Synergistic Herbal Composition for Enhancing Immunity and Respiratory Health

Abstract

The present invention discloses a synergistic herbal composition, which comprises a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing at least one metal salt or metal complex or metal chelate of a phytochemical selected from emblicanin A, chebulinic acid, gallic acid or a mixture thereof; and a second component, the second component being selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; and also provides a preparation process of the synergistic herbal composition, a treatment method, and uses of such a composition for improving immunity and respiratory health.

Description

Technical Field

[0001] The present invention relates to a synergistic herbal composition, said composition comprising: a first component, Terminalia chebula extract, said Terminalia chebula extract containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulinic acid, chebulic acid, gallic acid or mixtures thereof; and a second component, said second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; methods for preparing these compositions and methods for using these compositions to improve immunity and respiratory health. Background Art

[0002] Immunity: Immunity is the body's natural defense system against various bacterial / viral / fungal infections and diseases caused by different environmental stimuli. Host immunity is mainly divided into innate immunity and adaptive immune responses. Innate immune responses are rapid and non-specific to pathogens, and pathogens are mediated by innate immune cells such as myeloid cells, natural killer (NK) cells, innate lymphoid cells, and humoral systems such as defensins and complement. Adaptive immune responses are slower but specific to pathogens, accompanied by the recruitment of B-lymphocytes and T-lymphocytes, and the generation of long-lived immune memory. The innate immune system is the first responder within minutes to hours after infection. If the pathogen is not eliminated, the adaptive immune mechanism will be activated and specifically recognize and eliminate the pathogen. Recent studies have clearly demonstrated that there is a strong coordination network between the innate and adaptive immune systems, effectively responding to infections by amplifying the innate immune response (such as phagocytosis) and the bidirectional activation of B / T cell-mediated adaptive immune responses. However, if these immune responses are over-activated or inappropriately activated, they can damage host tissues and participate in the development of autoimmune diseases such as rheumatoid arthritis, allergies and cancer.

[0003] Many therapeutic effects of plant extracts are thought to be due to their broad immunomodulatory effects and impact on the human immune system. This effect of phytochemicals such as flavonoids, polysaccharides, lactones, alkaloids, diterpenoids and glycosides is attributed to their immunomodulatory properties. For example, curcumin is one of the most widely studied compounds due to its immunomodulatory properties. Epigallocatechin-3-gallate is one of the most active and abundant polyphenols in green tea (Camellia sinensis) and has been widely reported for its chemopreventive, anti-angiogenic, anti-invasive, anti-proliferative, anti-inflammatory and antioxidant effects in vitro and in vivo.

[0004] Respiratory health: The lungs are continuously exposed to the external environment, and the respiratory tract faces 10,000 liters of inhaled air every day. Most of the inhaled air contains harmless environmental components. However, the lungs are also faced with potential airborne pathogens, allergens, and environmental pollutants. This continuous exposure requires an effective and rapidly acting immune system. Therefore, the lung immune system contains extensive cellular and humoral defense mechanisms in the airways. The coordinated and complex interactions between resident airway epithelial cells and immune cells infiltrated with defensin, mucin, or collectin secretions form the outcome of host-pathogen, host-allergen, and host-particle interactions in the airway microenvironment. These interactions will further activate downstream immune responses by releasing mediators such as chemokines (CCL-2, CCL-20), cytokines (IL-1α, IL-1β), and lipid mediators (eicosanoids / leukotrienes). Conversely, excessive immune tolerance in the airways may lead to ineffective clearance of infectious agents such as influenza, tuberculosis, respiratory syncytial virus, Streptococcus pneumoniae, or coronaviruses, which may in turn lead to inflammatory lung diseases, bronchitis, pneumonia, and sepsis. Respiratory diseases are an important cause of morbidity and mortality globally, and the number of deaths worldwide due to these diseases is increasing.

[0005] Current synthetic drugs cannot completely cure these diseases. In contrast, due to their properties such as bronchodilation, mast cell stabilization, anti-inflammatory, anti-allergic, immune enhancement, immune regulation, and inhibition of inflammatory mediators (leukotrienes, cyclooxygenase, cytokines, etc.), many target-specific herbal alternatives have been recognized. Therefore, there is a need for better herbal formulations that have built-in immune stimulation and inflammation regulation effects on viral respiratory infections while still helping the immune system better cope with infections.

[0006] Patent application US20150283159A1 discloses a method for preventing or treating cancer, which includes: administering to an individual an effective amount of an anti-cancer agent containing ellagitannin, wherein the ellagitannin is selected from geraniin, casuarinin, eugenin, tellimagrandin I, 1,3-di-O-galloyl-4,6-O-hexahydroxydiphenoyl glucose, pu-erhoglucoside A, or any combination thereof.

[0007] PCT publication number WO2013 / 155175 discloses a hydrolysable tannic acid mixture rich in Terminalia chebula. An optimized water extraction method for Terminalia chebula is provided to maximize the content of bioactive hydrolysable tannic acids including chebulagic acid, chebulinic acid, and other low-molecular-weight hydrolysable tannic acids.

[0008] The PCT publication number WO / 2018 / 154602 discloses a preparation for treating and managing diabetes and related complications, which preparation comprises: a herbal ingredient, which comprises Salacia chinensis, Gymnema sylvestre, Phyllanthus emblica, Syzygium cumini, Curcuma longa, Commiphora mukul and Tinospora cordifolia or extracts thereof; and a mineral element, which comprises Shilajit and Bhasma. Wherein, the herbal ingredient further comprises at least one herb selected from the following group of herbs: Withania somnifera, Terminalia chebula, Terminalia bellirica, Andrographis paniculata, Boerhavia diffusa, Azadirachta indica, Aristolochia indica, Aegle marmelos, Cyperus rotundus, Smilax china, Trichosanthes dioica, Santalum album, Terminalia trifolia, Woodfordia fruticosa, Glycyrrhiza glabra, Mucuna pruriens, Myrica esculenta, Plumbago zeylanica, Inula racemosa, Zingiber officinale, Piper longum and Piper nigrum or extracts thereof.

[0009] Screening and isolating more specific immunomodulators from plant sources has the potential to counteract the side effects and high costs of synthetic compounds. Therefore, there is an urgent need to search for natural products from plant sources as new leads for developing effective and safe immunostimulants. Summary of the Invention

[0010] The main object of the present invention is to provide a synergistic herbal composition, comprising: a first component of Terminalia chebula extract, the Terminalia chebula extract containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulinic acid, chebulin, gallic acid or mixtures thereof, wherein the metal is selected from zinc, magnesium, calcium and potassium; and a second component, the second component being selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; for obtaining at least one health benefit selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health and improving respiratory health.

[0011] Another object of the present invention is to provide a method for obtaining at least one health benefit selected from enhancing immunity / triggering an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving human respiratory health; wherein the method comprises: administering to a human an effective dose of a composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from emblicanin A, chebulinic acid, and gallic acid or a mixture thereof in the extract; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents.

[0012] Another object of the present invention is to provide the use of a synergistic herbal composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from emblicanin A, chebulinic acid, and gallic acid or a mixture thereof in the extract; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents; for obtaining at least one health benefit selected from enhancing immunity / triggering an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving respiratory health.

[0013] The present invention provides a synergistic herbal composition, the synergistic herbal composition comprising: a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from extracts of chebulagic acid, chebulinic acid, and gallic acid or mixtures thereof, wherein the metal is selected from zinc, magnesium, calcium, and potassium; and a second component, the second component being selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; for obtaining at least one health benefit, the health benefit being selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving respiratory health.

[0014] On the other hand, the present invention provides a synergistic herbal composition, the synergistic herbal composition comprising: a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt of at least one phytochemical selected from extracts of chebulagic acid, chebulinic acid, and gallic acid or mixtures thereof; and a second component, the second component being selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers, and diluents.

[0015] On the other hand, the present invention provides a method for preparing a synergistic herbal composition, the synergistic herbal composition comprising: a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt of at least one phytochemical selected from extracts of chebulagic acid, chebulinic acid, and gallic acid or mixtures thereof; and a second component, the second component being selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata.

[0016] On the other hand, the present invention provides a method for obtaining at least one health benefit, characterized in that the health benefit is selected from enhancing immunity / initiating an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving human respiratory health; wherein the method comprises: administering to a human an effective dose of a composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing at least one metal salt or metal complex or metal chelate of a phytochemical selected from ellagic acid, chebulinic acid, and gallic acid or a mixture thereof in the extract; and a second component, the second component being selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents.

[0017] In yet another aspect, the present invention provides the use of a synergistic composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing at least one metal salt or metal complex or metal chelate of a phytochemical selected from ellagic acid, chebulinic acid, and gallic acid or a mixture thereof in the extract; and a second component, the second component being selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents; for obtaining at least one health benefit selected from enhancing immunity / initiating an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving respiratory health. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagrams of the chemical structures of ellagic acid, chebulinic acid, and gallic acid. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will now be described in detail in connection with certain preferred and optional embodiments so that various aspects of the present invention can be more fully understood and appreciated.

[0020] The terms "metal salt", "metal complex" and "metal chelate" described in the present invention are used interchangeably. Thus, the statements "Terminalia extract containing ellagic acid and / or chebulinic acid and / or gallic acid metal salt", "Terminalia extract containing ellagic acid and / or chebulinic acid and / or gallic acid metal complex", "Terminalia extract containing ellagic acid and / or chebulinic acid and / or gallic acid metal chelate" convey the same meaning and can be used interchangeably. The terms "improvement", "amelioration" and "betterment" used in the present invention convey the same meaning and can be used interchangeably. Unless stated to the contrary, any word "comprising", "including", "consisting" and "constituting" shall mean "including but not limited to", and shall not be construed as being restricted by any general statement following a specific or similar item.

[0021] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0022] The herbal sources used in the present invention are as follows:

[0023] 1) The Terminalia fruit raw material was collected from Mullapadu village, Krishna district, Andhra Pradesh, and the collected Terminalia fruit raw material was wild.

[0024] 2) The Withania somnifera root raw material was collected from Kamalapadu village, Anantapur district, Andhra Pradesh, and the collected Withania somnifera root raw material was cultivated.

[0025] 3) The Tinospora cordifolia stem raw material was collected from Mullapadu village, Krishna district, Andhra Pradesh, and the collected Tinospora cordifolia stem raw material was wild.

[0026] 4) The Andrographis paniculata aerial part raw material was collected from Mullapadu village, Krishna district, Andhra Pradesh, and the collected Andrographis paniculata aerial part raw material was wild.

[0027] Terminalia: Terminalia is a medium-sized tree belonging to the Combretaceae - Terminalia family. Terminalia is widely used in traditional medicine not only in India but also in other countries such as Asia and Africa. Terminalia fruit contains a high phenolic content, especially hydrolysable tannins. Ellagic acid, chebulinic acid and gallic acid are the main organic acids present in the fruit extract. The chemical structures of ellagic acid, chebulinic acid and gallic acid are as Figure 1 shown. Terminalia extracts, especially Terminalia fruit extracts, have been widely studied for their various pharmacological effects such as antioxidant activity, hepatoprotection, antidiabetes, nephroprotection, anti-allergy, immunomodulation, etc. The ethnomedical and traditional uses, pharmacology and safety studies of Terminalia have demonstrated the therapeutic uses of this single herb for various disease conditions.

[0028] It is estimated that 50% of all drug molecules used for drug treatment are administered in the form of salts. The formation of metal salts or metal ion complexes of organic acids is a key and desirable feature in drug development. Organic acid compounds have certain suboptimal physicochemical or biopharmaceutical properties, which can be overcome by pairing the organic acid with a counter metal ion to produce a salt or complex form of the compound. This process of metal ion salting or complexing is a simple way to overcome the undesirable characteristics of the parent compound by altering the properties of the organic acid.

