(12) Patent Application Publication (10) Pub. No.: US 2008/0175934 A1 Mitra Et Al

US 2008O175934A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0175934 A1 Mitra et al. (43) Pub. Date: Jul. 24, 2008

(54) NOVEL HERBAL COMPOSITION OF (21) Appl. No.: 11/965,239 EXTRACTS OF QUERCUS INFECTORIA, PROCESS FOR PREPARING THE SAME AND (22) Filed: Dec. 27, 2007 USE THEREOF (30) Foreign Application Priority Data (75) Inventors: Shankar Kumar Mitra, Bangalore Dec. 29, 2006 (IN) ...... 2831 (DELA2006 (IN); Ekta Saxena, Bangalore (IN); s Rangesh Paramesh, Bangalore Publication Classification (IN); Uddagiri Venkanna Babu, (51) Int. Cl Bangalore (IN); Sundaram we Ramachandran, Bangalore (IN); A6IR 36/49 (2006.01) Marikunte Venkata Ranganna, (52) U.S. Cl...... 424/771 Bangalore (IN) (57) ABSTRACT Correspondence Address: Disclosed is a novel herbal composition comprising extract of DARBY & DARBY P.C. Quercus infectoria having rich and effective concentration of P.O. BOX 770, Church Street Station as antioxidants and phenolic compounds. Further the present New York, NY 10008-0770 invention provides a process for preparing said composition and use thereof in cosmeceuticals, pharmaceuticals and (73) Assignee: Himalaya Global Holdings health drinks for treating oxidative stress and other life style Limited, Grand Cayman, KY (US) diseases. Patent Application Publication Jul. 24, 2008 Sheet 1 of 10 US 2008/O175934 A1

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Patent Application Publication Jul. 24, 2008 Sheet 7 of 10 US 2008/0175934 A1

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NOVEL, HERBAL COMPOSITION OF radicals and also the overall composition should improve the EXTRACTS OF QUERCUS INFECTORIA, bioavailability of the antioxidant active fraction. PROCESS FOR PREPARING THE SAME AND USE THEREOF PRIOR ART 0009 U.S. Pat. No. 6,534,046 to Golz-Bemer et al. dis FIELD OF THE INVENTION closed the preparation of plant derived anti-perspiration cos metics comprising the extracts of Equisetum arvense, Salvia 0001. This invention, in general relates to a novel herbal officinalis, Hamamelis Virginia and Quercus infectoria. composition comprising extracts of Quercus infectoria. More (0010 U.S. Pat. No. 6,264,926 to Farooqi et al. disclose a particularly, the present invention provides a novel herbal formulation useful as a natural herbal tooth powder compris composition comprising extract of Quercus infectoria having ing the extracts of Quercus infectoria, Zanthoxylum amatum high and effective concentration of antioxidants and phenolic and Zingiber officinalis etc. (0011 Kaur G. et al. (J. Ethnopharmacol. 2004, 90(2-3). compounds. The invention further provides a process for 285-92) have reported the anti-inflammatory activities of preparing said composition and use thereof. alcoholic extracts of galls of Quercus infectoria. 0012 Sawangiaroen N et al (J Ethnopharmacol 2005 BACKGROUND OF THE INVENTION 98(1-2) 67-72) have reported the antidiarrhoel effect of methanol extract of galls of Ouercus infectoria. 0002 Reactive Oxygen species and Reactive Nitrogen (0013 United States Patent application No. 20030138509 species (ROS/RNS) arise in the normal course of oxidative to Pushpangadan et al. teaches the preparation of fermented metabolism. These reactive species (ROS/RNS) influence herbal health drink from the plant Andrographis paniculata. multiple metabolic, signaling and transcriptional processes, 0014 U.S. Pat. No. 4,741,915 to Farret al. disclosed the which are involved in the normal function of cell. use of purified and hydrolyzed gallotannins derived from 0003 Oxidative stress is a condition that occurs when plant materials for inhibition of oxidation in foodstuffs. there is excess and prolonged imbalance between the genera 0015 U.S. Pat. No. 7,041,332 to Gaudout et al. teaches the tion of reactive oxygen species and reactive nitrogen species preparation of a phenolic rich fraction