotoxic T cells (% of lymphocyte population) in whole blood CD45+CD3+CD8+ cells are cytotoxic T cells, which respond to pathogens by cell division and secretion of apoptosis-promoting enzymes and kill infected cells. When expressed as a percentage of total white blood cells (CD45+ cells), non-immunized animals administered D-Gal+UP446 (200 mg / kg) had a significantly increased CD8+ cytotoxic T cells compared to both the non-immunized control group and the non-immunized D-gal group. The immunized D-Gal+UP446 (200 mg / kg) group had a significantly lower number of cytotoxic T cells than the non-immunized D-Gal+UP446 (200 mg / kg) group.

[0232]

Table 27

[0233] [Example 28] Effect of bioflavonoid composition on natural killer cells (% of lymphocyte population) in whole blood Two different natural killer cell markers, mouse CD49b and NKp46, were used to determine the percentage of natural killer cells in the leukocyte population. Natural killer cells are involved in the innate immune system. When activated, they secrete cytokines and granules, recruit immune cells, and directly cause cell death in cells infected with pathogens, so they are important for the immediate immune response against pathogens and are active in the early stage of systemic infections. CD49b is an integrin that is specifically present in most natural killer cells and a subset of T cells that are thought to be natural killer T (NKT) cells. NKp46 is a natural cytotoxic receptor that is exclusively present in natural killer cells and does not direct NKT cells. NK is generally CD45+CD3-CD49b+NKp46+, but NKT cells and NK-like T cells also express CD3, so they are excluded (Goh W)(Narni-Mancinelli E). When expressed as a percentage of total leukocytes (CD45+ cells), two weeks after influenza vaccination, in the immune D-Gal group, CD3-CD49b+ NK cells were significantly lower than in either the immune control group or the UP446 administration group (Table 28). From this, it was determined that D-Gal reduces the NK cell population and decreases the ability of the innate immune system to react to pathogens, but this effect is reversed by UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6.

[0234] Focusing on the CD3-NKp46+ population, non-immunized animals administered D-Gal+UP446 (100 mg / kg) had a significantly higher percentage of natural killer cells than the non-immunized D-gal group, and the immune D-Gal+UP446 (200 mg / kg) group had a significantly higher percentage of CD3-NKp46+ cells than the immune D-Gal group (Table 29). The immune D-Gal+UP446 (200 mg / kg) group also had significantly more NK cells than the non-immunized D-Gal+UP446 (200 mg / kg) group.

[0235] From these results, it was generally determined that administration of D-Gal+UP446 increased the natural killer cell population in both non-immunized and immunized animals compared to administration of D-Gal alone. Therefore, it is judged that UP446 helps to prime the immune system against pathogens by increasing the cell population involved in the immediate innate immune response.

[0236]

Table 28

[0237]

Table 29

[0238] [Example 29] Effect of the bioflavonoid composition on TCRγδ+ gamma-delta T cells (percentage of lymphocyte population) in whole blood When the CD4+ gamma-delta T cell population was represented as the total number of CD4+TCRγδ+ cells per 1 μL of blood, the cell count in non-immunized D-Gal+UP446 (200 mg / kg) was significantly higher than that in the non-immunized D-Gal group. The increase in CD4+TCRγδ+ cells in the D-Gal+UP446 (200 mg / kg) group is thought to indicate enhanced immune readiness or priming.

[0239]

Table 30

[0240] [Example 30] Effect of the bioflavonoid composition on cytokines GM-CSF and Il-12p70 in serum Serum collected from immunized mice two weeks after influenza vaccination was transported for cytokine profiling by Luminex technology. IL-12p70 and GM-CSF cytokines were detectable in all 10 samples from each group. The decrease in GM-CSF in the D-Gal+UP446 (100 mg / kg) group did not reach significance compared to the D-Gal group (p = 0.058), but the decrease in IL-12p70 in the D-Gal+UP446 (200 mg / kg) group reached statistical significance compared to the normal control group (p = 0.010). There was no difference between the D-Gal group and the D-Gal+UP446 (200 mg / kg) group, which was probably due to the variation within the D-Gal group.

[0241]

Table 31

[0242] [Example 31] Effect of Bioflavonoid Composition on Advanced Glycation End Products (AGEs) The mechanism by which D-gal induces an aging phenotype is mediated by free radicals, particularly the generation of advanced glycation end products. Therefore, we attempted to measure antioxidant enzyme concentrations and free radical concentrations to investigate whether UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, affects this aspect of the mouse model (Azman KF).

[0243] Advanced glycation end products (AGEs) in non-immune and immune serum samples were measured. The non-immune D-Gal+UP446 group was found to have significantly lower AGEs than the non-immune D-Gal group, and it was determined that administration of UP446 reduces reactive oxygen species under normal physiological conditions.

[0244]

Table 32

[0245] [Example 32] Effect of Bioflavonoid Composition on Glutathione Peroxidase Glutathione peroxidase neutralizes oxygen radicals and prevents oxidative damage to cell structures, proteins, and nucleic acids. Reactive oxygen species are used as second messengers in immune signal transduction (Ighodaro OM). Enhanced expression of antioxidant enzymes is an indicator of the ability to neutralize excessive reactive oxygen species.

[0246] The activity of glutathione peroxidase (GSH-Px) in the serum samples of immunized mice was measured. It was found that in the immunized D-gal+UP446 group, the glutathione peroxidase activity was significantly higher than that in the immunized D-gal group at both concentrations. Therefore, it was determined that the ability to neutralize reactive oxygen species was enhanced after administration of UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6.

