Standardized bioflavonoid composition for regulation of homeostasis of host defense mechanism

Abstract

To provide a bioflavonoid composition for establishment and regulation of homeostasis of a host defense mechanism.SOLUTION: A composition is provided that comprises at least one standardized bioflavonoid extract enriched for at least one free-B-ring flavonoid and at least one standardized bioflavonoid extract enriched for at least one flavan.SELECTED DRAWING: None

Description

[Background technology]

[0001] Aging is a natural phenomenon, a complex degenerative process that affects both physical and mental functions over time. Impaired host defense responses are one of the most common changes associated with aging. Understanding the underlying mechanisms of this decline in host defense responses in elderly individuals is the first step toward mitigating this decline. Chemically induced accelerated aging models, such as the D-galactose-induced thymus injury / immunosenescence mouse model, are a preferred option for studying the effects of aging on the immune system. In chemically induced animal aging models, animals exhibit immunosenescence, similar to the decline in host defense responses frequently observed in elderly individuals (Azman 2019). The D-galactose-induced aging model is one of the most widely used and well-validated animal models in anti-aging research. While D-galactose is converted to glucose in the body at normal concentrations, at high concentrations it is easily converted to aldoses and hydroperoxides, leading to the generation of oxygen-derived free radicals. It can also react with free amines in proteins and peptides to form advanced glycation end products (AGEs) through nonenzymatic glycation. The accumulation of these reactive oxygen species (ROS) and increased AGEs in this model leads to an imbalance in normal organ and host defense homeostasis, resulting in oxidative stress, systemic inflammation, impaired immune response, mitochondrial dysfunction, and apoptosis (e.g., of thymocytes), ultimately accelerating the aging process. These changes are among the naturally occurring pathological hallmarks of senescence and aging.

[0002] Sepsis is a severe organ dysfunction caused by dysregulation of the host defense response to infection, potentially leading to organ failure. Sepsis is primarily mediated by macrophages / monocytes and results from the overproduction of several early cytokines, such as TNF-α, IL-1, IL-6, and γ-interferon, as well as late mediators, such as HMGB1. High-mobility group box 1 protein (HMGB1) is a nuclear or cytoplasmic endogenous damage-associated molecular pattern (DAMP) protein that can be released or secreted from cells in response to damaging stimuli or cytokines. Nuclear HMGB1 is an architectural chromatin-binding factor responsible for maintaining genomic integrity, whereas extracellular HMGB1 released from activated or damaged cells is a mediator of inflammation and immune dysfunction in response to various stresses, such as oxidative damage and pathogen infection. HMGB1 is released from activated macrophages and monocytes in response to endogenous and exogenous inflammatory signals and is therefore a key mediator of sepsis (Wang et al., 1999), which can increase the imbalance of host defense mechanisms, leading to multiple organ failure and ultimately death. Late, sustained release of HMGB1 can be sufficient to trigger an inflammatory response, so that even patients who survive may continue to have an inflammatory response (Gentile and Moldawer, 2014).

[0003] When actively released from stimulated mononuclear cells or passively released from necrotic cells, HMGB1 acts as an alarmin (danger signal) that counteracts the imbalance in intracellular homeostasis with neighboring cells and activates the host immune response. HMGB1 plays an important role in activating the innate immune response by functioning as a chemokine that aids in the migration of immune cells to the site of infection and as a DAMP that activates other immune cells to secrete proinflammatory cytokines (Yang et al., 2001). When produced at low (optimal) concentrations, proinflammatory cytokines function to defend against viral or microbial invasion, but excessive production, such as in a "cytokine storm," can be harmful to the host by mediating harmful inflammatory responses. In most cases, this proinflammatory cytokine storm appears to result in acute systemic inflammatory syndrome in hosts with underlying diseases such as immunodeficiency or immunosuppression, or in elderly individuals. Even if patients survive, delayed inflammatory mediation may occur, potentially resulting in persistent inflammatory, immunosuppressive, and catabolic responses. HMGB1 not only functions as a chemoattractant for numerous cell types, including all inflammatory cells, but also induces inflammatory cells to secrete TNF-α, IL-1β, IL-6, IL-8, and macrophage inflammatory protein (MIP), suggesting its involvement in the "cytokine storm" through activation of NFκB signaling (Bianchi and Manfredi, 2007). Numerous studies have also demonstrated that extracellular HMGB1 can induce devastating inflammatory responses and 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 several hours after endotoxin stimulation both in vitro and in vivo, suggesting that it mediates late-stage inflammation. Indeed, administration of HMGB1-neutralizing antibodies 24 hours after the onset of sepsis confers protection against lethal endotoxemia, indicating that HMGB1 plays a major role as a late mediator of lethal sepsis (Wang et al., 1999).Clinically, a strong association between persistently high HMGB1 levels and the development of late-stage sepsis or death from sepsis has also been documented (Angus et al., 2007). Recently, several clinical trials have demonstrated that chloroquine and its analogue (hydroxychloroquine) are beneficial for clinical efficacy and viral clearance against COVID-19 (Andersson et al., 2020; Gao et al., 2020; Gautret et al., 2020). When tested in a mouse sepsis model, the antimalarial drug chloroquine prevented lethality, and its protective effect was mediated by the inhibition of HMGB1 release 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 alleviate hyperoxia-induced acute inflammatory lung injury by enhancing macrophage function through the suppression of HMGB1 accumulation in the airways (Patel et al., 2020). Based on the above, the natural bioflavonoid composition containing free B-ring flavonoids and flavans described in the patented subject matter of the present application has been confirmed to inhibit HMGB1 and NF-κB, prevent lethal sepsis, inhibit AGE formation, induce endogenous antioxidant enzymes, promote macrophage phagocytosis, increase bacterial clearance, and prevent acute lung injury. It is therefore possible to safely and historically apply the composition to maintain and protect respiratory and pulmonary health, and to prevent and treat pathologies such as viruses, microbial infections (e.g., COVID-19), and lung injury caused by PM2.5 air pollutants, atmospheric PM10 particles, air pollutants, photochemical smog, tobacco and e-cigarette smoke, and recreational marijuana smoke.

[0004] Acacia catechu Willd (Fabaceae: Leguminosae), commonly known as Kutch tree, Khair, and Khadira, is used as a traditional herbal medicine in India and other parts of Asia (Hazral et al., 2017). The plant is a deciduous tree of medium size (up to 15 m). The bark is dark gray-brown and peels into strips. The leaves are pinnate with a pair of spines at the base of the rachis. The flowers are pale yellow and arranged in tubular spikes. The legumes are glabrous, flattened, and elliptical. According to the Indian Ayurvedic Pharmacopoeia, the heartwood of Acacia catechu is pale pink, turning reddish-brown to almost black with age, surrounded by whitish sapwood, and is described as being resistant to cracking, tasteless, and astringent. Medium-sized trees, approximately 8 years old or older, are harvested for the extraction of Acacia catechu extract. Because the appearances of Acacia catechu (wood), Uncaria gambir (a climbing plant), and cashew nut shells (fruit) are very different, plant material sourcing and botanical authentication are primary supplier selection criteria. Acacia catechu is used in Ayurvedic medicine for the throat, mouth, and gums, and for coughs and diarrhea. Externally, it is used as an astringent and cooling agent for skin ulcers, swelling, and rashes. Powdered form is used for wound care. Acacia catechu has been shown to increase the number of antibody-producing cells in the spleen of animals, and may enhance the immune system, increase macrophage phagocytosis, and suppress the release of inflammatory cytokines (Sunil et al., 2019).

[0005] Scutellaria baicalensis Georgi (Lamiaceae), commonly known as Chinese skullcap (Huang Qin), is a traditional herbal medicine used in several Asian countries, as listed in the Chinese Pharmacopoeia. The plant is a perennial herb with creeping to erect stems and a purplish tint. It produces medium-sized, green leaves with short, lance-shaped, hairy petioles. From early summer to early autumn, it produces hairy flowers in racemes with dark blue lips at the top and pale blue below. The biennial roots are collected during spring or summer and air-dried for commercial use. According to the Chinese Pharmacopoeia, the roots are 8–25 cm long and 1–3 cm in diameter. They are brownish-yellow or dark yellow in color and have scattered traces of where they were dug. The upper part is shaggy with twisted vertical wrinkles or an irregular network, while the lower part has vertical stripes and fine wrinkles. The tissue is hard, brittle, and easily split. The cracks are yellow, and the core is reddish-brown. The center of the bark of old roots is dark brown or brownish-black and wilted or hollow. It has almost no odor and a bitter taste. Dried roots usually contain less than 10% of bioflavonoids, including baicalin. The roots used for Scutellaria extract are tested by TLC and HPLC methods based on the identification and quantification methods of the Chinese Pharmacopoeia.

[0006] Scutellaria has been recorded in the classical Chinese medical text, Shen Nong Ben Cao, since the Later Han Dynasty (c. 200 AD or approximately 2,200 years ago). A recent list of the top 30 herbs for the treatment of respiratory infections in Traditional Chinese Medicine (TCM) based on an analysis of two TCM databases (World Traditional Medicine Patent Database (WTM) and Saphron TCM database) ranked Scutellaria as the second most commonly used herb, with a frequency of 38% in all TCM compositions for the treatment of respiratory infections (Ge et al. 2010).

[0007] Scutellaria bark was included in a 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 Scutellaria plants (Zhong et al., 2006) was subsequently patented for the treatment of SARS and COVID-19 (Song et al., 2020). Recent scientific research on Scutellaria bark has identified bioflavonoids, particularly baicalin and baicalein, as bioactive components of this herb with biological functions related to antioxidant, anti-inflammatory, suppression of allergic responses, and antibacterial activity (Bejar et al., 2004) (Shen et al., 2021). Baicalin and baicalein also demonstrated potent antiviral activity by inhibiting proteins required for virus binding and germination, which are essential for infection (Yu et al., 2011). In mice infected with influenza A H1N1 virus (swine flu), extracts from Scutellaria Baicalensis modulated the inflammatory response to reduce disease severity, attenuated lung tissue damage, and ultimately increased survival (Zhi et al., 2019).

[0008] Flavonoids are a group of widely available natural products. Flavonoid intake has been shown to be inversely associated with the risk of nonvascular dementia. The mechanism of action is unclear, but it is speculated to be due to the antioxidant properties of flavonoids (Commenges et al. 2000). Polyphenolic flavones induce programmed cell death, differentiation, and growth inhibition in transformed colon cells by acting at the mRNA level on genes including cox-2, nuclear factor kappa BB (NFκB), and bcl-X(L) (Wenzel et al. 2000). The number of hydroxyl groups on the B ring of cox-2 has been reported to be important for the repression of cox-2 transcriptional activity (Mutoh et al. 2000).

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

[0010] Flavonoids are generally tested for activity randomly based on their availability. There are scattered claims that B-ring substitutions are required for specific biological activities, such as high-affinity binding to p-glycoprotein (Boumendjel et al. 2001), cardiotonic activity (Itoigawa et al. 1999), endothelial cell protection against linoleic acid hydroperoxide-induced toxicity (Kaneko and Baba 1999), COX-1 inhibitory activity (Wang 2000), and prostaglandin endoperoxide synthase (Kalkbrenner et al. 1992). Very few publications address the significance of the unsubstituted B-ring in free B-ring flavonoids. One example is the use of 2-phenylflavones, which inhibit NADPH quinone acceptor oxidoreductase, as potential anticoagulants (Chen et al. 2001).

[0011] There are various theories regarding the mechanism of action reported for the anti-inflammatory activity of various free B-ring flavonoids. The main bioactive free B-ring flavonoids in Scutellaria baicalensis have been reported to suppress proinflammatory cytokines (Liao et al., 2021). The anti-inflammatory activity of the free B-ring flavonoids chrysin (Liang et al., 2001), wogonin (Chi et al., 2001), and galangin (Raso et al., 2001) is related to the suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator-activated receptor γ (PPARγ), affecting degranulation and AA release (Tordera et al., 1994). Oroxylin, baicalein, and wogonin have been reported to inhibit 12-lipoxygenase activity without affecting cyclooxygenase activity (You et al., 1999). More recently, the anti-inflammatory activity of wogonin, baicalin, and baicalein has been reported to occur through the inhibition of inducible nitric oxide synthase and COX-2 gene expression induced by nitric oxide inhibitors and lipopolysaccharide (Chen et al. 2001). Oroxylin has also been reported to act 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 a well-documented bioactive flavonoid (Bae et al. 2020). Catechin and its isomer, epicatechin, are known to be effective in treating rheumatoid arthritis (IC). 50They inhibit prostaglandin endoperoxide synthase with an IC value of 40 μmol / L (Kalkbrenner et al. 1992). Five flavan-3-ol derivatives, including (+)-catechin and gallocatechin, isolated from four plant species, Atuna racemosa, Syzygium carynocarpum, Syzygium malaccense, and Vantanea peruviana, have inhibitory activity against COX-2 that is equal to or less than that of COX-1, with IC values ​​of 40 μmol / L. 50 The IC values ​​range from 3.3 μM to 138 μM (Noreen et al. 1998). (+)-Catechin isolated from the bark of Ceiba pentandra (kapok) has an IC 50 Commercially available pure (+)-catechin inhibits COX-1 with an IC value of approximately 183–279 μM depending on the experimental conditions (Noreen et al. 1998). 50 It inhibits COX-1 at a high level but is not selective for COX-2 (Noreen et al. 1998).

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

[0014] Supplementation of the diet of male Sprague-Dawley rats with green tea catechins resulted in a decrease in platelet phospholipase A2 activity and a significant decrease in platelet cyclooxygenase concentration (Yang et al. 1999). Catechin and epicatechin have been reported to slightly suppress cox-2 gene transcription in human colon cancer DLD-1 cells (IC 50 = 415.3 μM) (Mutoh et al. 2000). The neuroprotective effect of (+)-catechin derived from red wine is due to its antioxidant properties rather than its inhibitory effects on intracellular enzymes such as cyclooxygenase, lipoxygenase, or nitric oxide synthase (Bastianetto et al. 2000). Catechin derivatives purified from green and black tea, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and theaflavins, inhibited cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissue (Hong et al. 2001) and induced COX-2 expression and PGE2 production (Park et al. 2001).

[0015] A recent study on Acacia catechu extract and Scutellaria baicalensis extract was published regarding their suppressive effects on LPS-induced inflammatory responses via NF-κB, MAPK, and PI3K-Akt signaling pathways in type II alveolar epithelial cells ( Feng et al., 2019 ). U.S. Patent Nos. 9,061,039, 8,535,735, 7,972,632, and 7,192,611, entitled "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, and 7,108,868, entitled "Isolation of a Dual COX-2 and 5-Lipoxygenase Inhibitor from Acacia," respectively, describe the isolation, purification, and use of compositions containing Free-B-Ring flavonoids or flavans. Combination compositions of Free-B-Ring flavonoids and flavans based on COX / LOX dual inhibitors and their uses in joint care, mental acuity, oral care, skin care, etc. are described in U.S. Patent Nos. 9,849,152, 9,655,940, 9,061,039, 8,535,735, 7,674,830, and 7,514,469, entitled "Formulation of a mixture of Free-B-Ring flavonoids and flavans as a therapeutic agent," and U.S. Patent Nos. 8,652,535, 8,034,387, 7,695,No. 743, titled "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 Nos. 9,622,964 and 8,790,724, titled "Formulation of dual cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors for skin care," and U.S. Patent No. 8,945,518, titled "Formulation of Dual Eicosanoid System and Cytokine System Inhibitors for the Use in the Prevention and Treatment of Oral Diseases." No. 7,531,521, entitled "Formulation for prevention and treatment of carbohydrate-induced diseases and conditions," the disclosures of which are incorporated herein by reference in their entireties. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] U.S. Patent No. 9,061,039 [Patent Document 2] U.S. Patent No. 8,535,735 [Patent Document 3] U.S. Patent No. 7,972,632 [Patent Document 4] U.S. Patent No. 7,192,611 [Patent Document 5] U.S. Patent No. 9,168,242 [Patent Document 6] U.S. Patent No. 8,568,799 [Patent Document 7] U.S. Patent No. 8,124,134 [Patent Document 8] U.S. Patent No. 7,108,868 [Patent Document 9] U.S. Patent No. 9,849,152 [Patent Document 10] U.S. Patent No. 9,655,940 [Patent Document 11] U.S. Patent No. 9,061,039 [Patent Document 12] U.S. Patent No. 8,535,735 [Patent Document 13] U.S. Patent No. 7,674,830 [Patent Document 14] U.S. Patent No. 7,514,469 [Patent Document 15] U.S. Patent No. 8,652,535 [Patent Document 16] U.S. Patent No. 8,034,387 [Patent Document 17] U.S. Patent No. 7,695,743 [Patent Document 18] U.S. Patent No. 9,622,964 [Patent Document 19] U.S. Patent No. 8,790,724 [Patent Document 20] U.S. Patent No. 8,945,518 [Patent Document 21] U.S. Patent No. 7,531,521 [Non-patent literature]

[0017] [Non-Patent Document 1] Azman 2019 [Non-licensed document 2] Wang et al.,1999 [Non-licensed document 3] Gentile and Moldawer, 2014

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[0018] 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. [Brief explanation of the drawings]

[0019] [Figure 1] We present a host defense homeostasis concept that uses HMGB1 as a tipping point lever. [Figure 2] The novelty of the standardized composition for maintaining the homeostasis of the host defense mechanisms is demonstrated. [Figure 3] 1 shows a schematic diagram of the gate (⊥) through which bioflavonoid compositions are believed to interfere with the HMGB1 and NFκB pathways. [Figure 4] Cell viability after 24 hours of hyperoxia exposure in the presence of UP894-II is shown. *p<0.05 compared to room air control (0 hours). #P<0.05, ####P<0.001 compared to vehicle control. [Figure 5] This shows that UP894-II suppresses the decline in macrophage phagocytic function under hyperoxia. Each value represents the mean ± SEM of two independent experiments performed in duplicate for each group. Significance is compared with the 95% O2 (0 μg / ml) control group. [Figure 6] UP894-II inhibits hyperoxia-induced HMGB1 release in RAW264.7 cells. Each value represents the mean ± SEM of two independent experiments performed in duplicate. ***p<0.001 compared to room air control (RA). #p<0.05, ##P<0.01, ###P<0.001 compared to vehicle control. [Figure 7] H&E staining of lung tissue from LPS-challenged rats administered 250 mg / kg of UP446. A = normal control, B = vehicle control, C = sodium butyrate, D = UP446 (250 mg / kg). Magnification: 100x. [Figure 8] Fold change in lung HMGB1 expression in hACE2 transgenic mice infected with SARS-CoV-2 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Disclosed are compositions and methods for regulating host defense homeostasis, including 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 host defense homeostasis by modulating HMGB1, suppressing oxidative stress, and inducing mucosal immunity, particularly immune and respiratory immunoglobulin and T cell production. Also disclosed are methods for treating, managing, promoting, and protecting the phagocytic activity of macrophages, the first line of innate immune defense in mammals, and providing a critical host defense mechanism in populations increasingly exposed to pathogenic and oxidative stress caused by air pollution, viral and microbial infections such as SARS-CoV-2, particularly aging and hosts suffering from chronic inflammatory disorders, including chronic inflammatory disorders of the respiratory system, by administering an effective amount of the composition to the mammal in an amount of 0.01 mg to 500 mg per kg of body weight.

[0021] The subject matter of this application relates to the synergistic modulation of host defense homeostasis, using a standardized bioflavonoid composition containing free B-ring flavonoids and flavans to improve host immune function, respiratory health, and pulmonary function by regulating the extracellular protein HMGB1, suppressing oxidative stress, and inducing mucosal immunity, particularly the production of immunoglobulins and T cells. IgA is the second most abundant antibody in serum and is the first line of defense against pulmonary and systemic infections by inhibiting microbial and viral adhesion to epithelial cells and neutralizing bacteria, air pollutants, and viruses. It should be noted that the intended composition does not act or function by directly inhibiting microbial infection or viruses to achieve the intended effect. In one embodiment, the intended effect is to modulate the homeostasis of the host's self-defense mechanism so that the host's defense functions suppress microbial or viral infections.