obtained from the fruit and control by antioxidants. Reducing reactive oxygen spe of family rosaceae for use in cosmetic, dietary and nutraceu cies and reactive nitrogen species in the body is one of the tical preparation. primary target towards the protection of various diseases such 10016 U.S. Pat. No. 6,989,161 to Roufetal discloses a as aging process, cancer, diabetes and neurodegenerative dis composition comprising phytonutrients viz. lutein, lycopene, CaSCS. epigallocatechingallate (EGCG), ellagic acid, hesperidin and 0004 Dietary compounds such as vitamins C and E may quercetin. limit oxidative damage directly but along these other dietary constituents viz. carotenoids and polyphenols may also act SUMMARY OF THE INVENTION via indirect mechanisms such as endogenous antioxidant 0017. It is a principal object of the present invention to enzyme activity and thereby reduce the risk of a number of the provide a herbal composition comprising extract of Quercus age related disorders. infectoria having high and effective concentration of antioxi 0005 Polyphenols occur most commonly in foods of plant dant and phenolic compounds. origin have attracted much attention as potent antioxidants 0018. Another aspect of the present invention is to obtain due to their ability to scavenge free radical and form inert the extract using all parts of the plant Quercus Infectoria, phenoxy radical intermediates. A number of epidemiological preferably from the galls. studies have Suggested that consumption of polyphenol-rich 0019. Yet another aspect of the present invention is to foods reduces the risk of developing chronic diseases. obtain a herbal composition comprising antioxidant rich 0006 Although, these polyphenol antioxidants reported extract of Quercus Infectoria, wherein the antioxidant is pref for the prevention of various lifestyle diseases but only a few erably methyl gallate. findings show that they are active and majority of the results 0020 Still another aspect of the present invention is to show that they have no clinical efficacy. The vast literature on obtain the extract of Ouercus Infectoria employing solvents antioxidants Suggests that the failure of polyphenol antioxi selected from non-polar hydrocarbon, chlorinated Solvents, dant in clinical cases because it is almost impossible to elimi an ester and ketones, an alcohol and water, preferably, nate many active oxygen species produced in the body by selected from hexane, chloroform, ethyl acetate, acetone, single antioxidant molecule. methanol or ethyl alcohol. 0007 Polyphenol antioxidants derived from the plant ori 0021. Yet another aspect of the present invention is to gin have structurally phenol group as backbone and make it obtain the extract of the said herbal composition employing more lipid soluble. The bioavailability of these plant polyphe methanol with hexane and acetone preferably in 1:1 ratio, nols is very low in GI tract and therefore most of these wherein the said extract is characterized by having antioxi antioxidants have not sufficiently displayed efficacy in the dant concentration of more than 80%. body even though they have been reported as potent antioxi 0022. Still another aspect of the present invention is to dants in vitro. obtain the extract of the said herbal composition employing 0008 To overcome the above said difficulties in the prepa methanol with chloroform and methanol preferably in 1:1 ration of antioxidant composition that is effective both in ratio, wherein said extract is characterized by having phenolic vitro and clinical, it is necessary to select a group of polyphe compounds concentration of more than about 108 w/w nolic compounds rich composition that is selectively address 0023. In accordance with further aspect of the present the inhibition of many enzymatic pathways that produce free invention, there is provided an antioxidant and phenolic com US 2008/01 75934 A1 Jul. 24, 2008