[0247]

Table 33

[0248] [Example 33] Effect of bioflavonoid composition on protein expression of NFκB In the mice of the non-immunized group administered with 200 mg / kg of UP44, a statistically significant suppression of NFκB expression was observed. NFκB is a transcription factor involved in the activation of immune cells. Normally, it is inactivated by protein-protein interaction, but when the host defense response becomes active, it is stabilized, translocates into the nucleus, and is upregulated. The spleen homogenate was electrophoresed by SDS-PAGE, transferred, and the above protein was blotted. The band intensity was measured by densitometry and normalized to the β-actin loading control for each protein of interest. When comparing the semi-quantification of each protein of interest among the groups, it was found that in the non-immunized 200 mg / kg UP446+D-Gal group, the NFκB concentration was significantly lower than that in the D-Gal alone group. In the influenza vaccine immunized group, in the bioflavonoid composition UP446+D-Gal group, the expression of NFκB protein was significantly higher than that in the normal control group, and it was determined that the host defense mechanism was induced.

[0249]

Table 34

[0250] [Example 34] Effect of Bioflavonoid Composition on Protein Expression of HMGB1 Extracellular HMGB1 is an alarmin protein involved in enhancing the immune response. It is secreted from the nucleus, passes through the cytoplasm, and reaches the circulation. The spleen homogenate was electrophoresed by SDS-PAGE, transferred, and the above protein was blotted. The band intensity was measured by densitometry and normalized to β-actin loading control for each protein of interest. When comparing the semi-quantification of each protein of interest among each group, it was found that the non-immunized 200 mg / kg UP446 + D-gal group had a significantly lower HMGB1 concentration.

[0251]

Table 35

[0252] [Example 35] Effect of Bioflavonoid Composition on Hyperoxia-Induced Mortality in Mice Infected with Pseudomonas aeruginosa In this experiment, mice were acclimated for one week before induction. To investigate whether UP446, a bioflavonoid composition disclosed as the protected subject matter of this application, can reduce animal mortality and increase its survival rate, after oral administration of UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, at a dose of 250 mg / kg for 7 days, the mice were exposed to high oxygen (>90% oxygen for 72 hours), and this exposure was continued for 3 days. Then, Pseudomonas aeruginosa (PA) was inoculated into the mice. After bacterial inoculation, the mice were observed for 48 hours. Pre-exposure to high oxygen resulted in a significantly higher mortality rate (O2) compared to mice maintained in room air (Table 36, RA). Unexpectedly, in mice exposed to high oxygen for 48 hours, substantial mortality occurred 24 hours after PA inoculation. The mortality rate of mice inoculated with the same amount of PA and maintained in room air (RA) was 9%, while in mice administered the solvent under high oxygen for 2 days before PA inoculation, a mortality rate of 64% was observed. On the other hand, in mice prophylactically administered resveratrol (RES) and UP446 for 7 days before 2 days of high oxygen exposure and subsequent PA inoculation, the mortality rates 24 hours after inoculation were 27.3% and 28.6% respectively. These results suggest that UP446 protected the host from oxidative stress and microbial infection, leading to a decrease in mortality. In the LPS-induced animal sepsis tests of Examples 10 to 12, supplementation with UP446 resulted in a statistically significant decrease in mortality, and the survival rate data of this example confirmed by UP446 are consistent with the data described in Examples 10 to 12.

[0253]

Table 36

[0254] [Example 36] Effect of Bioflavonoid Composition on Acute Lung Injury Induced by Bacterial Infection and Worsened by Oxidative Stress To investigate the regulatory effect on natural host defense homeostasis, after orally administering the bioflavonoid composition UP446 to mice at a dose of 250 mg / kg for 7 days, (while continuing UP446 administration)> after exposing them to 90% O2 for 48 hours, Pseudomonas aeruginosa (PA) was inoculated. The mice were euthanized 24 hours after bacterial inoculation, the lungs were lavaged, and the total protein content in the bronchoalveolar lavage fluid was measured. When exposed to hyperoxia prior to microbial infection, significantly higher severity of acute lung injury occurred in these mice (O2) as judged from the occurrence of protein edema compared to mice maintained in room air (RA). The well-known antioxidant resveratrol (RES) significantly suppressed this effect. The decrease in the total protein content in the bronchoalveolar lavage fluid of mice in the UP446 administration group was statistically significant compared to control mice (O2) infected with microorganisms under hyperoxia and administered a solvent. These results suggest that UP446 can suppress acute lung injury induced by secondary bacterial infection and exacerbated by oxidative stress.

[0255]

Table 37

[0256] [Example 37] Effect of Bioflavonoid Composition on Bacterial Clearance in Lung Tissue It has been previously shown by Patel et al., 2013 that exposure to hyperoxia reduces host defense against bacterial infections and increases the bacterial burden in lung tissue after microbial infection. According to the results in Table 38, indeed, pre-exposure to hyperoxia (O2) increased the bacterial burden compared to mice maintained in room air (RA). In mice administered resveratrol and in mice administered UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, lung injury was significantly suppressed, and in these mice, the bacterial burden also significantly decreased. According to the data, the difference in the bacterial burden in lung tissue was statistically significant compared to microbial infection control mice (O2) administered a solvent under hyperoxia. These results suggest that UP446 can regulate natural host defense homeostasis to reduce the bacterial burden in lung tissue.