[0022] The subject of protection of this application is designated as the homeostasis of host defense mechanisms in the lungs and the whole body. The subject of protection of this application is expected to maintain systemic mucosal homeostasis in the gastrointestinal and genitourinary tracts. However, data presented in the text of the subject of protection of this application confirm that its primary function is to protect the structural integrity and function of the respiratory system, primarily by regulating HMGB1 and inducing frontline respiratory mucosal immunity, such as immunoglobulin A (IgA). The lung-protective effect of the subject of protection of this application was evaluated in vivo in living hosts using a lipopolysaccharide (LPS)-induced acute lung injury model and a hyperoxic microbial infection model, as well as in vitro using hyperoxically compromised macrophages. A bioflavonoid composition containing free B-ring flavonoids and flavans was tested in hyperoxically compromised macrophages, and inhibiting HMGB1 release enhanced macrophage phagocytic activity (innate immune defense). In vivo corroboration of these results demonstrated that the bioflavonoid composition increased bacterial clearance in the airways and lungs, significantly suppressed HMGB1 accumulation in the airways, and reduced total protein in the lungs of mice exposed to hyperoxia and microbial infection, demonstrating its potential for respiratory and pulmonary protection. Similar respiratory and pulmonary protective effects of the protective agent were observed in an LPS-induced acute lung injury model, where supplementation with the bioflavonoid composition attenuated key signs of inflammation and reduced biomarkers and lung injury. The systemic host defense homeostasis effects of the protective agent were also evaluated in a lipopolysaccharide (LPS)-induced sepsis model and a D-galactose-induced accelerated aging model with and without influenza vaccine immunization. In all models tested, the protective agent containing free B-ring flavonoids and flavans demonstrated statistically significant improvements in host defense mechanisms, demonstrating its potential for use in restoring host defense homeostasis locally or systemically.

[0023] 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. As described below, the at least one standardized bioflavonoid extract enriched in at least one free-B-ring flavonoid and the at least one standardized bioflavonoid extract enriched in at least one flavan in the composition range from 1% to 98% by weight of each extract, with an optimal weight ratio of 80:20. Contemplated embodiments further include those in which the at least one standardized bioflavonoid extract enriched for at least one Free-B-ring flavonoid is enriched and standardized from Scutellaria root and the at least one standardized bioflavonoid extract enriched for at least one flavan is enriched and standardized from Acacia catechu heartwood.

[0024] The intended target of protection was a bioflavonoid composition containing a combination of free B-ring flavonoids and flavans. The composition demonstrated local inhibition of extracellular HMGB1 secretion in lung lavage fluid samples and systemic inhibition in spleen homogenates in hosts exposed to hyperoxia and microbial infection, and in a D-galactose-induced accelerated aging model. Objective evaluation of the composition of the present invention was conducted based on key immune or inflammatory response biomarkers, such as HMGB1 and NFκB, and in vivo changes associated with immunosenescence. The bioflavonoid composition containing free B-ring flavonoids and flavans was demonstrated to significantly enhance macrophage phagocytosis in vitro and reduce the inflammatory cytokines TNF-α, IL-1β, IL-6, CRP, and CINC3 by modulating HMGB1 and NFκB, while increasing survival rates in vivo. This suggests that the composition can be used to restore, regulate, and maintain host defense homeostasis. Similarly, bioflavonoid compositions containing Free B-ring flavonoids and flavans disclosed herein have also been shown to reverse immunosenescence as evidenced by stimulation of innate and adaptive immune responses (increased complement C3, increased CD3+ T cells, CD8+ cytotoxic T cells, CD3-CD49b+ natural killer cells, NKp46+ natural killer cells, and CD4+TCRγδ+ gamma delta T cells), enhanced antioxidant capacity (decreased advanced glycation end products, increased glutathione peroxidase), and protection of key immune organs such as the thymus from age-related functional decline and structural damage.

[0025] The intended compositions maintain immune homeostasis in mammals by optimizing or balancing the immune response; ameliorating immune decline due to aging and immune organ senescence; preventing chronic inflammation and inflammation-induced immune decline; helping to maintain a healthy immune response to influenza vaccination and COVID-19 vaccination; helping to maintain healthy immune function against viral and bacterial infections; or protecting the immune system from oxidative stress damage induced by air pollution. Further, intended embodiments include compositions that regulate HMGB1 as an attack trigger for endogenous or exogenous responses and shift the host defense response to restore homeostasis, where the HMGB1 is released by immune cells deteriorated by immunosenescence, inflammation, or oxidative stress, viruses or microorganisms, immune cells contaminated with air pollutants, host respiratory cells, or cardiovascular cells.

[0026] Most importantly, human clinical trials have demonstrated that supplementation with the novel bioflavonoid composition containing free B-ring flavonoids and flavans disclosed herein induces IgA, a major mucosal defense-related immunoglobulin. IgA is the most important antibody class present on mucosal surfaces in the respiratory tract and plays a role in protecting mucosal surfaces from the invasion of microorganisms and foreign antigens. In a randomized, double-blind, placebo-controlled human clinical trial, supplementation with the bioflavonoid composition disclosed herein significantly elevated the immunoglobulin IgA. IgA levels increased in subjects after 56 days of daily supplementation with UP446, a standardized bioflavonoid composition containing free B-ring flavonoids and flavans described herein, and in subjects immunized with an influenza vaccination on day 28 and supplemented for a total of 56 days. Increased IgA levels indicate enhanced mucosal protection at the entrances to the gastrointestinal, respiratory, and genitourinary tracts.

[0027] The combined benefits of these standardized bioflavonoid extracts from two medicinal plants, Scutellaria baicalensis and Acacia catechu, in the subject matter of the present application were also tested in vivo in an LPS-induced sepsis model, and an unexpected synergistic effect was observed, as described in the body of the subject matter of the present application. Generally, if the host defense mechanism is considered as a lever and the bioflavonoid composition containing free B-ring flavonoids and flavans is considered as a pivot point, host defense homeostasis or lung protection is achieved by downregulating catabolic HMGB1 on one side of the lever and promoting the induction of mucosal immunity, particularly IgA production, on the other side.

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

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

[0030] [ka] and wherein: R1, R2, R3, R4, and R5 are independently -H, -OH, -SH, OR, -SR, -NH2, -NHR, -NR2, -NR3 + X - and in some embodiments selected from the group consisting of carbon, oxygen, nitrogen, or sulfur glycosides, single or multiple sugar combinations (including but not limited to aldopentoses, methyl aldopentoses, aldohexoses, ketohexoses, and chemical derivatives thereof); R is an alkyl group having 1 to 10 carbon atoms; X is selected from the group of pharmaceutically acceptable counter anions, including, but not limited to, hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, and the like.

[0031] In intended embodiments, the at least one standardized bioflavonoid extract enriched for at least one Free-B-Ring flavonoid contains from 0.5% to 99.5% of one or more Free-B-Ring flavonoids. In other embodiments, the at least one standardized bioflavonoid extract enriched for at least one flavan contains from 0.5% to 99.5% of catechins.

[0032] In a preferred embodiment, 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.

[0033] As demonstrated in Example 1, free B-ring flavonoids were extracted from plants using organic or aqueous solvents. Extraction yields vary depending on the specific plant species and plant part being extracted, ranging from a low single digit percentage to approximately 25% of the total biomass. Free B-ring flavonoids in the extracts can be isolated, identified, and quantified using analytical methods that combine UV spectrophotometry or PDA detection with high-pressure column chromatography (HPLC). The free B-ring flavonoid content in solvent extracts ranged from a low of <1% to a high of >35% (Table 2 in Example 1). Furthermore, Example 2 demonstrated the enrichment and standardization of free B-ring flavonoids. By optimizing the extraction solvent and extraction conditions, neutralizing the extract solution, precipitating, and filtering the extract, the content of the desired free B-ring flavonoids was increased from approximately 35% to 60-90%. Example 2 produced RM405, which contained greater than 75% baicalin as the major free B-ring flavonoid from Scutellaria root. Standardized bioflavonoid extracts derived from the roots, stems, or whole plant of Scutellaria can be obtained by precipitating the basic aqueous extract solution after neutralization with an acidic solution, by recrystallization in water, or by column chromatography using various resins, and the bioflavonoids can be concentrated two to three times to a purity of 20% to 99% free B-ring flavonoids.

[0034] Flavans have the following general structure:

[0035] [ka] wherein: R1, R2, R3, R4, and R5 are independently H, -OH, -SH, -OCH3, -SCH3, -OR, -SR, -NH2, -NRH, -NR2, -NR3 + X -esters of the above substituents (including but not limited to gallates, acetates, cinnamates, hydroxycinnamates, trihydroxybenzoates, and caffeates); carbon-, oxygen-, nitrogen-, or sulfur-glycosides of single or multiple sugar combinations (including but not limited to aldopentoses, methyl aldopentoses, aldohexoses, ketohexoses, and chemical derivatives thereof); dimeric, trimeric, and other polymeric flavans; R is an alkyl group having 1 to 10 carbon atoms; X is selected from the group of pharmaceutically acceptable counter anions, including, but not limited to, hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, and carbonate.

[0036] In certain contemplated embodiments, the at least one standardized bioflavonoid extract enriched for at least one flavan comprises at least one of catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epitheaflavin, epicatechin gallate, gallocatechin gallate, theaflavin, theaflavin gallate, or combinations thereof.

[0037] Catechin is a flavan found primarily in Acacia catechu, Uncaria gambir, cashew nut shells, and green tea, and has the following structure:

[0038] [ka]

[0039] As demonstrated in Example 3, flavan extracts were prepared from various plants using organic, aqueous, and alcoholic solvent extraction. The catechin and epicatechin contents as total flavans in these plant extracts were quantified by HPLC, and the results are shown in Table 4. A standardized flavan extract (RM406) derived from Acacia catechu heartwood was prepared by aqueous extraction, followed by concentration, precipitation, and recrystallization to enrich and standardize the flavan content from approximately 10% to 65%. Standardized bioflavonoid extracts derived from Acacia catechu or Gambir heartwood, bark, whole plant, or cashew nut shells can be obtained by concentrating the plant extract solution followed by precipitation, recrystallization in an ethanol / water solvent, or column chromatography using various resins. Bioflavonoids can be enriched 2-8 times to 10%-99% flavan purity.

[0040] Example 4 demonstrated a method for preparing a bioflavonoid composition designated UP446 by combining two standardized extracts: an Acacia extract (RM406 in Example 3) with a total flavan content of >65% as catechin and epicatechin, and a Scutellaria extract (RM405 in Example 2) with a free B-ring flavonoid content of >75% as baicalin, baicalein, etc., in combination with maltodextrin as an excipient. The major and minor bioflavonoid contents as individual free B-ring flavonoids and flavans were quantified and shown in Table 5; the total bioflavonoid content was 86%. Table 6 shows four different bioflavonoid compositions derived from various free B-ring flavonoid sources, such as Scutellaria root (UP446) or stem (UP223), and various flavan sources, such as Acacia catechu heartwood (UP894-II) or gambir whole plant (UG0408). The blend ratios of these compositions varied depending on the bioflavonoid content of each standardized extract and were adjusted depending on the intended application and biological functionality. The present patent application utilizes UP446 and UP894-II to demonstrate the unexpected synergistic benefits of combining two different bioflavonoid species and their unexpected functionality in regulating host defense homeostasis, leading to improved immune function, respiratory health, and lung function protection.

[0041] Strict maintenance of host defense homeostasis is essential for human physiology to defend against invading microorganisms, viruses, fungi, and pollutants, remove dead cells, and initiate regenerative and regenerative functions. Overstimulation of the immune system can result in allergic reactions and destructive autoimmune diseases. Aging, oxidative stress, psychological stress, systemic inflammation, and many chronic diseases, such as diabetes, obesity, and metabolic syndrome, can shift the tipping point of host defense homeostasis, leading to a decline in host defense function. Well-known healthy lifestyle practices, including daily balanced nutrition, exercise, and stress management, as well as the supplementation of antioxidant, anti-inflammatory, and immunomodulatory (immunosuppressive or immunostimulatory, depending on the state of host defense imbalance) natural compounds and prescription antivirals, antibiotics, steroids, and DTHE, can exert beneficial balancing effects to restore host defense mechanisms in a favorable direction. Many polyphenols, including bioflavonoids, have been reported to suppress cytokine production, which is essential for initiating host defense responses against infection or vaccination, and therefore were classified as immunosuppressants. Therefore, the real-world use of polyphenols to support host defense mechanisms has not been proven in clinical trials.

[0042] Unfortunately, there is very little information available about the tipping points essential for maintaining host defense homeostasis, and whether there are key biological, physiological, and pathological pathways and biomarkers that serve as tipping point factors that can accelerate a downward spiral in host defense responses to pathogens. Identifying such tipping points is crucial. Even more important is finding active compounds to develop compositions that can shift the tipping points away from destructive trends and restore host defense homeostasis. We believe that HMGB1 is such a biomarker that acts as an alarmin for the decline in intracellular homeostasis and can support a strong biological response to viruses such as coronavirus and SARS-CoV-2, microbial infections, and PM2.5 pollutants, which lead to a devastating decline in host defense function.

[0043] The nuclear protein HMGB1 is overwhelmingly elevated (100-fold higher than in healthy controls) in the airways of animals and humans exposed to prolonged oxidative stress. HMGB1 was originally identified as a nuclear protein that regulates transcription by stabilizing nucleosome structure and mediating conformational changes in DNA. In contrast to its role in the nucleus, extracellular HMGB1 induces a pronounced inflammatory response. Interestingly, evidence has been accumulating from several animal models of pulmonary infection that high concentrations of extracellular HMGB1 in the airways can directly impair host defense mechanisms against bacterial and viral infections by reducing macrophage function.

[0044] Therefore, we evaluated the effects of bioflavonoid composition UP894-II (Table 6), which contains 70-80% free B-ring flavonoids and 15-20% flavans, on macrophages under hyperoxia stress. As shown in Example 5, UP894-II at 8-128 μg / mL did not alter macrophage viability after 24 hours of hyperoxia exposure (Figure 4). As demonstrated in Figure 5 of Example 6, UP894-II dose-relatedly and statistically significantly enhanced macrophage phagocytic activity at concentrations as low as 3.7 μg / mL. Surprisingly, this protection of macrophage phagocytic activity under oxygen stress by UP894-II closely correlated with the reduction in hyperoxia-induced HMGB1 release in UP894-II-treated macrophages, demonstrating an identical dose-dependent effect (Figure 6 of Example 7).

[0045] In other words, the bioflavonoid composition UP894-II disclosed in this application reduced HMGB1 levels or blocked its activity in the airways, thereby protecting the phagocytic activity of macrophages, the first line of innate immune defense, and providing an important host defense mechanism for populations increasingly exposed to pathogenic and oxidative stress caused by air pollution, viral and bacterial infections such as SARS-CoV-2, particularly those with chronic inflammatory disorders.

[0046] As described in the body of the protected subject matter of the present application, the objective dose-response effects of the bioflavonoid compositions containing free B-ring flavonoids and flavans disclosed herein were evaluated in multiple in vivo studies (e.g., an LPS-induced sepsis model in Examples 9-12, an LPS-induced acute lung injury model in Examples 13-21, and a hyperoxia-induced microbial infection acute lung injury model in Examples 35-39). The data presented in these examples of the protected subject matter of the present application demonstrate that oral administration of the standardized compositions to subjects with sepsis or acute lung injury resulted in significant host defense homeostatic effects.

[0047] Using the data obtained from the LPS-induced viability assays demonstrated in Examples 10 and 11, the Colby formula was used to evaluate and confirm the significant value of combining free B-ring flavonoids from Scutellaria extract with flavans from Acacia extract. According to the Colby method, a standardized formulation containing two or more ingredients is presumed to have unexpected synergistic effects when the observed values ​​are higher than the predicted values. The subject matter of the present application was intended to confirm the unexpected synergistic effects of bioflavonoid compositions in reducing mortality and increasing survival. As shown in Example 12, the combination of free B-ring flavonoid extract and flavan extract demonstrated unexpected synergistic effects in reducing mortality or increasing survival. The beneficial effects observed when administering the composition exceeded the expected effects of simply adding the effects of each of the components at the specified ratios (Table 13). At 144 hours after LPS challenge, only the bioflavonoid composition containing free B-ring flavonoids and flavans showed a statistically significant increase in survival rate (SR%) compared to normal controls (Table 10). Indeed, 24 hours after administration, no animals died in the bioflavonoid composition (100% survival), whereas the Scutellaria (RM405) and Acacia (RM406) single-administered groups experienced mortality rates of 15.4% and 30.8%, respectively (Table 10 in Example 11). While the beneficial uses of these medicinal plants have been reported, to the inventors' knowledge, this is the first time that administration of a combination of standardized extracts derived from these medicinal plants has produced the unexpected outcomes of reduced mortality and increased survival in LPS-induced sepsis. These unexpected outcomes, along with other favorable innate and adaptive immune responses, particularly the increase in IgA observed in human clinical trials and the reduction in extracellular HMGB1 as described in the subject matter of this application, make the bioflavonoid composition containing free B-ring flavonoids and flavans uniquely unique in directing the host immune response toward balanced activity and overall 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. Improved host defense homeostasis through balancing HMGB1 resulted in significant changes in the concentrations of biomarkers TNF-α (Example 14) and IL-1β (Example 15) in serum, IL-6 (Example 16), CRP (Example 19), IL-10 (Example 20), and total protein (Example 18) in bronchoalveolar lavage fluid (BAL), and CINC-3 (Example 17) in lung homogenates, which were subsequently confirmed by histological examination of lung tissue. In Example 21, animals administered the composition disclosed herein showed a statistically significant reduction in the overall severity of lung injury. In Examples 11 and 12, we evaluated the benefits of formulating free B-ring flavonoids from Scutellaria extract with flavans from Acacia extract compared to the administration of each medicinal plant alone in an LPS-induced sepsis model, and unexpected synergistic effects were observed. Data from the present application suggest that a bioflavonoid composition containing free B-ring flavonoids and flavans helps maintain homeostasis of the host defense mechanism by balancing and interrupting the vicious cycle involving upstream extracellular HMGB1 and subsequent NFκB signaling and cytokine storm. Therefore, these key features of the composition may lead to novel applications requiring 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, viral (e.g., COVID-19), and bacterial infections.

[0049] Direct instillation of LPS into the lungs is known to activate the innate immune response by causing alveolar macrophages to release significant amounts of HMGB1, partially via activation of NFκB, leading to increased production of primary cytokines such as TNF-α, IL-1β, and IL-6, as well as the proinflammatory protein CRP. These cytokines, acting alone or in concert, can cause significant pulmonary pathology and induce the activation of cytokine and chemokine cascades essential for disease pathology. For example, during acute inflammatory responses, chemotactic cytokines induce neutrophil chemoattractant 3 (CINC-3), which plays a key role in pulmonary recruitment of neutrophils in LPS-induced acute lung injury. Suppression of HMGB1 is a key turning point in immune homeostasis, controlling these key cytokines and chemotactic factors involved in the acute pulmonary inflammatory response. The balance of HMGB1 is a key phenomenon in pulmonary pathology and has important clinical relevance for cytokine storm intervention and reducing the severity of acute respiratory distress syndrome (ARDS).

[0050] Protein or fibrin leakage into the interstitial space is a key factor in pulmonary edema, and increased exudate is an indicator of disease severity. Administration of the composition reduced total protein in bronchoalveolar lavage fluid (BAF) in both LPS-induced acute lung injury and hyperoxia-exposed / PA-infected mice, demonstrating its potential for ameliorating pulmonary pathology. These significant changes in biomarkers in serum, BAL, and homogenate demonstrated that the administration strategy led to a statistically significant reduction in the overall severity of lung injury, which was subsequently confirmed by histopathological evaluation. Based on the reductions in HMGB1 and NFκB levels, increased airway and pulmonary bacterial clearance, reduced total lung protein, reduced cytokines, improved histopathological data, and IgA induction shown in this study, it is clear that the bioflavonoid composition indeed modulates the tipping point of immune homeostasis, suppressing the cytokine storm and reducing the severity of acute inflammatory lung injury.

[0051] Therefore, the subject matter of the present application was evaluated in hyperoxia-sensitized, Pseudomonas aeruginosa (PA)-infected mice, comparing the disclosed bioflavonoid composition containing free B-ring flavonoids and flavans with a positive control, resveratrol (Example 35). In this model, UP446 (Table 6), a bioflavonoid composition containing 60% or more free B-ring flavonoids and 10% or more flavans, was first tested for its ability to increase mouse survival after 7 days of treatment. Mice maintained in room air (RA) experienced a 9% mortality rate, whereas mice exposed to hyperoxia for 2 days prior to PA inoculation experienced a 64.29% mortality rate (Table 36). Meanwhile, mice inoculated with PA after 7 days of prophylactic treatment with resveratrol (RES) and UP446 prior to 2 days of hyperoxia exposure experienced a 27.27% and 28.57% mortality rate, respectively (Table 36). Next, the bioflavonoid composition was tested using a mouse model of oxidative stress / pulmonary infection-induced acute lung injury, combining PA-induced pulmonary infection and hyperoxia-induced oxidative stress, to examine the effect of UP446 in suppressing acute lung injury induced by pulmonary infection and exacerbated by oxidative stress (Example 36). The bioflavonoid composition containing free B-ring flavonoids and flavans statistically significantly improved: a) reduced airway HMGB1 accumulation (Table 40 in Example 39); b) increased airway and pulmonary bacterial clearance (Tables 38 and 39 in Examples 37 and 38); and c) improved lung injury, as reflected by reduced BAL total protein (Table 37 in Example 36), in mice exposed to hyperoxia and PA infection. This correlates with the significantly enhanced ability of UP446 to improve host defense against pulmonary microbial infection. Furthermore, UP446 improved host defense against bacterial infection in the lungs and airways. These effects play an important role in preventing septic shock and systemic inflammatory responses. Data from this study clearly demonstrate that UP446, a composition of free B-ring flavonoids and flavans, may be beneficial for a growing population whose host defenses are compromised by oxidative stress and viral or microbial infections.

