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1. WO2020222259 - SYNERGISTIC HERBAL COMPOSITIONS FOR SARCOPENIA

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“SYNERGISTIC HERBAL COMPOSITIONS FOR SARCOPENIA”

Technical field of the invention:

The invention relates to synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica for obtaining at least one health benefit selected from treating/alleviating Sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy. The invention also relates to a method of obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Background of the invention

Sarcopenia is age-associated deterioration of muscle mass and function caused by a wide range of physiological and pathological changes ranging from hormonal disorders to loss of subcellular homeostasis. Recent research indicates that mitochondrial dysregulation with advanced age plays a central role in the development of sarcopenia due to the multifactorial functions of this organelle in energy supply, redox regulation, crosstalk with nuclear gene expression and apoptosis (Marzeti, E. et al., Int. J. Biochem. Cell Biol., 2013, 45, 2288-2301). The decline in skeletal muscle mitochondrial capacity with aging has been extensively studied as a contributor to slower walking speed, fatigability, and sarcopenia. Release of pro-apoptotic factors, morphological alterations (fission, swelling), energy stress via reduced ATP, and increased mitochondrial reactive oxygen species (ROS) emission have all been reported during muscle atrophy in preclinical studies (Coen P. M. et al., Front. Physiol., 2019, 9, Article 1883). Treating sarcopenia involves therapeutic interventions to mitigate the loss of muscle mass via improving mitochondrial function and proteostatic maintenance. These therapies, which include targeting members of the transforming growth factor b (TGF-b) superfamily, testosterone, selective androgen receptor modulators (SARMS), and growth hormone (GH), among others, are currently or have been tested at various clinical trials (Motley, J. E. Calcif. Tissue Int., 2016, 98, 319-333). However, long term use of drug molecules against these targets would result in side effects and this is a major concern. In this regard, there is an urgent need to search for safer alternatives. Herbal products/formulations could be of greater use in this regard as they are known to possess required biological activity with improved safety.

EP 3453398A1 discloses composition containing Vigna angularis var. angularis for increasing muscle mass and enhancing muscular function or exercise performance through an effect of promoting mRNA or protein expression.

KR101919081B1 discloses a composition for improving muscular functions and for preventing or treating muscle diseases containing a human placenta extract as an active ingredient.

Hence, there is a continuous need in the art to provide alternative treatments comprising highly effective herbal compositions for the treatment of Sarcopenia.

Object of the invention:

Therefore, the object of the present invention is to provide synergistic and safe herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Another object of the invention is to provide a method of obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy, wherein the method comprises of treating the human with a synergistic and safe herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica.

Yet another objective of the invention is to provide use of the synergistic herbal compositions of the present inventions for obtaining at least one health benefit selected from treating/alleviating sarcopenia or controlling loss of muscle mass or improving exercise performance or stimulating muscle protein synthesis and/or improving recovery of muscle functionality after muscle atrophy.

Summary of the invention:

The present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Another aspect of the invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Other aspect of the invention provides a method of obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy, wherein the method comprises supplementing human with an effective dose of compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents.

Another aspect of the invention provides the use of synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Description of figures

Figure I. Bar diagram represents the mean± SE of body weights of the rats assigned to G1 - G5, measured at days 1, 5 and 12, as indicated. The groups G1 and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38

and Composition-39 respectively. n=7; p<0.05; # G2 vs. G1 using One way ANOVA followed by Dunnett’s test.

Figure II. Bar diagram represents the mean±SE of percentage change of body weights of the rats on day 12 (from day 1). The groups Gl and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38 and Composition-39 respectively. The negative percentage values above the bars indicate the % decrease of body weight from day 1; n=7, p<0.05; # G2 vs. Gl using One way ANOVA followed by Dunnett’s test.

Figure III. Bar diagram represents the mean±SD of grip strengths in G1-G5 rats at day 12. The groups G1 and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38 and Composition-39 respectively. n=7; p<0.05; # G2 vs. Gl; a (G3 vs. G2), and b (G4 vs. G2) indicate significance in inteigroup difference using One way ANOVA followed by Dunnett’s test.

Figure IV. Bar diagram represents the mean±SD of calf thickness of rats in GIGS at day 12. The groups Gl and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38 and Composition-39 respectively. n=7; p<0.05; # G2 vs. Gl; a (G3 vs. G2), b (G4 vs. G2) and c (G5 vs. G2) indicate significance in inteigroup difference using One way ANOVA followed by Dunnett’s test.

Figure V. Bar diagram represents improvements in weight (A) and thickness (B) of the gastrocnemius (GAS) muscles of rats supplemented with herbal compositions at day 12. The groups Gl and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38 and Composition-39 respectively. n=7; p<0.05; in panel A, numerical above the bars indicate the relative weight (in percent) of the GAS muscle. # G2 vs. Gl; b (G4 vs. G2) and c (G5 vs. G2) indicate significance in intergroup difference using One way ANOVA followed by Dunnett’s test.

Figure VI. Supplementation of herbal formulations improves gastrocnemius muscle fiber cross-sectional area in Dexamethasone induced sarcopenic rats. The groups Gl and G2 are vehicle control (CMC), and dexamethasone induced control respectively; the groups G3, G4 and G5 represent 0.1 mg/kg dexamethasone induced rats supplemented with 250mg/kg of Composition-37, Composition-38 and Composition-39 respectively. Panel A depicts representative photomicrographs showing Sirius Red stained cross-sections of the gastrocnemius muscles of the different group of animals, as indicated. The double-headed arrows indicate the representative diagonal distance in an individual muscle fiber. The bar diagram in panel B shows the mean±SD of cross-sectional areas (CSA, in mm2) of the muscle fibers in different groups, as indicated. The numericals in the above the bars indicate the relative CSA (in percent) of the GAS muscle fibers. n=7; c (G5 vs. G2) indicate significance (p<0.05) in intergroup difference using One way ANOVA followed by Dunnett’s test.

Detailed description of the invention:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

To address the problem and to provide a safe herbal composition(s) for sarcopenia, Mitochondrial Biogenesis, a cell based assay has been chosen to demonstrate the efficacy of the inventive compositions for treating/alleviating sarcopenia. Additionally, the efficacy of the herbal compositions in improving the parameters, due to muscle atrophy, such as percent body weight change, Grip strength, Calf and gastrocnemius muscle thickness measurements in Dexamethasone induced muscle atrophy in Sprague Dawley rats, were also evaluated.

The inventors of the current application randomly screened a large number of plant extracts and their fractions for increase of mitochondrial biogenesis and found that the extract(s) and fraction(s) derived from Syzygium cumini, Rubia cordifolia and Mangifera indica show potent dose dependent activities.