[0257] [Table 38]

[0258] [Example 38] Effect of Bioflavonoid Composition on Bacterial Clearance in Airways In the above example, it was shown that upon exposure to hyperoxia, host defense against bacterial infections decreased and the bacterial load in lung homogenates increased. According to the results in Table 39, the bacterial load in the airways significantly increased in mice pre-exposed to hyperoxia (O2) compared to mice maintained in room air (RA). In mice administered resveratrol (RES), lung injury was significantly suppressed, and the airway bacterial load was also significantly lower. Similarly, mice administered UP446 had a significantly lower bacterial load in the airways compared to bacterially infected mice exposed to hyperoxia and administered solvent alone. These differences in the airway bacterial load were statistically significant compared to control mice (O2) exposed to hyperoxia and administered solvent. These results suggest that UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, can modulate natural host defense homeostasis to reduce the bacterial load in the airways.

[0259] [Table 39]

[0260] [Example 39] Effect of Bioflavonoid Composition on the Accumulation of Extracellular HMGB1 in Airways Accumulation of extracellular HMGB1 in the airway may reduce innate immunity and lead to a decrease in the ability to eliminate invading pathogens and apoptotic neutrophils. As a result, there is a risk of acute respiratory infections, lung injury, and even death (Entezari et al., 2012; Patel et al., 2013). To investigate whether the suppression of acute lung injury by UP446 in bacterially infected mice exposed to hyperoxia was due to its effect on the accumulation of extracellular HMGB1 in the airway, the concentration of HMGB1 in the bronchoalveolar lavage fluid was measured. As previously shown, when these mice were exposed to hyperoxia for a long time and then infected with microorganisms, the accumulation of HMGB1 in the airway increased. When mice were exposed to hyperoxia and infected with microorganisms, the concentration of HMGB1 increased 4.8-fold. Pretreatment with resveratrol (RES) or UP446 could suppress this increase. When animals were pretreated with RES and UP446, the HMGB1 expression levels decreased by 74.9% and 71.6%, respectively, compared with mice that were exposed to hyperoxia, infected with bacteria, and administered a solvent. These data suggest that UP446, a bioflavonoid composition disclosed in the present application, can reduce the accumulation of airway HMGB1 in mice exposed to hyperoxia and infected with bacteria. This correlates with a significant enhancement of the ability of UP446 to improve the host defense mechanism against microbial infection in the respiratory system.

[0261]

Table 40

[0262] [Example 40] Effect of the bioflavonoid composition on HMGB1 in the lung tissue of hACE2 transgenic mice infected with SARS-CoV-2 hACE2 transgenic mice were given 10 5 TCID 50A disease model was induced by intranasal spraying of 50 μL of SARS-CoV-2 virus (Bao et al. 2020). Within 2 hours of the intranasal spray of SARS-CoV-2 virus, mice were orally administered UP894-II, a bioflavonoid composition shown in Example 4 and Table 6, at doses of 400 mg / kg and 200 mg / kg. Daily administration was maintained for a total of 5 days (i.e., 0 dpi to 4 dpi). Normal transgenic control mice not infected with the virus and the disease model (infected with the virus) were administered the solvent (0.5% CMC) alone in an amount of 10 mL / kg. Necropsy was performed at 5 dpi. The right whole lung was homogenized to monitor tissue HMGB1 protein expression.

[0263] Lung tissue was excised, snap-frozen in liquid nitrogen, and stored at -80 °C until homogenization. The tissue was suspended in lysis buffer at a concentration of 50 mg of tissue per 1 mL of lysis buffer and homogenized. The samples were placed on ice for 30 minutes and vortexed every 5 minutes. The samples were centrifuged for 30 minutes and the pellet was discarded. Proteins were quantified by BCA assay. Briefly, a 0 - 10 μg standard curve and BCA working solution (50:1 reagent A:B) were prepared. 20 μL of the sample volume was mixed with 200 μL of BCA working solution in a microplate and incubated at 37 °C for 30 minutes. The plate absorbance at 562 nm was read and the amount of protein was calculated based on the absorbance of the standard curve. 40 μg of protein from each sample was mixed with sodium dodecyl sulfate loading buffer and boiled at 95 - 100 °C for 5 minutes to obtain denatured and reduced protein samples.

[0264] Polyacrylamide gel was prepared, the prepared protein sample was loaded, and electrophoresis was carried out with Tris-glycine running buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS, pH 8.3). The gel was immersed in transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol) and transferred by the wet transfer method. The membrane was stained with Ponceau Red to visualize the protein and confirmed that it was sufficiently transferred. Briefly, the membrane was washed with Tris-buffered saline with Tween 20 (TBST) containing 0.1% Tween 20. The stock solution of Ponceau Red was diluted 10-fold and added. The membrane was incubated with an agitator for 5 minutes and then washed thoroughly with water until the bands became clear.

[0265] The membrane was blocked and incubated overnight at 4 °C with the primary antibody (100 - 3000-fold dilution) in TBST. The membrane was washed 3 times for 5 minutes each to remove unbound primary antibody. The membrane was incubated with the secondary antibody (2000-fold dilution) labeled with horseradish peroxidase (HRP) in TBST at room temperature with stirring for 1 hour. An ECL Western blot detection kit (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for chemiluminescence detection to analyze the immunoblot. ImageJ (version 1.41, NIH, Baltimore, MD, USA) was used to perform quantification of the image data.