[0052] As demonstrated in the accelerated aging model in Example 22, mice were administered D-galactose to induce an aging phenotype. Four weeks after D-galactose induction, the mice were administered UP446, a composition of free B-ring flavonoids and flavans disclosed herein, at two different concentrations for four weeks. Then, the mice were immunized with influenza vaccine and host defense mechanisms were measured using multiple assays to determine whether UP446 contributed to a balanced host defense phenotype similar to that of control mice. Significant outcomes are highlighted below. A) In Example 23 and Table 23, the thymic index of the normal control group and the groups treated with UP446 and D-Gal at both concentrations was significantly higher than that of the D-Gal group, indicating that UP446 contributed to the reversal of thymic involution, i.e., the age-related decrease in thymus size, which likely affects the body's ability to mount an immune response. B) Example 24 and Table 24 revealed significant changes in humoral immunity between immunization groups. Complement C3 was significantly increased in the D-Gal + UP446 (200 mg / kg) group compared to the D-Gal alone group, and it was determined that the humoral immune response after immunization was prolonged in the UP446-administered group compared to the D-Gal group. C) In Example 28, leukocyte counts in whole blood from various groups were measured and significant differences were found between the immunized mouse groups. The immunized UP446 + D-Gal group had increased CD49b+ cells (Table 28) and NKp46+ natural killer cells (Table 29) compared to the immunized D-Gal alone group. From these data, it was determined that UP446 promoted an expansion of the natural killer cell population, increasing the percentage of innate immune cells. D) Significant differences were also observed between non-immunized mouse groups. The D-Gal + UP446 group showed a strong trend toward 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. These data, demonstrated in Examples 25-30, suggest that the bioflavonoid composition UP446 disclosed herein primes the inactivated immune system in non-immunized mice, expanding immune cell populations and enhancing immune "readiness." E) Antioxidant enzymes and biomarkers were tested to monitor antioxidant pathways. The aging phenotype induced by the D-Gal model is based on an increase in advanced glycation end products (AGEs), resulting in oxidative stress and damage similar to levels seen in aged animals (Azman KF, 2019). Stimulating antioxidant pathways may counteract the effects of oxidative stress. First, in Example 31, AGE concentrations were measured in serum samples from immunized and non-immunized mice. AGEs in the serum of mice from the non-immunized D-Gal + UP446 group (both concentrations) were found to be lower than in the D-Gal alone group (Table 32). Therefore, it was determined that animals administered UP446 had lower free radical concentrations, specifically those contributing to the aging phenotype in the D-Gal model. Next, in Example 32, we focused on glutathione peroxidase (GSH-Px) activity in the serum of mice from immunized animals. Compared with the immune D-Gal group, the immune UP446 + D-Gal groups at both concentrations showed significantly higher GSH-Px activity (Table 33), suggesting that the ability to neutralize free radicals was enhanced in the UP446-treated animals. F) Protein concentrations in the spleens of immunized animals were also analyzed. The spleen is one of the major organs of the immune system. It contains a high concentration of white blood cells and regulates the concentration of immune cell types in the blood. In Example 33, we measured NFκB, an inflammatory transcription factor activated in response to inflammation, and found that NFκB levels were reduced in the D-Gal + UP446 high-dose group (Table 34). This suggests that reduced NFκB levels are one mechanism by which UP446 regulates inflammatory responses during host defense homeostasis. HMGB1 is an alarmin protein and transcription factor. Under non-inflammatory conditions, it is a nuclear protein, but upon nuclear export, it is secreted into the extracellular space, further amplifying inflammatory signals. As demonstrated in Example 34, HMGB1 levels were significantly reduced in the non-immunized D-Gal + UP446 high-dose group 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] Aging is characterized by the gradual deterioration of tissues and organs, partially reflected by impaired antioxidant defense system and immune system. According to the free radical theory of aging, oxidative damage (an imbalance between free radicals and antioxidants) is a major contributor to aging and age-related degenerative structural and functional disorders of tissues and organs (Azman and Zakaria 2019). Increased levels of advanced glycation end products (AGEs) are known to accelerate the aging process and are considered a key pathway in the aging mechanism in the D-galactose-induced accelerated aging model, which is characterized by a poor immune response and impaired antioxidant defense system. Using a D-galactose-induced animal model to mimic these natural phenomena, mice treated with D-Gal+ solvent exhibited increased oxidative stress, decreased antioxidant enzyme activity, and impaired immune response. In contrast, supplementation with a bioflavonoid composition containing free B-ring flavonoids and flavans reversed the age-related structural and functional changes. Supplementation with the bioflavonoid composition UP446 resulted in a statistically significant dose-related reduction in serum AGEs, with the highest reduction rate being 58% in the high-dose group (Table 32 in Example 31). Furthermore, the most effective cellular defense mechanism against oxidative damage primarily involves the action of endogenous enzymatic antioxidants, such as glutathione peroxidase (GSH-Px). Indeed, the bioflavonoid composition exerted a potent antioxidant boosting effect, resulting in a statistically significant increase in GSH-Px at all doses (Example 33 in Example 31). Taking into account the induction of mucosal immunity, the preservation of immune organs, the reduction of AGEs, and the increase in endogenous antioxidant enzymes, a bioflavonoid composition containing free B-ring flavonoids and flavans may prevent age-related immune dysregulation and antioxidant defense system dysfunction.

[0054] Supplementation of chemically aged mice with a bioflavonoid composition enhanced innate immunity. Natural killer cell activation and expansion are key immunoregulatory mechanisms for maintaining host defense homeostasis. Natural killer cells (NK) are key components of the innate immune system, known to rapidly respond to a wide range of pathological sensitizations; air pollutants; viral, microbial, and fungal infections; and cellular oxidative and hormonal insults without priming or prior activation. Natural killer cells (NK) monitor cell integrity, detecting changes in cell surface molecules and deploying their cytotoxic effector mechanisms. Natural killer (NK) cells function as cytotoxic lymphocytes and producers of immunoregulatory cytokines. After stimulation, NK cells produce large amounts of cytokines, primarily gamma interferon (IFN-γ) and tumor necrosis factor (TNF). These cytokines and other cytokines produced by NK cells act directly during early immune responses and are important modulators of late-stage adaptive immune responses mediated by T cells and B cells. The significant increase in NK cells in the intended protected subjects as a result of oral administration of the bioflavonoid composition clearly indicates that the protected subjects have a significant impact on innate immune regulation, suggesting that their immediate and effective immune-inducing activity is involved in laying the foundation for immune homeostasis. This activation of innate immunity as natural killer cells is another aspect by which the bioflavonoid composition induces a response to protect the respiratory tract and maintain mucosal homeostasis.

[0055] The observed induction levels of CD4+TCRγδ+ gamma delta T cells, known to play roles in immunoregulation, promoting immune surveillance, and immune homeostasis, confirmed the mucosal immunoregulatory and host defense homeostatic activities of the protected target of this application. γδ T cells are a unique T cell subpopulation that resides primarily at multiple intestinal sites in the body, including the lungs and intestine, where they migrate early in development and persist as resident cells. Due to their strategic anatomical location (the mucosal layers of the respiratory and gastrointestinal systems), γδ T cells provide a first line of defense based on their innate immune-like response in that they directly kill infected cells, recruit other immune cells, activate phagocytosis, and limit the translocation of pathogens or pollutants to systemic compartments. These cells are known to rapidly expand in population upon secondary sensitization and provide pathogen-specific defense. Their ideal location in the respiratory and intestinal tracts also helps maintain the integrity of the respiratory and intestinal epithelia. In general, the physiological roles of γδ T cells include protective immunity against extracellular and intracellular pathogens or pollutants, surveillance and regulation of innate and adaptive immune responses, tissue healing and epithelial cell maintenance, and regulation of physiological organ function. γδ T cells share characteristics with natural killer (NK) cells; both are typically considered components of innate immunity, recognize transformed / damaged cells, play prominent roles in antiviral defense, promote downstream adaptive immune responses, and are potent cytolytic lymphocytes. Furthermore, γδ T cells also function as antigen-presenting cells (Ribot et al., 2021; Bonneville et al., 2010). UP446, the bioflavonoid composition protected by the present application, induced these rapidly responding immune cells (γδ T cells and NK cells), resulting in mucosal immunomodulation and host defense homeostasis.

[0056] In summary, D-Gal mice administered UP446, a composition of free B-ring flavonoids and flavans, showed significant changes compared with the D-Gal alone group, suggesting that the host defense mechanisms of aging animals were reversed to resemble those of normal control mice, or at least that host defense system priming and activation were enhanced. The thymic index, serum complement, natural killer cell, and glutathione peroxidase activity in the immunized D-Gal + UP446 group were higher than those in the D-gal alone group, suggesting that the host defense system in the UP446-treated group responded better to vaccination than the D-Gal-induced aging alone group. The non-immunized D-Gal + UP446 group showed higher CD8+ cytotoxic T cells, natural killer cells, and CD4+ TCRγδ+ gamma delta T cells than the D-gal alone group, and AGE and NFκB concentrations were lower compared to the D-gal group, indicating that both innate and adaptive immune responses were primed and oxidative stress and inflammation were reduced. These results demonstrate that UP446, a composition of free B-ring flavonoids and flavans, is useful for priming the host defense system against infection, both during vaccination or infection and as a preventative measure.

[0057] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, which can result in a variety of clinical outcomes, from asymptomatic infection to lung injury, inflammation, respiratory distress, multiple organ failure, and death. Extracellular HMGB1, secreted in SARS-CoV-2-infected lungs, is considered a therapeutic target in severe lung inflammation in COVID-19 (Andersson et al. 2020). Herbal medicines have been investigated to treat SARS-CoV-2 viral adhesion, acute respiratory failure, and sepsis by inhibiting HMGB1 release (Wyganowska-Swiatkowska et al. 2020). Given the binding of human angiotensin I-converting enzyme 2 (hACE2) to the SARS-CoV-2 spike protein as a major viral entry point, a transgenic mouse model expressing human ACE2 was challenged with SARS-CoV-2 to improve the efficacy of model induction and intervention. As shown in Example 40, transgenic mice infected with SARS-CoV-2 virus and administered vehicle showed a statistically significant 2-fold increase in lung HMGB1 protein expression compared to uninfected normal transgenic control mice. In contrast, when transgenic mice infected with SARS-CoV-2 virus were supplemented with UP894-II, a bioflavonoid composition containing 70-80% free B-ring flavonoids and 15-20% flavans, HMGB1 protein expression in lung tissue was reduced to the level of uninfected normal control transgenic mice (Figure 8). This reduction in lung HMGB1 expression levels as a result of administration of the bioflavonoid composition was statistically significant compared to transgenic mice infected with SARS-CoV-2 and administered vehicle. HMGB1 is a major late alarmin known to activate a complex sequence of host immune responses, which, if unrepressed, can lead to cytokine storms, host defense homeostatic imbalance, and ultimately adverse clinical manifestations such as those seen in hospitalized COVID-19 patients.Based on the significant and statistically significant reduction in HMGB1 expression in the lung tissue of these transgenic mice infected with SRS-CoV2, it was determined that the bioflavonoid composition containing free B-ring flavonoids and flavans could improve host defense mechanisms, induce homeostatic equilibrium, and reduce the lethal cytokine storm caused by SARS-CoV-2 coronavirus infection and the associated damage to the lungs and other organs.

[0058] Perhaps the most significant primary outcome of host defense modulation from UP446, a unique bioflavonoid composition containing over 60% free B-ring flavonoids and over 10% flavans, was the change in serum IgA in healthy volunteers, as demonstrated by human clinical trials in Example 41. In a double-blind, placebo-controlled clinical trial, healthy middle-aged subjects (Table 42) were supplemented with 250 mg of UP446 twice daily or a placebo daily for 28 days before sensitizing their immune systems with an influenza vaccine (Table 41). Subjects continued to receive UP446 or placebo for an additional 28 days, and blood samples were collected at baseline, after 28 days of supplementation, and after 56 days of supplementation (28 days after vaccination) for host defense biomarker measurements. After 8 weeks of supplementation, subjects receiving the bioflavonoid composition UP446 showed significantly increased mucosal immune indices, such as immunoglobulin A, before and after influenza vaccination compared to the placebo group. In the UP446 supplementation group, the changes in IgA from day 0 to day 56 and from day 28 to day 56 were significantly higher than in the self-group comparison. Throughout the supplementation period, subjects who took the bioflavonoid composition UP446 showed a significant increase in IgA levels after influenza vaccination compared to the placebo group. These data clearly demonstrate that IgA, which is considered the major immunoglobulin in a healthy respiratory system and the most important immunoglobulin for mucosal defense, is one of the major indicators of improved homeostasis of the host defense mechanism in humans.

[0059] The respiratory system (i.e., lungs and upper airways) is frequently exposed to various pathogens and pollutants inhaled during breathing, and has a large mucosal surface area (400–500 m2), which is the most common site of inflow of these pathogens and pollutants. 2 ) Constantly challenged by numerous airborne microorganisms, particulates, pollutants, and environmental antigens, respiratory mucosal surfaces must engage robust nonspecific and specific defense mechanisms to protect against respiratory infection and injury. Beyond physiological defenses (coughing, sneezing, and mucociliary clearance) and the removal of particles and microorganisms by alveolar macrophages, the induction of mucosal humoral immune responses, more specifically, IgA production in the respiratory tract, is paramount to protecting the respiratory system. IgA is thought to cooperate with nonspecific innate immune factors and function as an effective first line of respiratory / pulmonary defense against foreign agents without inducing potentially harmful inflammatory responses. Indeed, bioflavonoid compositions containing free B-ring flavonoids and flavans address innate immune responses by enhancing macrophage phagocytic activity, while simultaneously promoting adaptive immune responses by stimulating mucosal immunity, particularly IgA production. IgA, the major class of immunoglobulin in the respiratory mucosa, is the most important immunoglobulin for respiratory and pulmonary defense, and is known to a) protect mucosal surfaces from the invasion of microorganisms and foreign antigens, b) neutralize bacterial products, c) eliminate pathogens or antigens that have invaded the mucosal surface via the IgA-mediated secretory pathway, d) aggregate microorganisms and disrupt bacterial motility, and e) neutralize viruses intracellularly by interacting with viral antigens during transcytosis and disrupting viral synthesis or assembly (Pilette et al., 2001). As described in the present application and demonstrated in human clinical trials, particularly in Example 41, supplementation with a bioflavonoid composition containing free B-ring flavonoids and flavans induces mucosal immunity, particularly by increasing IgA production and enhancing the phagocytic activity of hyperoxic macrophages in human clinical trials, suggesting that the primary role of the present application's protection is lung protection and maintaining mucosal immune homeostasis.

[0060] In summary, using both cell culture and animal models, we demonstrate that prolonged exposure to oxidative stress during oxygen therapy, routinely used to treat COVID-19 patients, dramatically releases HMGB1, shifting the immune response balance and inducing a decline in innate immunity, leading to impaired macrophage function and, consequently, impaired host defenses to remove pathogens invading the respiratory tract and lungs, leading to acute respiratory inflammation, lung injury, and ultimately death. Using these model systems, we demonstrate that HMGB1 is a novel cellular and molecular mechanism underlying oxidative stress-induced susceptibility to pulmonary infections. We also demonstrate that a bioflavonoid composition containing free B-ring flavonoids and flavans improves innate immunity and alleviates respiratory dysfunction by shifting HMGB1 in these hosts, as shown in Figures 1 and 2. Administration of a bioflavonoid composition containing free B-ring flavonoids and flavans suppressed the accumulation of extracellular HMGB1, improved respiratory function, strengthened innate immunity against bacterial and viral infections, and suppressed inflammatory responses by improving the homeostasis of host defense mechanisms.

[0061] The subject matter of the present application for regulating HMGB1 using free B-ring flavonoids and flavans can be expressed, but is not limited to, by a) inhibiting HMGB1 release or interfering with its action by targeting the active or passive release of HMGB1 by blocking cytoplasmic translocation or vesicle-mediated release, or by inhibiting intramolecular disulfide bond formation in the nucleus; b) directly targeting HMGB1 upon release and neutralizing its action; or c) 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, as shown in Figure 3. Inhibition of oxidative stress-mediated HMGB1 release during infection, inflammation, and cell death can be achieved by targeting 1) HMGB1 nuclear export by CRM1 in activated immune cells, 2) HMGB1 release by PARP1 in necrosis, 3) HMGB1 release by caspase 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 necrosis. The effects of bioflavonoid compositions containing free B-ring flavonoids and flavans can also be achieved by preventing HMGB1 clustering or self-association, which 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, particularly zinc (the inclusion of low-dose Zn2+ promotes HMGB1 tetramer formation), and redox environment (under oxidative conditions similar to the extracellular environment, HMGB1 exists primarily as a tetramer, whereas under reducing conditions such as the intracellular environment, more dimeric species exist).The bioflavonoid compositions prevent HMGB1 tetramer formation and interfere with the binding affinity of HMGB1 to TLRs and RAGE by altering the physicochemical microenvironment.

[0062] In the above and following descriptions, certain specific details are set forth to provide a thorough understanding of various embodiments of the present disclosure. However, it will be apparent to one skilled in the art that the subject matter of the present application can be practiced without limitation without these details.

[0063] In this application, all concentration ranges, percentage ranges, ratio ranges, or integer ranges should be understood to include all integer values ​​within the stated range, and fractions thereof (e.g., tenths or hundredths of an integer), where appropriate, unless otherwise specified. Also, all numerical ranges stated herein for all physical characteristics, such as polymer subunits, size, or thickness, should be understood to include all integers within the stated range, unless otherwise specified. As used herein, the terms "about," "comprising," "consisting of," and "consisting essentially of" refer to the mean ±20% of the specified range, value, or structure, unless otherwise specified. As used herein, the indefinite article should be understood to mean "one or more" of the listed elements. The use of alternatives (e.g., "or" or "and / or") should be understood to mean either one, both, or any combination thereof. Unless the context contradicts otherwise, throughout this specification and claims, the terms "comprises" and its variations, "include" and "including," and cognate terms such as "include" and "having" and variations thereof, are to be interpreted in their open and inclusive sense, i.e., meaning "including but not limited to."

[0064] References throughout this specification to "one embodiment" or "one embodiment" mean that the particular feature, structure, composition, or characteristic described in connection with this embodiment is included in at least one embodiment of the subject matter covered by this application. Thus, various uses of the phrase "in one embodiment" or "in one embodiment" throughout this specification do not necessarily all refer to the same embodiment.

[0065] The term "prodrug" also refers to a covalent bond of an active compound of the present disclosure to a carrier that is released in vivo when the prodrug is administered to a mammalian subject. Prodrugs of the compounds of the present disclosure can be prepared by modifying functional groups present in the compounds of the present disclosure so that they cleave, either by routine manipulation or in vivo, back to the parent compound of the present disclosure. Examples of prodrugs include those in which a hydroxy group, an amino group, or a mercapto group is attached to any group of the compounds of the present disclosure, and cleaves 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 acetate, formate, and benzoate derivatives of alcohol moieties in the compounds of the present disclosure, or amide derivatives of amine functional groups.

[0066] "Stable compound" and "stable structure" mean a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an efficacious therapeutic agent having a reasonable shelf life.

[0067] "Biomarker" or "marker" component or compound means one or more chemical components or compounds inherent to a plant, plant extract, or combination composition of two or three plant extracts disclosed herein, which are used to control the quality, consistency, integrity, safety, or biological function of the compositions of the invention.

[0068] "Mammal" includes humans as well as 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 wildlife.

[0069] "Optional" or "optionally" means that the element, component, event, or circumstance modified by the word may or may not be present; that element, component, event, or circumstance may or may not be present. For example, "optionally substituted aryl" means that the aryl group may or may not be substituted, and includes both substituted and unsubstituted aryl groups.

[0070] A "pharmaceutically or nutraceutical acceptable carrier, diluent, or excipient" includes any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. In intended embodiments, the composition further comprises a pharmaceutically or nutraceutical 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 the active compound in the at least one standardized bioflavonoid extract.

[0071] "Pharmaceutically or nutraceutical acceptable salts" includes both acid addition salts and base addition salts. "Pharmaceutically or nutraceutical acceptable acid addition salts" refers to salts that retain the biological effectiveness and properties of the free base, are not biologically or otherwise unsuitable, and are formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well 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, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, It refers to salts formed with organic acids such as 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, and undecylenic acid.