A brief summary on each of the plant material used in the preferred embodiment of the invention is provided herein below.

Rubia cordifolia·. It is most commonly known as Indian Madder and in ancient world it was popularly known as manjistha. It has reputation as an efficient blood purifier. It is a perennial climbing herb belonging to family mbiaceae and stems are slender, rough and become woody at base. Traditionally decoction of stem is used as vermifuge and in Tibetan system of medicine the stem is used for treatment of blood disorders. Different phytoconstituents such as anthraquinones and their glycosides, naphthoquinones, terpenes, bicyclic hexapeptides, iridoids, carboxylic acids (malic, citric, quinic, rosmarinic acid(s) and carbohydrates (xylose, ribose, fructose, glucose, sucrose, primverose) were isolated from various parts of R cordifolia. Pharmacologically, the dried stem is used in blood, skin and urogenital disorders, dysentery, piles, ulcers, inflammations erysipelas, skin diseases and rheumatism. Stems bark showed anti-bacterial activity (Verma, A. et al., International Journal of Pharmaceutical Sciences and Research, 2016, 7, 2720-2731).

Manjistin, Rubiadin and 2-methyl- 1, 3, 6-trihydroxy-9, 10-anthraquinone-3 -O-(6 0-acetyl)-a-rhamnosyl-(l-2)-b-glucoside are isolated from the extracts of Rubia cordifolia (H. Itokawa et al., Phytochemistry, 1989, 28(12), 3465-3468). The extracts used in the current invention are standardized to at least one of the markers by HPLC method of analysis.


Manjistin (Manjistin) Rubiadin 2-methyl-1.3.6-trihydroxy-9, 10-anthraquino»e- 3-O-(6,-O-acetyl)-a hannosyl- (1 -2 >b- glucoside

Syzygium cumini : It is a large evergreen tree (also known as Syzygium jambolanum and Eugenia cumini) which belongs to genus syzygium, family

Myrtaceae and kingdom Plantae. It was a native of India or the East Indies originally, but these trees are found growing throughout the Asian subcontinent,

Eastern Africa, South America etc. (Swami, S.B. et al., Food and Nutrition

Sciences, 2012, 3, 1100-1117). It mainly contains Phytoconstituents like gallic acid, cyanidin glycoside, glycoside jamboline, triterpenoids, tannins, gallitannins, essential oils, myricetine, b-sitosterol, myricyl alcohol etc. (Singh, A et al.,

International Journal of Innovative Pharmaceutical Sciences and Research, 2018,

6, 32-47). The fruit pulp contains Anthocyanins, delphinidin, petunidin, malvidin-diglucosides. Traditionally the jambul fruits, leaves, seeds, and bark are all used in ayurvedic medicine. Charaka used seeds, leaves and fruits in decoctions for diarrhoea and the bark as an astringent. Sushruta prescribed the fruit internally for obesity, vaginal discharges and menstrual disorders, cold infusion in intrinsic haemorrhage. Various Pharmacological activities have been reported on fruits such as lipid peroxidation inhibition activity, anti-cancer, stomachic, carminative, antiscorbutic and diuretic, radioprotective, etc.

Mangifera indica: M. indica, commonly known as mango and aam, belongs to Anacaidiaceae, family in kingdom -Planate . The fruit has thick yellow pulp, single seed and thick yellowish-red skin when ripe. The seed is solitary, ovoid or oblong, encased in a hard, compressed fibrous endocarp. It is native to tropical Asia and cultivated in the Indian subcontinent for over 4000 years. It is now found naturalized in most tropical countries. Mangifera indica seed consists of a tenacious coat enclosing the kernel. The chemical constituents in seed include polyphenols such quercetin, Kaempferol, gallic acid, tannin, xanthone and the seeds are reported to have various pharmacological activities like anti-diarrheal, anti-inflammatory, anti-bacterial, anti-diabetic, anti-fungal, anti-cancer, hepatoprotective etc. Seed kernel is used to treat hemorrhages and bleeding hemorrhoids, bums, and Asthma.

Pentagalloyl glucose ( 1 ,2,3,4,6-pentagalloylglucose) is isolated as one of the major gallotannin in Mangifera indica seed. The extracts from Mangifera indica used in the current invention are standardized to Pentagalloyl glucose by HPLC method of analysis (A.B.B. Kassi et al., Ini. J. Biol. Chem. Sci., 2014, 8, 1885-1895).


Chemical structure of Pentagalloyl glucose

Source of the herbs used in the invention as follows: - 1) Rubia cordifolia procured from the market.

2) Syzygium cumini collected from Ampani village of Kalahandi district, Odisha.

3) Mangifera indica collected from Gudavalli village of krishna district, Andhra Pradesh.

The dried Rubia cordifolia stem was pulverized and the powder was extracted separately with various solvents such as 50% aqueous ethanol, ethanol, water, 50% aqueous acetone, 50% aqueous methanol and ethyl acetate to obtain 50% aqueous ethanol extract (R.C-1), ethanol extract (R.C-2), water extract (R.C-3), 50% aqueous acetone extract (R..C-4), 50% aqueous methanol extract (R.C-5) and ethyl acetate extract (R..C-6) respectively.

Similarly, the dried Syzygium cumini whole fruit was pulverized and the powder was extracted separately with various solvents such as 50% aqueous ethanol, ethanol, water, 50% aqueous acetone and 50% aqueous methanol to obtain 50% aqueous ethanol extract (S.C-1), ethanol extract (S.C-2), water extract (S.C-3), 50% aqueous acetone extract (S.C-4) and 50% aqueous methanol extract (S.C-5) respectively.

In a similar manner, dried Mangifera indica seed kernel was pulverized and the powder was extracted separately with various solvents such as 90% aqueous ethanol, ethanol, 50% aqueous ethanol, water, acetone, 90% aqueous methanol and ethyl acetate to obtain 90% aqueous ethanol extract (M.I-1), ethanol extract (M.I-2), 50% aqueous ethanol extract (M.I-3), and water extract (M.I-4), acetone extract (M.I-5), 90% aqueous methanol extract (M.I-6) and ethyl acetate extract (M.I-7) respectively.

These extracts of Syzygium cumini, Rubia cordifolia and Mangifera indica were evaluated for their efficacy in increasing mitochondrial biogenesis using in vitro cellular model using rat skeletal (L6) cells. The extracts from all the three herbs were observed to potently increase mitochondrial biogenesis activity in L6 rat skeletal muscle cell lines, when compared to the control. For example, Syzygium cumini whole fruit 50% aqueous ethanol extract (S.C-1), Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1) and Mangifera indica seed kernel 90% aqueous ethanol extract (M.I-1) at the treatment concentrations of 75 ng/mL showed 11.58%, 12.67% and 10.11% increase in mitochondrial biogenesis respectively over the control.