[0266] As is clear from Fig. 8, transgenic mice infected with SARS-CoV-2 and administered a solvent showed a two-fold increase in lung HMGB1 protein expression compared to normal transgenic control mice not infected with the virus. The increase in lung HMGB1 concentration in the solvent-administered group was statistically significant compared to uninfected normal controls. In contrast, when transgenic mice infected with the SARS-CoV-2 virus were administered the bioflavonoid composition UP894-II at two doses, it was found that the expression of HMGB1 protein in lung tissue decreased to the level of uninfected normal control transgenic mice. These decreases in lung HMGB1 expression levels as a result of administration of both high and low doses of the bioflavonoid composition were statistically significant compared to transgenic mice infected with SARS-CoV-2 and administered a solvent. Since HMGB1 in lung tissue decreased, it was determined that the bioflavonoid composition disclosed in the present application improved the host defense mechanism and reduced the possibility of damage to the lungs and other organs associated with the lethal cytokine storm after SARS-Cov-2 coronavirus infection.

[0267] [Example 41] Evaluation of the Bioflavonoid Composition UP446 in a Human Clinical Trial Protocol: A randomized triple-blind placebo-controlled parallel-group clinical trial to investigate the test article for the support of immune function in healthy adults. The objective of this trial was to investigate the effect of UP446, which contains 60% or more free B-ring flavonoids and 10% or more flavans, and is composed of these in some embodiments and manufactured as shown in Examples 4 and Tables 5 and 6, as the test article (IP) on the support of immune function in healthy adults.

[0268] In a randomized triple-blind placebo-controlled parallel-group trial, the test article was evaluated for its effect of supporting immune function in a healthy adult population 28 days before and 28 days after influenza vaccination. This trial was conducted on men and women aged 40 to 80 years who had not yet received the influenza vaccine but had the intention to receive it, agreed to verbally report their influenza vaccination history, agreed to maintain their current lifestyle as much as possible throughout the entire trial period according to their respective abilities to maintain diet, medication, nutritional supplements, exercise, and sleep, and not to consume new nutritional supplements, were judged to be healthy by a certified investigator (QI) based on their medical history and test results, completed the questionnaires and diaries related to the trial, had the intention to complete the hospital visits, and signed a written informed consent to participate in this trial voluntarily.

[0269] FLUCELVAX(R)QUAD, Drug Identification Number (DIN) 02494248, is a quadrivalent influenza vaccine (QIV) designed for the prevention of influenza subtypes A and B and for immunization of adults and children 9 years of age and older.

[0270]

Table 41

[0271] The following subjects were excluded: 1. Women who were pregnant, lactating, or planning to become pregnant during the trial. 2. Participants known to be allergic to the active or inactive ingredients of UP446, placebo, or the influenza vaccine. 3. Participants who had not received the influenza vaccine before the baseline in September 2020 or before the 28-day vaccine inoculation. 4. Participants who self-reported being diagnosed with COVID-19 before the baseline or before the 28-day vaccine inoculation. 5. Participants who had received the COVID-19 vaccine. 6. Currently using prescription immunomodulators (including corticosteroids) such as immunosuppressants or immunostimulants within 4 weeks from the baseline. 7. Participants currently using nutritional supplements or herbal medicines related to boosting or regulating the immune system and having no intention of washout.

[0272]

Table 42

[0273]

Table 43

[0274] The subjects were expected to participate in the test for up to 56 days. The first visit (screening, -45 days to -4 days) was for informed consent, and the second visit (baseline, 0 day) was for eligibility confirmation and randomization, and the subjects were asked to participate in the test.

[0275] At the second visit (0 day), the third visit (28 days), and the fourth visit (56 days), the primary and secondary effects of the test and safety endpoints were evaluated. Demographic information and medical history were recorded at the screening visit. The subjects were given 250 mg of the bioflavonoid composition UP446 twice a day with meals every day until influenza vaccination (28 days), and continued to take 250 mg of UP446 twice a day for another 4 weeks (until 56 days).

[0276] The primary test outcome was to evaluate the difference between UP446 and placebo in the changes in immune parameters evaluated by lymphocyte populations (CD3+, CD4+, CD8+, CD45+, TCRγδ+, CD3 - CD16+56+) and immunoglobulins (IgG, IgM, and IgA) in the blood from baseline to 28 days and 56 days.

[0277] Statistical analysis was performed to obtain summary statistics including demographic characteristics and the mean, median, standard deviation, minimum, maximum, and proportion (in the case of categorical) of outcome measurements for the entire sample and each test group. When the assumption of normality was satisfied, analysis of variance (ANOVA) was used to test for differences in the means of continuous variables between the two intake groups (UP446 and placebo), and when the assumption of normality was not satisfied, the Kruskal–Wallis test was used. The chi-square test and Fisher's exact test (when the cell count was less than 5) were used as needed to investigate differences in categorical variables. Repeated measures analysis of variance (linear mixed effects model) was used to test for differences in the mean outcome values over time between the intake groups. The baseline value was included in each model as a covariate. Similarly, repeated measures analysis of variance (linear mixed effects model) was used to test for differences between the two intake groups in the mean of the outcome changes over time (from baseline at 28 days, 56 days, and from 28 days to 56 days), and the baseline value was included in each model as a covariate. Pairwise statistical significance was determined from the LMM (between and within groups). The Bonferroni correction was used for pairwise comparisons. Statistical significance was defined as a p-value ≤ 0.05. Analyses were performed using Statistical Analysis System software version 9.4 (SAS Institute Inc., Cary, NC, USA).