[0072] "Pharmaceutically or nutraceutical acceptable base addition salt" refers to a base addition salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise unsuitable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In certain embodiments, the inorganic salt is an ammonium, sodium, potassium, calcium, or magnesium salt. Salts derived from organic bases include salts of primary amines, secondary amines, tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as 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, etc. Particularly useful organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0073] Crystallization often results in the formation of solvates of the compounds of the present disclosure. As used herein, the term "solvate" refers to an aggregate containing one or more molecules of a compound of the present disclosure and one or more molecules of solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and the corresponding solvates. While the compounds of the present disclosure can be true solvates, the compounds of the present disclosure may simply retain incidental water or may be a mixture of water and some incidental solvent.

[0074] A "pharmaceutical composition" or "nutraceutical composition" refers to a formulation comprising a compound of the present disclosure and a vehicle generally accepted in the art for delivering the bioactive compound to a mammal (e.g., a human). For example, a pharmaceutical composition of the present disclosure may be formulated or used as the sole composition, or as an ingredient or active pharmaceutical ingredient (API) in a prescription drug, over-the-counter (OTC) drug, botanical, herbal, natural remedy, homeopathic remedy, or any other form of health care product reviewed and approved by a government agency. Exemplary nutraceutical compositions of the present disclosure may be formulated or used as the sole composition, or as a nutritional or bioactive ingredient in a food, functional food, beverage, bar, food flavor, medical food, dietary supplement, or herbal product. Vehicles generally accepted in the art include all carriers, diluents, or excipients pharmaceutically or nutraceutical acceptable for this purpose.

[0075] As used herein, "enriched" refers to a plant extract or other preparation in which the amount of one or more active compounds has been increased by at least 2-fold and up to about 1000-fold compared to the amount of the active compound present in the weight of the plant material or other raw material prior to extraction or other processing. In certain embodiments, the weight of the plant material or other raw material prior to extraction or other processing can be the dry weight, the wet weight, or a combination thereof. In certain embodiments, the standardized bioflavonoid extract has been enriched by solvent precipitation, neutralization, solvent partitioning, ultrafiltration, enzymatic digestion, column chromatography on silica gel, XAD, HP20, LH20, C-18, alumina oxide, polyamide, ion exchange resin, CG161 resin, or a combination thereof, either individually or in combination.

[0076] As used herein, the term "major active ingredient" or "major active ingredient" refers to one or more active compounds present in or concentrated in a plant extract or other preparation, which are capable of exerting at least one biological activity. In certain embodiments, the major active ingredient of a concentrated extract will be one or more active bioflavonoid compounds concentrated in the extract. Generally, the major active ingredient(s) in the bioflavonoid composition will directly or indirectly provide the majority (i.e., greater than 60%, or greater than 50%, or greater than 20%, or greater than 10%) of one or more measurable biological activities or effects relative to other extract components. In certain embodiments, the major active bioflavonoids may be minor components of the extract that constitute a minor percentage by weight (e.g., less than 50%, less than 25%, or less than 10%, or less than 5%, or less than 1% of the bioflavonoids in the extract) yet still provide the majority of the desired biological activity. All bioflavonoid compositions of the present disclosure containing a primary active ingredient such as baicalin as a free-B-ring flavonoid may further contain a secondary active ingredient, epicatechin as a flavan, which may or may not contribute to the pharmaceutical or nutraceutical activity of the concentrated composition, but does not contribute to the concentration of the primary active ingredient, and the secondary active ingredient may not be effective alone in the absence of the primary active ingredient.

[0077] An "effective amount" or "therapeutically effective amount" means an amount that, when administered to a mammal such as a human, results in: (1) stimulation of innate immunity; (2) enhancement of adaptive immunity, particularly an increase 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) suppression of immune cells, respiratory tract infections, and damage to immune cells against HMGB1-induced cytokine storms. (5) inhibiting oxidative stress, reducing NF-kB, reducing advanced glycation end products, increasing glutathione peroxidase, neutralizing reactive oxygen species, and acting as a powerful antioxidant to prevent the structural integrity and functional decline of the respiratory, pulmonary, and immune systems caused by oxidative stress; (6) maintaining homeostasis of innate and adaptive immune responses; (7) increasing the phagocytic index of macrophages in humoral and cellular immune responses; (8) promoting the expression of NF-kB and NFAT (9) inhibition of lymphocyte activation and inflammatory cytokine gene expression (IL-2, iNOS, TNF-α, COX-2, and IFN-γ); (10) reduction of inflammatory cytokine levels such as IL-1β, IL-6, and TNF-α; (11) downregulation of COX-2, NOS-2, and NF-κB expression; (12) inhibition of eicosanoid production by inhibiting phospholipase A2 and TXA2 synthase activity; (13) (13) suppression of Th1 and Th17 cell responses; (14) decreased neutrophil chemotaxis due to decreased expression of ICAM and VCAM; (15) inhibition of MAPK phosphorylation, adhesion molecule expression, and signal transduction and activator of transcription 3 (STAT-3); and (16) activation of the transcription factor NRF2 and induction of heme oxygenase-1.

[0078] "Biomarkers" related to host defense function and lung structural integrity and function that are modulated by compositions for modulating homeostasis of host defense mechanisms using various combinations of two to three plant extracts (for example, but not limited to, UP446 or UP894-2 containing free B-ring flavonoids and flavans as disclosed herein) include, but are not limited to, hemagglutinin inhibitory (HI) titers 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- These include 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, and LO13.

[0079] As used herein, "virus" includes, but is not limited to, highly pathogenic avian influenza (type A H5N1 virus strain), influenza A (H1N1, H3N2, H5N1), influenza B / Washington / 02 / 2019-like virus, influenza B / Phuket / 3073 / 2013-like virus, hepatitis A, B, C, and D viruses, coronaviruses SARS-CoV, SARS-CoV-2 (COVID-19), MERS-CoV (MERS), respiratory syncytial virus (RSV), enterovirus A71 (EV71), parainfluenza, and adenovirus.

[0080] As used herein, "microorganisms" includes, but is not limited to, pathogenic bacteria that infect the respiratory system, with the most common bacterial pathogens including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, and Moraxella catarrhalis, and the major pulmonary fungal pathogens in upper and lower respiratory tract infections including Aspergillus, Cryptococcus, Pneumocystis, Histoplasma capsulatum, and the like. These include Bacillus capsulatum, Blastomyces, Cryptococcus neoformans, Pneumocystis jiroveci, Candida spp., and endemic fungi, while the main bacterial pathogen of pharyngitis and tonsillitis is Streptococcus pyogenes. Bacterial infections can also develop after viral illnesses such as the common cold or influenza.

[0081] As used herein, the term "respiratory system and lungs" includes, but is not limited to, the airways, which transport air to the lungs and oxygen from the lungs to all of the host's other organs; the mouth and nose, which are openings that allow air to enter the host's respiratory system from outside the host's body; the sinuses, which are cavities between the bones of the host's head that help regulate the temperature and humidity of the air the host inhales; the pharynx (throat), which are tubes that transport air from the host's mouth and nose to the trachea (throat); the trachea, which is the passageway that connects the host's throat to the lungs; the bronchi, which are tubes that lead from the bottom of the host's throat to each of the lungs; the lungs, which are two-part organs that remove oxygen from the air and deliver it to the host's bloodstream; the bloodstream, which transports carbon dioxide to the lungs and oxygenates the lungs to all of the host's organs and other tissues; and the muscles and bones that help move the air inhaled by the host in and out of the host's lungs.

[0082] "Respiratory infection" includes symptoms of the common cold, including nasal congestion, runny nose, sneezing, mild fever, headache, sore throat, chest tightness, wheezing, dry cough and wheezing, fatigue, shortness of breath, congestion, hoarseness, pain and difficulty swallowing, swollen lymph nodes, and facial tenderness (especially under the eyes or bridge of the nose). Some warning signs that the common cold may be progressing from a viral infection to a bacterial infection include, but are not limited to, symptoms lasting for more than 10-14 days, a fever above 100.4°C, a worsening fever that does not improve for several days, white pus-filled spots on the tonsils, sinusitis with postnasal drip, stuffy / congestive nose, toothache, cough, greenish nasal discharge, facial tenderness (especially under the eyes or bridge of the nose), bad breath, fatigue, and fever.

[0083] "Lung infection" or "pneumonia" is the most common bacterial or viral lower respiratory tract infection. It can be caused by air pollutants, cigarette, e-cigarette, or recreational marijuana smoking. It is an infection that causes inflammation of the air sacs in one or both lungs, resulting in the accumulation of mucus or pus. Symptoms of pneumonia include, but are not limited to, a productive cough or pus-producing cough, fever, chills, difficulty breathing, severe chest pain, dehydration, fatigue, loss of appetite, sticky and sweaty skin, rapid or shallow breathing, shortness of breath, wheezing, a high heart rate, and low blood oxygen saturation. "Lung infection" or "pneumonia" can be diagnosed by chest x-ray, CT scan, blood test, and saliva culture. Resident macrophages function to protect the lungs from foreign pathogens and, triggered by the pathogen's inflammatory response, are responsible for the histopathological and clinical findings seen in pneumonia. Macrophages engulf these pathogens, trigger signaling molecules or cytokines such as TNF-α, IL-6, and IL-1, and recruit inflammatory cells such as neutrophils to the site of infection. Macrophages then present these antigens to T cells, triggering both cellular and humoral defense mechanisms, activating complement, and forming antibodies against these organisms. This results in inflammation of the lung parenchyma, making capillaries in the alveolar walls more susceptible to "peeling," leading to exudative congestion and the development of pneumonia.

[0084] The amount of a compound, extract, or composition of the present disclosure that constitutes a "therapeutically effective amount" or "nutritionally effective amount" will vary depending on the bioactive compound or nutritional component, the condition being treated and biomarkers of its severity, the mode of administration, the duration of administration or dietary supplementation, or the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art in light of their knowledge and this disclosure. In certain embodiments, an "effective amount" or "therapeutically effective amount" or "nutritionally effective amount" can be expressed as an amount relative to 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). The "effective amount" or "therapeutically effective amount" or "nutritionally effective amount" in animal studies can be extrapolated to a human equivalent daily dose using FDA guidelines, taking into account differences in total body surface area and body weight between animals and humans.

[0085] As used herein, a "dietary supplement" is a product that improves, promotes, enhances, manages, controls, maintains, optimizes, modifies, suppresses, inhibits, establishes or prevents the homeostasis, balance, natural state or biological process, or imbalance or decline or suppression or overstimulation of a specific pathological condition, biological function or phenotypic state or defense mechanism related to structural and functional integrity (i.e., not used to diagnose, treat, mitigate, cure or prevent disease). For example, with respect to host defense mechanisms, "dietary supplements" can be used as immune adjuvants specific for immunostimulants that enhance vaccine efficacy, enhance the phagocytic activity of macrophages, improve the innate killing activity of NK cells, regulate the production levels of inflammatory cytokines, reduce inflammation and tissue damage, induce antibody responses and production, enhance antibody-dependent cellular cytotoxicity, stimulate T cell proliferation, promote the generation of immunosuppressive regulatory T cells, protect immune cells and lung cells from HMGB1-induced cytokine storm damage, suppress uncontrolled activation of NFκB, and protect organs or tissues from oxidative stress, or to regulate, maintain, control, balance, suppress, or stimulate any component of adaptive or innate immunity. In certain embodiments, dietary supplements are a special category of dietary supplements, natural nutrients, foods, functional foods, or medical foods, and are not pharmaceuticals.

[0086] As used herein, "treating" or "treatment" refers to the treatment of a disease or condition in a mammal, such as a human, having the disease or condition, and includes (i) preventing the disease or condition from occurring in the mammal, particularly when the mammal is predisposed to the disease or condition but has not yet been diagnosed with the disease or condition; (ii) inhibiting the disease or condition, i.e., halting its development; (iii) alleviating or modifying the disease or condition, i.e., causing regression of the disease or condition; or (iv) alleviating symptoms caused by the disease or condition without addressing the underlying disease or condition (e.g., alleviating cough and fever, alleviating pain, reducing inflammation, reducing pulmonary edema, alleviating pneumonia); or (v) balancing the regulation of immune homeostasis or otherwise altering the phenotype of the disease or condition.

[0087] As used herein, the terms "disease" and "pathological condition" may be used interchangeably, or they may not be synonymous in the sense that a specific pathological condition or condition is not yet recognized as a disease because the causative agent is unknown (and thus the etiology has not yet been determined), but rather is recognized only as an undesirable condition or syndrome, although a specific set of symptoms may be noted by clinicians. A disease or condition may be acute, such as a viral infection (SARS, COVID-19, MERS, hepatitis, influenza) or a microbial infection, or chronic, such as lung damage caused by exposure to air pollution or smoke. A homeostatic imbalance may also result in a weakened immune system, leading to a disease or condition, predisposing a mammal to infection, or leading to secondary organ and tissue damage directly or indirectly related to viral or bacterial infection or air pollutants.

[0088] As used herein, the term "statistical significance" means a p-value of 0.050 or less when calculated using Student's t-test, indicating that a particular event or measurement is unlikely to have occurred by chance.

[0089] For administration purposes, the compounds of the present invention may be administered as crude compounds or may be formulated as pharmaceutical, nutraceutical, or food compositions. Pharmaceutical or nutraceutical compositions of the present invention contain a compound of the present invention with a pharmaceutical, nutraceutical, or conventional food-acceptable carrier, diluent, or excipient. The compound of the present invention is present in the composition in an amount effective to treat a particular relevant disease or condition or to supplement natural nutrition, i.e., sufficient to establish homeostasis of host defense mechanisms or promote innate or adaptive immunity or immune homeostasis in general, or any of the other relevant effects described herein, and generally non-toxic or tolerable to the host.

[0090] The compounds or compositions of the present disclosure, or their pharmaceutically or nutraceutical acceptable salts, can be administered in pure form or in suitable pharmaceutical or nutraceutical compositions by any of the administration methods acceptable for pharmaceuticals of similar uses. The pharmaceutical or nutraceutical compositions of the present disclosure can be prepared by adding suitable pharmaceutically or nutraceutical acceptable carriers, diluents, or excipients to the compounds of the present disclosure, and can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, beverages, suppositories, injections, inhalants, gels, creams, lotions, tinctures, sachets, instant drinks, masks, microspheres, and aerosols. The bioflavonoid compositions disclosed herein can also be formulated with other food ingredients into conventional foods, functional foods, nutritional foods, and medical foods. 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 herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0091] Pharmaceutical or nutraceutical compositions of the present disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a patient. The composition administered to a subject, patient, or mammal may take the form of one or more dosage units; for example, a single tablet may be a single dosage unit, or a container filled with an aerosol of a compound or extract of the present disclosure, or a composition of two or three plant extracts, may hold multiple dosage units. Actual methods for preparing such dosage forms are known or readily apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). In any event, the composition administered will contain a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically or nutraceutical acceptable salt thereof, to treat the relevant disease or condition in accordance with the teachings of the presently protected subject matter.

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

[0093] For oral administration, the pharmaceutical or nutraceutical composition may be in solid or liquid form, with semi-solid, semi-liquid, suspension and gel forms being included in dosage forms considered solid or liquid for this application.

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

[0095] When the pharmaceutical or nutraceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or an oil.

[0096] The pharmaceutical or nutraceutical composition may be in the form of a liquid, such as an elixir, tincture, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, to name two examples. For oral administration, useful compositions contain, in addition to the compound of the present application, one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. For compositions for injection, 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 may be added.

[0097] Liquid pharmaceutical or nutraceutical compositions of the present disclosure, whether in solution, suspension, or other formulation, may contain one or more of the following adjuvants: water for injection; saline solutions such as physiological saline; Ringer's solution; isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides usable as solvents or suspending media; sterile diluents such as polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose. Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multidose vials. Physiological saline is generally a useful adjuvant. Pharmaceutical or nutraceutical compositions for injection are sterile.

[0098] Liquid pharmaceutical or nutraceutical compositions of the present disclosure for parenteral or oral administration should contain an amount of a compound of the present disclosure such that a suitable dosage will be obtained.

[0099] The pharmaceutical or nutraceutical compositions of the present disclosure may be for topical administration, in which case the carrier may comprise a solution, emulsion, cream, lotion, ointment, or gel base, as appropriate. The carrier may comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, emulsifiers, and stabilizers. A thickener may also be added to pharmaceutical or nutraceutical compositions for topical administration. For transdermal administration, the compositions may comprise 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, in the form of a suppository that melts in the rectum and releases the drug. The composition for rectal administration can contain an oily carrier as a suitable non-irritating excipient. Such carriers include lanolin, cocoa butter, and polyethylene glycol.

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

[0102] The pharmaceutical or nutraceutical compositions of the present disclosure, whether solid or liquid, may include a substance that binds to the compounds of the present disclosure to aid in the delivery of said compounds. Suitable substances that can perform this function include monoclonal or polyclonal antibodies, proteins, or liposomes.

[0103] The solid or liquid pharmaceutical or nutraceutical compositions of the present disclosure can have reduced particle size, for example, to improve bioavailability. The size of the powders, granules, particles, microspheres, etc. in the compositions, with or without excipients, can be macro (e.g., visible or at least 100 μm in size), micro (e.g., can range from about 100 μm to about 100 nm in size), nano (e.g., can be 100 nm or smaller in size), 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 comprised of dosage units that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems, from colloidal systems to systems comprised of pressurized packages. Delivery can be achieved by a liquefied or compressed gas or an appropriate pump system that dispenses the active ingredient. Aerosols of the compounds of the present disclosure can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery can include the necessary container, activator, valve, inner container, etc., and can be integrated 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 prepared by methods well known in the pharmaceutical or nutraceutical fields. For example, pharmaceutical or nutraceutical compositions for injection administration can be prepared by adding sterile distilled deionized water to a compound of the present disclosure to form a solution. A surfactant may be added to aid in the formation of a uniform solution or suspension. A surfactant is a compound that interacts non-covalently with a compound of the present disclosure and aids in the dissolution or uniform suspension of the compound in an aqueous delivery system.

[0106] The compounds of the present disclosure, or pharmaceutically or nutraceutical acceptable salts thereof, are administered in therapeutically effective amounts, which will vary depending on a variety of factors, such as the activity of the particular compound employed, the metabolic stability and duration of action of the compound, the age, weight, general health, sex, and diet of the patient, the mode and timing of administration, rate of excretion, concurrent drug use, the severity of the particular disorder or condition, and the subject being treated.

[0107] The compounds of the present disclosure, or pharmaceutically or nutraceutical acceptable derivatives thereof, may also be administered simultaneously with, before, or after food, water, and one or more other therapeutic agents. Such combination therapy includes administering a single pharmaceutical or nutraceutical formulation containing a compound or extract of the present disclosure, or a composition of two or three plant extracts, and one or more other active agents, as well as administering a compound or extract of the present disclosure, or a composition of Free B-ring flavonoids and flavans comprising two or three plant extracts, and each active agent in a separate pharmaceutical or nutraceutical formulation. For example, a compound or extract of the present disclosure, or a composition of two or three plant extracts, and another active agent can be administered to a patient together in a single oral dosage composition, such as a tablet or capsule, or each active agent can be administered in a separate oral dosage formulation. When separate dosage formulations are used, the compound of the present disclosure and one or more other active agents can be administered at essentially the same time, i.e., simultaneously, or separately at different times, i.e., sequentially, and combination therapy is understood to include all of these regimens.

[0108] It is understood that the present disclosure permissible combinations of substituents or variables of the depicted formulae only if such combinations result in stable compounds.

[0109] It will also be apparent to those skilled in the art that the processes described herein may require the protection of functional groups of intermediate compounds with suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid groups. Suitable protecting groups for hydroxy groups include trialkylsilyl or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino groups include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto groups include C(O)—R″ (where R″ is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid groups include alkyl, aryl, or arylalkyl ester groups. Protecting groups can be added or removed according to standard techniques, which are known to those skilled in the art and are described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Hutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As will be appreciated by those skilled in the art, the protecting group may be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0110] It will also be apparent to those skilled in the art that such protected derivatives of the protected compounds of the present application may not have pharmacological activity in their native form, but may be metabolized in the body to form pharmacologically active compounds of the present disclosure after administration to a mammal. Such derivatives may therefore be referred to as "prodrugs." All prodrugs of the protected compounds of the present application are within the scope of the present disclosure.

[0111] Additionally, all compounds or extracts of the present disclosure that exist in free base or free acid form can be converted to their pharmaceutically or nutraceutical acceptable salts by treatment with an appropriate inorganic base, organic base, inorganic acid, or organic acid by methods known to those skilled in the art. Salts of the compounds of the present disclosure can be converted to their free base or free acid form by standard techniques.

[0112] In any of the above embodiments, compositions containing mixtures of extracts or compounds can be blended in a specific weight ratio. For example, without limitation, Scutellaria extract and Acacia extract, each containing bioflavonoids including baicalin and catechin, can be blended in a 4:1 weight ratio. In certain embodiments, the ratio (by weight) of two extracts or compounds of the present disclosure ranges from about 0.5:5 to about 5:0.5. Similar ranges apply when three or more (e.g., three, four, five) extracts or compounds are used. 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 another embodiment, an individual Free-B-ring flavonoid extract of a Scutellaria extract disclosed herein is combined with a flavan extract of Acacia in a non-limiting example, 4:1 blend ratio to form a composition designated UP446.