These individual extract(s) or their fraction(s) were then evaluated to explore the feasibility of obtaining a synergistic efficacy between the individual extract and/or fraction. Accordingly, the compositions C-l to C-5 were prepared by combining Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1) and Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1) in 3:1, 2:1, 1:1, 1:2, 1:3 ratios respectively. Similarly, the compositions C-6 to C-10 were prepared by combining Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1) and Mangifera indica seed kernel 90% aqueous ethanol extract (M.I-1) in 3:1, 2:1, 1: 1, 1:2, 1:3 ratios respectively. Further, the compositions C-ll to C-15 were prepared by combining Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1) and Mangifera indica seed kernel 90% aqueous ethanol extract (M.I-1) in 3:1, 2: 1, 1:1, 1:2, 1:3 ratios respectively. Similarly the compositions C-16 to C-36 were also prepared using various solvent extracts of Syzygium cumini, Rubia cordifolia and Mangifera indica as disclosed in experimental section. The compositions (compositions 1-36) were tested in vitro along with the corresponding individual ingredients to evaluate their efficacy in increasing mitochondrial biogenesis activity in comparison with the corresponding individual ingredients. The compositions unexpectedly showed better efficacy compared to the corresponding individual ingredients suggesting synergistic activity between the extracts of Syzygium cumini, Rubia cordifolia and Mangifera indica.

For example, Syzygium cumini 50% aqueous ethanol extract (S.C-1) at 75 ng/mL and Rubia cordifolia 50% aqueous ethanol extract (R.C-1) at 25 ng/mL concentration showed 11.58% and 4.22% increase in Mitochondrial biogenesis respectively over the control. The composition-1 containing Syzygium cumini 50% aqueous ethanol extract (S.C-1) and Rubia cordifolia 50% aqueous ethanol extract (R.C-1) in the ratio of 3:1 at 100 ng/mL showed 26.05% increase in Mitochondrial biogenesis, which is significantly better than the additive effect 15.80% (11.58%+4.22%) calculated from the increase in Mitochondrial biogenesis showed by the corresponding individual ingredients. The compositions-2 & 3, and composition-5 (C-2, C-3 and C-5) containing these two extracts (S.C-1 and R.C-1) at ratios 2:1, 1:1 and 1:3 respectively also exhibited synergism in increasing mitochondrial biogenesis, when compared to the increase shown by each of their corresponding individual ingredient concentrations as summarized in Table 3.

Similarly, Syzygium cumini 50% aqueous ethanol extract (S.C-1) at 75 ng/mL and Mangifera indica 90% aqueous ethanol extract (M.I-1) at 25 ng/mL concentration showed 11.58% and 3.37% increase in Mitochondrial biogenesis respectively over the control. The composition-6 containing Syzygium cumini 50% aqueous ethanol extract (S.C-1) and Mangifera indica 90% aqueous ethanol extract (M.I-1) in the ratio of 3:1 at 100 ng/mL showed 20.27% increase in Mitochondrial biogenesis, which is significantly better than the additive effect 14.95% (11.58%+-3.37%) calculated from the increase in Mitochondrial biogenesis showed by the corresponding individual ingredients. The composition-7 and composition- 10 (C-7 and C-10) containing these two extracts (S.C-1 and M.I-1) at ratios 2:1 and 1:3 respectively also exhibited synergism in increasing mitochondrial biogenesis, when compared to the increase shown by each of their corresponding individual ingredient concentrations as summarized in Table 4.

Similarly, Rubia cordifolia 50% aq ethanol extract (R.C-1) at 75 ng/mL and Mangifera indica 90% aq ethanol extract (M.I-1) at 25 ng/mL concentration showed 12.67% and 3.37% increase in Mitochondrial biogenesis respectively over the control. The composition- 11 containing Rubia cordifolia 50% aq ethanol extract (R.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1) in the ratio of 3: 1 at 100 ng/mL showed 22.15% increase in Mitochondrial biogenesis, which is significantly better than the additive effect 16.04% (12.67%+3.37%) calculated from the increase in Mitochondrial biogenesis showed by the corresponding individual ingredients. The compositions-12 & 14 and composition-15 (C-12, C-14 and C-15) containing these two extracts (R.C-1 and M.I-1) at ratios 2:1, 1:2 and 1:3 respectively also exhibited synergism in increasing mitochondrial biogenesis, when compared to the increase shown by each of their corresponding individual ingredient concentrations as summarized in Table 5.

The other randomly selected compositions among C16 to C36 containing various extracts of Syzygium cumirti, Rubia cordifolia and Mangifira indica also exhibited synergism in increasing mitochondrial biogenesis, when compared to the increase shown by each of their corresponding individual ingredient concentrations as summarized in Tables 6-7.

Hence, the said compositions (composition 1-36) unexpectedly showed better efficacy in increasing mitochondrial biogenesis when compared to their corresponding individual ingredients suggesting that these individual extract(s) and fiaction(s) have the tendency to show synergism when combined together.

Formulations: The present invention also provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fiaction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; are formulated using at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents, for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy; wherein the pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents are selected from monosaccharide’s such as glucose, dextrose, fructose, galactose etc.; Disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose etc.; Polycarbohydrates such as Starch and modified starch such as Sodium starch glycolate, pre-gelatinized starch, soluble starch, and other modified starches; Dextrins that are produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, Maltodextrin etc.; Polyhydric alcohols or sugar alcohols such as but not limited to Sorbitol, mannitol, inositol, xylitol, isomalt etc.; cellulose based derivatives such as but not limited to microcrystalline cellulose, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose etc.; silicates such as but not limited to neusilin, veegum, Talc, colloidal silicon dioxide etc.; metallic stearates such as but not limited to calcium stearate, magnesium stearate, zinc Stearate etc.; Organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid etc.; Fatty acid esters and esters of poly sorbate, natural gums such as but not limited to acacia, carrageenan, Guar gum, Xanthan gum etc.; vitamin B group, nicotinamide, calcium pantothenate, amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organic metal salts such as but not limited to sodium chloride, calcium chloride, dicalcium phosphate, zinc Sulphate, zinc chloride etc.; Natural pigments, flavors, Class I & Class P preservatives and aqueous, alcoholic, hydro-alcoholic, organic solutions of above listed ingredients alone or in combination.