[0029] Zinc is the second most abundant trace element in the human body, after iron. Moreover, zinc deficiency in humans is currently known to be an important malnutrition problem worldwide. Zinc is a key mineral that cells use to metabolize nutrients, and zinc deficiency during the growth period can lead to growth disorders. The epidermis, gastrointestinal tract, central nervous system, immune system, bones, and reproductive system are the organs that are most clinically affected by zinc deficiency. Zinc has been clinically reported to be effective in treating diarrhea, age-related macular degeneration, upper respiratory tract infections, acne, adjunctive treatment of depression, wound healing, etc., and is also known to be very important in immune function, DNA and protein production, and cell division. Magnesium, calcium, and potassium are also essential minerals required for various metabolic functions in the human body.

[0030] The inventors unexpectedly found that the Terminalia chebula fruit extract containing the metal salt of chebulinic acid and / or the metal salt of chebogenic acid and / or the metal salt of gallic acid increased water solubility, thereby improving bioavailability and further improving its therapeutic applications, such as enhancing immunity, respiratory system health, etc.

[0031] Therefore, the inventors of the present invention prepared various compositions, including a Terminalia chebula extract containing the metal salt of at least one phytochemical selected from chebulinic acid, chebogenic acid, and gallic acid or a mixture thereof in the extract; and at least one additional ingredient selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata.

[0032] For example, the dry fruits of Terminalia chebula were crushed, and the Terminalia chebula fruit powder was extracted with a 50% aqueous ethanol solution to obtain a 50% ethanol-water extract solution. This solution was treated with zinc oxide, and after several hours, the solution was filtered to remove insoluble zinc oxide, and the solution was concentrated. Chebulinic acid, chebogenic acid, and gallic acid in the dry powder (T.Ch.Zn-1) were estimated by HPLC analysis, and zinc in the dry powder (T.Ch.Zn-1) was estimated by ICP-MS. The results are listed in Table-3. For comparative evaluation, a 50% ethanol-water extract (T.Ch) of Terminalia chebula fruit without salt was also prepared. Similarly, as described in Examples 3-5, Terminalia chebula extracts containing magnesium salts, calcium salts, and potassium salts were also prepared from a 50% ethanol-water extract of Terminalia chebula fruit.

[0033] Terminalia chebula fruit extract is rich in chebulinic acid, chebulin and their metal salts :Chebulagic acid and chebulinic acid are hydrolyzable tannins, which are known to have various biological activities such as antioxidant, anti-inflammatory, and anti-tumor. An extract rich in chebulagic acid and chebulinic acid was prepared by eluting a 50% ethanol-water extract solution of Terminalia chebula Retz. through a resin column. Therefore, the 50% ethanol-water extract of Terminalia chebula Retz. was loaded onto a PA-800 resin column and eluted with water, 10% ethanol / water, and finally with ethanol. The ethanol fraction was evaporated to obtain a concentrated extract of Terminalia chebula Retz., and the concentrated extract of Terminalia chebula Retz. was treated with zinc oxide to obtain a concentrated extract containing zinc salts of chebulagic acid and / or zinc salts of chebulinic acid. Similarly, a concentrated extract of Terminalia chebula Retz. containing metal salts was also obtained through SP-700 and HP-20 resin columns (Example 2).

[0034] Surprisingly, the inventors found that the Terminalia chebula Retz. extract containing metal salts of chebulagic acid, metal salts of chebulinic acid, and metal salts of gallic acid has higher water solubility compared to the corresponding extract without salts, and the solubility data are shown in Table-4.

[0035] Formation of salts :The chemical structures of chebulagic acid, chebulinic acid, and gallic acid ( Figure 1 ) contain carboxylic acid groups and phenolic hydroxyl groups, which can form salts / complexes with metal ions. When the Terminalia chebula Retz. extract containing these phytochemicals is treated with metal oxides, metal hydroxides, metal carbonates, etc., metal salts or complexes or chelates of chebulagic acid, chebulinic acid, and gallic acid are formed. For example, treating the aqueous extract of Terminalia chebula Retz. with zinc oxide gives a Terminalia chebula Retz. extract containing zinc salts, complexes, or chelates of chebulagic acid, chebulinic acid, and gallic acid. The presence of zinc, as shown by ICP-MS analysis of the extract (Table 3), clearly indicates the formation of salts. Moreover, the Terminalia chebula Retz. extract containing zinc salts has higher water solubility than the Terminalia chebula Retz. extract without zinc salts (Table 4). In addition, the acidity (pH) of the Terminalia chebula Retz. extract containing zinc salts is 5.5 - 5.9, while that of the extract without salts is 3.9, which also confirms the formation of salts (Table 4).

[0036] To solve this problem and provide a safe herbal composition for enhancing immunity and respiratory health, the following cell-based assays were selected to evaluate the efficacy of the composition in improving immunity and respiratory health.

[0037] (i) Interleukin-2 (IL-2) production

[0038] (ii) Interferon-γ (IFN-γ) production

[0039] Interleukin-2: Interleukin-2 (IL-2) promotes T cell proliferation and differentiation in vitro and plays a crucial role in antigen-driven T cell clonal expansion in vivo. For in vivo immune activation, IL-2 functions in the proliferation and survival of T cells, as well as the differentiation of T cells into effector T cells. In chronic infections, IL-2 is also an important factor in the generation of memory T cells, which undergo secondary expansion upon re-encountering antigen. Alternatively, IL-2 can promote activation-induced cell death (AICD) of T cells, downregulating the immune response after antigen-specific T cell clonal expansion. IL-2 can also prime CD8+ T cells with non-infectious immunogens. Conversely, very high levels of IL-2 can bind to CD25 and differentiate T cells into regulatory T cells (Tregs), which will suppress excessive immune responses. In this regard, compounds that promote IL-2 production are crucial for enhancing the host immune system.

[0040] Interferon-γ: Interferon-γ (IFN-γ) has been shown to have profound effects on both innate and adaptive immunity, contributing to host protection. IFN-γ is produced by adaptive CD4+ Th1 T cells, CD8+ cytotoxic T cells, natural killer (NK) cells, B cells, NKT cells, and innate professional antigen-presenting cells (APCs) [monocytes / macrophages, dendritic cells (DCs)]. IFN-γ has a key role in the recognition and elimination of pathogens as it can coordinate multiple antibacterial and antiviral functions through cell-mediated immunity. IFN-γ can enhance the ability of antigen-recognizing APCs and amplify their antigen presentation to T cells, followed by increasing the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) and inducing antiviral responses. In the case of viral infections, INF-γ treatment can protect neurons from varicella-zoster virus and limit the proliferation of hepatitis C virus in HIV patients. Importantly, IFN-γ plays a crucial role in establishing a protective immune response against respiratory syncytial virus (RSV) infection. RSV is a major cause of upper and lower respiratory tract infections such as bronchiolitis and viral pneumonia. In this regard, compounds that promote INF-γ production will likely be beneficial for enhancing the host immune system and improving respiratory health.

[0041] Therefore, the efficacy of Terminalia chebula extract containing zinc salts in improving IL-2 production and IFN-γ production in a cell model was evaluated compared to Terminalia chebula extract without zinc salts. Unexpectedly, the Terminalia chebula extract containing zinc salts effectively increased the levels of the said cytokines; moreover, compared to the Terminalia chebula extract without zinc salts, the Terminalia chebula extract containing zinc salts showed better improvement in the production of IL-2 and IFN-γ.

[0042] For example, the determination of IL-2 production of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) showed that at a concentration of 10 μg / mL, the IL-2 production increased by 20.67%, while the Terminalia chebula extract (without zinc salt) showed an increase in IL-2 production of 7.11% at a concentration of 10 μg / mL. This is a surprising and unexpected result. Compared with the Terminalia chebula extract without zinc salt (Table-1), the Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) enhanced the IL-2 production activity.

[0043] Table-1: Percentage increase in IL-2 and INF-γ production of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and Terminalia chebula extract without salt (T.Ch)

[0044]

[0045] Similarly, the determination of IFN-γ production of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) showed that at a concentration of 10 μg / mL, the IFN-γ level increased by 18.36%, while the Terminalia chebula extract (without salt) showed an increase in IFN-γ level of 8.19% at a concentration of 10 μg / mL. This is a surprising and unexpected result. Compared with the Terminalia chebula extract without salt, the Terminalia chebula extract containing zinc salt enhanced the IFN-γ production activity (Table-1).

[0046] Therefore, the present invention provides a Terminalia chebula extract containing a metal salt of chebulinic acid, a metal salt of chebualic acid, and a metal salt of gallic acid; wherein, chebulinic acid can be in the range of 1.0 - 40%, chebualic acid can be in the range of 1.0 - 30%, gallic acid can be in the range of 1.0 - 10%, and the metal content can be in the range of 0.5 - 5.0%; wherein, the metal is selected from zinc, magnesium, calcium, and potassium.

[0047] Composition

[0048] Inspired by the improved efficacy of the zinc salt of Terminalia chebula extract in increasing the production of IL-2 and IFN-γ levels, the inventors prepared a composition comprising: a Terminalia chebula extract containing a zinc salt, in combination with at least one ingredient selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; to explore the efficacy of these compositions in enhancing immunity and respiratory health.

[0049] Accordingly, various solvent extracts of Withania somnifera, Tinospora cordifolia, or Andrographis paniculata were prepared using different solvents. For example, the dried roots of Withania somnifera were pulverized, and the resulting powder was extracted with a 60% aqueous ethanol solution, and the extract was concentrated to obtain a 60% aqueous ethanol extract (W.S-1). Similarly, the dried root powder of Withania somnifera was extracted with other solvents such as ethanol, water, an 80% aqueous methanol solution, and an 80% aqueous acetone solution to obtain an ethanol extract (W.S-2), a water extract (W.S-3), an 80% aqueous methanol extract (W.S-4), and an 80% aqueous acetone extract (W.S-5), respectively. These extracts of Withania somnifera roots were standardized to total withanolides by HPLC analysis method, and the results are summarized in Table 5.

[0050] Similarly, the dried stem material of Tinospora cordifolia was pulverized, and the resulting powder was extracted with water, a 50% aqueous ethanol solution, and ethanol to obtain a water extract of Tinospora cordifolia (T.C-1), a 50% aqueous ethanol extract of Tinospora cordifolia (T.C-2), and an ethanol extract of Tinospora cordifolia (T.C-3), respectively. These extracts of Tinospora cordifolia were standardized to 8-hydroxycolumbin by HPLC analysis method, and the results are summarized in Table 6. Similarly, the dried whole plant of Andrographis paniculata was pulverized, and the resulting powder was extracted with a 70% aqueous ethanol solution, water, and ethanol to obtain a 70% aqueous ethanol extract of Andrographis paniculata (A.P-1), a water extract of Andrographis paniculata (A.P-2), and an ethanol extract of Andrographis paniculata (A.P-3), respectively. These extracts of Andrographis paniculata were standardized to andrographolide by HPLC analysis method, and the results are summarized in Table 7.

[0051] Subsequently, the inventors prepared two compositions: (a) Composition-3, which contained Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% aqueous ethanol extract of Withania somnifera (W.S-1) in a ratio of 1:1; (b) Composition-3A, which contained Terminalia chebula extract (T.Ch) and 60% aqueous ethanol extract of Withania somnifera (W.S-1) in a ratio of 1:1 as a comparative composition without zinc salt. The efficacy of these two compositions in increasing the production of IL-2 and IFN-γ in an in vitro cell model was tested, and the results are summarized in Table-2.