[0113] In other embodiments, various combinations of individual extracts of the genera Scutellaria and Acacia were made into such formulations (e.g., but not limited to, UP446, UP223, UP894-II, or UG0408) and evaluated in vitro, ex vivo, or in vivo models for perceived biological functional advantages / disadvantages and unexpected synergistic / antagonistic effects, effective modulation of host defense homeostasis, and reduction of organ damage caused by cytokine storm, oxidative stress, and sepsis. Due to the diversity of chemical constituents in each extract, the different mechanisms of action from the different types of bioactive flavonoid compounds in each extract, and the potential to improve ADME of the bioflavonoid compounds in the compositions to maximize biological and nutritional output, the best compositions containing specific blend ratios of individual extracts of flavan or free B-ring flavonoids were selected based on unexpected synergistic effects measured in in vitro, ex vivo, or in vivo models.

[0114] In any of the above embodiments, compositions 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 certain embodiments, compositions containing Scutellaria root extract powder or Acacia heartwood extract can contain 0.1% to 99.9%, or about 10% to about 40%, or about 60% to about 80% Free-B-ring flavonoids, 0.1% to 99.9%, or about 1% to about 10%, or about 5% to about 50% flavans, or a combination thereof. In certain embodiments, a composition comprising a Scutellaria Free-B-Ring Flavonoid extract powder or an Acacia flavan extract can contain from about 0.01% to about 99.9% baicalin or catechin, or can contain 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% or 95% baicalin or catechin.

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

[0116] Conversion products of the compounds disclosed herein are also contemplated. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily through enzymatic processes. Thus, contemplated compounds are those produced by a method comprising administering the compound or composition of interest to a mammal for a period of time sufficient to yield a metabolic product of the compound or composition. Such products are generally identified by administering a detectable dose of a compound of the present disclosure, with or without radiolabeling, to an animal, such as a rat, mouse, guinea pig, dog, cat, pig, sheep, horse, monkey, or human, and isolating the conversion product from urine, blood, or other biological sample after allowing sufficient time for metabolism to occur.

[0117] The intended compounds, medicinal compositions, and compositions can further comprise, or consist of, at least one pharmaceutically, nutraceutical, or cosmetically acceptable carrier, diluent, or excipient. As used herein, the term "pharmaceutically, nutraceutical, or cosmetically acceptable carrier, diluent, or excipient" includes any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. The intended compounds, medicinal compositions, and compositions can further comprise, or consist of, at least one pharmaceutically, nutraceutical, or cosmetically acceptable salt. As used herein, the term "pharmaceutically, nutraceutical, or cosmetically acceptable salt" includes acid addition salts and base addition salts.

[0118] The desired compositions containing Free B-ring flavonoids and flavans include 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, Zanthoxylum bark extract, Philodendron bark extract, hops extract, Boswellia extract, rosehip extract, Sophora extract, Withania somnifera (Ashwagandha), Bupleurum falcatum (Bupleurum), Radix bupleuri (Radix glycyrrhizae), Radix glycyrrhizae (Radix Glycyrrhiza), Fructus Forsythiae (Forsythia), Panax quinquefolium (American ginseng), Panax ginseng CAMeyer (Ginseng), Lentinula edodes (Shiitake mushroom), Inonotus obliquus (Bush mushroom), Lentinula edodes (Shiitake mushroom), Lycium barbarum (Lycium barbarum), Phellinus linteus (Meshimakobu) (fruiting body), Trametes versicolor (Trametes versicolor) (fruiting body), Cyamopsis tetragonolobus (Cluster bean), Trametes versicolor (Trametes versicolor), Cladosiphon okamuranus Tokida (Okinawa Mozuku), Undaria pinnatifida (Wakame), Mentha 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, Pycnogenol, 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 The composition may comprise, further comprise, or consist of at least one other active ingredient, adjuvant, excipient, or carrier selected from one or more of salt, silica, histidine, copper gluconate, CMC, β-cyclodextrin, cellulose, dextrose, saline, water, oils, UCII, shark and bovine cartilage, mushrooms, seaweed, yeast, brown algae, agave nectar, brown seaweed, fermentable dietary fiber, cereals, sea cucumber, agave, artichoke, asparagus, leek, garlic, onion, rye, barley kernel, wheat, pear, apple, guava, quince, plum, gooseberry, orange, and other citrus fruits.

[0119] The contemplated compositions containing Free-B-Ring flavonoids and flavans can also comprise, or consist of, at least one other naturally occurring phenolic active ingredient. In certain embodiments, the at least one bioactive ingredient can comprise, or consist of, a plant powder or plant extract, or the like. Plant species containing the immunosuppressive natural phenolic compounds include, but are not limited to, Piper longum Linn (Piper longum), Coptis chinensis Franch (Coptis chinensis), Angelica sinensis (Oliv.) Diels (Angelica sinensis), Toxicodendron vernicifluum (Rhus vernicifluum), Glycyrrhiza glabra (Licorice), Curcuma longa (Turmeric), Salvia rosmarinus (Rosemary), Rosmarinus officinalis (Rosemary), Zingiber officinalis (Zingiber officinalis), and the like. officinalis (ginger), Polygala tenuifolia (polygala), Morus alba (mulberry), Humulus lupulus (hops), Lonicera japonica (honeysuckle), Salvia officinalis L.: Sage), Centella asiatica (Centella asiatica), Boswellia carteri, Mentha longifolia (Mentha longifolia), Picea crassifolia (Picea crassifolia), Citrus nobilis Lour, Citrus aurantium L (Bitter orange), Camellia sinensis L (Camellia sinensis L), Pueraria mirifica, Pueraria lobata (Kudzu), Glycine max (Soybean), Capsicum species, Fallopia japonica Many phenolic compounds can also be found in various fruit trees and vegetables, such as tomatoes, cruciferous vegetables, grapes, blueberries, raspberries, mulberries, apples, and chili peppers.

[0120] The free-B-ring flavonoids may be one or more 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 and norwogonin glycoside, galangin, or any combination thereof. Free-B-ring flavonoids that can be used in accordance with the methods of the present application include compounds represented by the general structure above. The standardized free-B-ring bioflavonoids in the composition are synthesized, metabolized, biodegraded, biotransformed, or biosynthesized from small carbon units by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, or microbacteria.

[0121] One or more Free B-ring flavonoids may be present in the genera Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum, and the like. The active ingredient is enriched and standardized from a higher plant genera including at least one species of the genera: Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus, and Alpinia, or a combination thereof.

[0122] One or more free B-ring flavonoids may be present in Scutellaria baicalensis (scutellaria), Scutellaria barbata (scutellaria), Scutellaria orthocalyx, Scutellaria lateriflora (blue skullcap), Scutellaria galericulata (marsh skullcap), Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria gallericulata (marsh skullcap), Scutellaria galericulata (Marsh skullcap), Scutellaria indica (Scutellaria indica), Scutellaria sessilifolia, Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria orientalis, Oroxylum indicum, Passiflora caerulea (Passion flower), Passiflora incarnata (Passion flower), Pleurotus ostratus ostreatus (oyster mushroom), Lactarius deliciosus (red mushroom), Suillus bellini (Suillusbellinii), chamomile, carrot, mushroom, honey, propolis, passion flower, and Indian trumpet flower, or a combination thereof.

[0123] Flavans are comprised of one or more of catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epitheaflavin, epicatechin gallate, gallocatechin gallate, theaflavin, and theaflavin gallate, or any combination thereof. Flavans that can be used in accordance with the methods of the present application include compounds represented by the general structure above. The standardized flavan bioflavonoids in the composition are synthesized, metabolized, biodegraded, biotransformed, or biosynthesized from small carbon units by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, or microbacteria.

[0124] The flavans of interest of the present application are isolated from one or more plants selected from the Acacia genus of plants. In a preferred embodiment, the plant is Acacia catechu (black catechu), Senegalia catechu, Acacia concinna (sompoi), Acacia farnesiana (gum arabic), Acacia Senegal (gum arabic), Acacia speciosa, Acacia arabica (pseudo-arabic gum), Acacia caesia, Acacia pennata (chaom), Acacia sinuata, Acacia mearnsi (gum arabic), Acacia mearnsii (Black Wattle), Acacia picnantha (Golden Wattle), Acacia dealbata (Dealbata), Acacia auriculiformis (Acacia), Acacia holoserecia (Strap Wattle), Acacia mangium (Acacia mangium), Anacardium occidentale (Cashew Nut Shell), Uncaria gambir (White Catechu), Uncaria rhynchophylla (Uncaria rhynchophylla), Camellia sinensis (Tea Plant), Camellia assamica (Camellia assumica (Assam tea), Euterpe oleracea (Acai), Caesalpinia decapetala (Caesalpinia decapetala), Delonix regia (Delonix regia), Ginkgo biloba (Ginkgo biloba), Acer rubrum (Acer rubrum)rubrum (Red Maple), Cocos nucifera (Coconut Palm), Limonium brasiliense (Limonium Brasiliense), Acerola bagasse, Vitellaria paradoxa (Shea Butter Tree), Vitis vinifera (European Grape), Lawsonia inermis (Henna), Artocarpus heterophyllus (Jackfruit), Medicago sativa (Medicago sativa), Lotus japonicus (Lotus grass), Lotus uliginosus (Lotus grass), Eisenia bicyclis (Eisenia In some embodiments, the bioactive ingredient is selected from the group consisting of: Arame (Eisenia bicyclis), Hedysarum sulfurescens, Robinia pseudoacacia (Robinia pseudoacacia), apple, apricot, prune, cherry, grape leaf, strawberry, legume, lemon, tea, black tea, green tea, rooibos tea, barley kernel, green algae (Acetabularia ryukyuensis), red algae (Chondrococcus hornemannii), chocolate (cocoa), green coffee beans, or a combination thereof.

[0125] In certain embodiments, the Free-B-Ring flavonoid or flavan compounds or extracts of the present disclosure can be isolated from plant or marine sources, such as those mentioned in the Examples and elsewhere in this application. Plant parts suitable for isolating the compounds include leaves, bark, trunks, trunk bark, stems, stem bark, twigs, tubers, roots, rhizomes, root bark, bark surfaces, shoots, seeds, fruits, stamens, pistils, calyxes, stamens, petals, sepals, carpels (pistils), flowers, stem cells, or any combination thereof. In certain related embodiments, the compounds or extracts are isolated from plant sources and synthetically modified to include any of the specified substituents. In this regard, synthetic modification of compounds isolated from plants can be carried out using numerous techniques known in the art, including but not limited to total organic synthesis, metabolism, biodegradation, biotransformation, biotransformation, biosynthesis from small carbon units by transgenic microorganisms, P450 enzymes, glycosyltransferases or enzyme combinations, microbacteria, and such techniques are well within the knowledge of one of ordinary skill in the art.

[0126] Other embodiments of the subject matter of the present application relate to methods of using standardized bioflavonoid compositions (such as, but not limited to, UP446 or UP894-II as exemplified in the examples of the present disclosure) containing Free B-ring flavonoids and flavans and various combinations of two to three plant extracts to regulate the homeostasis of host defense mechanisms, including, but not limited to, methods of optimizing or balancing the immune response; helping to maintain healthy immune function against viral and bacterial infections; protecting the immune system from oxidative stress damage induced by air pollution; methods for protecting normal, healthy lung function from viral infections, bacterial infections, and air pollution; methods for supporting a healthy inflammatory response; methods for maintaining healthy levels of cytokines and cytokine responses to infection; and methods for preventing and treating lung diseases, including 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 and CINC3; methods for controlling oxidative responses and alleviating oxidative stress; methods for enhancing antioxidant capacity by increasing SOD and NRf2; methods for reducing advanced glycation end products; methods for increasing glutathione peroxidase; methods for neutralizing reactive oxygen species and preventing damage to the structural integrity and functional decline of the respiratory tract, lungs, and immune system caused by oxidative stress; methods for maintaining lung cleansing and detoxification capacity; methods for protecting lung structural integrity and oxygen exchange capacity; methods for maintaining respiratory passage and enhancing alveolar oxygen absorption capacity; methods for treating oxidative stress. methods for reducing lung damage caused by pulmonary edema; methods for promoting pulmonary microcirculation and protecting normal coagulation function; methods for increasing the activity and number of white blood cells; methods for enhancing natural killer (NK) cell function; methods for increasing the number of T lymphocytes and B lymphocytes; methods for increasing CD4+ and CD8+ cell counts; methods for increasing CD3+, CD4+NKp46+ natural killer cells, TCRγδ+ gamma delta T cells, CD4+TCRγδ+ gamma delta T cells and CD8+ cell counts; methods for protecting and promoting macrophage phagocytic activity; methods for supporting or promoting normal antibody production;A method for maintaining a healthy lung microbiome or symbiotic system in the respiratory tract; a method for relieving or alleviating cold / flu-like symptoms including, but not limited to, body aches, sore throat, cough, minor throat and bronchial irritation, nasal congestion, sinus congestion, sinus pressure, runny nose, sneezing, hyposmia, hypotaste, muscle aches, headache, fever, and chills; a method for loosening phlegm (mucus) and thinning bronchial secretions to make them easier to cough; a method for reducing the severity of bronchial irritation; a method for reducing the severity of lung injury or edema or inflammatory cell infiltration caused by viral infections, microbial infections, and air pollution. methods for supporting the bronchial system and comfortable breathing throughout 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, enhancing and rejuvenating the repair and regeneration functions of the lungs and the entire respiratory system; [Example]

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

[0128] [Table 1]

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

[0130] [Table 2]

[0131] Example 2: Preparation of a standardized plant-derived free B-ring flavonoid extract Scutellaria baicalensis roots were washed with water and thinly sliced ​​into small pieces. The washed and thinly sliced ​​roots were placed in an extractor and extracted twice with hot water at 90-95°C. Approximately 8 L of water was added per 1 kg of roots, and the extract was performed at 90-95°C for approximately 1 hour. After the extract solution was collected, the roots were extracted again with 6 L / kg of water at 90-95°C for an additional 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 approximately 2 with aqueous hydrochloric acid or sulfuric acid. After allowing the acidic solution to stand for approximately 2 hours, the precipitate was filtered and washed with purified water. The precipitated extract was dried at 80-90°C. The dried powder was milled and blended. The extraction yield was 1 kilogram of concentrated bioflavonoid extract from 10-15 kg of roots. Bioflavonoid content was quantified by HPLC as in Example 1 above, producing a standardized extract designated RM405 with a baicalin content of 75% or greater and loss on drying of less than 5%. The particle size of RM405 was controlled to ensure 80% passing through an 80-mesh filter. Potential heavy metal contamination as lead, arsenic, Pb, Cd, and Hg was analyzed by ICP-MS. Potential contamination by coliforms, mold, and yeast, as well as total viable counts, were also measured to meet USP / AOAC / KFDA requirements.

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

[0133] Example 3: Preparation of a standardized bioflavonoid extract from Acacia catechu and cashew nut shells Acacia catechu (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, ground cashew nut (Anacardium occidentale) shell powder (60 g) was placed in a 100 ml stainless steel tube and extracted twice with 70% ethanol in DI water using an ASE350 automated extractor at 80°C and 1500 psi. The extract solution was automatically filtered and collected. The combined organic extract solutions were 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] The amount of free catechin in bioflavonoid extracts derived from Acacia catechu heartwood or cashew nut shell was quantified using a Hitachi HPLC / PDA system with a C18 reverse-phase column (Phenomenex, USA, Luna 5 μm, 250 mm x 4.6 mm) at 275 nm UV absorbance was measured at 35°C with mobile phase A: 0.1% aqueous phosphoric acid and mobile phase B: acetonitrile at a flow rate of 1.0 ml / min (Table 3). A catechin reference standard was purchased from Sigma-Aldrich. The reference standard was dissolved in MeOH:0.1% H3PO4 (1:1) supplemented with catechin (C1251) at a concentration of 0.5 mg / ml and epicatechin (E1753) at a concentration of 0.1 mg / ml. Test samples were prepared in volumetric flasks at a concentration of 2 mg / ml in 50% methanol / 0.1% H3PO4, sonicated until dissolved (approximately 10 minutes), cooled to room temperature, mixed thoroughly, and filtered through a 0.45 μm nylon syringe filter. HPLC analysis was performed by injecting a 20 μL sample into the HPLC.

[0136] [Table 3]

[0137] Catechin and epicatechin were used as standards, and chemical components were quantified based on retention time and PDA data. The catechin quantification results from Acacia catechu extract are shown in Table 4. As shown in Table 4, the flavan extract produced by solvent extraction with 80% methanol / water had the highest concentration of flavan components. The bioflavonoid content in the 70% ethanol extract of cashew nut shells was 9.4% catechin and 6.1% epicatechin.

[0138] [Table 4]

[0139] The bark was removed from Acacia catechu heartwood, washed with water, and sliced ​​into small pieces. The washed and sliced ​​heartwood was placed in an extractor and extracted twice with hot water at approximately 115°C. Approximately 4 L of water was added per kg of Acacia catechu, and extraction was performed at 105-115°C for approximately 5 hours. The extract solution was filtered and concentrated under reduced pressure at 50-60°C. The concentrated solution was then kept at approximately 5°C for 7-10 days, after which the precipitate was filtered, the wet cake was frozen, and dried at approximately -20°C for 1 day. The dried powder was crushed, sieved, dried at 90°C for 10 hours, and then blended. The final extract to heartwood ratio was approximately 1 kg of bioflavonoid extract from 20 kg of Acacia catechu heartwood. Bioflavonoid content was quantified by HPLC as follows, resulting in a standardized extract designated RM406 with a total catechin and epicatechin content of 65% or greater and loss on drying of less than 5%. The particle size of RM406 was controlled to ensure 80% passing through an 80-mesh filter. Potential heavy metal contamination as lead, arsenic, Pb, Cd, and Hg was analyzed by ICP-MS. Potential contamination by coliforms, mold, and yeast, as well as total viable counts, were also measured to meet USP / AOAC / KFDA requirements.

[0140] Standardized bioflavonoid extracts derived from the heartwood, bark, or whole plant of Uncaria catechu or Uncaria gambir, or cashew nut shells, can be obtained by concentrating the plant extract solution followed by precipitation, by recrystallization in an ethanol / water solvent, or by column chromatography using various resins, and the bioflavonoids can be concentrated 2-10 times to a purity of 10%-99%.

[0141] Example 4: Formulation of a standardized bioflavonoid composition A bioflavonoid composition designated UP446 was formulated using two standardized extracts: an Acacia extract (RM406 in Example 3) with a total flavan content of >65% as catechin and epicatechin, and a Scutellaria extract (RM405 in Example 2) with a free B-ring flavonoid content of >75% as baicalin, baicalein, etc., and maltodextrin as an excipient. The ratio of flavans to free B-ring flavonoids can be adjusted based on the indication and functionality. The amount of excipients is adjusted based on the actual active ingredient content in each ingredient. A blending schedule for each batch of formulation should be prepared based on the formulation specifications and the QC of each batch of ingredients. A 2-5% excess of active ingredient is recommended to meet formulation specifications. The blend table for one batch of UP446 (Lot # G1702) is shown below, with a blend ratio of Free-B-Ring Flavonoid Extract:Flavan Extract:Maltodextrin of 80:17:3.

[0142] [Table 5]

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

[0144] A bioflavonoid composition designated UP894-II was formulated using a standardized extract derived from Acacia heartwood extract containing >90% total flavan content as catechin and epicatechin, and a standardized extract derived from Scutellaria root extract containing >90% free B-ring flavonoid content as baicalin, baicalein, etc. The blend ratio of free B-ring flavonoid extract to flavan extract was 4:1, with a baicalin content of 70-80% and total catechins of 15-20% (Table 6).

[0145] [Table 6]

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

[0147] RAW264.7 cells were maintained in room air (21% oxygen O) or exposed to 95% O 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 vehicle. Cell viability was measured by MTT assay as described by the manufacturer.

[0148] Compared with the TO control, which was the reading at the time of seeding, significantly more viable cells were observed in the T24 room air control group. Cell viability was significantly reduced in the O2 control group (95% O2) compared with the room air control group. Administration of the solvent, DMSO, at concentrations of 0.16% and 0.32% did not affect cell viability under O2. To examine whether the formulation UP894-II could improve macrophage function impaired by oxidative stress, we first constructed a dose curve of this formulation on cell viability under normal culture conditions or hyperoxic conditions. The attached graph (Figure 4) shows representative results from three independent experiments. UP894-II at doses below 128 μg / ml did not significantly alter cell viability compared to the DMSO control group. Therefore, we tested UP894-II for its effect on enhancing macrophage function at doses below 128 μg / ml.