Evaluation of herbal compositions in dexamethasone-induced skeletal muscle atrophy (sarcopenia) in rats: Sarcopenia is the degenerative loss of skeletal muscle mass (0.5-1% loss per year after the age of 50), quality, and strength associated with aging. Sarcopenia is primarily characterized first by muscle atrophy (a reduction in muscle mas(s), along with a reduction in muscle tissue quality, characterized by such factors as replacement of muscle fibers with fat, an increase in fibrosis, changes in muscle metabolism, oxidative stress, and degeneration of the neuromuscular junction and leading to progressive loss of muscle function and frailty. The most important causative factors of Sarcopenia arc aging, prolonged chemotherapy, lack of proper nutrition. For example, a prolonged use of corticosteroids such as Dexamethasone in the treatment of

various pathological conditions including rheumatic problems, skin diseases, severe allergies, asthma, chronic obstructive lung disease etc. results in developing Sarcopenia conditions such as severe muscle atrophy and loss of muscle mass. In an in vivo proof-of-concept study, we have evaluated tire herbal compositions for their ability to ameliorate the typical Sarcopenia conditions in a Dexamethasone-induced Sarcopenia model in Sprague Dawley rats. The effectiveness of the inventive formulation to modulate the body weight, body weight change, grip strength, calf muscle thickness, gastrocnemius muscle weight and thickness and gastrocnemius muscle fiber sizes is summarized in figures I to

VI.

Effect on body weight change: Figure I depicts the mean body weight of the experimental groups at day 12 and the Figure II demonstrates the percent body weight changes at day 12 (from the day 1) in the treatment groups supplemented with herbal compositions. Dexamethasone treatment (G2) significantly reduced the mean body weight (p<0.001) in comparison with normal control (Gl) rats (figure 1); and the change in body weight in G2 is also significant (compared with Gl) at day 12 from beginning of the experiment (day 1). The composition-37, composition-38 and composition-39 supplemented groups showed improvements in their body weight, when compared with the Dexamethasone induced group (G2).

Efficacy of herbal compositions to improve Grip strength: Figure IP demonstrates that supplementation of the herbal compositions improves grip strengths in dexamethasone treated rats. The rats in G2 showed significant reduction (p<0.05) in grip strength, compared with the vehicle control group (Gl). The rats supplemented with composition-37 (G3) and composition-38 (G4) showed significant improvements (p<0.05) in grip strength, compared with the G2 rats, while composition-39 (G5) exhibited moderate increase in grip strength.

Efficacy of herbal compositions supplementation in increasing calf muscle thickness: Figure IV demonstrates that a 12-days supplementation of the herbal compositions enhanced the calf thickness of the dexamethasone treated rats. Dexamethasone treatment (G2) caused significant reduction in calf size (p<0.05), when compared with the vehicle control rats (Gl). The animals in the treatment groups G3, G4 and G5 supplemented with the herbal compositions-37, 38 and 39 respectively showed significantly increased calf sizes in comparison with G2.

The efficacy of the herbal compositions in increasing the gastrocnemius muscle weight and thickness: The results summarized in Figure 5A demonstrates that at the end of the study, the rats supplemented with the compositions had increased gastrocnemius (GAS) weight and improved muscle sizes in comparison with the Dexamethasone treated rats (G2). Dexamethasone treatment significantly (p<0.05) reduced the GAS muscle weight and size in G2 rats. Figure 5A and 5B demonstrate the improvements in muscle weight and size, respectively, in herbal composition supplemented rats, at the end of the study. In comparison with G2, Composition-39 supplementation (G5) significantly improved GAS weight and size, and composition-38 supplemented rats (G4) showed significant improvement (p<0.05) in size and moderate increase in GAS weight. Composition-37 supplemented (G3) rats showed moderate improvements in GAS weight and size at the end of the study.

The efficacy of the herbal formulations supplementation in increasing the gastrocnemius muscle fiber sizes: Figure 6A depicts the representative photomicrographs of the Sirius red stained cross-sections of the gastrocnemius muscle tissues of the rats in groups Gl to G6. Further, the morphometric analysis revealed that the average cross-sectional area (CSA) of G2 was reduced to 87.7% from 100%, the arbitrary value assigned to CSA of the muscle fibers in Gl animals. In comparison with the G2 rats (87.7%), the muscle fiber CSA in G3, G4 and G5 were increased to 89.4%, 107.3%, and 117.1% (p<0.05), respectively. These observations clearly suggest that supplementation of the herbal

formulations increased gastrocnemius muscle fiber sizes in the Dexamethasone induced Sarcopenia rats. Together, these observations strongly indicate that the foimulations alleviated the muscle atrophy and other conditions associated with Sarcopenia in the in vivo preclinical model.

Therefore, in an important embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

In another embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fiaction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the concentration of the first ingredient in the composition varies in the range of 10%-90% by weight and the concentration of the second ingredient varies in the range of 90%-10% by weight.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the concentration of extract, fraction, phytochemical and mixtures thereof derived from Rubia cordifolia in the composition varies in the range of 10%-90% by weight and concentration of the extract, fraction, phytochemical and mixtures thereof derived from Mangifera indica in the composition varies in the range of 10%-90% by weight.

In an exemplary embodiment, as demonstrated in the invention encompasses stem extracts / fractions of Rubia cordifolia and seed kernel extracts/fractions of Mangifera indica in 2:1 ratio along with suitable excipients/caniers.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the concentration of extract, fraction, phytochemical and mixtures thereof derived from Syzygium cumini in the composition varies in the range of 10%-90% by weight and concentration of the extract, fraction, phytochemical and mixtures thereof derived from Rubia cordifolia in the composition varies in the range of 10%-90% by weight.

In an exemplary embodiment, as demonstrated in the invention encompasses fruit extracts/fractions, phytochemical(s) and mixtures thereof of Syzygium cumini and stem extracts/fractions of Rubia cordifolia in 2: 1 ratio along with suitable excipients/caniers.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the concentration of extract, fraction, phytochemical and mixtures thereof derived from Syzygium cumini in the composition varies in the range of 10%-90% by weight and concentration of the extract, fraction, phytochemical and mixtures thereof derived from Mangifera indica in the composition varies in the range of 10%-90% by weight.