pounds rich herbal composition, which is obtained by a pro yeast Malassezia furfur (MTCC-1374) causing dandruff and cess, comprising the steps of (a) pulverizing the shade dried Malassezia pachydermatis causing ear infection. galls of Quercus Infectoria to coarse powder, (b) Subjecting 0033. In one another preferred aspect of the present inven the resultant coarse powder of said dried galls of Quercus tion is to evaluate the antioxidant and free radical scavenging Infectoria into an extraction vessel in presence of a solvent activity of the said herbal composition by employing DPPH either alone or in combination thereof, (c) filtering the result method, Ferric reducing Power Assay Method, and determin ant plant extracts employing Suitable filter, (d) drying the ing Nitric oxide scavenging activity. resultant solvent extracts to form a concentrate. 0034. Yet another aspect of the present invention is to 0024. Further aspect of the present invention is the prepare the extracts of Quercus Infectoria by employing per obtained extract is characterized by having antioxidant prin colation method and hot soxhlet method. ciple concentration of more than 80% and phenolic principle 0035 Another aspect of the present invention is to prepare concentration of more than about 108% w/w. the various extracts such as methanol extract, water extract, 0025. In yet another aspect, the present invention is acetone extract, ethyl acetate and acetone (1:1) extract, chlo endowed with a process for preparing a herbal composition roform and methanol (8:2) extract in order to obtain 20% of comprising extract of Quercus Infectoria having high and the raw materials. effective concentration of antioxidant and phenolic prin 0036. It is another aspect of the present invention is to ciples, alone or in combination with other effective herbs and identify the various active fractions of the methanolic extract pharmaceutically acceptable excipients, wherein the said of Quercus infectoria with varying degree of activity. process is comprised of: (a) pulverizing the shade dried galls 0037. In one another aspect of the present invention is to of Quercus Infectoria to coarse powder, (b) Subjecting the estimate the percentage concentration of methyl gallate resultant coarse powder of said dried galls of Quercus Infec present in the various active fractions of Quercus infectoria toria into an extraction vessel in presence of a solvent either by HPLC standardization. alone or in combination thereof (c) filtering the resultant 0038. It is still another aspect of the present invention is to plant extracts employing Suitable filter, (d) drying the result estimate the presence of total phenolic compounds in various ant solvent extracts to form a concentrate, (d) characterizing solvent fractions and semi purified fractions of extract of the resultant extract by analyzing presence of concentration Quercus infectoria. of antioxidant and phenolic compounds, (f) mixing the ana 0039. One another aspect of the present invention is to lyzed resultant extract with pharmaceutically acceptable employ the column chromatography for purifying the active excipients to prepare the said herbal composition. compounds of various active fractions of Quercus infectoria. 0026. It is another aspect of the present invention wherein; 0040. Additional aspect of the present invention is to pre the extraction is performed employing any suitable hot or pare an anti stress health drink comprising water extract of cold extraction techniques, preferably, percolation, macera Withania somnifera (1.0 g), Emblica officinalis (1.0 g), Vitis tion or soxhlet method. vinifera (3.0 g) and methanol extract of galls of Quercus 0027. One another preferred aspect of the present inven infectoria (AXT-3) (0.6 g). tion is to effectively incorporate the said herbal composition 0041 Further aspect of the present invention is to prepare comprising extract of galls of Quercus Infectoria in cosmet the energy health drink comprising the water extracts of ics, pharmaceuticals and functional foods such as energy Camellia sinensis (6.0 g.). Withania somnifera (2.0 g). health drinks to reduce oxidative stress that is associated with Emblica officinalis (2.0 g), Vitis vinifera (6.0 g) and methanol ageing, cardiovascular disease, cancer, immunological disor extract of galls of Quercus infectoria (AXT-3) (0.6 g.) ders, dementia, diabetes and macular degeneration and other lifestyle diseases or as natural preservative, antioxidant or as BRIEF DESCRIPTION OF DRAWINGS antimicrobial agent. 0042. Further objects of the present invention together 0028. One another aspect of the present invention is to with additional features contributing thereto and advantages assess the antioxidant and antimicrobial activity of methanol accruing there from will be apparent from the description of extract of galls of Quercus Infectoria. preferred embodiments of the present invention which are 0029. It is yet another aspect of the present invention is to shown in the accompanying drawing figures. use the said herbal composition for preventing oxidative 0043 FIG. 1. DPPH radical scavenging activity of Ouer StreSS. cus infectoria extracts (ICso-g/ml values) AXT-01, 02, 16. 0030. It is yet another preferred aspect of the present 17, 18. invention is to evaluate the said herbal composition for anti 0044 FIG. 2. DPPH radical scavenging activity of Ouer fungal activity employing fungal strains such as Trycophyton cus infectoria extracts (ICso-lug/ml values) AXT-03.04.05. rubrum (MTCC296), Candida albicans (MTCC 741), Trico 06, 07, 08, O9, 10, 11, 12, 13, 14, 15, 21, 24. phyton gypseum (ATCC 8125TM), Tricophyton mentagro 004.5 FIG. 3. DPPH radical scavenging activity of Ouer phytes (ATCC 52018). cus infectoria extracts (ICso-lug/ml values) AXT-22, 23. 0031. It is still another preferred aspect of the present 0046 FIG. 4. DPPH radical scavenging activity of Ouer invention is to appraise the said herbal composition for anti cus infectoria extracts (ICsoug/ml values) AXT-19, 20. bacterial activity employing bacterial strains such as Escheri 0047 FIG. 5. Reducing power assay of Quercus infectoria chia coli (MTCC 443), Pseudomonas aeruginosa (MTCC extracts (ICso-lug/ml values) AXT-01, 02, 16, 17, 18. 741), Salmonella typhi (MTCC 733), Staphylococcus aureus 0048 FIG. 6. Reducing power assay of Quercus infectoria (MTCC96), Helicobacter pylori (ATCC No-51653TM), Nies extracts (ICso-lug/ml values) AXT-03.04.05, 06, 07, 08, 09. seria gonorrhoeae (ATCC 49226). 10, 11, 12, 13, 14, 15, 21, 24. 0032. Another aspect of the present invention is to evalu 0049 FIG. 7. Reducing power assay of Quercus infectoria ate the inhibitory activity of said herbal composition against extracts (ICso-lug/ml values) AXT-22, 23. US 2008/01 75934 A1 Jul. 24, 2008