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

[0150] RAW264.7 cells were maintained in room air (21% O) or exposed to 95% O for 24 hours in the presence of UP894-II (0–100 μg / ml), a standardized bioflavonoid composition shown in Example 4 and Table 6. Cells were then incubated with FITC-labeled latex minibeads for 1 hour and stained with phalloidin and DAPI to visualize the actin cytoskeleton and nucleus, respectively. To quantify phagocytic activity, at least 200 cells per group were counted, and the number of beads per cell was expressed as a percentage of the 21% O (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 cell viability assays.

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

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

[0153] RAW264.7 cells were maintained in room air (21% O) or exposed to 95% O 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. HMGB1 concentrations in the culture medium were analyzed by Western blot analysis. The blots are images showing the HMGB1 concentrations in each group, with the bar graphs below every two lanes corresponding to the HMGB1 concentrations.

[0154] HMGB1 release was significantly increased in the hyperoxia control group (95% O2) compared with the room air control group (21% O2). DMSO, the vehicle, did not significantly alter HMGB1 release compared with the hyperoxia control group. In contrast, administration of UP894-II resulted in a dose-related, statistically significant decrease (75.9%-89.7%) in HMGB1 concentrations measured at 3.7 μg / ml, 11.1 μg / ml, and 33.3 μg / ml (Figure 6).

[0155] Particles emitted from ambient air pollution are known to cause exogenous oxidative stress in biological systems through the generation of reactive oxygen species (ROS), leading to a weakening of host defenses and inflammation, resulting in lung injury. HMGB1, in cooperation with ROS, plays a key role in lung injury pathology, causing alveolar macrophage apoptosis and inhibiting alveolar macrophage phagocytosis, in part through activation of NF-kB, leading to the upregulation of proinflammatory cytokines and chemokines, resulting in a cytokine storm. The interaction of these factors may result in harmful pathological changes in the lung during pollution-induced lung injury, viral, or bacterial infection. As an example of this interaction, prolonged exposure to oxidative stress during oxygen therapy, which is routinely used to treat COVID-19 patients, may reduce innate immunity and macrophage function, resulting in a reduced ability to eliminate invading pathogens and potentially leading to acute inflammatory lung injury. Therefore, for the growing population exposed to oxidative stress caused by cytokine storm, such as COVID-19 patients and those with inflammatory disorders, reducing HMGB1 levels or blocking its activity in the airways may be an important therapeutic and preventative strategy. Therefore, the data presented in this example suggest that the standardized bioflavonoid composition UP894-II may be used for these novel uses through these well-defined mechanisms, in addition to its previously reported important uses. The present application provides proof of concept and demonstrates the efficacy of the standardized composition in multiple disease models, as described in the following examples.

[0156] [Example 8] Animals and husbandry CD-1 mice and Sprague-Dawley rats were purchased from a USDA-approved vendor. Eight-week-old male CD-1 mice and SD rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Upon arrival, the animals were acclimated and used for the study. The animals were housed in a temperature-controlled room (71–72°F) with a 12-hour light / dark cycle and provided with free access to food and water.

[0157] Animals were housed in polypropylene mouse cages, 3–5 per cage, and individually identified by unique tail numbers. Each cage was fitted with a mouse or rat wire-bar lid and filter top (Allentown, NJ). Project number, test article name, dose level, group name, animal number, and sex were written on cage cards and attached to each cage for identification. Harlan T7087 soft corncob bedding was used and changed at least twice weekly. Animals had free access to fresh water and commercially available rodent chow diet #T2018 from Harlan (Harlan Teklad, 370W, Kent, WA).

[0158] [Example 9] Lipopolysaccharide (LPS)-induced sepsis model This model uses animal survival as an endpoint measure (Wang et al., 1999). Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram-negative bacteria and is a major contributor to the initiation of systemic inflammatory processes that can lead to endotoxic shock. Endotoxic shock is a condition primarily mediated by macrophages / monocytes and results from the overproduction of several early cytokines, including TNF-α, IL-1, IL-6, and gamma interferon (IFN-γ), as well as the late mediator HMGB1. After administration of a median lethal dose of LPS (25 mg / kg) in phosphate-buffered saline (PBS; Lifeline, Lot No. 07641), animals develop endotoxemia, and HMGB1 is detectable in serum within 8 hours, reaching a peak plateau level 16–32 hours after LPS administration. Without treatment, mice begin to die within 24 hours. In this study, mice were monitored for 4 days after LPS injection. Survival rates were compared for LPS plus sodium butyrate (SB; Aldrich, St. Louis, MO; lot number MKCG7272), LPS plus vehicle (0.5% CMC; Spectrum, New Brunswick, NJ; lot number 1IJ0127), and LPS plus UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6. The following groups were tested:

[0159] [Table 7]

[0160] In this model, mice were pretreated with UP446, the bioflavonoid composition shown in Example 4, for 1 week (7 days) before receiving a lethal dose of 25 mg / kg LPS (derived from E. coli 055:B5; Sigma, St. Louis, MO; lot number 081275) intraperitoneally with 10 mL / kg PBS. Animals were observed hourly. Sodium butyrate was selected as a positive control for this study given that this compound ameliorated LPS-induced injury in mice by suppressing HMGB1 release (Li et al., 2018).

[0161] Example 10: Standardized bioflavonoid composition improved animal survival under lethal endotoxin doses.

[0162] Three hours after intraperitoneal injection of LPS, mice began to show early signs of endotoxemia. Mice's exploratory behavior progressively decreased, accompanied by piloerection, decreased mobility, lethargy, and diarrhea. These signs and symptoms appeared to be present in all treatment groups, but were more severe in the vehicle-treated group.

[0163] Two mice in the vehicle-treated group and one mouse in the positive control sodium butyrate (SB) group were found to have died 24 hours after LPS injection. The survival rates for these groups were determined to be 62.5% and 75%, respectively (Table 8). Mice treated with UP446, the standardized bioflavonoid composition described in Example 4 and Table 6, had a 100% survival rate 24 hours after LPS injection. Mice treated with UP446, SB, and vehicle had survival rates of 87.5%, 62.5%, and 50%, respectively, 34 hours after LPS injection. Perhaps the most striking results were observed in the UP446-treated mice 48 hours after LPS injection. At this time point, only 12.5% ​​of vehicle-treated mice survived, while 75% of UP446-treated mice survived. Even in the positive control sodium butyrate group, half of the mice had died at this time point. On day 3 (72 hours after LPS injection), the survival rates of the UP446, SB, and vehicle groups were 62.5%, 50%, and 12.5%, respectively. All mice in the vehicle control group had died 82 hours after LPS injection, resulting in a survival rate of 0%.

[0164] On the other hand, mice treated with UP446 and SB showed a 50% survival rate, which remained unchanged at 96 and 120 hours after LPS injection. These survival rates were statistically significant for both UP446 (p = 0.001) and SB (p = 0.01) compared with vehicle-treated animals (Table 8). Surviving animals in these groups showed a gradual improvement in their overall health. Mice appeared physically well and gradually resumed normal behavior.

[0165] [Table 8]

[0166] Example 11: Comparison of a standardized bioflavonoid composition and its components in an LPS-induced sepsis model The benefits of combining free B-ring flavonoids from Scutellaria extract with flavans from Acacia extract in a specific ratio to form UP894-II, as demonstrated in Example 4, were evaluated in lipopolysaccharide (LPS)-induced endotoxemia. Scutellaria extract RM405, containing 60% or more baicalin, as shown in Example 3, and Acacia extract RM406, containing 10% or more catechins, as 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 prior to LPS injection. On day 8, mice were intraperitoneally (ip) injected with 25 mg / kg LPS dissolved in 10 mL / kg PBS. Mice in the UP894-II group received a daily dose of 250 mg / kg UP894-II. All mice received daily treatment with each substance throughout the study, which ended on day 6 after LPS injection. After intraperitoneal administration of a median lethal dose of LPS (25 mg / kg), animals are expected to develop sepsis within a few hours. Without treatment, mice will begin to die within 24 hours. Animals were observed hourly. In this study, mice were monitored for 6 days after LPS injection.

[0167] [Table 9]

[0168] Survival rates were compared for LPS + sodium butyrate (SB), LPS + vehicle (0.5% CMC), LPS + UP894-II, LPS + Scutellaria extract (RM405), and LPS + Acacia extract (RM406). Normal control animals were intraperitoneally administered PBS alone, and vehicle controls were gavaged with 0.5% CMC alone. Given that sodium butyrate (SB) ameliorated LPS-induced injury in mice by inhibiting HMGB1 release (Li et al., 2018), this compound was selected as the positive control for this study.

[0169] The survival and mortality rates of the composition (UP894-II) were compared with equivalent doses of the individual extracts in the same formulation, and potential additive, antagonistic, or synergistic effects of the combinations were investigated using Colby's formula (Colby, 1967). For blends of these plant extracts to produce unexpected synergistic effects, the observed inhibition values ​​must exceed the calculated values.

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

[0171] Twenty-four hours after LPS injection, four mice in the vehicle-treated and Acacia extract (RM406 shown in Example 4)-treated groups and two mice in the positive control, SB, and Scutellaria extract (RM405 shown in Example 3) groups were found to have died. The survival rates of these groups at this time point were determined to be 69.2% for the vehicle and Acacia extract (RM406) groups and 84.6% for the Scutellaria extract (RM405) and SB groups (Table 10). The survival rate of UP894-II-treated mice 24 hours after LPS injection was 100%. Thirty-six hours after LPS injection, the survival rates of mice treated with UP894-II, Scutellaria extract (RM405), vehicle, SB, and Acacia extract (RM406) were 84.6%, 61.5%, 53.9%, 53.9%, and 53.9%, respectively. The most striking results were observed in mice treated with UP894-II 48 hours after LPS injection; at this time, the survival rate of vehicle-treated mice was only 15.4%, while that of UP894-II-treated mice was 69.2%. At 48 hours after LPS injection, mice treated with Scutellaria extract (RM405), Acacia extract (RM406), and SB had survival rates of 46.2%, 38.5%, and 46.2%, respectively.

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

[0173] [Table 10]

[0174] [Table 11]

[0175] [Table 12]

[0176] The survival rate of vehicle-treated mice remained at 15.4% from 48 hours after LPS injection through the remainder of the study. In contrast, mice treated with Acacia extract (RM406) continued to die until 96 hours after LPS injection. By the end of the 7-day observation period, the survival rate of the Acacia extract (RM406) group was only 7.7%. On the other hand, mice treated with UP894-II and Scutellaria extract (RM405) maintained survival rates of 53.9% and 30.8%, respectively, from 3 days after LPS injection through the remainder of the observation period. The positive control, sodium butyrate (SB) group, completed the study with a survival rate of 30.8%. Only the UP894-II group had a statistically significant survival rate compared to the vehicle control (p=0.01). Surviving animals in this group demonstrated a gradual improvement in their overall health. The mice appeared physically well and gradually resumed normal exploratory behavior.

[0177] Example 12: Unexpected synergistic effects observed with standardized bioflavonoid compositions.

[0178] Using an LPS-induced survival test, we evaluated possible synergistic or unexpected effects of extracts derived from Scutellaria and Acacia when used in combination at specific ratios using the Colby assay. When mice were administered 250 mg / kg of UP894-II, the standardized bioflavonoid composition shown in Example 4 and Table 6, survival rates were higher than theoretically predicted at each time point (Table 13). For example, the predicted survival rates at 24 and 144 hours after LPS injection were 95.3% and 36.1%, respectively, while the observed survival rates for UP894-II were 100% and 53.9%, respectively. These results suggest that the combination of two standardized extracts, a free-B-ring flavonoid extract and a flavan extract derived from Scutellaria and Acacia, at specific ratios is significantly more advantageous in extending the life of test subjects during sepsis than the use of either Acacia or Scutellaria extract alone. The predicted mortality rates at these time points were also examined using the Colby assay. The observed mortality rates in mice treated with UP894-II were found to be much lower than the predicted values, confirming that the combination therapy resulted in a favorable survival prognosis for these subjects (Table 13).

[0179] For example, the predicted mortality rate for mice treated with UP894-II was 41.4% 24 hours after LPS injection, but was actually zero. Furthermore, while 97.6% of subjects were expected to die at the end of the observation period, the actual mortality rate for UP894-II was only 46.2%. Thus, in this survival study, the benefit of combining Scutellaria extract and Acacia extract was assessed using the Colby equation. This method predicts unexpected synergistic effects of a formulation containing two or more bioflavonoid extracts if the actual measured value of a given endpoint is greater than the calculated predicted value.

[0180] [Table 13]

[0181] Using the viability and mortality values ​​for Scutellaria extract (RM405, shown in Example 3) (200 mg / kg) and Acacia extract (RM406, shown in Example 4) (50 mg / kg) at 24, 36, 48, 60, 72, 96, 120, and 144 hours after LPS injection, calculated viability and mortality values ​​were compared to the observed viability values ​​for the composite UP894-II (250 mg / kg) at these specific time points. In this study, an unexpected synergistic effect was observed with the combination of Scutellaria extract (RM405) and Acacia extract (RM406). The beneficial effect of UP894-II administration exceeded the sum of the effects of its components at all time points tested. At the end of the observation period (i.e., 7 days after LPS injection and 14 days after oral administration of the extracts and compositions), the survival rates of the UP894-II, Scutellaria extract (RM405) and Acacia extract (RM406) groups 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 patient survival during sepsis.

[0182] Example 13: Effect of a standardized bioflavonoid composition (UP446) on attenuating lipopolysaccharide (LPS)-induced acute inflammatory lung injury in rats - Study design This study was designed to evaluate the direct effect of oral administration of UP446, a bioflavonoid composition containing free B-ring flavonoids and flavans as described in Example 4, at 250 mg / kg (high dose) and 125 mg / kg (low dose) in alleviating LPS-induced acute lung injury. Acute lung injury is a clinical syndrome resulting from damage to alveolar and capillary epithelial cells, leading to diffuse lung injury similar to that seen in acute respiratory distress syndrome (ARDS). In this study, the test substance was orally administered to Sprague-Dawley rats for 7 days prior to LPS challenge. On day 8, 1 hour after oral administration, each rat was intratracheally instilled with 10 mg / kg LPS dissolved in 0.1 mL / 100 g PBS. Normal control rats received an identical volume of PBS alone intratracheally.

[0183] [Table 14]

[0184] LPS induces systemic and pulmonary responses, leading to the accumulation of inflammatory immune cells such as neutrophils and macrophages and proinflammatory 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, resulting in interstitial pulmonary edema, alveolar pulmonary edema, and epithelial cell injury.

[0185] Surviving animals were sacrificed 24 hours after intratracheal LPS administration. At necropsy, bronchoalveolar lavage fluid (BAL) was collected by intratracheal instillation of 1.5 mL of PBS into the right lung lobe followed by gentle aspiration at least three times. The collected lavage fluids were pooled, centrifuged at 1500 rpm for 10 minutes at 4°C, and used to measure cytokine (e.g., IL-6) and lung protein concentrations. 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 certified pathologist for histopathological evaluation. Serum collected at necropsy was used to measure cytokines such as TNF-α and IL-1β. All animals survived for 24 hours after sensitization following intratracheal instillation of 10 mg / kg LPS. Measurements of key cytokines and chemoattractants thought to be involved in the pathology of acute pulmonary infection, as well as data from histopathological analysis, are summarized in the Examples below.

[0186] [Example 14] Bioflavonoid composition demonstrated a dose-related, statistically significant reduction in serum TNF-α.

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

[0188] As can be seen from Table 15, a statistically significant increase in serum TNF-α was observed in vehicle-treated rats sensitized to intratracheal LPS. This increase was significantly reduced when rats were treated with UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6. A statistically significant dose-related reduction was observed in rats orally treated with 250 mg / kg and 125 mg / kg UP446. These reductions in serum TNF-α levels were calculated relative to the vehicle control and were found to be 90.7% and 69.8% for the 250 mg / kg and 125 mg / kg UP446 groups, respectively. The positive control, sodium butyrate (SB), also demonstrated a statistically significant (67.9%) reduction in serum TNF-α levels.

[0189] [Table 15]

[0190] [Example 15] Standardized bioflavonoid composition demonstrated a dose-related, statistically significant reduction in serum IL-1β.

[0191] The amount of IL-1β present in undiluted rat serum was measured using the Rat IL-1β Quantikine ELISA Kit (Product No. RLB00) from RandD Systems as follows: Undiluted serum was added to an IL-1β antibody-coated microplate. After 2 hours at room temperature, IL-1β in the serum was allowed to bind to the plate, and the plate was thoroughly washed. 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 enzyme substrate was added to the plate. After 30 minutes of development at room temperature, a stop solution was added, and the absorbance was read at 450 nm. The IL-1β concentration was calculated based on the absorbance readings of the IL-1β standard curve.

[0192] Again, a dose-related, statistically significant reduction in IL-1β was observed in rats treated with UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6. A statistically significant increase in serum IL-1β levels was observed in vehicle-treated rats with LPS-induced acute lung injury. UP446-treated rats showed an 81.2% and 61.8% reduction in IL-1β levels at oral doses of 250 mg / kg and 125 mg / kg, respectively (Table 16). The sodium butyrate (SB) group showed a 65.3% reduction in serum IL-1β levels. These reductions were statistically significant in both the UP446 and SB groups.

[0193] [Table 16]

[0194] [Example 16] Standardized bioflavonoid composition demonstrated a dose-related, statistically significant reduction in IL-6 concentrations in bronchoalveolar lavage fluid (BAL).

[0195] The amount of IL-6 present in undiluted rat bronchoalveolar lavage fluid (BAL) was measured using the Rat IL-6 Quantikine ELISA Kit (Product No. R6000B) from RandD Systems as follows: Undiluted BAL was added to an IL-6 antibody-coated microplate. After 2 hours at room temperature, IL-6 in the BAL was allowed to bind to the plate, and the plate was thoroughly washed. 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 enzyme substrate was added to the plate. After 30 minutes of development at room temperature, a stop solution was added, and the absorbance was read at 450 nm. IL-6 concentrations were calculated based on the absorbance readings of the IL-6 standard curve.

[0196] Consistent with the TNF-α and IL-1β data, UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, demonstrated a dose-related, statistically significant reduction in BAL IL-6 levels. A high dose (250 mg / kg) of UP446 reduced BAL IL-6 levels by 74.6%, while a low dose of the bioflavonoid composition reduced BAL IL-6 levels by 58.3% (Table 17). The reductions were statistically significant at both the high and low doses of UP446 compared to vehicle-treated acute lung injury rats. The sodium butyrate (SB) group demonstrated a statistically insignificant 37.7% reduction in BAL IL-6 compared to vehicle-treated disease models.

[0197] [Table 17]

[0198] Example 17: Administration of a standardized bioflavonoid composition resulted in a statistically significant reduction 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, also known as CXCL2, and acts by binding to CXCR1 and CXCR2. It is produced primarily by macrophages, monocytes, and epithelial cells and is responsible for chemotaxis to sources of inflammation and activation of neutrophils.

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

[0201] Daily oral administration of 250 mg / kg UP446 for 1 week resulted in a statistically significant reduction in cytokine-induced neutrophil chemoattractant-3 (CINC-3) levels in LPS-induced acute lung injury (Table 18). Normal control rats intratracheally administered PBS alone had CINC-3 levels near zero. In contrast, vehicle-administered rats with intratracheal LPS-induced acute lung injury had mean CINC-3 levels in lung homogenates of 563.7 ± 172.9 pg / mL. In rats administered 250 mg / kg UP446, this level decreased to a mean value of 360.8 ± 110.7 pg / mL. This 36% reduction in CINC-3 levels in rats administered 250 mg / kg UP446 was statistically significant compared to the vehicle-administered disease model. The low-dose UP446 and sodium butyrate (SB) groups showed only a slight decrease in CINC-3 concentration in lung homogenates compared to vehicle-treated rats, at only 10.5% and 17.7%.

[0202] [Table 18]

[0203] Example 18: A standardized bioflavonoid composition reduced total protein in bronchoalveolar lavage fluid (BAL).

[0204] Total protein in bronchoalveolar lavage fluid (BAL) was measured using the ThermoFisher Scientific Pierce BCA Protein Assay Kit (Product No. 23225) as follows: BAL was diluted 5-fold, mixed with bicinchoninic acid (BCA) reagent in a microplate, and incubated at 37°C for 30 minutes. Absorbance was read at 580 nm, and protein concentration in BAL was calculated based on the absorbance readings of a bovine serum albumin standard curve.

[0205] In vehicle-treated rats with LPS-induced acute lung injury, the total lung protein concentration from BAL was found to be three-fold elevated compared with normal control rats. Daily oral administration of 250 mg / kg and 125 mg / kg UP446 to rats for one week reduced the total BAL protein content by 45.1% (p = 0.06 vs. vehicle) and 36.6% (p = 0.21), respectively, compared with vehicle-treated rats with LPS-induced acute lung injury (Table 19). The positive control sodium butyrate (SB) group showed a 30.2% (p = 0.27) reduction in BAL total protein concentration compared with vehicle-treated rats with LPS-induced acute lung injury.