In an exemplary embodiment, as demonstrated in the invention encompasses fruit extracts/fractions, phytochemical(s) and mixtures thereof of Syzygium cumini and seed kernel extracts/fractions of Mangifera indica in 2: 1 ratio, along with suitable excipients/caniers.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the extracts or fractions are standardized to at least one phytochemical reference marker compound or biological active marker in the extract or fraction; wherein phytochemical marker compound or total of phytochemical compounds is in the concentration range of 0.1% to 99% by weight of the extract.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the said compositions are standardized to at least one phytochemical compound of Rubia cordifolia, which include but not limited to Munjistin (Manjistin), Rubiadin and 2-methyl- 1 ,3 ,6-trihydroxy-9, 10-anthraquinone-3 -0-(6 '-O-acetyl)-a-rhamnosyl-( 1 -2)-b-glucoside; wherein the phytochemical is in the concentration range of 0.05% to 10% by weight of the composition.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the said compositions are standardized to at least one phytochemical compound of Mangifera indica, which include but not limited to Pentagalloyl glucose; wherein the Pentagalloyl glucose is in the concentration range of 0.05% to 10% by weight of the composition

In another embodiment, the invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica ; wherein the extract or fraction is obtained from at least one plant part selected from the group comprising leaves, stems, tender stems, tender twigs, aerial parts, whole fruit, fruit peel rind, seeds, flower heads, root, bark, hardwood or whole plant or mixtures thereof.

In another embodiment, the invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the extract or fraction is produced using at least one solvent selected from the group comprising C1-C5 alcohols like ethanol, methanol, n-propanol, isopropyl alcohol; ketones like acetone, methyl isobutyl ketone, chlorinated solvents like methylene dichloride and chloroform, water and mixtures thereof; C1-C7 hydrocarbons such as hexane; esters like ethyl acetate and the like and mixtures thereof.

In the other embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents for obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

In another embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents for obtaining at least one health benefit selected from treating/alleviating Sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy; wherein the pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents are selected from monosaccharide’s such as glucose, dextrose, fructose, galactose etc.; Disaccharides such as but not limited to sucrose, maltose, lactose, lactulose, trehalose, cellobiose, chitobiose etc.; Polycarbohydrates such as Starch and modified starch such as Sodium starch glycolate, pre -gelatinized starch, soluble starch, and other modified starches; Dextrins that are produced by hydrolysis of starch or glycogen such as yellow dextrin, white dextrin, Maltodextrin etc.; Polyhydric alcohols or sugar alcohols such as but not limited to Sorbitol, mannitol, inositol, xylitol, isomalt etc.; cellulose based derivatives such as but not limited to microcrystalline cellulose, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose etc.; silicates such as but not limited to neusilin, veegum, Talc, colloidal silicon dioxide etc.; metallic stearates such as but not limited to calcium stearate, magnesium stearate, zinc Stearate etc.; Organic acids such as citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid etc.; Fatty acid esters and esters of poly sorbate, natural gums such as but not limited to acacia, carrageenan, Guar gum, Xanthan gum etc.; vitamin B group, nicotinamide, calcium pantothenate, amino acids, proteins such as but not limited to casein, gelatin, pectin, agar; organic metal salts such as but not limited to sodium chloride, calcium chloride, dicalcium phosphate, zinc Sulphate, zinc chloride etc.; Natural pigments, flavors, Class I & Class P preservatives and aqueous, alcoholic, hydro-alcoholic, organic solutions of above listed ingredients alone or in combination.

In another embodiment, the present invention provides synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; wherein the composition(s) are formulated into a dosage form selected from dry powder form, liquid form, beverage, water dispersible formulation, food product, dietary supplement or any suitable form such as a tablet, a capsule, a soft chewable or gummy bear.

In another embodiment of the invention, the composition(s) as disclosed above can be formulated into nutritional/dietary supplements that can be contemplated/made into the dosage form of healthy foods, or food for specified health uses such as solid food like chocolate or nutritional bars, water dispersible beverage, semisolid food like cream, jam, or gel or beverage such as refreshing beverage, lactic acid bacteria beverage, drop, candy, chewing gum, gummy candy, yoghurt, ice cream, pudding, soft adzuki bean jelly, jelly, cookie, tea, soft drink, juice, milk, coffee, cereal, snack bar and the like.

A method of obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy in a human is disclosed herein, wherein the method comprises supplementing the human in need with an effective dose of a composition comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents.

In another embodiment, the present invention provides a method of obtaining at least one health benefit selected from treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy in humans or animals, wherein the method comprises supplementing humans or animals with an effective dose of a compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica ; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents; wherein supplementing the human or animal with a suitable dose is in the form of tablets, capsules, controlled release tablets or using controlled release polymer-based coatings by the techniques including nanotechnology, microencapsulation, colloidal carrier systems and other drug delivery systems for obtaining the desired therapeutic benefit.

In another embodiment, the present invention provides use of synergistic herbal compositions comprising at least two herbal ingredients selected from the extract(s), fraction(s), phytochemical(s) and mixtures thereof derived from Syzygium cumini, Rubia cordifolia and Mangifera indica; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents for treating/alleviating sarcopenia, preventing muscle loss, improving muscle mass, improving exercise performance, stimulating muscle protein synthesis and improving recovery of muscle functionality after muscle atrophy.

Those of ordinary skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed herein, but is intended to cover modifications within the objectives and scope of the present invention as defined in the specification. The presented examples illustrate the invention, but they should not be considered to limit the scope of the invention in any way.

Examples

Example 1: Preparation of Rubia cordifolia extracts

(a) 50% aqueous ethanol extract: The dried Rubia cordifolia stem (100 g) was pulverized and the powder raw material was added to 50% aqueous ethanol (700 mL) at rt. The mixture was stirred at ambient temperature for 1 h and the extract was filtered through celite. The extraction process was repeated twice with 50% aqueous ethanol (2 x 500 mL) under similar conditions. The combined extracts were filtered and evaporated under reduced pressure to obtain extract concentrate, which was further subjected to freeze drying to give the 50% aqueous ethanol extract of Rubia cordifolia as a pale brown color powder (R.C-1; 16.4 g).

(b) Ethanol and water extracts: The ethanol extract (R.C-2; 6.3 g) and water extract (R.C-3; 9.7 g) were obtained from 100 g raw material by adopting similar procedure using ethanol and water as extraction solvents respectively.

(c) 50% aqueous acetone extract: The 50% aq acetone extract (R.C-4; 12.8 g) was obtained from 100 g raw material by adopting similar procedure using 50% aq acetone as extraction solvent.

(d) 50% aqueous methanol extract: The 50% aq methanol extract (R.C-5; 11.8 g) was obtained from 100 g raw material by adopting similar procedure using 50% aq methanol as extraction solvent.

(e) Ethyl acetate extract: The ethyl acetate extract (R.C-6; 1.2 g) was obtained from 100 g raw material by adopting similar procedure using ethyl acetate as extraction solvent.

Standardization: The above Rubia cordifolia stem extracts were standardized to Munjistin (manjistin), Rubiadin and 2-methyl- 1, 3, 6-trihydroxy-9, 10-

anthraquinone-3-O-(6’-O-acetyl)-a-rhamnosyl-(l-2)-b-glucoside by analytical HPLC method and the results are summarized in Table 1.