[0278] In the pre-clinical data report, statistically significant outcomes were observed for primary endpoints such as immunoglobulin A (IgA) from the oral intake of the standardized bioflavonoid composition shown in Example 4 and Table 6. As is clear from Table 43, after 8 weeks of intake, subjects who took the bioflavonoid composition UP446 showed a statistically significant increase in immunoglobulin A (IgA), a mucosal immune index, compared to subjects who took the placebo from day 28 to day 56 (P = 0.0260). Among the participants who took UP446, the change in IgA before and after vaccination was 0.08755 g / L higher than that of the participants who took the placebo (p = 0.0260). Within the group, subjects supplemented with UP446 showed a statistically significant increase in IgA of 0.05720 g / L on average from day 0 to day 56 (p = 0.0412) and 0.06280 g / L from day 28 to day 56 (p = 0.0252). These data clearly show that IgA, which is the main immunoglobulin of the healthy respiratory system and is considered the most important immunoglobulin for mucosal defense, is an important activity of the bioflavonoid composition in the regulation of the human host defense mechanism.

[0279]

Table 44

[0280] For secondary outcomes, the differences between UP446 and placebo at days 28 and 56 were evaluated for the following: 1. The number of confirmed COVID-19 infections; 2. The number of confirmed influenza cases; 3. The impact of COVID-19 on quality of life as evaluated by a COVID-19 impact on quality of life questionnaire; 4. Use of over-the-counter cold and influenza medications. Also, for the following, the differences between UP446 and placebo at day 56 were evaluated: 1. The number of hospitalizations due to COVID-19; 2. The number of hospitalizations due to influenza.

[0281] Other outcomes investigated changes from baseline to measurements at 28 and 56 days for the following items and evaluated the differences between the standardized bioflavonoid composition UP446, shown in Example 4 and Table 6, and placebo. 1. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP); 2. Hematological parameters: white blood cell (WBC) count and fractions (neutrophils, lymphocytes, monocytes, eosinophils, basophils), reticulocyte count, red blood cell (RBC) count, hemoglobin, hematocrit, platelet count, RBC indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW)); 3. Complement C3 and C4 proteins; 4. Mean overall severity index measured by the area under the curve (AUC) of the daily symptom scores of the Modified Wisconsin Upper Respiratory Symptom Survey (WURSS)-24; 5. Mean symptom severity score measured by the AUC of the daily severity symptom scores of the WURSS-24; 6. Number of healthy days evaluated by the Modified WURSS-24 questionnaire (defined as the number of days in which the answer to the question "How do you feel today?" was 0 (disease-free)); 7. Number of sick days evaluated by the Modified WURSS-24 questionnaire (defined as the number of days in which the answer to the question "How do you feel today?" was any number from 1 to 7 (sick)); 8. Frequency of general upper respiratory tract infection (UTRI) symptoms evaluated by the Modified WURSS-24 questionnaire; 9. Duration of general UTRI symptoms evaluated by the Modified WURSS-24 questionnaire; 10. Severity of general UTRI symptoms evaluated by the Modified WURSS-24 questionnaire; 11. Vitality and quality of life evaluated by the Vitality and Quality of Life (QoL) questionnaire.

[0282] Blood samples were collected from each subject and stored for subsequent analysis to analyze the differences between the standardized bioflavonoid composition and placebo shown in Example 4 and Table 6 for changes in the following items from baseline to 28 and 56 days. 1. Cytokines (GM-CSF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, TNF-α, TNF-β / LTA150). 2. High-mobility group box 1 (HMGB1) protein, nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf-2). 3. Oxidative stress evaluated by 8-isoprostaglandin F2α, catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), and advanced glycation end products (AGE). 4. Hemagglutination inhibition (HI) titer against specific virus strains.

[0283] In addition to the efficacy analysis, safety evaluation is also performed by testing each blood sample for the following attributes. 1. Clinical chemistry parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, creatinine, electrolytes (Na+, K+, Cl-), estimated glomerular filtration rate (eGFR), glucose; 2. Incidence of pre- and post-expression adverse events; 3. Vital signs (blood pressure (BP) and heart rate (HR)).

[0284]

Table 45

Claims

1. A bioflavonoid composition for reducing the expression level of high-mobility group box 1 protein (HMGB1), comprising: at least one bioflavonoid extract with a high concentration of at least one free B-ring flavonoid and at least one bioflavonoid extract with a high concentration of at least one flavan, wherein the at least one bioflavonoid extract with a high concentration of at least one free B-ring flavonoid is derived from the root of Scutellaria baicalensis and is concentrated by extraction with water at a temperature of 90 to 95 °C, and the at least one bioflavonoid extract with a high concentration of at least one flavan is derived from the heartwood of Acacia catechu and is concentrated by (i) extraction with water at a temperature of 105 to 115 °C, (ii) extraction with a mixture of water and methanol, (iii) extraction with methanol, or (iv) extraction with a mixture of methanol and tetrahydrofuran.

2. The composition according to claim 1, wherein the at least one bioflavonoid extract with a high concentration of at least one free B-ring flavonoid and the at least one bioflavonoid extract with a high concentration of at least one flavan in the composition are in the range of 1% to 98% based on the weight of each extract, and the optimal weight ratio of the free B-ring flavonoid to the flavan is 80:

20.