[0206] [Table 19]

[0207] Example 19: Standardized bioflavonoid composition demonstrated a statistically significant reduction in CRP in bronchoalveolar lavage fluid (BAL).

[0208] The amount of CRP present in a 1:1000 dilution of rat BAL was measured using the Abcam C-reactive protein (PTX1) Rat ELISA Kit (product number ab108827) as follows: A 1:1000 dilution of BAL was added to a microplate coated with CRP antibody. After 2 hours at room temperature on a plate shaker, CRP in the BAL was allowed to bind to the plate, and the plate was thoroughly washed. Biotinylated C-reactive protein antibody was added to the plate and allowed to bind for 1 hour at room temperature on a plate shaker. Washing was repeated, and streptavidin-peroxidase conjugate was added to the plate. After a 30-minute incubation at room temperature, washing was repeated, and a chromogenic substrate was added. After 10 minutes of development at room temperature, stop solution was added, and the absorbance was read at 450 nm. The concentration of CRP was calculated based on the absorbance readings of the CRP standard curve.

[0209] A statistically significant 5.6-fold increase in BAL CRP levels was observed in vehicle-treated rats with LPS-induced acute lung injury compared with normal control rats. Oral administration of UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, at a dose of 250 mg / kg to rats for one week reduced BAL CRP levels by 42.4% compared with vehicle-treated disease models (Table 20). This reduction was statistically significant (p≦0.05). The positive control sodium butyrate (SB) group and the low-dose UP446 group showed a slight, but not statistically significant, decrease in CRP levels compared with vehicle-treated disease rats.

[0210] [Table 20]

[0211] Example 20: Standardized bioflavonoid composition demonstrated a statistically significant reduction in IL-10 in bronchoalveolar lavage fluid (BAL).

[0212] The amount of IL-10 present in undiluted BAL was measured using the Rat IL-10 Quantikine ELISA Kit (Product No. R1000) from RandD Systems as follows: Undiluted BAL was added to an IL-10 antibody-coated microplate. After 2 hours at room temperature, IL-10 in the serum was allowed to bind to the plate, and the plate was thoroughly washed. An enzyme-labeled IL-10 antibody was added to the plate and allowed to bind for 2 hours at room temperature. Washing was repeated, and enzyme substrate was added to the plate. After 30 minutes of development at room temperature, a stop solution was added, and the absorbance was read at 450 nm. IL-10 concentrations were calculated based on the absorbance readings of the IL-10 standard curve.

[0213] Anti-inflammatory IL-10 levels were measured in BAL samples from diseased rats that were orally administered UP446 at doses of 250 mg / kg and 125 mg / kg daily for 7 days prior to challenge and then sacrificed 24 hours after intratracheal instillation of LPS. IL-10 levels often correspond to the severity of the host's infection and inflammatory response during infection or injury. As shown in Table 21, vehicle-treated rats had a significantly elevated IL-10 level (80-fold) compared with normal control rats, indicating a high level of acute lung injury. In contrast, rats treated with UP446 showed a dose-related decrease in BAL IL-10 levels. The percent reductions were calculated to be 73.6% and 49.2% for 250 mg / kg and 125 mg / kg UP446, respectively. At the high dose (250 mg / kg), the percent reduction was statistically significant, p ≤ 0.05. At least in this particular model, the reduction in anti-inflammatory cytokines resulting from the administration of UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, may be explained by the fact that reducing disease severity may have an effect on attenuating the host inflammatory response, and therefore on inflammation via upstream mechanisms, presumably via HMGB1 secretion. Supporting this hypothesis, UP446 statistically significantly reduced pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, leading to a significant reduction in the inflammatory response, making the need for anti-inflammatory cytokines, such as IL-10, less important to the host. Indeed, IL-10 levels in normal controls were near zero, suggesting that the induction of anti-inflammatory cytokines is based on the presence or severity of acute lung injury. The significant reduction in IL-10 by the free B-ring flavonoid and flavan composition demonstrated the establishment of a host defense mechanism.

[0214] [Table 21]

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

[0216] H&E-stained lung tissue was used to evaluate the severity of lung injury resulting from intratracheal LPS. The left lung lobe was used for histopathological analysis. As can be seen from Table 22 and Figure 7, vehicle-treated rats showed statistically significant increases in lung injury severity (3.5-fold increase), pulmonary edema (2.5-fold increase), and polymorphonuclear (PMN) leukocyte infiltration (2.4-fold increase) caused by intratracheal LPS. Daily oral administration of a high dose of 250 mg / kg UP446 to rats for 1 week resulted in a statistically significant 20.8% reduction in the overall severity of lung injury compared to vehicle-treated rats with LPS-induced acute lung injury. Similarly, a strong trend toward a reduction in pulmonary edema (23.3% reduction, p = 0.08) was observed with the high dose of UP446 compared to vehicle-treated rats. The positive control sodium butyrate (SB) group and the low-dose UP446 group showed slight changes in histopathological evaluation compared with the vehicle-treated diseased rats.

[0217] [Table 22]

[0218] [Example 22] D-galactose-induced immunosenescence model as a response to endogenous and exogenous challenge triggers Systemic administration of D-galactose induces accelerated immune cell senescence, affecting immune responses during sensitization in a similar manner to aging mice. These phenomena are presumed to resemble the immune response profile of elderly individuals. A novel protective target, UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, was tested in this experimental aging mouse model to demonstrate its immunostimulatory effects. Experimental CD-1 mice (12 weeks old) were purchased and used for the accelerated aging study after two weeks of acclimation. Mice were randomly assigned to four immunized and four non-immunized groups. The immunized groups were: G1 = normal control + vehicle (0.5% CMC), G2 = D-galactose + vehicle, G3 = D-galactose + 200 mg / kg UP446, and G4 = D-galactose + 100 mg / kg UP446. The non-immunized groups were G1 = normal control + vehicle (0.5% CMC), G2 = D-galactose + vehicle, G3 = D-galactose + 200 mg / kg UP446, and G4 = D-galactose + 100 mg / kg UP446. Ten mice were assigned to each treatment group.

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

[0220] Two doses of UP446 were administered orally via gavage daily for 6 weeks, from weeks 5 to 10. At necropsy (i.e., 14 days after immunization), whole blood (1 mL) was collected, and 110 μL was aliquoted for flow cytometry immunopanel analysis (shipped on ice 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 in two tubes for cytokine analysis (shipped overnight by FedEx to Sirona DX, Portland, OR). The thymus and spleen of each animal were weighed, and thymus and spleen indices were calculated. Representative images of the thymus and spleen were taken from each group. At necropsy, spleens were stored on dry ice and transferred to -80°C for future use. Thymus glands fixed in paraformaldehyde and sucrose were sent to Nationwide Histology for senescence-associated β-galactosidase staining and analysis.

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

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

[0223] [Table 23]

[0224] [Example 24] Bioflavonoid composition increased complement C3.

[0225] At the end of the study, serum samples were collected and evaluated for markers of humoral immunity, including the C3 component of the complement system. As can be seen from Table 24, the immune-normal control group had significantly lower complement C3 levels than the non-immunized control group. Both immunized D-Gal + UP446 groups had significantly higher complement C3 levels than the immune control group. The non-immunized D-Gal + UP446-treated group tended to have elevated complement C3 levels compared to the non-immunized D-Gal group, and the immunized group treated with D-Gal + 200 mg / kg UP446 (the standardized bioflavonoid composition shown in Example 4 and Table 6) had significantly elevated complement C3 levels compared to the immune D-Gal group, demonstrating that UP446 enhanced humoral immunity in immunosenescent animals responsive to vaccination.

[0226] [Table 24]

[0227] Example 25: Effect of 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), non-immunized animals administered 200 mg / kg UP446 + D-Gal tended to have a higher percentage of circulating T cells than the D-gal group, indicating that UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, enhanced CD3+ T cell expansion or differentiation in non-immunized animals.

[0228] [Table 25]

[0229] Example 26: Effect of 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 dividing cells and secreting cytokines. Expressed as a percentage of total leukocytes (CD45+ cells), immunized animals receiving D-Gal showed significantly lower percentages of circulating helper T cells than controls 2 weeks after influenza vaccination. The immunized D-Gal and D-Gal + UP446 (200 mg / kg) groups also showed significantly lower CD4+ helper T cells than non-immunized animals.

[0230] [Table 26]

[0231] Example 27: Effect of bioflavonoid compositions on CD8+ cytotoxic T cells (% of lymphocyte population) in whole blood CD45+CD3+CD8+ cells are cytotoxic T cells that respond to pathogens by dividing cells and secreting proapoptotic enzymes, killing infected cells. When expressed as a percentage of total leukocytes (CD45+ cells), non-immunized animals treated with D-Gal + UP446 (200 mg / kg) had significantly elevated CD8+ cytotoxic T cells compared with both the non-immunized control and non-immunized D-gal groups. The immunized D-Gal + UP446 (200 mg / kg) group had significantly lower numbers 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 distinct natural killer cell markers, murine CD49b and NKp46, were used to determine the percentage of natural killer cells in leukocyte populations. Natural killer cells (NKCs) are involved in the innate immune system. Upon activation, they secrete cytokines and granules, recruit immune cells, and directly induce cell death in pathogen-infected cells, making them important for the immediate immune response to pathogens and active early in systemic infections. CD49b is an integrin specifically present on most natural killer cells and a subset of T cells thought to be natural killer T (NKT) cells. NKp46 is a natural cytotoxicity receptor present exclusively on natural killer cells and does not direct NKT cells. NK cells are generally CD45+CD3-CD49b+NKp46+, but NKT cells and NK-like T cells also express CD3, excluding them (Goh W) (Narni-Mancinelli E). Expressed as a percentage of total white blood cells (CD45+ cells), two weeks after influenza vaccination, the immunized D-Gal group had significantly lower CD3-CD49b+ NK cells than both the immunized control group and the UP446-treated group (Table 28). This indicates that D-Gal reduces the NK cell population and impairs the ability of the innate immune system to respond to pathogens, an effect that 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 treated with D-Gal + UP446 (100 mg / kg) had a significantly higher percentage of natural killer cells than the non-immune D-gal group, and the immunized D-Gal + UP446 (200 mg / kg) group had a significantly higher percentage of CD3-NKp46+ cells than the immunized D-Gal group (Table 29). The immunized D-Gal + UP446 (200 mg / kg) group also had significantly higher NK cells than the non-immune D-Gal + UP446 (200 mg / kg) group.

[0235] These results generally indicate that administration of D-Gal plus UP446 increases natural killer cell populations compared with administration of D-Gal alone in both non-immunized and immunized animals, suggesting that UP446 helps prime the immune system against pathogens by increasing cell populations involved in the immediate innate immune response.

[0236] [Table 28]

[0237] [Table 29]

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

[0239] [Table 30]

[0240] [Example 30] Effect of bioflavonoid composition on serum cytokines GM-CSF and Il-12p70 Serum collected from immunized mice 2 weeks after influenza vaccination was transported for cytokine profiling by Luminex technology. IL-12p70 and GM-CSF cytokines were detected 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 with the D-Gal group (p = 0.058), whereas the decrease in IL-12p70 in the D-Gal + UP446 (200 mg / kg) group reached statistical significance (p = 0.010) compared with the normal control group. There was no difference between the D-Gal and D-Gal + UP446 (200 mg / kg) groups, likely due to 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 via the generation of free radicals, particularly advanced glycation end products. Therefore, we sought to determine whether UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, could affect this aspect in a mouse model by measuring antioxidant enzyme and free radical concentrations (Azman KF).

[0243] Advanced glycation end products (AGEs) were measured in non-immune and immune serum samples. AGEs were significantly lower in the non-immune D-Gal + UP446 group than in the non-immune D-Gal group, suggesting 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, preventing oxidative damage to cell structures, proteins, and nucleic acids. Reactive oxygen species are used as second messengers in immune signaling (Ighodaro OM). Increased expression of antioxidant enzymes is an indicator of the ability to neutralize excess reactive oxygen species.

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

[0247] [Table 33]

[0248] [Example 33] Effect of bioflavonoid composition on NFκB protein expression Statistically significant suppression of NFκB expression was observed in non-immunized mice treated with 200 mg / kg UP44. NFκB is a transcription factor involved in immune cell activation. Normally inactivated by protein-protein interactions, it is stabilized, translocated to the nucleus, and upregulated during host defense responses. Spleen homogenates were subjected to SDS-PAGE, transferred, and the proteins were blotted. Band intensities were measured by densitometry and normalized to a β-actin loading control for each protein of interest. Comparing the semiquantitative values ​​of each protein of interest between groups, the non-immunized 200 mg / kg UP446 + D-Gal group showed significantly lower NFκB levels than the D-Gal alone group. In the influenza vaccine-immunized group, the bioflavonoid composition UP446 + D-Gal group showed significantly higher NFκB protein expression than the normal control group, indicating the induction of host defense mechanisms.

[0249] [Table 34]

[0250] [Example 34] Effect of bioflavonoid composition on HMGB1 protein expression Extracellular HMGB1 is an alarmin protein involved in enhancing immune responses. It is secreted from the nucleus, passes through the cytoplasm, and enters the circulation. Spleen homogenates were run on SDS-PAGE, transferred, and the proteins were blotted. Band intensities were measured by densitometry and normalized to the β-actin loading control for each protein of interest. Semiquantitative comparison of each protein of interest between groups revealed significantly lower HMGB1 levels in the non-immunized 200 mg / kg UP446 + D-gal group.

[0251] [Table 35]

[0252] Example 35: Effect of bioflavonoid composition on hyperoxia-induced mortality in mice infected with Pseudomonas aeruginosa In this study, mice were allowed to acclimate for one week before challenge. To investigate whether the bioflavonoid composition UP446 disclosed in the present application could reduce animal mortality and increase survival rates, mice were orally administered 250 mg / kg of the standardized bioflavonoid composition UP446 shown in Example 4 and Table 6 for 7 days, then exposed to hyperoxia (>90% oxygen for 72 hours). This exposure continued for three days, and then challenged with Pseudomonas aeruginosa (PA). After challenge, mice were observed for 48 hours. Pre-exposure to hyperoxia resulted in significantly higher mortality (O2) compared with mice maintained at room air (Table 36, RA). Unexpectedly, mice exposed to hyperoxia for 48 hours experienced substantial mortality 24 hours after PA challenge. Mice maintained at room air (RA) and challenged with the same amount of PA experienced a 64% mortality rate, compared with a 9% mortality rate in mice maintained at room air (RA) and challenged with the same amount of PA. On the other hand, mice receiving 7 days of prophylactic administration of resveratrol (RES) and UP446 prior to 2 days of hyperoxia exposure and subsequent PA inoculation had mortality rates of 27.3% and 28.6%, respectively, 24 hours after inoculation. These results suggest that UP446 protects the host from oxidative stress and microbial infection, leading to reduced mortality. In the LPS-induced animal sepsis studies described in Examples 10-12, UP446 supplementation resulted in a statistically significant reduction in mortality. The survival data observed with UP446 in this example are consistent with those described in Examples 10-12.

[0253] [Table 36]

[0254] [Example 36] Effect of bioflavonoid composition on acute lung injury induced by bacterial infection and exacerbated by oxidative stress To investigate the modulatory effect of innate host defense homeostasis, mice were orally administered the bioflavonoid composition UP446 at a dose of 250 mg / kg for 7 days, then exposed to >90% O2 for 48 hours (while continuing UP446 administration), followed by inoculation with the microorganism Pseudomonas aeruginosa (PA). 24 hours after bacterial inoculation, mice were euthanized, their lungs were lavaged, and the total protein content of the lung lavage fluid was measured. Pre-exposure to hyperoxia prior to microbial infection in these mice (O2) resulted in significantly higher levels of acute lung injury, as determined by the development of protein edema, compared with mice maintained on room air (RA). Resveratrol (RES), a well-known antioxidant, significantly inhibited this effect. The reduction in total protein content in the lung lavage fluid of UP446-treated mice was statistically significant compared with vehicle-administered control mice (O2) infected with the microorganism under hyperoxia. 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 Patel et al. (2013) previously demonstrated that exposure to hyperoxia reduces host defenses against bacterial infection and increases bacterial burden in lung tissue after microbial infection. The results in Table 38 indicate that pre-exposure to hyperoxia (O2) indeed increases bacterial burden compared to mice maintained on room air (RA). Mice treated with resveratrol and mice treated with UP446, a standardized bioflavonoid composition shown in Example 4 and Table 6, showed significantly reduced lung injury, and these mice also had significantly reduced bacterial burden. The data showed that the difference in bacterial burden in lung tissue was statistically significant compared to vehicle-treated control mice (O2) infected with a microorganism under hyperoxia. These results suggest that UP446 can modulate innate host defense homeostasis to reduce bacterial burden in lung tissue.

[0257] [Table 38]

[0258] [Example 38] Effect of bioflavonoid composition on bacterial clearance in the respiratory tract The above examples demonstrate that exposure to hyperoxia reduces host defenses against bacterial infection and increases bacterial load in lung homogenates. According to the results in Table 39, pre-exposure of mice to hyperoxia (O2) significantly increased airway bacterial load compared to mice maintained on room air (RA). Resveratrol (RES)-treated mice significantly reduced lung injury and also had significantly lower airway bacterial load. Similarly, UP446-treated mice had significantly lower airway bacterial load compared to bacterially infected mice exposed to hyperoxia and administered vehicle alone. These differences in airway bacterial load were statistically significant compared to control mice exposed to hyperoxia and administered vehicle (O2). These results suggest that UP446, the standardized bioflavonoid composition shown in Example 4 and Table 6, can modulate innate host defense homeostasis to reduce airway bacterial load.

[0259] [Table 39]

[0260] [Example 39] Effect of bioflavonoid composition on the accumulation of extracellular HMGB1 in the airways Accumulation of extracellular HMGB1 in the airways may impair innate immunity, leading to a reduced ability to eliminate invading pathogens and apoptotic neutrophils. This can result in acute respiratory infection, lung injury, and even death (Entezari et al., 2012; Patel et al., 2013). To investigate whether UP446 suppressed acute lung injury in hyperoxia-exposed, bacterially infected mice through its effect on extracellular HMGB1 accumulation in the airways, we measured HMGB1 levels in lung lavage fluid. As previously shown, prolonged exposure to hyperoxia followed by microbial infection increased airway HMGB1 accumulation in these mice. Exposure of mice to hyperoxia and subsequent microbial infection increased HMGB1 levels by 4.8-fold. This increase could be prevented by pretreatment with resveratrol (RES) or UP446. Pretreatment of animals with RES and UP446 reduced HMGB1 expression levels by 74.9% and 71.6%, respectively, compared to mice exposed to hyperoxia and infected with bacteria and treated with vehicle. These data suggest that the bioflavonoid composition UP446 disclosed herein can reduce airway HMGB1 accumulation in mice exposed to hyperoxia and infected with bacteria. This correlates with a significant enhancement in the ability of UP446 to improve host defense mechanisms against microbial infection in the respiratory system.

[0261] [Table 40]

[0262] [Example 40] Effect of bioflavonoid composition on lung tissue HMGB1 in hACE2 transgenic mice infected with SARS-CoV-2 hACE2 transgenic mice 5 TCID 50A disease model was induced by infection with SARS-CoV-2 virus via intranasal spray at a volume of 50 μL / kg (Bao et al. 2020). Within 2 hours of SARS-CoV-2 virus intranasal spray, 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., from 0 dpi to 4 dpi). Normal transgenic control mice not infected with the virus and the disease model (infected with the virus) were administered vehicle (0.5% CMC) alone at a volume of 10 mL / kg. Necropsy was performed at 5 dpi. Right whole lungs were homogenized to monitor tissue HMGB1 protein expression.

[0263] Lung tissue was excised, flash-frozen in liquid nitrogen, and stored at -80°C until homogenization. Tissue was suspended in lysis buffer at a concentration of 50 mg tissue per mL of lysis buffer and homogenized. Samples were placed on ice for 30 minutes, vortexing every 5 minutes. Samples were centrifuged for 30 minutes, and the pellet was discarded. Protein was quantified using the BCA assay. Briefly, a 0-10 μg standard curve and BCA working solution (50:1 Reagent A:B) were prepared. A 20 μL sample volume was mixed with 200 μL of BCA working solution in a microplate and incubated at 37°C for 30 minutes. 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 gels were prepared, and the prepared protein samples were loaded and run in Tris-glycine running buffer (25 mM Tris base, 190 mM glycine, 0.1% SDS, pH 8.3). The gels were immersed in transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol) and transferred using the wet transfer method. The membranes were stained with Ponceau Red to visualize proteins and confirm complete transfer. Briefly, the membranes were washed with Tris-buffered saline (TBST) supplemented with 0.1% Tween 20. Ponceau Red stock solution was diluted 10-fold and added. The membranes were incubated for 5 minutes on an agitator and then thoroughly washed with water until bands were clearly visible.