Table 1: Details of Rubia cordifolia extracts

S. No. Extract Solvent for Weight of HPLC

code extraction the product Munjistin Rubiadin Anthraquinone diglycoside

1 R.C-1 50% aqueous 16.4 g 5.49% 1.98% 0.59%

ethanol

2 R.C-2 Ethanol 6.3 g 0.93% 4.50% 0.06%

3 R.C-3 Water 9.7 g 0.27% 0.07% 0.03%

4 R.C-4 50% aqueous 12.8 g 5.21% 1.11% 0.05%

acetone

5 R.C-5 50% aqueous 11.8 g 1.91% 0.37% 0.03%

methanol

6 R.C-6 Ethyl acetate 1 2 g 1.15% 5.69% 0.36%

Example 2: Preparation of Syzygium cumini extracts

(a) 50% aqueous ethanol extract: The dried Syzygium cumini whole fruit (100 g) was pulverized and the powder raw material was added to 50% aqueous ethanol (700 mL) at rt. The mixture was stirred at ambient temperature for 1 h and the extract was filtered through celite. The extraction process was repeated twice with 50% aqueous ethanol (2 x 500 mL) under similar conditions. The combined extracts were filtered and evaporated under reduced pressure to obtain extract concentrate, which was further subjected to freeze drying to give the 50% aqueous ethanol extract of Syzygium cumini as apale brown color powder (S.C-1; 22.1 g).

(b) Ethanol and water extracts: The ethanol extract (S.C-2; 8.9 g) and water extract (S.C-3; 22.5 g) were obtained from 100 g raw material by adopting similar procedure using ethanol and water as extraction solvents respectively. (c) 50% aqueous acetone extract: The 50% aq acetone extract (S.C-4; 31.2 g) was obtained from 100 g raw material by adopting similar procedure using 50% aq acetone as extraction solvent.

(d) 50% aqueous methanol extract: The 50% aq methanol extract (S.C-5; 26.0 g) was obtained from 100 g raw material by adopting similar procedure using 50% aq methanol as extraction solvent.

Example 3: Preparation of Mangifera indica extracts

(a) 90% aqueous ethanol extract: The dried Mangifera indica seed kernel (100 g) was pulverized and the powder raw material was added to 90% aqueous ethanol (700 mL) at rt. The mixture was stirred at ambient temperature for 1 h and the extract was filtered through celite. The extraction process was repeated twice with 90% aqueous ethanol (2 x 500 mL) under similar conditions. The combined extracts were filtered and evaporated under reduced pressure to obtain extract concentrate, which was further subjected to freeze drying to give the 90% aqueous ethanol extract of Mangifera indica as a pale brown color powder (M.I-1; 11.4 g).

(b) Ethanol and 50% aq ethanol extracts: The ethanol extract (M.I-2; 13.0 g) and 50% aqueous ethanol extract (M.I-3; 24.1 g) were obtained from 100 g raw material by adopting similar procedure using ethanol and 50% aq ethanol as extraction solvents respectively.

(c) Water extract: The water extract (M.I-4; 21.5 g) was obtained from 100 g raw material by adopting similar procedure using water as extraction solvent.

(d) Acetone extract: The acetone extract (M.I-5; 14.6 g) was obtained from 100 g raw material by adopting similar procedure using acetone as extraction solvent.

(e) 90% aqueous methanol extract: The 90% aq methanol extract (M.I-6; 13.0 g) was obtained from 100 g raw material by adopting similar procedure using 90% aq methanol as extraction solvent.

(f) Ethyl acetate extract The ethyl acetate extract (M.I-7; 11.2 g) was obtained from 100 g raw material by adopting similar procedure using ethyl acetate as extraction solvent.

Standardization: The above Mangifera indica seed kernel extracts are standardized to pentagalloyl glucose, a gallotannin, by analytical HPLC method and the results were summarized in Table 2.

Table 2: Details of Mangifera indica extracts


Example 4: Preparation of compositions of Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1) and Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1)

Composition-1 (C-1): The composition- 1 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1) in the ratio of 3: 1.

Composition-2 (C-2): The composition-2 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1) in the ratio of 2: 1.

Composition-3 (C-3): The composition-3 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1) in the ratio of 1 : 1.

Composition-4 (C-4): The composition-4 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1) in the ratio of 1:2.

Composition-5 (C-5): The composition-5 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1) in the ratio of 1:3.

Example 5: Preparation of compositions of Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1) and Mangifera indica seed kernel 90% aqueous ethanol extract (M.I-1)

Composition-6 (C-6): The composition-6 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1) in the ratio of 3: 1.

Composition-7 (C-7): The composition-? was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1) in the ratio of 2: 1.

Composition-8 (C-8): The composition-8 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1) in the ratio of 1 : 1.

Composition-9 (C-9): The composition-9 was prepared by combining Syzygium cumini 50% aq ethanol extract (S.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1) in the ratio of 1:2.

Composition- 10 (C-10): The composition- 10 was prepared by combining Syzygium cumini 50% ethanol extract (S.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 1:3.

Example 6: Preparation of compositions of Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1) and Mangifera indica seed kernel 90% aqueous ethanol extract (M.I-1)

Composition- 11 (C- 11): The composition- 11 was prepared by combining Rubia cordifolia 50% ethanol extract (R.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 3: 1.

Composition- 12 (C-12): The composition-12 was prepared by combining Rubia cordifolia 50% ethanol extract (R.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 2: 1.

Composition- 13 (C-13): The composition- 13 was prepared by combining Rubia cordifolia 50% ethanol extract (R.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 1: 1.

Composition- 14 (C-14): The composition-14 was prepared by combining Rubia cordifolia 50% ethanol extract (R.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 1:2.

Composition- 15 (C-15): The composition- 15 was prepared by combining Rubia cordifolia 50% ethanol extract (R.C-1) and Mangifera indica 90% ethanol extract (M.I-1) in the ratio of 1:3.

Example 7: Preparation of compositions of Syzygium cumini fruit 50% aqueous acetone extract (S.C-4) and Rubia cordifolia stem ethanol extract (R.C-2)

Composition- 16 (C-16): The composition- 16 was prepared by combining Syzygium cumini 50% aq acetone extract (S.C-4) and Rubia cordifolia ethanol extract (R.C-2) in the ratio of 3: 1.

Composition- 17 (C-17): The composition- 17 was prepared by combining Syzygium cumini 50% aq acetone extract (S.C-4) and Rubia cordifolia ethanol extract (R.C-2) in the ratio of 1: 1.

Composition- 18 (C-18): The composition- 18 was prepared by combining Syzygium cumini 50% aq acetone extract (S.C-4) and Rubia cordifolia ethanol extract (R.C-2) in the ratio of 1:3.