0050 FIG.8. Reducing power assay of Quercus infectoria prepared by percolation method. The yields of solvent extracts (ICso-lug/ml values) AXT-19, 20. extracts that are considerably more than 20% are summarized 0051 FIG. 9. Lipid peroxidation (in vitro) assay of Ouer in the table-1. cus infectoria extracts (ICso-lug/ml values) AXT-01, 03, 10, 31, 33. TABLE 1 0052 FIG. 10. NO (Nitric Oxide) scavenging activity of SI Quercus infectoria extracts (ICso-g/ml values) AXT-03, No Code No Solvents Used Source Yield (%) 05, O7, 09, 11, 16, 17, 19, 20, 21, 23, 28, 30, 31. 1 AXT-1 Methanol Plant material 82.O DETAILED DESCRIPTION OF THE INVENTION 2 AXT-2 Water Plant material 6O.O 3 AXT-16 Ethyl acetate:Acetone (1:1) Plant material 6.3.3 0053. The present invention involves the selection of herb 4 AXT-17 Chloroform:Methanol (8:2) Plant material 21.5 and subjecting the herb for solvent extraction by various 5 AXT-18 Acetone Plant material 69.5 methods and Screening of these solvent extracts for the anti oxidant activity and antimicrobial activity. Preparation of a composition using the bioactive extract for pharmaceuticals, EXAMPLE-3 cosmeceuticals and health drinks as an antioxidant to treat and prevent various life style disease Such as stress, ageing, Solvent-Solvent Fractionation of Methanol Extract cardiovascular disease, cancer, immunological disorders, (AXT-1) dementia, diabetes and macular degeneration and also pre vents microbial contamination as natural preservative. 0058. About 1 Kg of methanol extract was macerated with 0054 Quercus infectoria of the family Fagaceae is a small different solvents alone or combination thereofas mentioned tree or shrub and about 2-5 m. high, leaves are very rigid, 4-6 in the table-2 to obtain a purified active fraction with potent cm. long, very rigid, often glabrescent with spinous teeth, antioxidant and antimicrobial activities. The yields of active mostly distributed in the temperate regions of the northern fractions obtained from the solvent-solvent fractionation are hemisphere and extending to Sub-tropical and tropical given in table-2. America and Asia at high altitudes. Around, 23 evergreen are found in the Himalayan region of India TABLE 2 0055 Gall nut is used externally for its astringent effect; it S Code Yield is used in ointments for the treatment of piles, and in plasters. No No Solvents Used Source (%) The tannic and gallic acids extracted from the galls are often 1 AXT-3 Hexane:acetone(1:1) Methanol extrac 7.0 used in dysentery and diarrhoea and as a gargle. 2 AXT-4 Hexane:acetone (6:4) Methanol extrac 4.0 3 AXT-5 Chloroform:methanol (9:1) Methanol extrac 7.5 EXAMPLE-1 4 AXT-6 Chloroform:methanol (1:1) Methanol extrac 70.O 5 AXT-7 Dichloromethane:acetone (7:3) Methanol extrac 2.5 Preparation of Quercus infectoria Extract by Perco 6 AXT-8 Dichloromethane:acetone (1:1) Methanol extrac 46.3 lation Method 7 AXT-9 Chloroform:acetone (1:1) Methanol extrac 4.2 8 AXT-10 Ethyl acetate:acetone (1:1) Methanol extrac 88.2 9 AXT-11 Toluene:methanol (9:1) Methanol extrac 2.O 0056. The shade dried galls of Quercus infectoria was 10 AXT-12 Ethyl acetate Methanol extrac 69.2 pulverized to coarsepowder and about 1 Kg each of powdered 11 AXT-13 Ethyl acetate:methanol (7.5:2.5) Methanol extrac 94.0 material placed in different percolators and the material was 12 AXT-14 Ethyl acetate:methanol (1:1) Methanol extrac 96.O soaked in n-hexane, dichloromethane, chloroform, ethyl 13 AXT-15 Ethyl acetate:acetone (6:4) Methanol extrac 80.0 14 AXT-21 Dichloromethane:methanol (1:1) Methanol extrac 82.O acetate, acetone, ethanol, methanol and water either alone or 15 AXT-24 Ethyl acetate:acetone (2.5:7.5) Methanol extrac 88.0 in combination thereof at room temperature for 24 to 48 h. 16 AXT-25 Acetone Methanol extrac 8O then plant extracts were drained out from the percolator and filtered through whatmann no. 1 filter paper. The percolation of the residual material was again carried out with respective Solvents and the combined solvent extracts concentrated to EXAMPLE-4 dryness on rotatory evaporator or on Steam bath at optimum temperature and under reduced pressure. Solvent-Solvent Fractionation of Water Extract (AXT-2) EXAMPLE-2 0059. About 1 Kg each of water extract was suspended in Preparation of Quercus infectoria Extract by Hot dichloromethane:acetone (7:3) and dichloromethane:acetone Soxhilation Method (1:1) to obtain semipurified fractions AXT-22 and AXT-23 respectively. The yields of AXT-22 and AXT-23 fractions are 0057 The shade dried galls of Quercus infectoria was pulverized to coarse powder and about 1 Kg each of the given in table-3. powdered material subjected to hot-soxlation in different SOXhlet apparatus using solvents n-hexane, dichloromethane, TABLE 3 chloroform, ethyl acetate, acetone, ethanol and methanol SI Yield either alone or combination thereof at optimum temperature No Code No Solvents Used Source (%) until extraction is completed, then plant extracts were filtered 1 AXT-22 Dichloromethane:acetone (7:3) Water extract S.O through whatmann no. 1 filter paper and concentrated to 2 AXT-23 Dichloromethane:acetone (1:1) Water extract 8.0 dryness on rotatory evaporator or on Steam bath at optimum temperature. All extracts were qualitatively similar to extracts US 2008/01 75934 A1 Jul. 24, 2008