[0052] Table-2: Efficacy of compositions containing Terminalia chebula extract containing zinc salt and 60% aqueous ethanol extract of Withania somnifera, or compositions of conventional extract without salt and 60% aqueous ethanol extract of Withania somnifera

[0053]

[0054] As can be seen from the above table, at a concentration of 10 μg / mL, the IL-2 production of Composition-3 increased by 39.78%. Composition-3 contains Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and Withania somnifera 60% ethanol-water extract (W.S-1) in a ratio of 1:1. Although the IL-2 production of a similar composition without zinc salt (comp-3A) increased by 19.78% at a concentration of 10 μg / mL, compared with the corresponding control without zinc salt (Table-2), the enhancement of the IL-2 production activity of Composition-3 is a surprising and unexpected result. Similarly, at 10 μg / mL, the IFN-γ production of Composition-3 increased by 41.08%. While the IFN-γ production of a similar composition without zinc salt (comp-3A) increased by 27.08% at a concentration of 10 μg / mL. This is also a surprising and unexpected improvement. Therefore, compared with the compositions of Terminalia chebula extract without zinc salt and other herbal extracts, the compositions containing Terminalia chebula extract with zinc salt and other herbal extracts such as Withania somnifera show better efficacy in increasing the production of IL-2 and IFN-γ levels.

[0055] After this surprising result, various compositions summarized in Examples 22-32 were prepared. The compositions include: Terminalia chebula extract, which contains a metal salt of at least one phytochemical. The Terminalia chebula extract is selected from extracts of chebulinic acid, chebulin, gallic acid or a mixture thereof, wherein the metal is selected from zinc, magnesium, calcium and potassium; and is combined with at least one extract derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata.

[0056] Then, these compositions (C1-C35) were tested for their efficacy in increasing the production of IL-2 and IFN-γ and lymphocyte proliferation in an in vitro cell model compared with the corresponding individual components. Unexpectedly, these compositions showed synergistic activity.

[0057] For example, zinc salt of Terminalia chebula Retz. extract (T.Ch.Zn-1) at a concentration of 7.5 μg / mL and 60% ethanol-water extract of Withania somnifera (W.S-1) at a concentration of 2.5 μg / mL showed that the production of IL-2 increased by 15.50% and 5.83%, respectively. Composition-1 (C-1) containing zinc salt of Terminalia chebula Retz. extract (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 3:1, at a concentration of 10 μg / mL, the production of IL-2 increased by 31.04%, which was significantly higher than the additive effect of 21.33% (15.50% + 5.83%) calculated from the increase in IL-2 production shown by the corresponding individual components. As summarized in Table 8, Compositions-2 to 5 (C-2 to C-5) containing these two extracts (T.Ch.Zn-1 and W.S-1), where the ratios of T.Ch.Zn-1 and W.S-1 are 2:1, 1:1, 1:2, and 1:3, also showed a synergistic effect compared to the increase in IL-2 production shown by each corresponding individual component concentration in each composition. Similarly, as summarized in Table 8, other compositions (C-6 to C-8) containing zinc salt of Terminalia chebula Retz. extract (T.Ch.Zn-2) and 60% ethanol-water extract of Withania somnifera (W.S-1) also showed a synergistic increase in IL-2 production.

[0058] Similarly, Compositions (C-9 to C-14) containing zinc salt-containing Terminalia chebula Retz. extract (T.Ch.Zn-1) and aqueous extract of Tinospora cordifolia (T.C-1) or 70% ethanol-water extract of Andrographis paniculata (A.P-1), Compositions (C-15 to C-23) containing magnesium salt-containing Terminalia chebula Retz. extract (T.Ch.Mg) and ethanol extract of Withania somnifera (W.S-2) or 50% ethanol-water extract of Tinospora cordifolia (T.C-2) or aqueous extract of Andrographis paniculata (A.P-2), Compositions (C-24 to C-32) containing calcium salt-containing Terminalia chebula Retz. extract (T.Ch.Ca) and 80% methanol-water extract of Withania somnifera (W.S-4) or ethanol extract of Tinospora cordifolia (T.C-3) or ethanol extract of Andrographis paniculata (A.P-3), and Compositions (C-33 to C-35) containing potassium salt-containing Terminalia chebula Retz. extract (T.Ch.K) and 80% aqueous acetone extract of Withania somnifera (W.S-5) also showed a synergistic improvement in IL-2 production (Tables 9 - 12).

[0059] In addition, the compositions (1-35) also showed a greater increase in INF-γ production than the corresponding individual components. For example, the zinc salt of Terminalia chebula extract (T.Ch.Zn-1) at a concentration of 7.5 μg / mL and the 60% ethanol water extract of Ashwagandha (WS-1) at a concentration of 2.5 μg / mL showed an increase in INF-γ production of 13.77% and 5.26%, respectively. Composition-1 (C-1) containing the above two extracts in a ratio of 3:1 increased INF-γ production by 24.96% at a concentration of 10 μg / mL, which was significantly higher than the additive effect of 19.03% (13.77% + 5.26%) calculated from the increase in INF-γ production exhibited by the corresponding individual components. As summarized in Table 13, compositions-2 to 5 (C-2 to C-5) containing the two extracts in other ratios also showed a synergistic effect compared to the increase in INF-γ production exhibited by each of the corresponding individual component concentrations in each composition. Likewise, other compositions (C-6 to C-8) containing a zinc salt of a Terminalia chebula extract (T.Ch.Zn-2) and a 60% ethanol aqueous extract of Ashwagandha (WS-1) also showed a synergistic increase in the amount of INF-γ produced (Table 13). Similarly, other compositions (C-9 to C-35) comprising a Terminalia chebula extract containing a metal salt and an extract derived from Ashwagandha, Tinospora cordifolia extract or Andrographis paniculata also showed a synergistic increase in the amount of INF-γ produced (Tables 14-17).

[0060] Furthermore, these combinations (1-35) were screened for their ability to increase lymphocyte proliferation and interestingly, all of them showed a better increase in lymphocyte proliferation than the corresponding individual components.

[0061] Lymphocyte proliferation: The proliferation of lymphocytes, especially antigen-specific T cells, is essential for mediating protective immunity against bacterial / viral pathogens and creating immune memory. Peripheral blood mononuclear cells (PBMCs) serve as an important cell population for proliferation assays. There are different methods to assess cell proliferation: a. 3 H-thymidine incorporation into the DNA of dividing cells, b. Fluorescent dye dilution assay using CFSE or its derivative Oregon Green (OG), c. Estimation of Ki67 labeling. Ki67 is a nuclear protein that plays a role in regulating cell division. It is expressed in all active phases of cell division but not in quiescent cells and during DNA repair. Intracellular Ki67 expression directly in vitro or after in vitro cell culture has been used to measure specific T cell responses induced by vaccination or turnover of these cells in individuals with chronic viral infections such as HIV infection. Ki67 assessment is often an important tool for evaluating the efficacy of compounds that promote PBMC proliferation, especially T cells.

[0062] Lymphocytes are the main cellular components of the immune system and are responsible for generating an adaptive immune response in the host body. Increasing the number of lymphocytes by enhancing the proliferation of precursor hematopoietic cells is crucial for establishing a strong cell-mediated immune response against bacterial or viral infections in the body.

[0063] For example, zinc salt of Terminalia chebula extract (T.Ch.Zn-1) at a concentration of 7.5 μg / mL and 60% ethanol-water extract of Withania somnifera (W.S-1) at a concentration of 2.5 μg / mL showed that lymphocyte proliferation increased by 10.04% and 4.21%, respectively. Composition-1 (C-1) containing the above two extracts in a ratio of 3:1, at a concentration of 10 μg / mL, showed a 20.03% increase in lymphocyte proliferation, which was significantly better than the additive effect of 14.25% (10.04% + 4.21%) calculated from the increase in lymphocyte proliferation shown by the corresponding individual components. As summarized in Table 18, compositions-2 to 5 (C-2 to C-5) containing these two extracts in other ratios also showed a synergistic effect compared to the increase in lymphocyte proliferation shown by each corresponding individual component concentration in each composition, as shown in Table 18. Similarly, other compositions (C-6 to C-8) containing zinc salt of Terminalia chebula extract (T.Ch.Zn-2) and 60% ethanol-water extract of Withania somnifera (W.S-1) also showed a synergistic increase in lymphocyte proliferation (Table 18). Similarly, compositions (C-9 to C-35), which contain a Terminalia chebula extract containing a metal salt of at least one phytochemical selected from chebulagic acid, chebulinic acid, and gallic acid or a mixture thereof; and an additional extract selected from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata, also showed a synergistic increase in lymphocyte proliferation (Tables 19 - 21).

[0064] Regulating the inflammatory and immune responses in rats: The regulation of the inflammatory and immune responses in rats by these compositions was evaluated; these compositions contain a Terminalia chebula extract containing a metal salt of at least one phytochemical selected from chebulagic acid, chebulinic acid, and gallic acid or a mixture thereof; and an additional extract selected from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata.

[0065] Cluster of differentiation 3 (CD3) is a multimeric protein complex and a unique co-receptor of the T cell lineage. It is expressed early in T cell maturation. CD3-positive cells (CD3+) are involved in the activation of CD4+ naive T cells (helper T cells) and CD8+ naive T cells (cytotoxic T cells). Once triggered, CD4+ T cells differentiate into functional subsets - T helper type 1 cells (TH1) and T helper type 2 cells (TH2). TH1 cells and TH2 cells produce cytokines IFN-γ and IL-4, respectively. Helper T cells are crucial for initiating immune protection against various intracellular and extracellular infections caused by viruses and bacteria. CD8+ T cells mediate their effector functions by producing cytokines such as IFN-γ and TNF-α and / or through the cytolytic process. In summary, different subsets of T lymphocytes are components of the host cellular immune system and are very important in preventing or maintaining immune defense against infection or disease.

[0066] Immunoglobulin G (IgG) is a class of antibodies that accounts for approximately two-thirds of the total antibody amount in the body. Immunoglobulins are crucial for generating humoral immune responses and are essential for host defense against infection. When stimulated by an infected antigen response, plasma B cells produce IgG. Essentially, IgG helps neutralize pathogens in phagocytes.

[0067] In the above context, compounds that increase the numbers of T cell subsets (CD3+, CD4+, and CD8+) and raise the IgG level in the body have potential value in enhancing host immune defense by triggering cellular and humoral immunity against infection.

[0068] IL-6 is a pro-inflammatory cytokine produced by cells of the innate immune system (such as macrophages, dendritic cells, mast cells, neutrophils). In many inflammatory conditions, the level of IL-6 in serum increases. Therefore, IL-6 is considered a general marker of inflammation. Different stimuli, including allergens, respiratory viruses, cause an inflammatory response in lung epithelial cells and raise the serum IL-6 level. Thus, compounds that reduce the IL-6 level in serum will be beneficial in alleviating systemic inflammation, including lung inflammation caused by exposure to allergens or microbial pathogens.

[0069] Therefore, biomarkers that regulate inflammatory and immune responses were explored in experimental rats. These observations showed that rats supplemented with the composition of the present invention exhibited a synergistic improvement in regulating biomarkers such as CD3 + numbers, CD4 + +CD8 + numbers, IL-6, and IgG (Table 22 - 24).