Example 8: Preparation of compositions of Syzygium cumini fruit 50% aqueous methanol extract (S.C-5) and Mangifera indica seed kernel 50% aqueous ethanol extract (M.I-3)

Composition- 19 (C-19): The composition- 19 was prepared by combining Syzygium cumini 50% methanol extract (S.C-5) and Mangifera indica 50% ethanol extract (M.I-3) in the ratio of 3: 1.

Composition-20 (C-20): The composition-20 was prepared by combining Syzygium cumini 50% methanol extract (S.C-5) and Mangifera indica 50% ethanol extract (M.I-3) in the ratio of 1: 1.

Composition-21 (C-21): The composition-21 was prepared by combining Syzygium cumini 50% methanol extract (S.C-5) and Mangifera indica 50% ethanol extract (M.I-3) in the ratio of 1:3.

Example 9: Preparation of compositions of Rubia cordifolia stem water extract (R.C-3) and Mangifera indica seed kernel acetone extract (M.I-5)

Composition-22 (C-22): The composition-22 was prepared by combining Rubia cordifolia water extract (R.C-3) and Mangifera indica acetone extract (M.I-5) in the ratio of 3: 1.

Composition-23 (C-23): The composition-23 was prepared by combining Rubia cordifolia water extract (R.C-3) and Mangifera indica acetone extract (M.I-5) in the ratio of 1: 1.

Composition-24 (C-24): The composition-24 was prepared by combining Rubia cordifolia water extract (R.C-3) and Mangifera indica acetone extract (M.I-5) in the ratio of 1:3.

Example 10: Preparation of compositions of Rubia cordifolia stem 50% aqueous acetone extract (R.C-4) and Mangifera indica seed kernel 90% aqueous methanol extract (M.I-6)

Composition-25 (C-25): The composition-25 was prepared by combining Rubia cordifolia 50% acetone extract (R.C-4) and Mangifera indica 90% methanol extract (M.I-6) in the ratio of 3: 1.

Composition-26 (C-26): The composition-26 was prepared by combining Rubia cordifolia 50% acetone extract (R.C-4) and Mangifera indica 90% methanol extract (M.I-6) in the ratio of 1: 1.

Composition-27 (C-27): The composition-27 was prepared by combining Rubia cordifolia 50% acetone extract (R.C-4) and Mangifera indica 90% methanol extract (M.I-6) in the ratio of 1:3.

Example 11: Preparation of compositions of Rubia cordifolia stem ethanol extract (R.C-2) and Mangifera indica seed kernel 50% aqueous ethanol extract (M.I-3) Composition-28 (C-28): The composition-28 was prepared by combining Rubia cordifolia ethanol extract (R.C-2) and Mangifera indica 50% aq ethanol extract (M.I-3) in the ratio of 3:1.

Composition-29 (C-29): The composition-29 was prepared by combining Rubia cordifolia ethanol extract (R.C-2) and Mangifera indica 50% aq ethanol extract (M.I-3) in the ratio of 1: 1. Composition-30 (C-30): The composition-30 was prepared by combining Rubia cordifolia ethanol extract (R.C-2) and Mangifera indica 50% aq ethanol extract (M.I-3) in the ratio of 1:3.

Example 12: Preparation of compositions of Rubia cordifolia stem ethyl acetate extract (R.C-6) and Mangifera indica seed kernel water extract (M.I-4)

Composition-31 (C-3 1): The composition-31 was prepared by combining Rubia cordifolia ethyl acetate extract (R.C-6) and Mangifera indica water extract (M.I-4) in the ratio of3:l.

Composition-32 (C-32): The composition-32 was prepared by combining Rubia cordifolia ethyl acetate extract (R.C-6) and Mangifera indica water extract (M.I-4) in the ratio of 1:1.

Composition-33 (C-33): The composition-33 was prepared by combining Rubia cordifolia ethyl acetate extract (R.C-6) and Mangifera indica water extract (M.I-4) in the ratio of 1:3.

Example 13: Preparation of compositions of Rubia cordifolia stem 50% aqueous methanol extract (R.C-5) and Mangifera indica seed kernel ethyl acetate extract (M.I-7)

Composition-34 (C-34): The composition-34 was prepared by combining Rubia cordifolia 50% methanol extract (R.C-5) and Mangifera indica ethyl acetate extract (M.I-7) in the ratio of 3: 1.

Composition-35 (C-35): The composition-35 was prepared by combining Rubia cordifolia 50% methanol extract (R.C-5) and Mangifera indica ethyl acetate extract (M.I-7) in the ratio of 1: 1.

Composition-36 (C-36): The composition-36 was prepared by combining Rubia cordifolia 50% methanol extract (R.C-5) and Mangifera indica ethyl acetate extract (M.I-7) in the ratio of 1:3.

Example 14: Mitochondrial Biogenesis Assay

Mitochondrial biogenesis assay was performed by using an in-cell ELISA kit obtained from Abeam (Cambridge, UK; Cat# abl 10217) following manufacturer’s protocol. The levels of two mitochondrial proteins were measured simultaneously in each well. The two proteins are each subunits of a different oxidative phosphorylation enzyme complex, one protein being subunit I of Complex IV (COX-I), which is mitochondrial-DNA (mtDNA)-encoded, and the other being the 70 kDa subunit of Complex P (SDH-A), which is nuclear DNA (nDNA)-encoded. For the assay, rat skeletal (L6) cells (ATCC, Manassas, VA; Cat# CRL-1458) in DMEM medium (Sigma Cat# D7777) supplemented with 10%FBS were seeded (10000 cells/well) in a 96 well poly L-lysine coated plate and incubated overnight at 37°C in a C02 incubator. Next day, the medium was removed and cells were treated with respective samples at different concentrations. The treatment was repeated every day up to 3rd day. The culture wells receiving 0.2% DMSO were considered as vehicle control. After the treatment period, cells were fixed with 4% paraformaldehyde and probed with primary antibodies (COX-1 and SDH-A). Alkaline phosphatase (AP) and horse radish peroxidase (HRP)- labeled secondary antibodies were used for SDH-A and COX-1 respectively. Finally, AP/HRP development solution was added and measured the absorbance at 405nm and 600nm in a multi-mode spectrophotometer (Spectramax 2e). (COX-I) to (SDH-A) signal ratio was

calculated for all the treatments for analysis of the effect of treatments. The percent increase of mitochondrial biogenesis over control was calculated using the following formula:


The results are presented in Tables 3-7.