3. The composition according to claim 1, wherein the at least one bioflavonoid extract with a high concentration of at least one free B-ring flavonoid contains 0.5% to 99.5% of one or more free B-ring flavonoids.

4. The composition according to claim 1, wherein the at least one bioflavonoid extract with a high concentration of at least one flavan contains 0.5% to 99.5% of catechin.

5. The composition according to claim 1, wherein the free B-ring flavonoid comprises at least one of baicalin, baicalein, baicalein glycoside, wogonin, wogonin glucuronide, wogonin glycoside, oroxylin, oroxylin glycoside, oroxylin glucuronide, chrysin, chrysin glycoside, chrysin glucuronide, scutellarin and scutellarin glycoside, norwogonin, norwogonin glycoside, galangin, or a combination thereof.

6. The composition according to claim 1, wherein the at least one bioflavonoid extract with a high concentration of at least one flavan comprises at least one of catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epiteafllavin, epicatechin gallate, gallocatechin gallate, theaflavin, theaflavin gallate, or a combination thereof.

7. The composition according to claim 1, wherein the at least one bioflavonoid extract with increased concentration of at least one free B-ring flavonoid is concentrated and derived from a higher plant genus further comprising Desmos, Achyrocline, Oroxyllum, Buchenavia, Anaphalis, Cotula, Gnaphlium, Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus, Alpinia, or a combination thereof.

8. The at least one bioflavonoid extract with an increased concentration of at least one free B-ring flavonoid is selected from Scutellaria baicalensis (Skullcap), Scutellaria barbata, Scutellaria orthocalyx, Scutellaria lateriflora (Blue Skullcap), Scutellaria galericulata (Marsh Skullcap), Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria galericulata (Marsh Skullcap), Scutellaria indica (Indian Skullcap), Scutellaria sessilifolia, Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria orientalis, Oroxylum indicum, Passiflora caerulea (Passionflower), Passiflora incarnata (Maypop), Pleurotus ostreatus (Oyster Mushroom), Lactarius deliciosus (Saffron Milk Cap), Suillus belliniiThe composition according to claim 1, which is highly concentrated and derived from a plant species further comprising bellinii), chamomile, carrot, mushroom, honey, propolis, passion flower, Indian trumpet flower, or a combination thereof.

9. The at least one bioflavonoid extract with at least one flavan concentrated at a high level is derived from Acacia catechu (Acacia catechu: black catechu), Senegalia catechu, Acacia concinna (Acacia concinna: Sompoy), Acacia farnesiana (Acacia farnesiana: mimosa), Acacia Senegal (Acacia Senegal: gum arabic), Acacia speciosa, Acacia arabica (Acacia arabica: false gum arabic), Acacia caesia, Acacia pennata (Acacia pennata: cha om), Acacia sinuata, Acacia mearnsii (Acacia mearnsii: black wattle), Acacia picnanta (Acacia picnanta: golden wattle), Acacia dealbata (Acacia dealbata: silver wattle), Acacia auriculiformis (Acacia auriculiformis: earleaf acacia), Acacia holosericea (Acacia holosericea: strap wattle), Acacia mangium, Anacardium occidentale (Anacardium occidentale: cashew nut shell), Uncaria gambir (Uncaria gambir: Gambier catechu), Uncaria rhynchophylla (Uncaria rhynchophylla: hook climber), Camellia sinensis (Camellia sinensis: tea plant), Camellia assamica (Camellia assamica: Assam tea), Euterpe oleracea (Euterpe oleracea: acai), Caesalpinia decapetala (Caesalpinia decapetala: peacock flower), Delonix regia (Delonix regia: royal poinciana), Ginkgo biloba (Ginkgo biloba: ginkgo), Acer rubrum (Acer rubrum: red maple), Cocos nucifera (Cocosnucifera: coconut), Limonium Brasiliense, Acerola barbatus, Vitellaria paradoxa: shea butter tree), Vitis vinifera: European grape), Lawsonia inermis: henna), Artocarpus heterophyllus: jackfruit), Medicago sativa: alfalfa), Lotus japonicus: Miyakogusa), Lotus uliginosus: Nebikimiyakogusa), Eisenia bicyclis: arame), Hedysarum sulfurescens, Robinia pseudoacacia: false acacia), apple, apricot, plum, cherry, grape leaves, strawberry, legumes, lemon, tea, black tea, green tea, rooibos tea, barley grains, green algae (Caulerpa ryukyuensis), red algae (Chondrococcus hornemannii), chocolate (cocoa), raw coffee beans, or a combination thereof, and is highly concentrated, the composition according to claim 1, which is derived from a plant species further comprising

10. The composition according to claim 1, wherein the at least one bioflavonoid extract with increased concentration of at least one free B-ring flavonoid and the at least one bioflavonoid extract with increased concentration of at least one flavan are extracted and concentrated from a plant part further comprising leaves, bark, trunk, trunk bark, stem, stem bark, twig, tuber, root, rhizome, root bark, bark surface, shoot, seed, nut, pericarp, fruit, fruiting body, mushroom, androecium, gynoecium, calyx, stamen, petal, sepal, carpel (gynoecium), flower, stem cell, cell culture tissue, or any combination thereof.