EXAMPLE-5

Solvent-Solvent Fractionation of Semipurified Ethyl Column: Reverse Phase C-18 Acetate Fraction (AXT-12) Mobile Phase: Solvent A:Solvent B (20:80) Solvent A: 0.05% phosphoric acid in ACN 0060. The ethyl acetate fraction obtained at the yield of Solvent B: 0.1% phosphoric acid in water 69.2% from the methanol extract was further fractionated Flow rate: 1 ml/min Detection: UV was 240 mm with dichloromethane:acetone (7:3) and dichloromethane: acetone (1:1) to yield AXT-19 and AXT-20 respectively. The yields of these fractions are given in table-4 TABLE 6 TABLE 4 SINC Code No (% of Methyl gallate) Code 1 AXT-1 11.6 SINo No Solvents Used Source Yield (%) 2 AXT-2 O.2 3 AXT-3 79.9 1 AXT-19 Dichloromethane:acetone(7:3) Ethyl acetate 9.O 4 AXT-4 7O.O fraction 5 AXTS 64.7 2 AXT-20 Dichloromethane:acetone(1:1) Ethyl acetate 1O.O 6 AXT-6 3O4 fraction 7 AXT-7 S8.9 8 AXT-8 37.7 9 AXT-9 48.0 10 AXT-10 32.4 EXAMPLE-6 11 AXT-11 724 12 AXT-12 27.3 13 AXT-13 21.5 Column Chromatography of Methanol Extract 14 AXT-14 28.8 (AXT-1) 15 AXT-15 3O.O 16 AXT-16 7.5 17 AXT-17 15.9 0061 The column chromatography was performed over 18 AXT-18 6.O silica gel (60-120 mesh). About 2.5 Kg of silica gel was 19 AXT-19 58.3 suspended in chloroform and packed the column. About 300 2O AXT-20 36.5 21 AXT-21 31.8 g of methanol extract was prepared as slurry using silica gel 22 AXT-22 64.6 and poured on to the column. The column was then eluted 23 AXT-23 77.8 with chloroform, chloroform:methanol with increasing 24 AXT-24 30.2 polarity to obtain purified active fraction. The details of col 25 AXT-2S 25.8 umn fractions and their yields are summarized in the table-5.

TABLE 5 EXAMPLE-8 SI. No. Fraction No Solvent system Yield (g) Quantitative Estimation of Phenolic Compounds 1 AXT-11 Chloroform 5 0064. The phenolics present in the extracts and fractions 2 AXT-12 Chloroform:methanol (95:5) 7 of Quercus infectoria was quantitatively measured using tan 3 AXT-13 Chloroform:methanol (90:10) 50 4 AXT-1 f4 Chloroform:methanol (85:15) 15 ninc acid as standard. The greenish blue colour produced 5 AXT-1 S Chloroform:methanol (80:20) 100 during reaction of phenolic compounds with potassium ferri 6 AXT-1, 6 Methanol 100 cyanide and ferric chloride was measured at 720 nm. The results are summarized in the table-7.

TABLE 7 EXAMPLE-7 S. Total phenols Standardization of Extracts and Fractions by TLC No Code No (Tannic acid) wiw and HPLC 1 AXT-1 90.09 2 AXT-2 74.OS 0062 All extracts and fractions were prepared at a con 3 AXT-3 97.72 4 AXT-4 90.45 centration of 100 mg in 5 mL of the respective solvent and 5 AXTS 82.13 about 50 ul of the sample solution were spotted on precoated 6 AXT-6 108.31 (e-Merck) silica gel TLC plates. The TLC plates were air 7 AXT-7 87.22 dried and placed in the mobile Phase: (1) Dichloromethane: 8 AXT-8 97.02 Methanol (85:15) and (2) Hexane:Ethylacetate (30:70). TLC 9 AXT-9 91.91 10 AXT-10 83.96 plates were air dried and sprayed with 1% alcoholic ferric 11 AXT-11 96.37 chloride Solution to visualize as dark blue spots confirming 12 AXT-12 98.53 the presence of phenolic compounds. 13 AXT-13 83.97 14 AXT-14 80.2 0063 All extracts and fractions were also subjected to 15 AXT-15 89.52 HPLC analysis for standardization purpose taking methyl 16 AXT-16 78.22 gallate as principle marker. The results are Summarized in 17 AXT-17 92.91 table-6. The HPLC conditions are as follows. US 2008/01 75934 A1 Jul. 24, 2008