Table-3: % Increase in Mitochondrial biogenesis of the compositions containing Syzygium cumini 50% aq ethanol extract (S.C-1) and Rubia cordifolia 50% aq ethanol extract (R.C-1)

Table-4: % Increase in Mitochondrial biogenesis of the compositions containing Syzygium cumini 50% aq ethanol extract (S.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1)

Table-5: % Increase in Mitochondrial biogenesis of the compositions containing Rubia cordifolia 50% aq ethanol extract (R.C-1) and Mangifera indica 90% aq ethanol extract (M.I-1)

Table-6: % Increase in Mitochondrial biogenesis of the compositions containing Syzygium cumini 50% aq acetone extract (S.C-4) and Rubia cordifolia ethanol extract (R.C-2); Syzygium cumini 50% aq methanol extract (S.C-5) and Mangifera indica 50% aq ethanol extract (M.I-3) and Rubia cordifolia water extract (R.C-3) and Mangifera indica acetone extract (M.I-5)

Table-7: % Increase in Mitochondrial biogenesis of the compositions containing Rubia cordifolia 50% aq acetone extract (R.C-4) and Mangifera indica 90% aq methanol extract (M.I-6); Rubia cordifolia ethanol extract (R.C-2) and Mangifera indica 50% aq ethanol extract (M.I-3) and Rubia cordifolia ethyl acetate extract (R.C-6) and Mangifera indica water extract (M.I-4)

Example 15: Fonnulation of the compositions

Composition-37 (C-37): To a mixture of Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1; 60.0 g) in 30% aqueous ethanol (100 mL) was added Mangifira indica seed kernel 90% aqueous ethanol extract (M.I-1; 30.0 g) and stirred for 30 min. Then Glucidex 12D (Maltodextrin; 8.0 g) was added to the mixture and stirred for 30 min and dried under reduced pressure at ambient temperature. The dried flakes were sieved through mesh (#40) after addition of Syloid (2.0 g) to give the composition-37.

Composition-38 (C-38): To a suspension of Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1; 53.33 g) in water (50 mL) was added Ultrasperse A (10.0 g) and stirred for 30 min. Then Glucidex 12D (Maltodextrin; 8.0 g) was added to the mixture and stirred for 30 min. A solution of Mangifera indica seed kernel 90% aq ethanol extract (M.I-1; 26.67 g) in ethanol (20 mL) was added to the above mixture, stirred for 30 min and dried under reduced pressure at ambient temperature. The dried flakes were sieved through mesh (#40) after addition of Syloid (2.0 g) to give the composition-38.

Composition-39 (C-39): To a mixture of Syzygium cumini fruit 50% aqueous ethanol extract (S.C-1; 60.0 g) in 30% aqueous ethanol (100 mL) was added Rubia cordifolia stem 50% aqueous ethanol extract (R.C-1; 30.0 g) and stirred for 30 min. Then Glucidex 12D (Maltodextrin; 8.0 g) was added to the mixture, stirred for 30 min and dried under reduced pressure at ambient temperature. The dried flakes were sieved through mesh (#40) after addition of Syloid (2.0 g) to give the composition-39.

Example 16: Evaluation of efficacy of the herbal compositions-37, 38 and 39 to improve the parameters associated sarcopenia in Dexamethasone(DEXA) induced muscle atrophy in Sprague Dawley rats

Induction: Healthy male Sprague-Dawley rats were randomly assigned to five groups (n=7) viz. G1 (control), G2 (Dexa), G3 (Dexa + composition-37;

250mg/kg), G4 (Dexa + composition-38; 250mg/kg) and G5 (Dexa + composition-39; 250mg/kg). From day 1 to day 4, the rats in G3 to G5 received respective supplements; in parallel, the G1 and G2 rats received carboxymethyl cellulose (CMC, 0.5% in water) supplements. The rats in all groups received the supplements through oral gavage. From day 5 to day 12 of the study, the rats in G2 to G5 received 0.1 mg/kg dexamethasone through i.p. route, while the G1 animals received the equivalent volume of sterile normal saline (i.p.). The rats in G1 to G5 received CMC or respective supplements throughout the study. Rats’ body weight was measured on days 1, 5 and 12; grip strength and calf thickness were measured on day 12. The animals were euthanized by CO2 asphyxiation; their gastrocnemius muscles were collected and weighed. Thickness of the gastrocnemius muscle at the mid-central region was measured, and small pieces of the muscle tissues were preserved in 10% buffered formalin for tissue morphometric analysis.

Estimation of percent body weight change: Animal weighing balance (Mettler Toledo, USA) was used to assess their body weights. Body weights were measured on the days 1, 5 and 12. The percent body weight change of the rats was estimated using the following formula:

Percent change in body wt. on day 12 (from day 1) = [(body weight on day 12-body weight on day 1) x 100] / (body weight on day 1). The composition-37 (G3), composition-38 (G4) and composition-39 (G5) showed improvements in their body weight, when compared with the dexamethasone induced group (G2) as summarized in figures I & II.

Estimation of Grip strength: At the end of study, the grip strength of individual animals was measured using a Grip strength meter (Laboratory enterprises; Mumbai; INDIA). Rats were allowed to grasp the grasping bar and pulled back gently in a horizontal plane by the tail with gradually increasing force till the pulling force overcomes the grip strength of the animal. The force applied at the moment the rat leaves its grasp on the grasping bar while pulling by the tail was recorded as grip strength in Newtons (N). Three trials were conducted and their average was recorded for each animal. The composition-37 (G3) and composition-38 (G4) showed significant improvements in grip strength, compared with the G2 rats, while the composition-39 (G5) demonstrated increase in grip strength as depicted in figure IP.

Calf and gastrocnemius muscle thickness measurements: The calf and the isolated gastrocnemius muscles from each animal were measured using a Vernier Caliper (Fisher Scientifics, USA), on day 12. Each measurement was repeated thrice and their average value was recorded. The results showed that all the three compositions improved calf thickness as summarized in figure IV and improved in muscle weight (A) and size (B) as shown in figure V.

Histopathological investigation: The mid-central portions of the gastrocnemius muscles were fixed in 10% buffered-formalin, embedded in paraffin. The fixed tissues were sectioned at 5mm thickness in a microtome. The tissue sections were stained with sirius red and examined under a light microscope as summarized in figure VIA. Pixel sizes of the muscle fibers were estimated using Axiovision 4.8 software (Carl Zeiss GmbH, Jena, Germany). A conversion scale of 0.1694 mm per pixel was used to estimate the area of each fiber and represented in mm2. As depicted in figure VIB, these compositions increased gastrocnemius muscle fiber sizes in the Dexamethasone induced sarcopenic rats.

Statistical analysis: The intergroup difference analyses were performed using One-way analysis of variance (ANOVA), and Dunnett’s test was performed to identify the significance using GraphPad Prism Software. P<0.05 was considered as significant.