[0070] CD3 +and CD4 + +CD8 + Increase in numbers: The compositions of the present invention show a synergistic effect in increasing the numbers of CD3 + and CD4 + +CD8 + in experimental animals. For example, compared with LPS-induced rats supplemented with LPS, the 60% ethanol-water extract of Withania somnifera (W.S-1) and the zinc-containing Terminalia chebula extract (T.Ch.Zn-1) increased the numbers of CD3 + by 7.79% and 7.17% respectively. Compared with LPS-induced rats, Composition-36 containing the above two extracts in a ratio of 1:1 and an excipient showed an increase in the number of CD3 + by 16.51% (Table 22), which is significantly higher than the efficacy exhibited by the corresponding individual components, indicating that the 60% ethanol-water extract of Withania somnifera (W.S-1) and the zinc-containing Terminalia chebula extract (T.Ch.Zn-1) have an in vivo synergistic effect in increasing the number of CD3 + Similarly, compared with LPS-induced rats supplemented with LPS, the 60% ethanol-water extract of Withania somnifera (W.S-1) and the zinc-containing Terminalia chebula extract (T.Ch.Zn-1) increased the numbers of CD4 + +CD8 + by 8.05% and 7.28% respectively (Table 23). Composition-36 containing the above two extracts in a ratio of 1:1 and an excipient showed an 18.39% increase compared to the LPS-induced group (G2), which is significantly higher than the corresponding individual components, indicating that these two extracts have a synergistic effect in increasing the number of CD4 + +CD8 +

[0071] Reduction of IL-6 level and increase of IgG level: The compositions of the present invention also show a synergistic effect in reducing the IL-6 level and increasing the IgG level in experimental animals. For example, compared with the IL-6 level found in LPS-induced rats, the 60% ethanol-water extract of Withania somnifera (W.S-1) and the zinc-containing Terminalia chebula extract (T.Ch.Zn-1) showed a 18.3% and 15.5% reduction respectively in the IL-6 level. Compared with LPS-induced rats, Composition-36 containing the above two extracts in a ratio of 1:1 and an excipient showed a 29.4% reduction, which is significantly greater than the reduction by the corresponding individual components, indicating that these two extracts have a synergistic effect in reducing serum IL-6. Similarly, Composition-36 showed a significantly higher increase in the IgG level than the corresponding individual components, indicating that W.S-1 and T.Ch.Zn-1 have a synergistic effect in increasing the IgG level, as shown in Table-24.

[0072] ​Thus, these compositions (C1 to C35) unexpectedly showed better efficacy in increasing the production of IL-2 and IFN-γ, as well as lymphocyte proliferation, compared to the corresponding individual components. Accordingly, the composition comprises a first component, Terminalia chebula extract, which contains a metal salt of at least one phytochemical selected from the group consisting of chebulagic acid, chebulinic acid, gallic acid, or a mixture thereof; and a second component selected from the group consisting of extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; which tend to exhibit a synergistic effect when combined together.

[0073] Method: A method for preparing a synergistic composition, the composition comprising a first component, Terminalia chebula extract, which contains a metal salt of at least one phytochemical selected from the group consisting of chebulagic acid, chebulinic acid, gallic acid, or a mixture thereof; and a second component selected from at least one of extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; wherein the method comprises the following steps:

[0074] (i) Extracting dried Terminalia chebula fruit powder with a suitable solvent;

[0075] (ii) Optionally, eluting the extract in step (i) through a resin column to obtain a concentrated extract;

[0076] (iii) Treating the solution in step (ii) with a metal compound;

[0077] (iv) Filtering the solution;

[0078] (v) Evaporating the solvent and drying the residue to obtain Terminalia chebula extract containing a metal salt;

[0079] (vi) Mixing the Terminalia chebula extract containing a metal salt with at least one extract derived from Withania somnifera or Tinospora cordifolia or Andrographis paniculata in the presence of a pharmaceutically or nutritionally or dietetically acceptable excipient, carrier, and diluent;

[0080] (vii) Drying the product under vacuum to obtain the composition.

[0081] Suitable solvents used in the method for preparing the composition are selected from, but not limited to: C1-C5 alcohols such as ethanol, methanol, and n-butanol; water and their mixtures. Metals used in the method for preparing the composition are selected from zinc, magnesium, calcium, and potassium, and the metal compounds used for preparing these compositions are in the form of their metal salts, metal oxides, metal hydroxides, or carbonates. Examples include zinc oxide, zinc carbonate, zinc hydroxide, magnesium oxide, magnesium carbonate, magnesium hydroxide, calcium hydroxide, calcium carbonate, potassium hydroxide, and potassium carbonate. The resin columns used in the method for preparing the composition are selected from PA-800, SP-700, and HP-20.

[0082] Formulation: The present invention also provides a synergistic herbal composition, the synergistic herbal composition comprising a first component of Terminalia chebula extract, the Terminalia chebula extract containing a metal salt of at least one phytochemical selected from emblicanin, chebulinic acid, and gallic acid or a mixture thereof in the extract; and a second component selected from at least one of extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; the composition can be formulated with at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers, and diluents.

[0083] The synergistic herbal composition comprises a first component, Terminalia chebula extract containing a metal salt; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; and contains at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers and diluents; for obtaining at least one health benefit selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections and improving respiratory / lung health; wherein, the pharmaceutically or nutritionally or dietetically acceptable excipients, carriers and diluents are selected from monosaccharides such as glucose, dextrose, fructose, galactose, etc.; disaccharides such as sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose, etc. but not limited thereto; polycarbohydrates such as starch and modified starches such as sodium starch glycolate, pregelatinized starch, soluble starch and other modified starches; dextrins produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin, etc.; polyols or sugar alcohols such as sorbitol, mannitol, inositol, xylitol, isomaltulose, etc. but not limited thereto; cellulose-based derivatives such as microcrystalline cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, etc. but not limited thereto; silicates such as magnesium aluminum silicate, aluminum magnesium silicate, talc, colloidal silica, etc. but not limited thereto; metal stearates such as calcium stearate, magnesium stearate, zinc stearate, etc. but not limited thereto; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, etc.; fatty acid esters and polysorbates; natural gums such as gum arabic, carrageenan, guar gum, xanthan gum, etc. but not limited thereto; vitamin B group; niacinamide; calcium pantothenate; amino acids; proteins such as casein, gelatin, pectin, agar, etc. but not limited thereto; inorganic metal salts such as sodium chloride, calcium chloride, dicalcium phosphate, zinc sulfate, zinc chloride, etc. but not limited thereto; natural pigments; flavors; class I and class II preservatives; and aqueous solutions, alcoholic solutions, hydroalcoholic solutions, organic solutions of the above components alone or in combination.

[0084] The foregoing indicates that the synergistic herbal composition comprises a first component, Terminalia chebula extract, which contains a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulinic acid, chebulin, and gallic acid or mixtures thereof in an extract, wherein the metal is selected from zinc, magnesium, calcium, and potassium; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; these synergistic herbal compositions unexpectedly show better efficacy in increasing the production of IL-2 and IFN-γ, as well as lymphocyte proliferation, compared to the corresponding individual components. Accordingly, the compositions can be used to improve immunity, lung function, and respiratory health.

[0085] Accordingly, in an important embodiment, the present invention provides a synergistic composition comprising a first component, Terminalia chebula extract, which contains a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulinic acid, chebulin, and gallic acid or mixtures thereof in an extract, wherein the metal is selected from zinc, magnesium, calcium, and potassium; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; for obtaining at least one health benefit selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving respiratory health.

[0086] In a preferred embodiment, the present invention provides a synergistic composition comprising a first component, Terminalia chebula extract, which contains a zinc salt of at least one phytochemical selected from the group consisting of chebulinic acid, chebulin, and gallic acid or mixtures thereof in an extract; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; for improving immunity and respiratory / lung health.

[0087] In a preferred embodiment, the present invention provides a synergistic composition comprising a first component, Terminalia chebula extract, which contains a magnesium salt of at least one phytochemical selected from the group consisting of chebulinic acid, chebulin, and gallic acid or mixtures thereof in an extract; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; for improving immunity and respiratory / lung health.

[0088] In a preferred embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a calcium salt of at least one phytochemical selected from emblicanin A, chebulinic acid, gallic acid or mixtures thereof in the extract; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; for improving immunity and respiratory / lung health.

[0089] In a preferred embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a potassium salt of at least one phytochemical selected from emblicanin A, chebulinic acid, gallic acid or mixtures thereof in the extract; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; for improving immunity and respiratory / lung health.

[0090] In another embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt, metal complex or metal chelate of at least one phytochemical selected from emblicanin A, chebulinic acid, gallic acid or mixtures thereof in the extract; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; wherein, emblicanin A is in the range of 1.0 - 40%, chebulinic acid is in the range of 1.0 - 30%, gallic acid is in the range of 1.0 - 10%, and the metal is in the range of 0.5 - 5.0%; wherein, the metal is selected from zinc, magnesium, calcium and potassium.

[0091] In another embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt, metal complex or metal chelate of at least one phytochemical selected from emblicanin A, chebulinic acid, gallic acid or mixtures thereof in the extract; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; wherein, the weight percentage concentration of the Terminalia chebula extract containing the metal salt in the composition varies in the range of 10% - 90%, and the second extract, fraction or phytochemical derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata varies in the weight percentage concentration in the composition in the range of 90% - 10%.

[0092] In another embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component of Terminalia chebula extract, the Terminalia chebula extract containing at least one phytochemical metal salt or metal complex or metal chelate selected from the extracts of chebulagic acid, chebulinic acid, gallic acid or mixtures thereof; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; wherein the extract or fraction is obtained from at least one plant part selected from the group consisting of leaves, stems, shoots, twigs, aerial parts, whole fruits, pericarp, seeds, flower heads, roots, barks, hardwoods, rhizomes or whole plants or mixtures thereof.

[0093] In another embodiment, the present invention provides the synergistic herbal composition as described above, wherein the extract, fraction, phytochemical or mixture thereof is prepared using at least one solvent selected from: C1-C5 alcohols, selected from ethanol, methanol, n-propanol, isopropanol; ketones, selected from acetone, methyl isobutyl ketone; chlorinated solvents, selected from dichloromethane and chloroform; water and mixtures thereof; C1-C7 hydrocarbons such as hexane; esters such as ethyl acetate and the like and mixtures thereof.

[0094] In another embodiment, the present invention provides the synergistic herbal composition as described above; wherein the extract, fraction or mixture thereof is standardized to at least one phytochemical reference marker compound or pharmacological activity marker in the composition; wherein the weight percentage concentration range of the phytochemical marker compound or phytochemical compound group in the extract is from 0.01% to 90%.

[0095] In another embodiment, the present invention provides the synergistic herbal composition as described above, wherein the Withania somnifera root extract or fraction is standardized to total withanolides; wherein the weight percentage concentration range of total withanolides in the composition is from 0.01% to 10%.

[0096] In another embodiment, the present invention provides the synergistic herbal composition as described above, wherein the Tinospora cordifolia stem extract or fraction is standardized to 8-hydroxycolumbin; wherein the weight percentage concentration range of 8-hydroxycolumbin in the composition is from 0.01% to 5%.

[0097] In another embodiment, the present invention provides the synergistic herbal composition as described above, wherein the Andrographis paniculata whole plant extract or fraction is standardized to andrographolide, wherein the weight percentage concentration range of total andrographolide in the composition is from 1.00% to 40%.

[0098] In another embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulagic acid, chebulinic acid, gallic acid, or mixtures thereof in the extract; and a second component, the second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and further optionally comprising at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers, and diluents.

[0099] In another embodiment, the present invention provides a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from the group consisting of chebulagic acid, chebulinic acid, gallic acid, or mixtures thereof in the extract; and a second component, the second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and further optionally comprising at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers, and diluents; wherein the pharmaceutically or nutritionally or dietetically acceptable excipients, carriers, and diluents are selected from: monosaccharides such as glucose, dextrose, fructose, galactose, etc.; disaccharides such as sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose, etc. but not limited thereto; polycarbohydrates such as starch and modified starches such as sodium starch glycolate, pregelatinized starch, soluble starch, and other modified starches; dextrins produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, maltodextrin, etc.; polyols or sugar alcohols such as sorbitol, mannitol, inositol, xylitol, isomaltulose, etc. but not limited thereto; cellulose-based derivatives such as microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc. but not limited thereto; silicates such as magnesium aluminum silicate, aluminum magnesium silicate, talc, colloidal silica, etc. but not limited thereto; metal stearates such as calcium stearate, magnesium stearate, zinc stearate, etc. but not limited thereto; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, etc.; fatty acid esters and polysorbates; natural gums such as gum arabic, carrageenan, guar gum, xanthan gum, etc. but not limited thereto; vitamin B group; nicotinamide; calcium pantothenate; amino acids; proteins such as casein, gelatin, pectin, agar, etc. but not limited thereto; inorganic metal salts such as sodium chloride, calcium chloride, dicalcium phosphate, zinc sulfate, zinc chloride, etc. but not limited thereto; natural pigments; flavors; class I and class II preservatives; and aqueous solutions, alcoholic solutions, hydroalcoholic solutions, and organic solutions of the above components alone or in combination.