EXAMPLE-10 TABLE 7-continued MIC Assay for Antibacterial Activity S. Total phenols No Code No (Tannic acid) wiw (0075. The test organisms Escherichia coli (MTCC 443), 18 AXT-18 92.20 Pseudomonas aeruginosa (MTCC 741), Salmonella typhi 19 AXT-19 98.55 (MTCC 733), Staphylococcus aureus (MTCC 96) were 2O AXT-20 93.65 21 AXT-21 94.71 obtained from IMTECH, Chandigarh, India. 22 AXT-22 96.33 0076 Preparation of Samples for Screening 23 AXT-23 94.40 0077. About 100 mg of test sample (extract) was weighed 24 AXT-24 96.32 25 AXT-2S 93.04 in to a sterile screw capped tube and dissolved in 1 ml of sterile distilled water in case of water soluble extracts, other wise, extracts were dissolved in 100 ul of DMSO and then subsequently in 900 ul of sterile distilled water. EXAMPLE-9 (0078 Media Preparation (0079) Mueller Hinton Agar (Himedia M173, 38 g.) was MIC Assay for Antifungal Activity by Agar Dilution suspended in 1 Litre of distilled water and heated to dissolve Method the medium completely. Mueller Hinton Broth (Himedia M391) 21 g. was suspended in 1 Litre distilled water and 0065. The test organisms Trycophyton rubrum (MTCC autoclaved at 15 lbs pressure at 121°C. for 15 minutes. Miller 296) and Candida albicans (MTCC 741) were procured from Hinton Agar was cooled to 55° C. IMTECH, Chandigarh, India and Tricophyton gypseum (ATCC 8125TM), Tricophyton mentagrophytes (ATCC Inoculum Preparation 52018), were procured from USA. These are dermatophytes causing infections of hair, nails and skin in humans. These are 0080. The overnight culture oftest bacteria was suspended cosmopolitan in distribution from the agar plate into 2 ml of sterile saline and adjusted the turbidity of the suspension to McFarlands turbidometer stan 0066 Preparation of Test Samples (Extracts) for Screen dard 0.5. Dilution (/100") of the adjusted inoculum was pre ing pared in Muller Hinton Broth. This inoculum contained 0067. About 100 mg of test sample (extract) was weighed approximately 1 x 1' Organisms per ml. in to a sterile screw capped tube and dissolved in 1 ml of 0081 Procedure sterile distilled water in case of water soluble extracts, other I0082. The required amount of the extract was taken in the wise, extracts were dissolved in 100 uL of DMSO and then petri plate and mixed it with 4 ml of the molten Mueller subsequently in 900 u, of sterile distilled water. Hinton agar. (To achieve 1 mg/ml concentration in the plate 0068 Media Preparation diluted 40 ul of extract (100 mg/ml). A range of concentra 0069 Sabouraud chloremphenicol Agar (Himedia) was tions was prepared with the extracts in duplicates. A plate for used for cultivation of fungal strains. The sabouraud chlorem negative control (media control), and a plate for positive phenicol (65 grams) was suspended in 1 Litre distilled water, control without extract were kept to solidify. Inoculate was spotted with 20 ul of adjusted inoculum on the surface of the heated to boil to dissolve the media completely and auto agar plate. The plates were incubated for 48 hours at 37° C. claved at 15 lbs at 121° C. for 15 minutes. Observed the plates for visual growth and MIC were calcu 0070 Inoculum Preparation lated. The results are given in table-8. 0071. A seven day old pure culture of the fungi was taken for the test. EXAMPLE-11 0072 Candida albicans—The 24 hrs culture of Candida albicans was suspended from agar plate into 2 ml of sterile Assay for Gram-Negative Rod Helicobacter pylori saline. The turbidity of the suspension was adjusted to McFar (ATCC No-51653TM) land's turbidometer standard 0.5. 0073 Procedure I0083 Media Preparation 0074 The required amount of the test sample was taken in I0084) Mueller Hinton Agar (Himedia M173) 38 g was the petri plate and mixed it with 4 ml of the molten sabouraud suspended in 1 Litre of distilled water. Tryptic soya broth chloremphenicol Agar. (To achieve 1 mg/ml concentrations in (Himedia) 21 g was put in 1 Litre distilled water. About 10% the plate dilute 40 uL of the extract (100 mg/ml). A range of defibrinated sheep blood was autoclaved at 15 lbs pressure at concentrations with the extracts in duplicate was prepared. A 121° C. for 15 minutes. These were autoclaved separately. plate for negative control (media control), a plate for positive Autoclaved 10% defibrinated sheep blood was added to control without extract were saved and allowed to solidify. miller Hinton Agar. The medium was brought to 55° C. The plates were marked on the bottom for the names of the 0085 Inoculum Preparation organisms viz. Trycophyton rubrum, Tricophyton gypseum, I0086. The fully grown test bacteria (5 days) was sus and Tricophyton mentagrophytes. A Small amount of mycelia pended from the agar plate into 2 ml of sterile Saline and were picked with sterile inoculation stab and inoculated on adjust the turbidity of the suspension to McFarlands turbi the surface of the plate. The plates were incubated at room dometerstandard 0.5. Prepared Moo" dilution of the adjusted temp for 7 days and observed for visible growth and MIC inoculum in Tryptic soya Broth. This inoculum contain were calculated. The results are summarized in the table-8 approximately X Organisms per ml. US 2008/01 75934 A1 Jul. 24, 2008