11. Both the at least one bioflavonoid extract with the at least one free B-ring flavonoid concentrated in the composition and the at least one bioflavonoid extract with the at least one flavan concentrated in the composition are extracted with a solvent containing a supercritical fluid of CO ２ a supercritical fluid, water, acidic water, basic water, acetone, methanol, ethanol, propenol, butanol, an alcohol / water mixture, a mixed organic solvent, or a combination thereof, according to claim 1.

12. The composition according to claim 1, wherein both the at least one bioflavonoid extract with an increased concentration of the at least one free B-ring flavonoid and the at least one bioflavonoid extract with an increased concentration of the at least one flavan are further modified by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, microbacteria, or combinations thereof, by synthesis, metabolism, biodegradation, in vivo conversion, in vivo transformation, or biosynthesis from small carbon units.

13. The composition according to claim 1, wherein both the at least one bioflavonoid extract with an increased concentration of the at least one free B-ring flavonoid and the at least one bioflavonoid extract with an increased concentration of the at least one flavan are concentrated individually or in combination by solvent precipitation, neutralization, solvent partitioning, ultrafiltration, enzymatic digestion, column chromatography using silica gel, XAD, HP20, LH20, C-18, alumina oxide, polyamide, ion exchange resin, or CG161 resin as a carrier, or combinations thereof.

14. The composition according to claim 1, wherein the composition contains a pharmaceutically or nutritionally acceptable active ingredient, adjuvant, carrier, diluent, or excipient, and the pharmaceutical or nutritional supplement formulation further contains 0.1 weight percent (wt%) to 99.9 weight percent of an active compound in the at least one bioflavonoid extract.

15. The active ingredient, adjuvant, excipient or carrier is Cannabis sativa seed oil or CBD / THC, turmeric extract or curcumin, Terminalia extract, willow bark extract, Aloe vera leaf gel powder, Poria cocos extract, rosemary extract, rosmarinic acid, devil's claw root extract, cayenne pepper extract or capsaicin, American ginseng bark extract, philodendron bark extract, hop extract, Boswellia extract, rose hip extract, green tea extract, Sophora extract, Withania somnifera (Ashwagandha), Bupleurum falcatum (Mishima saiko), Radix Bupleuri (saiko), Radix Glycyrrhiza, Fructus Forsythiae (Rengyou), Panax quinquefolium (American ginseng), Panax ginseng C. A. Meyer (Otanen ginseng), Lentinula edodes (Shiitake), Inonotus obliquus (Kabananatake), Lentinula edodes (Shiitake), Lycium barbarum (Nagabakuco), Phellinus linteus (Meshima kobu) (fruiting body), Trametes versicolor (Kawaratake) (fruiting body), Cyamopsis tetragonolobus (cluster bean), Trametes versicolor (Kawaratake), Cladosiphon okamuranus Tokida (Okinawa mozuku), Undaria pinnatifidapinnatifida: wakame), extracts of the genus Mentha or peppermint, extracts of ginger or black ginger, green tea or grape seed polyphenols, omega 3 or omega 6 fatty acids, krill oil, gamma-linolenic acid, citrus bioflavonoids, acerola concentrate, astaxanthin, picnogenol, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B, vitamin A, L-lysine, calcium, manganese, zinc, amino acid chelate minerals, amino acids, boron and boron glycinate, silica, probiotics, camphor, menthol, calcium salts, silica, histidine, copper gluconate, CMC, beta-cyclodextrin, cellulose, dextrose, physiological saline, water, oils, shark and bovine cartilage, or combinations thereof, the composition according to claim 14.

16. The composition according to claim 1, which is formulated as tablets, hard capsules, soft gel capsules, powders, granules, compressed tablets, pills, gums, chewing gums, sachets, wafers, bars, liquids, tinctures, aerosols, semi-solids, semi-liquids, solutions, emulsions, creams, lotions, ointments, or gel bases.

17. The composition according to claim 1, which is effective against respiratory diseases and conditions.

18. The composition according to claim 1, which is administered via oral, topical, suppository, intravenous, intradermal, intragastric, intramuscular, intraperitoneal, or intravenous routes.

19. The composition according to claim 1, which reduces the expression level of HMGB1 in a mammal by administering an effective amount of the composition that is 0.01 mg to 500 mg per kg of the mammal's body weight.

20. The composition according to claim 1, which maintains the immunological homeostasis of a mammal by optimizing or balancing the immune response; improves immunodeficiency due to aging and immune organ senescence; prevents immunodeficiency due to chronic inflammation and inflammation; helps maintain a healthy immune response to influenza vaccination or COVID-19 vaccination; helps maintain a healthy immune function against viral and bacterial infections; or protects the immune system from oxidative stress damage induced by air pollution.

21. The composition according to claim 1, wherein the composition reduces the expression level of HMGB1 as an endogenous or exogenous response attack trigger, shifts the host defense response to restore homeostasis, and the HMGB1 is released by immune cells, viruses or microorganisms, immune cells contaminated with air pollutants, host respiratory cells, or cardiovascular cells deteriorated by immune aging, inflammation, or oxidative stress.

22. The composition according to claim 1, which targets the active or passive release of HMGB1 by preventing cytoplasmic translocation or vesicle-mediated release, or inhibits the formation of intramolecular disulfide bonds in the nucleus, thereby inhibiting HMGB1 release or interfering with its action, or directly targets HMGB1 at the time of release and neutralizes its action, or blocks the HMGB1 pattern recognition receptor or inhibits its signal transduction, or changes the physicochemical microenvironment to prevent the formation of HMGB1 tetramers and interfere with the binding affinity of HMGB1 for TLR and RAGE, or prevents the clustering or self-association of HMGB1, thereby reducing the expression level of HMGB1.