[0100] In another embodiment, the present invention provides a method for preparing a synergistic composition, the synergistic composition comprising a first component, Terminalia chebula extract, the Terminalia chebula extract containing at least one metal salt of a phytochemical selected from extracts of chebulagic acid, chebulinic acid, gallic acid or mixtures thereof; and a second component selected from extracts, fractions, phytochemicals or mixtures thereof derived from Withania somnifera, Tinospora cordifolia and Andrographis paniculata; wherein the method comprises the following steps:

[0101] (i) Extracting dried Terminalia chebula fruit powder with a suitable solvent;

[0102] (ii) Optionally, eluting the extract in step (i) through a resin column to obtain a concentrated extract;

[0103] (iii) Treating the extract solution obtained in step i or ii with a metal compound;

[0104] (iv) Filtering the solution;

[0105] (v) Evaporating the solvent and drying the residue to obtain Terminalia chebula extract containing a metal salt;

[0106] (vi) Mixing the Terminalia chebula extract containing a metal salt with at least one extract derived from Withania somnifera or Tinospora cordifolia or Andrographis paniculata in the presence of at least one component selected from pharmaceutically or nutritionally or dietetically acceptable excipients, carriers and diluents;

[0107] (vii) Drying the product under vacuum to obtain the composition.

[0108] In another embodiment, the present invention provides a method for preparing the above composition, wherein the suitable solvent used in the method for preparing the composition is selected from, but not limited to: C1-C5 alcohols such as ethanol, methanol, n-butanol; water and mixtures thereof.

[0109] In another embodiment, the present invention provides a method for preparing the above composition, wherein the metal is selected from zinc, magnesium, calcium and potassium; and the metal compounds used for preparing these compositions are in the form of their metal salts, metal oxides, metal hydroxides or carbonates. Examples include zinc oxide, zinc carbonate, zinc hydroxide, magnesium oxide, magnesium carbonate, magnesium hydroxide, calcium hydroxide, calcium carbonate, potassium hydroxide, potassium carbonate.

[0110] In another embodiment, the present invention provides a method for preparing the above composition, wherein the resin column is selected from PA-800, SP-700 and HP-20.

[0111] In another embodiment, the present invention provides a method for obtaining at least one health benefit selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving human respiratory health; wherein the method comprises: administering to a human an effective dose of a composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from extracts of chebulagic acid, chebulinic acid, and gallic acid or mixtures thereof; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents.

[0112] In another embodiment of the present invention, there is provided the use of a synergistic composition comprising a first component, an extract of Terminalia chebula, the extract of Terminalia chebula containing a metal salt or metal complex or metal chelate of at least one phytochemical selected from extracts of chebulagic acid, chebulinic acid, and gallic acid or mixtures thereof; and a second component selected from extracts, fractions, phytochemicals, or mixtures thereof derived from Withania somnifera, Tinospora cordifolia, and Andrographis paniculata; and optionally further comprising at least one component selected from pharmaceutically, nutritionally, or dietetically acceptable excipients, carriers, and diluents; for obtaining at least one health benefit selected from enhancing immunity / eliciting an immune response / restoring the immune system, enhancing innate immunity, enhancing adaptive immunity, enhancing cellular immunity, enhancing humoral immunity, strengthening natural defenses, improving protection against airway inflammation and microbial infections, preventing viral respiratory infections, improving lung function / health, and improving respiratory health.

[0113] In another embodiment of the present invention, the above composition is formulated into a dosage form selected from dry powder form, liquid form, beverage, food, dietary supplement, or any suitable form, such as tablets, capsules, soft chewables, or gummy bears.

[0114] In another embodiment of the present invention, the above composition can be formulated into a nutritional / dietary supplement, and the nutritional / dietary supplement can be expected to be made into a health food or a food dosage form for specific health uses. For example, solid foods such as chocolate or nutritional bars, semi-solid foods such as cream or jam, or gels, or beverages such as energy drinks, lactic acid bacteria drinks, drops, candies, chewing gums, gummies, yogurt, ice cream, pudding, soft red bean jelly, jelly, biscuits, tea, soft drinks, fruit juices, milk, coffee, cereals, snack bars, etc.

[0115] Those of ordinary skill in the art will understand that the above embodiments can be changed without departing from their broad inventive concepts. Therefore, it should be understood that the present invention is not limited to the specific embodiments or examples disclosed herein, but is intended to cover modifications within the scope and objectives of the invention as defined in the specification. The embodiments given are for illustrative purposes only and should not be construed as limitations of the present invention. There may be various variations without departing from the essence of the present invention.

[0116] Examples

[0117] Example 1: Terminalia chebula extract (T.Ch.Zn-1) containing zinc ellagitannate of Terminalia chebula and / or zinc chebulate and / or zinc gallate

[0118] To dry Terminalia chebula whole fruit powder (500 g), 50% aqueous ethanol solution (5 L) was added, and the mixture was extracted for 16 h at ambient temperature. The mixture was filtered through diatomaceous earth, and the extraction process was repeated with 50% aqueous ethanol solution (2 × 3 L) for 2 h under similar conditions. Zinc oxide (8.1 g) was added to the combined 50% aqueous ethanol extract, and the mixture was stirred at room temperature for 2 h. The mixture was filtered, and the filtrate was evaporated under vacuum to obtain a brown solid product (T.Ch.Zn-1, 178 g).

[0119] Example 1A: Terminalia chebula extract (T.Ch)

[0120] For comparison, the salt-free Terminalia chebula extract (T.Ch) was prepared using the following procedure. To dry Terminalia chebula whole fruit powder (50 g), 50% aqueous ethanol solution (500 mL) was added, and the mixture was extracted for 16 h at ambient temperature. The mixture was filtered through diatomaceous earth, and the extraction process was repeated with 50% aqueous ethanol solution (2 × 300 mL) for 2 h under similar conditions. The combined 50% aqueous ethanol extract was evaporated under reduced pressure to obtain a brown solid product (T.Ch, 23 g).

[0121] Example 2: Concentrated Terminalia chebula extract containing zinc ellagitannate of Terminalia chebula and / or zinc chebulate and / or zinc gallate

[0122] Method 1 (T.Ch.Zn-2): Add 50% aqueous ethanol solution (500 mL) to dry Terminalia chebula whole fruit powder (50 g), and extract the mixture at ambient temperature for 16 h. Filter the mixture through diatomaceous earth, and repeat the extraction process for 2 h with 50% aqueous ethanol solution (2 × 300 mL) for the remaining residue under similar conditions. Evaporate the combined 50% ethanol water extract under reduced pressure to obtain a concentrated extract. Add water (1000 mL) to the concentrated extract, and load the solution onto a 230 mL PA-800 resin at a flow rate of 20 mL per minute. After loading the extract, elute the chromatographic column successively with water (230 mL), 10% ethanol / water (230 mL), and finally with ethanol (350 mL). Collect and combine the fractions eluted with ethanol separately. Evaporate the combined ethanol fractions under reduced pressure to obtain a concentrated extract, and then dilute it with water (200 mL). Add zinc oxide (808 mg) to this mixture, and stir for 2 h at ambient temperature. Filter the mixture, and evaporate the filtrate under reduced pressure to obtain a brown solid product (T.Ch.Zn-2, 7.35 g).

[0123] Method 2: Replace the PA-800 resin with SP-700 resin, and prepare the Terminalia chebula extract containing zinc salt from Terminalia chebula (50 g) as described in Method 1 to obtain a brown solid product (8.0 g).

[0124] Method 3: Replace the PA-800 resin with HP-20 resin, and prepare the Terminalia chebula extract containing zinc salt from Terminalia chebula (50 g) as described in Method 1 to obtain a brown solid product (6.8 g).

[0125] Example 3: Terminalia chebula extract (T.Ch.Mg) containing Terminalia chebula ellagic acid magnesium salt and / or Terminalia chebula acid magnesium salt and / or gallic acid magnesium salt

[0126] Prepare the Terminalia chebula extract containing magnesium salt from Terminalia chebula (50 g) as described in Example 1 or 2 by replacing zinc oxide with magnesium oxide (667 mg) to obtain a brown solid product (T.Ch.Mg, 18.3 g).

[0127] Example 4: Terminalia chebula extract (T.Ch.Ca) containing Terminalia chebula ellagic acid calcium salt and / or Terminalia chebula acid calcium salt and / or gallic acid calcium salt

[0128] Prepare the Terminalia chebula extract containing calcium salt from Terminalia chebula (50 g) as described in Example 1 or 2 by replacing zinc oxide with calcium oxide (556 mg) to obtain a brown solid product (T.Ch.Ca, 18.7 g).

[0129] Example 5: Terminalia chebula extract (T.Ch.K) containing Terminalia chebula ellagic acid potassium salt and / or Terminalia chebula acid potassium salt and / or gallic acid potassium salt

[0130] By replacing zinc oxide with potassium carbonate (1.13 g), a Terminalia chebula Retz extract containing potassium salt was prepared from Terminalia chebula Retz (50 g) as described in Example 1 or 2, and a brown solid product (T.Ch.K, 18.0 g) was obtained.

[0131] Alternatively, a Terminalia chebula Retz extract containing a metal salt of chebulinic acid and / or Terminalia chebula Retz and / or gallic acid can also be prepared from an aqueous extract of the whole fruit of Terminalia chebula Retz and zinc oxide or magnesium oxide or calcium oxide or potassium carbonate.

[0132] Example 6: Standardization of Terminalia chebula Retz Extract Containing Metal Salt

[0133] Chebulinic acid, chebulin, and gallic acid in the above Terminalia chebula Retz extract containing metal salt were analyzed by HPLC. The concentrations of metals such as zinc, magnesium, calcium, and potassium were analyzed by ICP-MS, and the results are summarized in Table 3.

[0134] Table 3: Analysis Data of Crayfish Extract Containing Metal Salt

[0135] Example # Chebulinic acid (1) Chebulin (2) Gallic acid (3) Sum of 1 + 2 + 3 Metal 1A 10.05％ 4.90％ 2.6％ 17.55％ --- 1 10.26％ 4.80％ 2.81％ 17.87％ Zinc: 1.0% 2 (Method 1) 20.03％ 10.41％ 0.87％ 31.31％ Zinc: 2.79% 2 (Method 2) 18.56％ 8.98％ 0.72％ 28.26％ Zinc: 2.45% 2 (Method 3) 22.26％ 12.35％ 0.89％ 35.50％ Zinc: 2.86% 3 10.33％ 5.44％ 3.10％ 18.87％ Magnesium: 1.2% 4 11.14％ 5.73％ 2.97％ 19.84％ Calcium: 1.3% 5 8.79％ 4.12％ 3.11％ 16.02％ Potassium: 1.8%

[0136] Example 7: Solubility and pH Value Data of Terminalia chebula Retz Extract Containing Metal Salt

[0137] The solubility of the above Terminalia chebula Retz extract containing metal salt in water was evaluated by gradually increasing the volume of water, and its pH value was measured. The results are summarized in Table 4.