0087 Procedure (0096. Media Preparation 0088. The required amount of the extract was taken in the 0097 Sabouraud Dextrose Agar-Emmons modified (Hi petriplate and mixed it with 4 ml of the molten Mueller media) (23.5 g) was suspended in 500 ml distilled water. Hinton agar--10% defibrinated sheep blood. (To achieve 1 Heated to boil to dissolve the medium completely and steril mg/ml concentration in the plate diluted 40 micro liter of ized by autoclaving at 15 lbs pressure, 121°C. for 15 minutes. extract (100 mg/ml). A range of concentrations were prepared After cooling by bringing to 55° C. added few drops of sterile with the extracts in duplicates. A plate for negative control corn oil and mixed the medium (media control), and a plate for positive control without (0098. Inoculum Preparation extract were kept to solidify. Inoculate was spotted with 20 ul (0099. The fresh culture of M. furfur (incubated for 4 or 5 of adjusted inoculum on the Surface of the agar plate. The days) from the Agar plate was suspended into 2 ml of sterile plates were incubated for 5 days in anaerobic jar at 37° C. saline. The turbidity of the suspension was adjusted to McFar Observed the plates for visual growth and MIC were calcu lands turbidometer standard 0.5. lated. The results are given in table-8. 0100 Procedure 0101 Sterile petriplates (50 mm) were labeled with the EXAMPLE-12 test sample number and percentage of the sample. The required amount of the test sample was added into petriplates Assay for Gram-Negative Cocci Niesseria gonor and the molten medium to make up the Volume to 4 ml in rhoeae (ATCC 49226) duplicates. A plate as negative control without inoculation of I0089 Media Preparation test organism, and a plate as positive control with test organ 0090 GC Agar Base (Himedia) (7.2 g) was prepared in ism were saved. The contents were mixed gently and allowed distilled water (100 ml) to make a double strength base. to solidify. Adjusted inoculate 20 microlitre was spotted on Heated to boiling to dissolve the medium completely. The the surface of the media plates. The plates were incubated for same was sterilized by autoclaving at 15 lbs pressure (121° 7 days at room temp and observed for visible growth by C.) for 15 minutes. And allowed to cool up to 55° C. asepti comparing with positive control. MIC were calculated and cally. Separately prepared Haemoglobin (FD002 Himedia) results given in table-8. (2%) was added. The vitamin Growth supplements (FD025 Himedia) were added to increase the selectivity of the EXAMPLE-14 medium. MIC Assay for Yeast Malassezia pachydermatis 0091 Inoculum Preparation 0092. About 48 hours incubated culture kept at 37°C. with 0102 The test organism Malassezia pachydermatis is 5% CO was suspended in to 2 ml of sterile saline. The yeast causing ear infections in canines (MTCC-1369) pro inoculum was adjusted to McFarland standard 0.5. Prepared cured from IMTECH Chandigarh, India. The organism was !/100 dilution of this in Trypric Soya Broth (DIFCO). used to test ear infections. 0093 Procedure (0103 Media Preparation 0094. The media was kept at 55° C. on hot plate to keep 0104 Sabouraud Dextrose Agar-Emmons modified (Hi molten. The required amount of test sample (extract) was media) (23.5 g) was suspended in 500 ml distilled water. taken in sterile 50 mm petri plate and labeled with the name Heated to boil to dissolve the medium completely and steril and concentration of the drug. The test sample was diluted ized by autoclaving at 15 lbs pressure, 121°C. for 15 minutes. with molten medium in duplicates (To get final concentration After cooling by bringing to 55° C. added few drops of sterile of the drug at 1 mg/ml dissolve 40 uL of the extract in 4 ml of corn oil and mixed the medium uniformly. the molten medium) One plate for positive control without 0105. Inoculum Preparation drug and a plate for media control were kept. The plates were 0106 The fresh culture of M. pachydermatis (incubated allowed to solidify and then were inoculated with 20LL of the for 7 days) from the agar plate was suspended into 2 ml of adjusted inoculum incubated for 48 hours at 37° C. in a sterile saline. The turbidity of the suspension was adjusted to anaerobic chamber with 5% CO. According to instructions McFarlands turbidometer standard 0.5. of Gas pack (BBL). After incubation plates were observed 01.07 Procedure for visual growth by comparing with control. Results were (0.108 Sterile petriplates (50 mm) were labeled with the recorded. MIC were calculated as the lowest concentration of test sample number and percentage of the sample. The the drug showing no growth. The results are Summarized in required amount of the test sample was added into petriplates table-8. and the molten medium to make up the Volume to 4 ml in duplicates. A plate as negative control without inoculation of EXAMPLE-13 test organism, and a plate as positive control with test organ ism were saved. The contents were mixed gently and allowed MIC Assay for Yeast to solidify. Adjusted inoculate 20 uL was spotted on the 0095. The test organisms Malassezia firfiur (MTCC surface of the media plates. The plates were incubated for 7 1374) is lipophilic yeast found on skin and body surface. The days at room temp and observed for visible growth by com organism was used to test antidandruff activity. This is pro paring with positive control. MIC (mg/ml conc) were calcu cured from IMTECH, Chandigarh, India. lated and results are given in table-8. TABLE 8 Organisms Tested AXT-5 AXT-7 AXT-8 AXT-9 AXT-10 AXT-11 AXT-17 AXT-19 AXT-2O AXT-21 AXT-22 AXT-23

Neisseria O.O2S O.OSO 0.100 O.OSO O.OSO O.OSO O.100 O.OSO O.100 O.100 O.O2S O.OSO gonorrhoeae Trichophyton 2 2 2 2 2 1 2 2 2 1 2 2 rubrum US 2008/01 75934 A1 Jul. 24, 2008 7

TABLE 8-continued Organisms Tested AXT-5 AXT-7 AXT-8 AXT-9 AXT-10 AXT-11 AXT-17 AXT-19 AXT-2O AXT-21 AXT-22 AXT-23 Trichophyton 2 2 1 1 2 2 2 1 1 1 2 2 mentagrophytes Microsporum 2 2 1 1 2 2 2 2 1 1 2 2 gypselin Entaneba O.2SO O.2SO O.200 O.200 O.2SO O.2SO O.200 O.200 O.2SO O.2SO O.200 O.200 Coi Staphylococcus 0.500 O.4SO O4SO O.SOO O.4SO OSOO O.4SO O4SO O.SOO O4SO OSOO O.SOO (iiietis Saimoneiia O.2SO O.200 OSOO O.SOO O.2SO O.200 O.SOO OSOO O.SOO O.1SO O.1SO O.1SO Typhi Pseudomonas 2 O.SOO 1 1 2 2 2 1 1 1 2 2 aeruginosa Helicobacter OSOO O.SOO OSOO 1 1 2 2 2 O.SOO 1 1 1 pylori Candida O.2SO O.200 OSOO O.SOO O.2SO O.200 O.SOO OSOO O.SOO O.1SO O.1SO O.1SO albicans Maieihsia OSOO O.4SO O4SO O.SOO O.4SO OSOO O.4SO O4SO O.SOO O4SO OSOO O.SOO Furfur Maia thesia OSOO O.4SO O4SO O.SOO O.4SO OSOO O.4SO O4SO O.SOO O4SO OSOO O.SOO Pachydermatis