[0265] The membrane was blocked and incubated overnight with primary antibody (1:100–3,000 dilutions) in TBST at 4°C. Unbound primary antibody was removed by washing the membrane three times for 5 minutes each. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000 dilution) in TBST at room temperature for 1 hour with agitation. Immunoblots were analyzed using the ECL Western Blot Detection Kit (GE Healthcare Life Sciences, Piscataway, NJ, USA) for chemiluminescence detection. Quantification of image data was performed using ImageJ (version 1.41, NIH, Baltimore, MD, USA).

[0266] As shown in Figure 8, transgenic mice infected with SARS-CoV-2 and administered vehicle showed a two-fold increase in lung HMGB1 protein expression compared to normal transgenic control mice that were not infected with the virus. The increase in lung HMGB1 levels in the vehicle-treated group was statistically significant compared to uninfected normal controls. In contrast, administration of two doses of the bioflavonoid composition UP894-II to transgenic mice infected with SARS-CoV-2 virus reduced HMGB1 protein expression in lung tissue to the level of uninfected normal control transgenic mice. These decreases in lung HMGB1 expression levels resulting from 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 vehicle. Based on the reduction in HMGB1 in lung tissue, it was determined that the bioflavonoid composition disclosed herein improved host defense mechanisms and reduced the potential for lung and other organ damage associated with a lethal cytokine storm following SARS-Cov-2 coronavirus infection.

[0267] Example 41: Evaluation of bioflavonoid composition UP446 in human clinical trials Protocol: A randomized, triple-blind, placebo-controlled, parallel-group clinical trial to investigate the effectiveness of a test article (IP) containing, and in some embodiments consisting of, at least 60% Free B-ring flavonoids and at least 10% flavans in supporting immune function in healthy adults. The purpose of this study was to investigate the effectiveness of the test article (IP), UP446, containing, and in some embodiments consisting of, at least 60% Free B-ring flavonoids and at least 10% flavans, prepared as described in Example 4 and Tables 5 and 6, in supporting immune function in healthy adults.

[0268] This randomized, triple-blind, placebo-controlled, parallel-group study evaluated the effectiveness of the test product in supporting immune function in healthy adults 28 days before and 28 days after influenza vaccination. The study enrolled men and women aged 40 to 80 years who had not yet received the influenza vaccine but were willing to receive it, agreed to be verbally informed of their influenza vaccination history, agreed to maintain their current lifestyle as much as possible throughout the study period depending on their ability to maintain diet, medications, dietary supplements, exercise, and sleep, and agreed to not take any new dietary supplements, were deemed healthy by a qualified investigator (QI) based on medical history and laboratory results, were willing to complete the study-related questionnaires and diaries and complete clinic visits, and provided written informed consent to freely participate in the study.

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

[0270] [Table 41]

[0271] The following subjects were excluded: 1. Women who were pregnant, nursing, or planning to become pregnant during the study. 2. Participants with known allergies to UP446, placebo, or the active or inactive ingredients of the influenza vaccine. 3. Participants who had not received an influenza vaccine prior to baseline or day 28 vaccination in September 2020. 4. Participants who self-reported a diagnosis of COVID-19 prior to baseline or day 28 vaccination. 5. Participants who have received a COVID-19 vaccine. 6. Currently using prescription immunomodulatory agents such as immunosuppressants or immunostimulants (including corticosteroids) within 4 weeks of baseline. 7. Participants currently using dietary supplements or herbal medicines associated with boosting or modulating the immune system and are not willing to wash out.

[0272] [Table 42]

[0273] [Table 43]

[0274] Subjects were expected to participate in the study for up to 56 days. Subjects were invited to participate at Visit 1 (screening, days -45 to -4) for informed consent and at Visit 2 (baseline, day 0) for eligibility confirmation and randomization.

[0275] The study's primary and secondary efficacy and safety endpoints were assessed at visits 2 (day 0), 3 (day 28), and 4 (day 56). Demographic information and medical history were recorded at the screening visit. Subjects were instructed to take 250 mg of the bioflavonoid composition UP446 twice daily with meals (morning and evening) until influenza vaccination (day 28), and then continue to take 250 mg of UP446 twice daily for an additional 4 weeks (day 56).

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

[0277] Statistical analyses were performed to obtain summary statistics, including means, medians, standard deviations, minimums, maximums, and percentages (if categorical), for demographic characteristics and outcome measures for the entire sample and each study group. Analysis of variance (ANOVA) was used to test for differences in means of continuous variables between the two treatment groups (UP446 and placebo) if the assumption of normality was met; if not, the Kruskal-Wallis test was used. Differences in categorical variables were examined using chi-square tests and (if cell counts were <5) Fisher's exact tests, as appropriate. Repeated-measures analysis of variance (linear mixed-effects models) was used to test for differences in mean outcome measures over time between treatment groups. Baseline values ​​were included as covariates in each model. Repeated-measures analysis of variance (linear mixed-effects models) was also used to test for differences in mean change in outcomes over time (baseline to 28 days, 56 days, and 28 to 56 days) between the two treatment groups, with baseline values ​​included as covariates in each model. Pairwise statistical significance was determined from LMM (between and within groups). 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] Preliminary clinical data reports showed statistically significant outcomes in primary endpoints, such as immunoglobulin A (IgA), from oral intake of the standardized bioflavonoid composition shown in Example 4 and Table 6. As can be seen from Table 43, after 8 weeks of intake, subjects who took bioflavonoid composition UP446 showed a statistically significant increase in immunoglobulin A (IgA), an indicator of mucosal immunity, compared to subjects who took placebo from days 28 to 56 (P=0.0260). Participants who took UP446 had a 0.08755 g / L higher change in IgA from pre- to post-vaccination compared to participants who took placebo (p=0.0260). Within the group, subjects supplemented with UP446 showed a statistically significant increase in IgA, with a mean of 0.05720 g / L (p=0.0412) from day 0 to 56 and 0.06280 g / L (p=0.0252) from day 28 to 56. These data clearly demonstrate that IgA, the major immunoglobulin in a healthy respiratory system and considered to be the most important immunoglobulin for mucosal defense, is a key activity of bioflavonoid compositions in modulating human host defense mechanisms.

[0279] [Table 44]

[0280] Secondary outcomes were assessed for differences between UP446 and placebo at 28 and 56 days in the following areas: 1. Number of confirmed COVID-19 cases; 2. Number of confirmed influenza cases; 3. Impact of COVID-19 on quality of life; Impact of COVID-19 on quality of life assessed by questionnaire; and 4. Use of over-the-counter cold and flu medications. Additionally, differences between UP446 and placebo were assessed for the following areas at 56 days: 1. Number of COVID-19 hospitalizations; and 2. Number of influenza hospitalizations.

[0281] Other outcomes were assessed by examining the change from baseline to measurements at 28 and 56 days for the following parameters, and evaluating the differences between UP446, the standardized bioflavonoid composition 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 differential (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. Modified Wisconsin Upper Respiratory Symptom Inventory (Modified Wisconsin Upper Respiratory Symptom Inventory); 4. Mean global severity index measured by the area under the curve (AUC) of the daily symptom score of the World Under the Sleep Survey (WURSS)-24; 5. Mean symptom severity score measured by the AUC of the daily severity symptom score of the WURSS-24; 6. Number of healthy days assessed by the modified WURSS-24 questionnaire (defined as the number of days when 0 (disease-free) was answered to the question "How are you feeling today?"); 7. Number of sick days assessed by the modified WURSS-24 questionnaire (defined as the number of days when 0 (disease-free) was answered to the question "How are you feeling today?"); 1. Vitality and quality of life (QoL) were assessed using the modified WURSS-24 questionnaire.

[0282] Blood samples were collected from each subject and stored for subsequent analysis to analyze the difference between the standardized bioflavonoid composition shown in Example 4 and Table 6 and placebo for changes from baseline to days 28 and 56 in the following: 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), and nuclear factor erythroid 2-related factor 2 (Nrf-2). Oxidative stress assessed by 3.8-isoprostaglandin F2α, catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), and advanced glycation end products (AGEs). 4. Hemagglutinin inhibition (HI) titer against specific virus strains.

[0283] In addition to efficacy analyses, safety assessments will also be 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-event adverse events; 3. Vital signs (blood pressure (BP) and heart rate (HR)).

[0284] [Table 45] TIFF2025148423000049.tif234169TIFF2025148423000050.tif234168TIFF2025148423000051.tif229168TIFF2025148423 000052.tif242167TIFF2025148423000053.tif228169TIFF2025148423000054.tif236167TIFF2025148423000055.tif25165

Claims

1. A bioflavonoid composition for establishing and regulating homeostasis of host defense mechanisms, 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.

2. 2. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched in at least one Free-B-ring flavonoid and the at least one standardized bioflavonoid extract enriched in at least one flavan in the composition are in the range of 1% to 98% by weight of each extract, with an optimal weight ratio of 80:

20.

3. 2. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched in at least one Free-B-ring flavonoid is enriched and standardized from the root of Scutellaria baicalensis, and the at least one standardized bioflavonoid extract enriched in at least one flavan is enriched and standardized from the heartwood of Acacia catechu.

4. 10. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched in at least one Free-B-Ring flavonoid comprises from 0.5% to 99.5% of one or more Free-B-Ring flavonoids.

5. 10. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched in at least one flavan comprises from 0.5% to 99.5% catechins.

6. 2. The composition of 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.

7. 2. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched for at least one flavan comprises at least one of catechin, epicatechin, catechin gallate, gallocatechin, epigallocatechin, epigallocatechin gallate, epitheaflavin, epicatechin gallate, gallocatechin gallate, theaflavin, theaflavin gallate, or a combination thereof.

8. The at least one standardized bioflavonoid extract enriched in at least one Free-B-ring flavonoid is selected from the group consisting of Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphallus, Cotula, Glycyrrhiza, and the like. naphalium), Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys s), Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephros ia), Breadfruit (Artocarpus), Fig (Ficus), Pityrogramma, Notholaena, Pine (Pinus), Elm (Ulmus), Alpinia, or combinations thereof.

9. The at least one standardized bioflavonoid extract enriched in at least one Free-B-ring flavonoid is selected from the group consisting of Scutellaria baicalensis (scutellaria), Scutellaria barbata (botanical mustard), Scutellaria orthocalyx, Scutellaria lateriflora (blue skullcap), Scutellaria galericulata (marsh skullcap), Scutellaria viscidula, Scutellaria amoena (scutellaria), ... barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata (botanical mustard), Scutellaria barbata ( amoena, Scutellaria rehderiana, Scutellaria likiangensis, Scutellaria galericulata (Marsh skullcap), Scutellaria indica (Scutellaria indica), Scutellaria sessilifolia, Scutellaria viscidula, Scutellaria amoena, Scutellaria rehderiana rehderiana), Scutellaria likiangensis, Scutellaria orientalis, Oroxylum indicum, Passiflora caerulea (passion flower), Passiflora incarnata (passion flower), Pleurotus ostreatus (oyster mushroom), Lactarius deliciosus (red mushroom), Suillus bellini (suillusbellinii), chamomile, carrot, mushroom, honey, propolis, passion flower, Indian trumpet flower, or a combination thereof.

10. The at least one standardized bioflavonoid extract enriched in at least one flavan is selected from the group consisting of Acacia catechu (black catechu), Senegalia catechu, Acacia concinna (sompoy), Acacia farnesiana (gum arabic), Acacia Senegal (gum arabic), Acacia speciosa, Acacia arabica (pseudo-arabic gum), Acacia caesia (gum arabic), Acacia oleracea ... caesia), Acacia pennata (Chaom), Acacia sinuata (Acacia sinuata), Acacia mearnsii (Black Wattle), Acacia pycnantha (Golden Wattle), Acacia dealbata (Dealbata), Acacia auriculiformis (Acacia auriculiformis), Acacia holoserecia (Strap Wattle), Acacia mangium (Acacia mangium, Anacardium occidentale (cashew nut shell), Uncaria gambir (white catechu), Uncaria rhynchophylla (unclear vine), Camellia sinensis (tea plant), Camellia assumica (Assam tea), Euterpe oleracea (acai), Caesalpinia decapetala (Caesalpinia decapetala), Delonix regia (Delonix regia (Delonix regia), Ginkgo biloba (Ginkgo biloba), Acer rubrum (Red maple), Cocos nucifera (Cocosnucifera (coconut palm), Limonium brasiliensis (Limonium brasiliensis), Acerola bagasse, Vitellaria paradoxa (shea butter tree), Vitis vinifera (European grape), Lawsonia inermis (henna), Artocarpus heterophyllus (jackfruit), Medicago sativa (alfalfa), Lotus japonicus (lotus grass), Lotus uliginosus (Lotus 10. The composition of claim 1, wherein the composition is derived from and enriched in plant species including: Eisenia bicyclis (Arame), Hedysarum sulfurescens, Robinia pseudoacacia (Robinia pseudoacacia), apple, apricot, prune, cherry, grape leaf, strawberry, legume, lemon, tea, black tea, green tea, rooibos tea, barley kernel, green algae (Acetabularia ryukuyuensis), red algae (Chondrococcus hornemannii), chocolate (cocoa), green coffee beans, or combinations thereof.

11. 2. The composition of claim 1, wherein the at least one standardized bioflavonoid extract enriched in at least one Free-B-ring flavonoid and the at least one standardized bioflavonoid extract enriched in at least one flavan are extracted and enriched from plant parts including leaves, bark, trunks, trunk bark, stems, stem bark, twigs, tubers, roots, rhizomes, root bark, surface bark, shoots, seeds, nuts, pericarp, fruits, fruiting bodies, mushrooms, stamens, pistils, calyxes, stamens, petals, sepals, carpels (pisces), flowers, stem cells, cell culture tissue, or any combination thereof.

12. The standardized bioflavonoid extract in the composition is ２ 10. The composition of claim 1, wherein the composition is extracted with any suitable solvent, including a supercritical fluid of 100% or more of the above, water, acidic water, basic water, acetone, methanol, ethanol, propenol, butanol, an alcohol-water mixture, a mixed organic solvent, or a combination thereof.

13. 2. The composition of claim 1, wherein the standardized bioflavonoid extract is synthesized, metabolized, biodegraded, biotransformed, biotransformed, or biosynthesized from small carbon units by a transgenic microorganism, a P450 enzyme, a glycosyltransferase or enzyme combination, a microbacterium, or a combination thereof.

14. 10. The composition of claim 1, wherein the standardized bioflavonoid extract is enriched by solvent precipitation, neutralization, solvent partitioning, ultrafiltration, enzymatic digestion, column chromatography on silica gel, XAD, HP20, LH20, C-18, alumina oxide, polyamide, ion exchange resin, CG161 resin, or combinations thereof, either individually or in combination.

15. 10. The composition of claim 1, wherein the composition comprises a pharmaceutically or nutraceutical acceptable active ingredient, adjuvant, carrier, diluent, or excipient, and the pharmaceutical or nutraceutical formulation further comprises from about 0.1 weight percent (wt%) to about 99.9 wt% of the active compound in the at least one standardized bioflavonoid extract.

16. The active ingredient, adjuvant, excipient, or carrier may be selected from the group consisting of 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, Zanthoxylum arvense bark extract, Philodendron bark extract, hops extract, Boswellia extract, rosehip extract, green tea extract, Sophora extract, Withania somnifera (Ashwagandha), Bupleurum falcatum extract, falcatum (Bupleurum falcatum), Radix bupleuri (Bupleurum falcatum), Radix glycyrrhizae (Radix glycyrrhizae), Fructus forsythiae (Forsythia forsythia), Panax quinquefolium (American ginseng), Panax ginseng C.A. Meyer (Panax ginseng), Lentinula edodes (Shiitake mushroom), Inonotus obliquus (Inonotus obliquus (Bulrush mushroom), Lentinula edodes (Shiitake mushroom), Lycium barbarum (Lycium barbarum), Phellinus linteus (Meshimakobu) (fruiting body), Trametes versicolor (Trametes versicolor) (fruiting body), Cyamopsis tetragonolobus (Cluster bean), Trametes versicolor (Trametes versicolor), Cladosiphon okamuranus Tokida Tokida: Okinawan mozuku), Undaria pinnatifida (Undaria10. The composition of claim 1, comprising: Undaria pinnatifida (Undaria pinnatifida), Mentha 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, pycnogenol, 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, saline, water, oils, shark and bovine cartilage, or combinations thereof.

17. 10. The composition of claim 1, wherein the composition is formulated as a tablet, hard capsule, softgel capsule, powder or granules, compressed tablet, pill, gum, chewing gum, sachet, wafer, bar, liquid, tincture, aerosol, semi-solid, semi-liquid, solution, emulsion, cream, lotion, ointment, or gel base.

18. 10. The composition of claim 1, wherein the composition is effective against respiratory diseases and conditions.

19. 10. The composition of claim 1, wherein the composition is administered by oral, topical, suppository, intravenous, intradermal, intragastric, intramuscular, intraperitoneal, or intravenous route.

20. 10. The composition of claim 1, wherein the composition treats, manages, or promotes homeostatic regulation of host defense mechanisms in a mammal by administering an effective amount of the composition that is between 0.01 mg and 500 mg per kg of body weight of the mammal.

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

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

23. The composition of claim 1, wherein the composition inhibits HMGB1 release or interferes with its action by targeting active or passive release of HMGB1 by blocking cytoplasmic translocation or blocking vesicle-mediated release, or by inhibiting intramolecular disulfide bond formation in the nucleus, or by directly targeting HMGB1 upon release and neutralizing its action, or by 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 signaling, or by altering the physicochemical microenvironment to prevent the formation of HMGB1 tetramers, interfere with the binding affinity of HMGB1 to TLR and RAGE, or prevent the clustering or self-association of HMGB1.

24. 10. The composition of claim 1, wherein the composition supports a healthy inflammatory response; maintains healthy levels of cytokines and cytokine responses to infection; maintains healthy levels of complement C3 and C4 proteins, cytokines and cytokine responses to 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.

25. The composition of claim 1, wherein the composition controls 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, lung and immune systems caused by oxidative stress.

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

27. 2. The composition of 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.

28. 10. The composition of claim 1, wherein the composition supports or enhances a mammal's normal antibody IgG, IgM, IgA, hemagglutinin inhibitory (HI) titers against specific viral strain production, and the like.

29. 10. The composition of claim 1, wherein the composition neutralizes, suppresses, prevents, or ameliorate infections from viruses including highly pathogenic avian influenza (H5N1 strain A virus), influenza A (H1N1, H3N2, H5N1), influenza B / Washington / 02 / 2019-like virus, influenza B / Phuket / 3073 / 2013-like virus, hepatitis A, B, C, and D viruses, coronaviruses SARS-CoV, SARS-CoV-2 (COVID-19), MERS-CoV (MERS), respiratory syncytial virus (RSV), enterovirus A71 (EV71), parainfluenza, and adenovirus.

30. The composition is effective against Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Moraxella catarrhalis, and / or Clostridium moniliforme. catarrhalis, Aspergillus, Cryptococcus, Pneumocystis, Histoplasma capsulatum, Blastomyces, Cryptococcus neoformans, Pneumocystis jiroveci, Candida spp. and Streptococcus pyogenes 10. The composition of claim 1, wherein the composition neutralizes, inhibits, prevents, or relieves respiratory tract infections from microbial infections, including S. pyogenes.

31. 10. The composition of claim 1, wherein the composition neutralizes, inhibits, prevents, or reverses respiratory damage caused by atmospheric PM2.5 particles, atmospheric PM10 particles, air pollutants, photochemical smog, tobacco, e-cigarette smoke, and recreational marijuana smoke.

32. The composition of claim 1, wherein the composition has the effects of maintaining a healthy lung microflora or symbiotic system in the respiratory tract of mammals; maintaining lung cleansing and detoxification capabilities; protecting lung structural integrity and oxygen exchange capacity; maintaining respiratory passage and enhancing alveolar oxygen absorption capacity; protecting normal and healthy lung function from viral infections, bacterial infections and air pollution; reducing lung damage caused by oxidative stress; and promoting pulmonary microcirculation and protecting normal coagulation function.

33. The compositions relieve or alleviate cold / flu-like symptoms in mammals, including, but not limited to, body aches, sore throat, cough, minor throat and bronchial irritation, nasal congestion, sinus congestion, sinus pressure, runny nose, sneezing, loss of smell, loss of taste, muscle aches, headache, fever, and chills; loosen phlegm (mucus) and thin bronchial secretions to make them easier to cough; reduce the severity of bronchial irritation; reduce the severity of lung injury or edema or inflammatory cell infiltration caused by viral infection, microbial infection, and air pollution; relieve cold / flu or pollution symptoms. The composition of claim 1 has the effects of supporting the bronchial system and comfortable breathing through the respiratory system; preventing or treating pulmonary fibrosis; reducing the duration or severity of the common cold / flu; reducing the severity or duration of viral and bacterial infections of the respiratory system; preventing, treating or curatively treating respiratory infections caused by viruses, microorganisms and air pollutants; managing, treating or preventing or reversing the progression of respiratory infections; and promoting, enhancing and rejuvenating the repair and regeneration functions of the lungs and the entire respiratory system.

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