[0138] Table 4: Solubility and pH Value Data of Terminalia chebula Retz Extract Containing Metal Salt

[0139] Example # Extract code Solubility of 1.0 g product in water pH value (1% solution) 1A (Conventional extract) T.Ch > 100 mL 3.9 1 T.Ch.Zn-1 10 mL 5.5 2 (Method 1) T.Ch.Zn-2 20 mL 5.6 3 T.Ch.Mg 10 mL 5.9 4 T.Ch.Ca 10 mL 5.5 5 T.Ch.K 10 mL 5.8

[0140] Example 8: 60% Ethanol-Water Extract of Withania somnifera (W.S-1)

[0141] 60% ethanol aqueous solution (300 L) was added to dry Withania somnifera root powder (50 Kg), and the mixture was extracted for 16 h at ambient temperature. The mixture was filtered, and the extraction process was repeated 2 h with 60% ethanol aqueous solution (2 × 200 L) under similar conditions. The combined 60% ethanol-water extract was evaporated under reduced pressure to obtain a brown solid product (W.S-1, 8.9 Kg).

[0142] Example 9: Ethanol Extract of Withania somnifera (W.S-2)

[0143] By adopting a similar procedure and using ethanol as the extraction solvent, an ethanol extract (W.S-2, 2.55 g) was prepared from 100 g of raw material.

[0144] Example 10: Aqueous Extract of Withania somnifera (W.S-3)

[0145] By adopting a similar procedure and using water as the extraction solvent, a water extract (W.S-3; 15.8 g) was prepared from 100 g of the raw material.

[0146] Example 11: Withania somnifera 80% methanol-water extract (W.S-4)

[0147] By adopting a similar procedure and using an 80% methanol-water solution as the extraction solvent, an 80% methanol-water extract (W.S-4; 14.5 g) was prepared from 100 g of the raw material.

[0148] Example 12: Withania somnifera 80% acetone-water extract (W.S-5)

[0149] By adopting a similar procedure and using an 80% acetone-water solution as the extraction solvent, an 80% acetone-water extract (W.S-5; 9.5 g) was prepared from 100 g of the raw material.

[0150] Example 13: Standardization of Withania somnifera extracts

[0151] Various extracts of Withania somnifera were standardized to total withanolides by HPLC method (USP method), and the results are summarized in Table 5.

[0152] Table 5: Details of Withania somnifera extracts

[0153]

[0154]

[0155] Example 14: Tinospora cordifolia water extract (T.C-1)

[0156] Water (250 mL) was added to dry powdered Tinospora cordifolia stems (100 g), and the mixture was extracted for 3 h at ambient temperature. The mixture was filtered and the extraction process was repeated twice with water (5 × 250 mL) under similar conditions. The combined water extracts were evaporated under reduced pressure to give a brown solid product (T.C-1; 7.5 g).

[0157] Example 15: Tinospora cordifolia 50% ethanol-water extract (T.C-2)

[0158] By adopting a similar procedure and using a 50% ethanol-water solution as the extraction solvent, a 50% ethanol-water extract (T.C-2; 8.3 g) was prepared from 100 g of the raw material.

[0159] Example 16: Tinospora cordifolia ethanol extract (T.C-3)

[0160] By following a similar procedure, using ethanol as the extraction solvent, an ethanol extract (T.C-3; 2.1 g) was prepared from 100 g of the raw material.

[0161] Example 17: Standardization of Tinospora cordifolia Extract

[0162] Various extracts of Tinospora cordifolia were standardized to 8-hydroxycolumbin by HPLC analysis, and the results are summarized in Table 6.

[0163] Table 6: Details of Tinospora cordifolia Extracts

[0164] Example # Extract code Extraction solvent 8-Hydroxytetrandrine obtained by HPLC method 14 T.C-1 Water 0.25％ 15 T.C-2 50% aqueous ethanol solution 0.40％ 16 T.C-3 Ethanol 0.55％

[0165] Example 18: 70% Ethanol-Water Extract of Andrographis paniculata (A.P-1)

[0166] To the dried powder of whole Andrographis paniculata plants (50 g) was added 70% aqueous ethanol solution (300 mL), and the mixture was extracted for 3 h at ambient temperature. The mixture was filtered, and the extraction process was repeated twice with 70% aqueous ethanol solution (2 × 200 mL). The combined 70% aqueous ethanol solution layer was evaporated to a minimum volume, and hexane (50 mL) was added. The mixture was stirred for 30 minutes, and the hexane layer was separated. The solution was washed again with hexane (50 mL). Then the aqueous layer was extracted with ethyl acetate (3 × 50 mL), and the combined ethyl acetate layers were washed with water (50 mL). The ethyl acetate layer was evaporated under reduced pressure to obtain a green solid product (A.P-1; 1.2 g).

[0167] Example 19: Water Extract of Andrographis paniculata (A.P-2)

[0168] To the dried powder of whole Andrographis paniculata plants (100 g) was added water (700 mL), and the mixture was extracted for 1 h at ambient temperature. The mixture was filtered, and the extraction process was repeated twice with water (2 × 500 mL) under similar conditions. The combined water extracts were evaporated under reduced pressure to obtain a light brown solid product (A.P-2; 12.9 g).

[0169] Example 20: Ethanol Extract of Andrographis paniculata (A.P-3)

[0170] By following a similar procedure, using ethanol as the extraction solvent, an ethanol extract (A.P-3; 6.9 g) was prepared from 100 g of the raw material.

[0171] Example 21: Standardization of Andrographis paniculata Extracts

[0172] By HPLC analysis, various extracts of Andrographis paniculata were standardized to andrographolide (the sum of andrographolide, neoandrographolide, paniculide A, and 14-deoxy-11,12-dehydroandrographolide), and the results are summarized in Table 7.

[0173] Table 7: Details of Andrographis paniculata Extracts

[0174]

[0175]

[0176] Example 22: Preparation of various compositions of Terminalia chebula extract containing zinc salt and 60% ethanol-water extract of Withania somnifera

[0177] Composition-1 (C-1) : Composition-1 was prepared by mixing Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 3:1.

[0178] Composition 2 (C-2) : Composition 2 was prepared by mixing Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 2:1.

[0179] Composition 3 (C-3) : Composition 3 was prepared by mixing Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:1.

[0180] Composition 4 (C-4) : Composition 4 was prepared by mixing Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:2.

[0181] Composition 5 (C-5) : Composition 5 was prepared by mixing Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:3.

[0182] Composition for comparison (Composition-3A): This composition-3A (C-3A) was prepared by mixing Terminalia chebula extract without zinc salt (T.Ch) and 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:1.

[0183] Example 23: Preparation of various compositions of concentrated Terminalia chebula extract containing zinc salt and 60% ethanol-water extract of Withania somnifera

[0184] Composition 6 (C-6): Composition 6 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 2:1.

[0185] Composition 7 (C-7) : Composition 7 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:1.

[0186] Composition 8 (C-8) : Composition 8 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 60% ethanol-water extract of Withania somnifera (W.S-1) in a ratio of 1:2.

[0187] Example 24: Preparation of various compositions of the extract of Terminalia chebula Retz. containing zinc salt and the water extract of Tinospora cordifolia

[0188] Composition-9 (C-9) : Composition 9 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the water extract of Tinospora cordifolia (T.C-1) in a ratio of 2:1.

[0189] Composition-10 (C-10) : Composition 10 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the water extract of Tinospora cordifolia (T.C-1) in a ratio of 1:1.

[0190] Composition-11 (C-11) : Composition -11 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the water extract of Tinospora cordifolia (T.C-1) in a ratio of 1:2.

[0191] Example 25: Preparation of various compositions of the extract of Terminalia chebula Retz. containing zinc salt and the 70% ethanol-water extract of Andrographis paniculata (A.P-1)

[0192] Composition-12 (C-12) : Composition 12 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 70% ethanol-water extract of Andrographis paniculata (A.P-1) in a ratio of 2:1.

[0193] Composition-13 (C-13) : Composition 13 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 70% ethanol-water extract of Andrographis paniculata (A.P-1) in a ratio of 1:1.

[0194] Composition-14 (C-14) : Composition 14 was prepared by mixing the extract of Terminalia chebula Retz. containing zinc salt (T.Ch.Zn-1) and the 70% ethanol-water extract of Andrographis paniculata (A.P-1) in a ratio of 1:2.

[0195] Example 26: Preparation of various compositions of Terminalia chebula Retz. extract containing magnesium salt and Withania somnifera (L.) Dunal ethanol extract

[0196] Composition-15 (C-15) : Composition - 15 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the ethanol extract of Withania somnifera (L.) Dunal (W.S - 2) in a ratio of 2:1.

[0197] Composition-16 (C-16) : Composition - 16 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the ethanol extract of Withania somnifera (L.) Dunal (W.S - 2) in a ratio of 1:1.

[0198] Composition-17 (C-17) : Composition - 17 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the ethanol extract of Withania somnifera (L.) Dunal (W.S - 2) in a ratio of 1:2.

[0199] Example 27: Preparation of various compositions of Terminalia chebula Retz. extract containing magnesium salt and Tinospora cordifolia (Willd.) Miers 50% ethanol - water extract

[0200] Composition-18 (C-18) : Composition 18 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the 50% ethanol - water extract of Tinospora cordifolia (Willd.) Miers (T.C - 2) in a ratio of 2:1.

[0201] Composition-19 (C-19) : Composition 19 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the 50% ethanol - water extract of Tinospora cordifolia (Willd.) Miers (T.C - 2) in a ratio of 1:1.

[0202] Composition-20 (C-20) : Composition 20 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the 50% ethanol - water extract of Tinospora cordifolia (Willd.) Miers (T.C - 2) in a ratio of 1:2.

[0203] Example 28: Preparation of various compositions of Terminalia chebula Retz. extract containing magnesium salt and Andrographis paniculata (Burm.f.) Nees water extract

[0204] Composition-21 (C-21) : Composition - 21 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the water extract of Andrographis paniculata (Burm.f.) Nees (A.P - 2) in a ratio of 2:1.

[0205] Composition-22 (C-22) : Composition - 22 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salt (T.Ch.Mg) and the water extract of Andrographis paniculata (Burm.f.) Nees (A.P - 2) in a ratio of 1:1.

[0206] Composition-23 (C-23) : Composition-23 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing magnesium salts (T.Ch.Mg) and the aqueous extract of Andrographis paniculata (A.P-2) in a ratio of 1:2.

[0207] Example 29: Preparation of various compositions of the extract of Terminalia chebula Retz. containing calcium salts and the 80% methanol-water extract of Withania somnifera

[0208] Composition-24 (C-24) : Composition-24 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the 80% methanol-water extract of Withania somnifera (W.S-4) in a ratio of 2:1.

[0209] Composition-25 (C-25) : Composition-25 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the 80% methanol-water extract of Withania somnifera (W.S-4) in a ratio of 1:1.

[0210] Composition-26 (C-26) : Composition-26 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the 80% methanol-water extract of Withania somnifera (W.S-4) in a ratio of 1:2.

[0211] Example 30: Preparation of various compositions of the extract of Terminalia chebula Retz. containing calcium salts and the ethanol extract of Tinospora cordifolia

[0212] Composition-27 (C-27) : Composition-27 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the ethanol extract of Tinospora cordifolia (T.C-3) in a ratio of 2:1.

[0213] Composition-28 (C-28) : Composition-28 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the ethanol extract of Tinospora cordifolia (T.C-3) in a ratio of 1:1.

[0214] Composition-29 (C-29) : Composition-29 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the ethanol extract of Tinospora cordifolia (T.C-3) in a ratio of 1:2.

[0215] Example 31: Preparation of various compositions of the extract of Terminalia chebula Retz. containing calcium salts and the ethanol extract of Andrographis paniculata

[0216] Composition-30 (C-30) : Composition-30 was prepared by mixing the concentrated extract of Terminalia chebula Retz. containing calcium salts (T.Ch.Ca) and the ethanol extract of Andrographis paniculata (A.P-3) in a ratio of 2:1.