23. The composition of claim 1, wherein the composition supports a healthy inflammatory response; maintains healthy levels of cytokines and cytokine responses against infection; maintains healthy levels of complement C3 and C4 proteins, cytokines and cytokine responses against infection; reduces, regulates and maintains TNF-α, IL-1β, IL-6, GM-CSF, IFN-α, IFN-γ, IL-1α, IL-1RA, IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, TNF-β / LTA, CRP, and CINC3.

24. The composition of claim 1, wherein the composition controls the oxidative response and alleviates oxidative stress in the respiratory system; enhances antioxidant capacity by increasing SOD and NRF2; reduces advanced glycation end products; increases glutathione peroxidase; neutralizes reactive oxygen species and prevents damage to the structural integrity and functional decline of the respiratory system, lungs and immune system caused by oxidative stress.

25. The composition of claim 1, wherein the composition minimizes or prevents age-related chronic diseases caused by AGE and AGE-RAGE interactions, for example, in the case of diabetes, preventing diabetic complications and diabetic microvascular complications; in the case of cardiovascular diseases, preventing coronary atherosclerotic arteriosclerosis and the severity of coronary artery diseases; in the case of kidney diseases, preventing renal insufficiency and end-stage renal diseases; in the case of obesity, preventing hypothalamic dysfunction; reducing cancer onset, progression, migration, invasion and metastasis; in the case of enteric microbiota-related diseases, preventing systemic endotoxemia, inflammation and multiple organ injury; in the case of neurodegenerative diseases, preventing neuron death and degeneration; in the case of Alzheimer's disease, preventing neuron apoptosis and neurodegeneration; in the case of Parkinson's disease, preventing neurodegeneration; in the case of liver diseases, preventing the onset and progression of non-alcoholic fatty liver disease, inflammatory liver injury, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

26. The composition according to claim 1, wherein the composition improves innate immunity; improves adaptive immunity; increases the activity and number of white blood cells; enhances natural killer (NK) cell function; increases the number of T lymphocytes and B lymphocytes; increases the number of CD3+, CD4+NKp46+ natural killer cells, TCRγδ+ gamma delta T cells, CD4+TCRγδ+ helper gamma delta T cells and CD8+ cells; and protects and promotes macrophage phagocytic activity.

27. The composition according to claim 1, wherein the composition supports or promotes the hemagglutination inhibition (HI) titer against normal mammalian antibodies IgG, IgM, IgA, and the production of specific virus strains.

28. The composition according to claim 1, wherein the composition neutralizes, suppresses, prevents, and recovers from the infection of viruses including highly pathogenic avian influenza (type A H5N1 virus strain), type A influenza (H1N1, H3N2, H5N1), type B influenza / Washington / 02 / 2019-like virus, type B influenza / Puket / 3073 / 2013-like virus, hepatitis A, B, C, and D viruses, coronavirus SARS-CoV, SARS-CoV-2 (COVID-19), MERS-CoV (MERS), respiratory syncytial virus (RSV), enterovirus A71 (EV71), parainfluenza, and adenovirus.

29. The composition according to claim 1, which neutralizes, inhibits, prevents, and restores the respiratory system from microbial infections including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Moraxella catarrhalis, Aspergillus spp., Cryptococcus spp., Pneumocystis spp., Histoplasma capsulatum, Blastomyces spp., Cryptococcus neoformans, Pneumocystis jirovecii, Candida spp., and Streptococcus pyogenes.

30. The composition according to claim 1, which neutralizes, inhibits, prevents, and restores damage to the respiratory system caused by PM2.5 particles in the atmosphere, PM10 particles in the atmosphere, air pollutants, photochemical smog, tobacco, smoke from e-cigarettes, and smoke from recreational marijuana.

31. The composition according to claim 1, which maintains a healthy lung microbiota or symbiotic system in the respiratory tract of mammals; maintains lung purification and detoxification ability; protects lung structural integrity and oxygen exchange ability; maintains respiratory passage and enhances alveolar oxygen absorption ability; protects normal and healthy lung function from viral infection, bacterial infection, and air pollution; reduces lung damage caused by oxidative stress; and promotes pulmonary microcirculation and protects normal coagulation function.

32. The composition according to claim 1, which alleviates or reduces cold / flu-like symptoms including pain in the body of a mammal, sore throat, cough, mild throat and bronchial irritation, nasal congestion, sinus congestion, sinus pressure, runny nose, sneezing, reduced sense of smell, reduced sense of taste, muscle pain, headache, fever and chills; loosens phlegm (mucus), thins bronchial secretions to make coughing easier; reduces the severity of bronchial irritation; reduces the severity of lung damage or edema or inflammatory cell infiltration caused by viral infection, microbial infection and air pollution; supports the bronchial system and comfortable breathing throughout the cold / flu or pollution season; prevents or treats pulmonary fibrosis; reduces the duration or severity of the common cold / flu; reduces the severity or duration of viral and bacterial infections of the respiratory system; prevents or treats or cures respiratory infections caused by viruses, microorganisms and air pollutants; manages or treats or prevents or reverses the progression of respiratory infections; and has the effect of promoting, enhancing and rejuvenating the repair and regeneration functions of the lungs and the entire respiratory system.

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