[0217] Composition-31 (C-31) : Composition-31 was prepared by mixing the concentrated extract of Terminalia chebula containing calcium salts (T.Ch.Ca) and the ethanol extract of Andrographis paniculata (A.P-3) in a ratio of 1:1.

[0218] Composition-32 (C-32) : Composition-32 was prepared by mixing the concentrated extract of Terminalia chebula containing calcium salts (T.Ch.Ca) and the ethanol extract of Andrographis paniculata (A.P-3) in a ratio of 1:2.

[0219] Example 32: Preparation of various compositions of the extract of Terminalia chebula containing potassium salts and the 80% acetone-water extract of Withania somnifera

[0220] Composition-33 (C-33) : Composition-33 was prepared by mixing the concentrated extract of Terminalia chebula containing potassium salts (T.Ch.K) and the 80% acetone-water extract of Withania somnifera (W.S-5) in a ratio of 2:1.

[0221] Composition-34 (C-34) : Composition-34 was prepared by mixing the concentrated extract of Terminalia chebula containing potassium salts (T.Ch.K) and the 80% acetone-water extract of Withania somnifera (W.S-5) in a ratio of 1:1.

[0222] Composition-35 (C-35) : Composition-35 was prepared by mixing the concentrated extract of Terminalia chebula containing potassium salts (T.Ch.K) and the 80% acetone-water extract of Withania somnifera (W.S-5) in a ratio of 1:2.

[0223] Concentrated extract containing zinc ellagate of Terminalia chebula and / or zinc chebulate

[0224] Example 33: Preparation of the composition

[0225] Composition 36 (C-36): At room temperature, the extract of Terminalia chebula containing zinc ellagate, zinc chebulate and zinc gallate (45 g, T.Ch.Zn-1) was slowly added to water (100 mL), and stirred for 20 - 30 minutes to obtain a homogeneous solution. Then amylopectin (3 g) and maltodextrin (5 g) were successively added to the suspension and stirring was continued for 10 - 15 minutes. A solution of the 60% ethanol-water extract of Withania somnifera (45 g, W.S-1) in 60% ethanol aqueous solution (100 mL) was slowly added to the above suspension, and stirring was continued for 20 - 30 minutes. The resulting slurry was dried under vacuum to obtain a flaky composition. These flakes were uniformly mixed with colloidal silica (2 g) in a polyethylene lid or any suitable stirrer, and then the material was pulverized to obtain a finely powdered composition (Composition-36).

[0226] Example 34: Determination of the production amount of interleukin-2 (IL-2)

[0227] In a 96-well plate, an equal amount of Jurkat cells (0.1×10 6 cells / well) were seeded with 200 μL of RPMI medium supplemented with 10% FBS. The cells were pretreated with test samples at different concentrations. Cells containing 0.2% DMSO were used as the blank control. The plate was incubated in a CO2 incubator at 37 °C for 2 hours. After appropriate incubation, the plate was kept in a CO2 incubator at 37 °C, and the cells were induced with a combination of phorbol 12-myristate 13-acetate (PMA, 7.5 nM) and phytohemagglutinin A (PHA, 0.5 μg / ml) for 4 hours, except for the blank control. The plate was centrifuged at 270×g for 5 minutes, and the cell-free culture supernatant was collected. According to the manufacturer's instructions, an ELISA kit (enzyme-linked immunosorbent assay kit, R&D systems catalog number DY202) was used for IL-2 quantification. The absorbance at 450 nm was measured in a SpectraMax2e microplate reader. The percentage increase in IL-2 production was calculated using the following formula.

[0228]

[0229] The results are shown in Tables 8 - 12.

[0230] Table 8: Percentage increase in IL-2 production of the combination of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and Withania somnifera 60% ethanol-water extract (W.S-1), the combination of Terminalia chebula concentrated extract containing zinc salt (T.Ch.Zn-2) and Withania somnifera 60% ethanol-water extract (W.S-1)

[0231]

[0232]

[0233] Table 9: Percentage increase in IL-2 production of the combination of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and Tinospora cordifolia aqueous extract (T.C-1) or Andrographis paniculata 70% ethanol-water extract (A.P-1)

[0234]

[0235] Table 10: Percentage increase in IL-2 production of the combination of Terminalia chebula extract containing magnesium salt (T.Ch.Mg) and Withania somnifera ethanol extract (W.S-2) or Tinospora cordifolia 50% ethanol-water extract (T.C-2) or Andrographis paniculata aqueous extract (A.P-2)

[0236]

[0237]

[0238] Table 11: Percentage increase in IL-2 production of compositions of Terminalia chebula extract containing calcium salts (T.Ch.Ca) and Withania somnifera 80% methanol-water extract (W.S-4) or Tinospora cordifolia ethanol extract (T.C-3) or Andrographis paniculata ethanol extract (A.P-3)

[0239]

[0240] Table 12: Percentage increase in IL-2 production of compositions of Terminalia chebula extract containing potassium salts (T.Ch.K) and Withania somnifera 80% acetone-water extract (W.S-5)

[0241]

[0242] Example 35: Determination of interferon-γ (IFN-γ) production

[0243] Human blood was collected from the peripheral vein of a healthy volunteer using a syringe containing EDTA at a final concentration of 2 mM. Plasma was separated by centrifugation at 150×g for 10 minutes, and the remaining blood was diluted 1:3 with RPMI medium supplemented with 10% FBS and 2 mM EDTA. In the dark, 30 mL of blood was carefully layered over 15 mL of Ficoll / Lymphoprep in a 50 mL Falcon tube and centrifuged for 30 minutes at a centrifugal force of 350×g and an acceleration of 9 without using the brake. The buffy coat (the interface between the medium and Ficoll) containing peripheral blood mononuclear cells (PBMCs) was carefully collected in 25 mL of cold 0.01 M phosphate-buffered saline (PBS) and centrifuged at 1200 rpm for 10 minutes. Residual red blood cells found in the PBMC pellet were removed by treatment with ACK lysis buffer (Gibco, USA; catalog number A10492-01) and washed with fresh 0.01 M PBS. PBMCs were seeded at a density of 0.1x10 6A 96-well plate with cells / well was treated with test samples at different concentrations. Cells containing 0.2% DMSO were used as the blank control. The plate was cultured in a CO2 incubator at 37°C for 2 hours. Finally, the plate was kept in a CO2 incubator at 37°C, and cells were induced with a combination of phorbol 12-myristate 13-acetate (PMA, 7.5 nM) and phytohemagglutinin A (PHA, 2 μg / ml) for 4 hours, except for the blank control. The plate was centrifuged at 270×g for 5 minutes, and 120 μL of cell-free supernatant was collected. IFN-γ was quantified using an ELISA kit (R&D systems catalog number DY202) according to the manufacturer's instructions. Absorbance was measured at 450 nm in a SpectraMax2e microplate reader. The percentage increase in IFN-γ production was calculated using the following formula.

[0244]

[0245] The results are shown in Table 13-17.

[0246] Table 13: Percentage increase in IFN-γ production of compositions of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and Withania somnifera 60% ethanol-water extract (W.S-1), compositions of concentrated Terminalia chebula extract containing zinc salt (T.Ch.Zn-2) and Withania somnifera 60% ethanol-water extract (W.S-1)

[0247]

[0248]

[0249] Table 14: Percentage increase in IFN-γ production of compositions of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and aqueous extract of Tinospora cordifolia (T.C-1) or 70% ethanol-water extract of Andrographis paniculata (A.P-1)

[0250]

[0251] Table 15: Percentage increase in IFN-γ production of compositions of Terminalia chebula extract containing magnesium salt (T.Ch.Mg) and ethanol extract of Withania somnifera (W.S-2) or 50% ethanol-water extract of Tinospora cordifolia (T.C-2) or aqueous extract of Andrographis paniculata (A.P-2)

[0252]

[0253] Table 16: Percentage increase in IFN-γ production of compositions of Terminalia chebula extract containing calcium salt (T.Ch.Ca) and 80% methanol-water extract of Withania somnifera (W.S-4) or ethanol extract of Tinospora cordifolia (T.C-3) or ethanol extract of Andrographis paniculata (A.P-3)

[0254]

[0255] Table 17: Percentage increase in IFN-γ production of the composition of Terminalia chebula extract containing potassium salts (T.Ch.K) and Withania somnifera 80% acetone-water extract (W.S-5)

[0256]

[0257] Example 36: Lymphocyte proliferation assay

[0258] The same number of human peripheral blood mononuclear cells (PBMC; 0.1x10 6 ) were seeded into each well of a 96-well plate and treated with different concentrations of the test sample in a CO2 incubator at 37 °C for 72 hours. At the end of the incubation period, the cells were treated again with the corresponding concentration of the test sample in a CO2 incubator at 37 °C for 5 hours. Cells cultured with 0.2% DMSO were used as the blank control. The treated plates were centrifuged at 180 g for 10 minutes, the cell pellets were collected in FACS (flow cytometry) buffer, transferred to a "V"-bottom 96-well plate, and subjected to flow cytometry staining. Intracellular staining was performed using the BD Cytofix / CytopermPlus fixation / permeabilization kit (with the protein transport inhibitor BD GolgiPlug TM (BD Biosciences catalog number 555028)) according to the manufacturer's protocol. Ki67 expression was detected using Alexa 488 anti-mouse / human Ki67 antibody (catalog number 151204; Biolegend). Finally, the cells were suspended in staining buffer and Ki67-positive cells were obtained in a BD FACSVerse flow cytometer. The percentage increase in lymphocyte proliferation in the wells treated with the test sample was calculated using the following formula.

[0259]

[0260] The results are shown in Tables 18 - 21.

[0261] Table 18: Percentage increase in lymphocyte proliferation of the composition of Terminalia chebula extract containing zinc salts (T.Ch.Zn-1) and Withania somnifera 60% ethanol-water extract (W.S-1), and the composition of concentrated Terminalia chebula extract containing zinc salts (T.Ch.Zn-2) and Withania somnifera 60% ethanol-water extract (W.S-1)

[0262]

[0263] Table 19: Percentage increase in lymphocyte proliferation of the composition of Terminalia chebula extract containing zinc salt (T.Ch.Zn-1) and aqueous extract of Tinospora cordifolia (T.C-1) or 70% ethanol aqueous extract of Andrographis paniculata (A.P-1)

[0264]

[0265]

[0266] Table 20: Percentage increase in lymphocyte proliferation of the composition of Terminalia chebula extract containing magnesium salt (T.Ch.Mg) and ethanol extract of Withania somnifera (W.S-2) or 50% ethanol aqueous extract of Tinospora cordifolia (T.C-2) or aqueous extract of Andrographis paniculata (A.P-2)

[0267]

[0268] Table 21: Percentage increase in lymphocyte proliferation of the composition of Terminalia chebula extract containing calcium salt (T.Ch.Ca) and 80% methanol aqueous extract of Withania somnifera (W.S-4) or ethanol extract of Tinospora cordifolia (T.C-3) or ethanol extract of Andrographis paniculata (A.P-3)

[0269]

[0270]

[0271] Example 37: In vivo study on the regulation of inflammation and immune response in rats

[0272] In an acute lung injury model of 8 - 9 week - old male BALB / c mice weighing between 25 and 30 grams induced by bacterial lipopolysaccharide (lipopolysaccharide LPS from Escherichia coli O111:B4; Merck catalog number L2630 - 25MG), the regulatory effects of the test sample on inflammatory and immune responses were evaluated. The animals were randomly assigned to five groups. Each group contained eight animals (n = 8). Each group of animals received oral supplementation with 0.5% carboxymethyl cellulose (CMC) or the test sample for 7 days. Group 1 and Group 2 (G1 and G2) received CMC; Group 3 (G3), Group 4 (G4), and Group 5 (G5) received W.S - 1, T.Ch.Zn - 1, and comp - 36 at 100 mg / kg body weight (BW), respectively. On the 7th day, except for G1, each animal received an intraperitoneal (i.p.) injection dose of LPS (5 mg / kg BW) 1 hour after administration of the control / test sample. G1 animals received an intraperitoneal injection of sterile phosphate - buffered saline (PBS). Eight hours after LPS induction, blood samples were collected from each mouse by retro - orbital puncture under mild anesthesia. Then, the animals were euthanized with an overdose of thiopental, followed by exsanguination and autopsy. Spleens were collected for analysis of immune cells by flow cytometry (FACS). Serum biomarkers were measured from the blood samples.

[0273] Biomarker analysis (IL - 6 and total IgG): IL - 6 (Merck catalog number RAB0308) and total IgG (AbCam catalog number ab157719) enzyme - linked immunosorbent assay (ELISA) kits were used to measure IL - 6 and total IgG in serum samples of experimental animals, respectively. The assay procedures followed the manufacturer's protocols. Briefly, 100 μL of serum samples or standards were added to each well of a pre - coated 96 - well ELISA plate and incubated at room temperature for 2.5 hours. After incubation, the plate was washed with 1X wash buffer. 100 μL of detection antibody was added to each well and incubated with gentle shaking at room temperature for 1 hour. The washing step was repeated, and 100 μL of streptavidin solution was added; the plate was sealed and incubated with gentle shaking at room temperature for 45 minutes. After washing, TMB substrate was added; the plate was sealed and incubated with gentle shaking in the dark at room temperature for 30 minutes. 50 μL of stop solution was added, and the absorbance at 450 nm was measured in a microplate reader (Spectramax2e; Molecular Devices, San Jose, CA). The levels of IL - 6 and total IgG were quantified using the standard curves generated for each analyte.

[0274] Immune cell populations in spleen cell preparations: Using 1X RPMI medium in Gently crush the spleen of experimental mice on a 100 μm sterile cell filter (Corning, catalog number 352360) to obtain a single cell suspension of splenocytes. Count the number of splenocytes, and place 0.3 x 106 cells into a "V"-bottom 96-well plate using FACS buffer, and perform flow cytometry staining. Briefly, the cells were washed with FACS buffer and incubated with 0.3 μg FITC anti-mouse CD3 antibody (Biolegend, catalog number 100204), 70 ng PE anti-mouse CD4 antibody (Biolegend, catalog number 100408), and 70 ng APC anti-mouse CD8a antibody (Biolegend, catalog number 100712) together in the dark for 30 minutes. After incubation, the cells were washed with FACS buffer and fixed with 100 μL BD Cytofix fixation buffer (BD Biosciences, catalog number 554655) in the dark for 20 minutes. After incubation, the cells were washed with FACS buffer and resuspended in the same buffer, and samples were collected on a BD FACSVerse flow cytometer for analysis. Record the percentages of CD3, CD4, and CD8 positive cells in the splenocyte preparation.

[0275] The results are listed in Tables 22 - 24.

[0276] Table 22: Percentage increase in the number of CD3 + for Withania somnifera 60% ethanol-water extract (W.S-1), Terminalia chebula extract containing zinc salt (T.Ch.Zn-1), and their 1:1 ratio composition

[0277] Test sample <![CDATA[Average CD3 + Quantity (%)]]> <![CDATA[CD3 + Relative quantity of control]]> % increase compared to LPS control Control (G1) 36.4 100 NA LPS (G2) 32.1 88 NA LPS + W.S-1 (G3) 34.6 95.1 7.79 LPS + T.Ch.Zn-1 (G4) 34.4 94.5 7.17 LPS + Composition-36 (G5) 37.4 102.8 16.51

[0278] Table 23: CD4 + +CD8 + Percentage increase in the number of

[0279]

[0280]

[0281] Table 24: Percentage decrease in IL-6 and percentage increase in IgG caused by Withania somnifera 60% ethanol-water extract (W.S-1), Terminalia chebula extract containing zinc salt (T.Ch.Zn-1), and their 1:1 ratio composition

[0282]

Claims

1. A synergistic herbal composition for enhancing immunity, characterized in that, The active ingredients in the composition are: a first ingredient, Terminalia chebula extract, which is a metal salt containing chebulinic acid, chebualic acid and gallic acid; and a second ingredient selected from extracts derived from Withania somnifera, Tinospora cordifolia or Andrographis paniculata. Among them, the metal is selected from zinc, magnesium, calcium and potassium. The Terminalia chebula extract is obtained by extracting Terminalia chebula fruits with a 50% ethanol aqueous solution; the Withania somnifera extract is obtained by extracting the roots of Withania somnifera with a 60% ethanol aqueous solution, ethanol, an 80% methanol aqueous solution or an 80% acetone aqueous solution; the Tinospora cordifolia extract is obtained by extracting the stems of Tinospora cordifolia with water, a 50% ethanol aqueous solution or ethanol; the Andrographis paniculata extract is obtained by extracting the whole plant of Andrographis paniculata with water, a 70% ethanol aqueous solution or ethanol. The mass ratio of the Terminalia chebula extract to the second ingredient in the composition is 3:1, 2:1, 1:1, 1:2 or 1:

3.

2. The synergistic herbal composition according to claim 1, characterized in that, Among them, the improvement of immunity means the improvement of cellular immunity and humoral immunity.

3. The synergistic herbal composition according to claim 1, wherein The Terminalia chebula extract is standardized to contain: chebulinic acid in a weight percentage range of 8.79 - 22.26%, chebualic acid in a weight percentage range of 4.12 - 10.41%, gallic acid in a weight percentage range of 0.72 - 3.11%; metal ions in a weight percentage range of 0.5 - 5.0%.

4. The synergistic herbal composition according to claim 1, characterized in that, Among them, the Withania somnifera extract is standardized to total withanolides; the Tinospora cordifolia extract is standardized to 8-hydroxycolumbin; the Andrographis paniculata whole plant extract is standardized to andrographolide.

5. The synergistic herbal composition according to claim 4, wherein Among them, the weight percentage concentration of total withanolides varies within the range of 1.8% to 3.1%; the weight percentage concentration of 8-hydroxycolumbin varies within the range of 0.25% to 0.55%; the weight percentage concentration of andrographolide varies within the range of 4.33% to 33.5%.

6. The synergistic herbal composition according to claim 1, wherein It also contains excipients selected from pharmaceutically or dietetically acceptable ones.

7. The synergistic herbal composition according to claim 6, wherein The excipient is a diluent.

8. The synergistic herbal composition according to claim 6, wherein Among them, The pharmaceutically or dietetically acceptable excipients are selected from one or more of monosaccharides, disaccharides, polycarbohydrates, dextrins produced by hydrolysis of starch or glycogen, polyols, cellulose-based derivatives, silicates, metal stearates, organic acids, fatty acid esters, natural gums, vitamin B group, nicotinamide, calcium pantothenate, amino acids, proteins, pectin, agar, inorganic metal salts, natural pigments, flavors, class I and class II preservatives, and aqueous solutions and organic solutions of one or more combinations of the above components.

9. The synergistic herbal composition according to claim 8, wherein Among them, The monosaccharide is selected from one or more of glucose, dextrose, fructose, and galactose; the disaccharide is selected from one or more of sucrose, maltose, lactose, lactulose, trehalose, cellobiose, and chitobiose; the polycarbohydrate is starch; the dextrin is selected from one or more of yellow dextrin, white dextrin, and maltodextrin; the polyol is sugar alcohol; the cellulose-based derivative is selected from one or more of microcrystalline cellulose, hydroxypropyl methylcellulose, and hydroxyethyl cellulose; the silicate is selected from one or more of magnesium aluminum silicate, talc, and colloidal silica; the metal stearate is selected from one or more of calcium stearate, magnesium stearate, and zinc stearate; the organic acid is selected from one or more of citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and L-ascorbic acid; the fatty acid ester is polysorbate; the natural gum is selected from one or more of gum arabic, carrageenan, guar gum, and xanthan gum; the protein is selected from one or more of casein and gelatin; the inorganic metal salt is selected from one or more of sodium chloride, calcium chloride, dicalcium phosphate, zinc sulfate, and zinc chloride; the organic solution is a hydroalcoholic solution.

10. The synergistic herbal composition according to claim 9, wherein The starch is modified starch, and the hydroalcoholic solution is an alcoholic solution.

11. The synergistic herbal composition according to claim 10, characterized in that, The modified starch is selected from one or more of sodium starch glycolate, pregelatinized starch, and soluble starch; the sugar alcohol is selected from one or more of sorbitol, mannitol, inositol, xylitol, and isomaltulose.

12. The synergistic herbal composition according to any one of claims 1 to 11, characterized in that, Wherein the composition is formulated into a food in dry powder form or liquid form, and the dosage form of the food is selected from one of tablets, capsules, and soft chewables.

13. The synergistic herbal composition according to claim 12, wherein The food in liquid form is a beverage.

14. The synergistic herbal composition according to any one of claims 1 to 11, characterized in that, The composition is formulated into a dietary supplement, and the dosage form of the dietary supplement is selected from one of solid food, semi-solid food, and beverage.

15. The synergistic herbal composition according to claim 14, characterized in that, The solid food is selected from one of chocolate, chewing gum, gummy candy, cookies, and oatmeal; the semi-solid food is selected from one of cream, jam, yogurt, ice cream, and jelly; the beverage is selected from one of milk, tea, coffee, lactic acid bacteria beverage, and fruit juice.

16. The synergistic herbal composition according to any one of claims 1 to 11, characterized in that, Wherein the composition is formulated into a controlled-release tablet by one or more of the technologies of nanotechnology, microencapsulation, and colloidal carrier system, using a coating based on a controlled-release polymer.

17. A method for preparing a synergistic herbal composition for enhancing immunity, characterized in that, The active ingredients in the composition are: the first ingredient, Terminalia chebula extract, which is a metal salt containing chebulinic acid, chebulin, and gallic acid; and the second ingredient, which is selected from extracts derived from Withania somnifera, Tinospora cordifolia, or Andrographis paniculata; the mass ratio of the Terminalia chebula extract to the second ingredient in the composition is 3:1, 2:1, 1:1, 1:2, or 1:3; wherein, the method comprises the following steps: (i) Extract dried Terminalia chebula fruit powder with 50% aqueous ethanol solution; (ii) Elute the extract in step (i) through a resin column to obtain a concentrated extract; (iii) Treat the extract solution obtained in step i or ii with a metal compound; the metal compound is selected from zinc oxide, zinc carbonate, zinc hydroxide, magnesium oxide, magnesium carbonate, magnesium hydroxide, calcium hydroxide, calcium carbonate, potassium hydroxide, and potassium carbonate; (iv) Filter the solution; (v) Evaporate the solvent and dry the residue to obtain the Terminalia chebula extract containing metal salts; wherein the metal is selected from zinc, magnesium, calcium and potassium; (vi) Extract Withania somnifera roots with 60% aqueous ethanol solution, ethanol, 80% aqueous methanol solution or 80% aqueous acetone solution to obtain the extract of Withania somnifera; extract Tinospora cordifolia stems with water, 50% aqueous ethanol solution or ethanol to obtain the extract of Tinospora cordifolia; extract Andrographis paniculata whole plants with water, 70% aqueous ethanol solution or ethanol to obtain the extract of Andrographis paniculata; in the presence of excipients selected from pharmaceutically or dietetically acceptable ones, mix the Terminalia chebula extract containing metal salts with at least one extract derived from Withania somnifera or Tinospora cordifolia or Andrographis paniculata; (vii) Dry the product under vacuum to obtain the composition.

18. The method for preparing the synergistic herbal composition according to claim 17, wherein, The resin column is selected from PA-800, SP-700 and HP-20.

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