EXAMPLE-15 respective solvents to obtain lower dilutions) in 1.0 ml of deionized water was mixed with phosphate buffer (2.5 ml, Antioxidant Activity of Extracts and Fractions of 0.2M, pH 6.6) and 1% potassium ferricyanide (2.5 ml). The Quercus Infectoria mixture was incubated at 50° C. for 20 min. Aliquots of 0109 All extracts and fractions prepared from the galls of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, Ouercus infectoria was screened for antioxidant activity in which was then centrifuged at 1036xg for 10 min. The upper three in vitro models viz. DPPH methods, reducing power layer of solution (2.5 ml) was mixed with distilled water (2.5 assay and NO scavenging activity and one ex vivo Lipid ml) and a freshly prepared FeCls solution (0.5 ml, 0.1%). The peroxidation assay methods. absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The EXAMPLE-16 results are shown in FIG. 5-8.

Free Radical Scavenging Activity by DPPH Method EXAMPLE-18 0110. The free radical scavenging activity of the test sample was measured in terms of hydrogen donating or radi Scavenging Activity Against Nitric Oxide cal-scavenging ability using the stable radical DPPH. Nitrite Detection Method 0111 Procedure 0112 1 ml of 0.1 mM solution of DPPH in ethanol was 0116 Sodium nitroprusside in aqueous solution at physi added to 3.0 ml of test sample solution in water at different ological pH spontaneously generates nitric oxide, which concentrations (The stock sample solutions was serially interacts with oxygen to produce nitrite ions that can be diluted with respective solvents to obtain lower dilutions). estimated by use of Greiss reagent. Scavengers of nitric oxide Thirty minutes later, the absorbance was measured at 517 nm. compete with oxygen leading to reduce production of nitric Lower absorbance of the reaction mixture was indicated oxide. higher free radical scavenging activity. Butylated hydroxy 0117 Procedure 0118 1.0 ml Sodium nitroprusside (5 mM) in 20 mM toluene (BHT) was used as standard. The capability to scav phosphate-buffered saline (PBS) pH 7.4 was mixed with 1.0 enge the DPPH radical was calculated. The antioxidant activ ml of different concentrations of test samples (extracts) (The ity of the test samples was expressed as ICs. The ICs value stock test sample solutions was serially diluted with respec is defined as the concentration (in ug/ml) of extracts that tive solvents to obtain lower dilutions) and incubated at 25°C. inhibited the formation of DPPH radicals by 50%. The results for 150 min. The samples from the above were reacted with 1 of are shown in FIG. 1-4 ml of Greiss reagent. The absorbance of the chromophore formed during the diazotization of nitrite with sulphanil EXAMPLE-17 amide and Subsequent coupling with napthylethylenediamine Free Radical Scavenging Activity by Reducing was read at 546 nm and referred to the absorbance of standard Power Assay Solutions of potassium nitrite, treated in the same way with 0113. The reducing power of test sample (extract) was Griess reagent. The percentages of NO scavenging activity of determined in Fe" Fe" redox system according to the test samples (extracts) are shown in FIG. 10. method of Oyaizu (1986). EXAMPLE-19 0114 Procedure 0115 Various concentrations of the test samples (extracts) 0119) All herbal antistress drink were prepared using the (The stock test sample solutions was serially diluted with said antioxidant composition, AXT-3 along with water US 2008/01 75934 A1 Jul. 24, 2008

extracts of Withania somnifera, Emblica officinalis, Vitis vin with hexane and acetone (1:1) solvent mixture and wherein ifera, Sugar syrup, Citric acid, Ascorbic acid and Sodium said fraction is characterized by having antioxidant concen benzoate as per the formula given below. tration of more than about 80%. 7. The herbal composition according to claim 5, wherein said extract is obtained by fractionation of methanol extract 1. Withania somnifera 1.0 g with chloroform and methanol (1:1) solvent mixture. 2. Emblica officinalis 1.0 g 8. The composition according to claim 1, wherein said 3. Vitis vinifera 3.0 g composition is effectively incorporated in cosmetics, phar 4. AXT-3 0.6 g. 5. Citric acid 15.0 g maceuticals and functional foods such as energy health drinks 6. Ascorbic acid 2.4 g to reduce oxidative stress that is associated with ageing, car 7. Sodium benzoate 1.0 g diovascular disease, cancer, immunological disorders, 8. Sugar syrup 1.0 L dementia, diabetes or macular degeneration and other lif 9. Flavour (passion fruit) 10.0 g estyle diseases or as natural preservative or as antimicrobial 10. DM water (q.s.) 2.0 L agent. 9. The herbal composition according to claim 1, wherein said composition is effectively used as antistress herbal EXAMPLE-20 drinks. 0120 All herbal Energy drink was prepared using the said 10. The herbal composition according to claim 9, wherein antioxidant composition AXT-3 along with water extracts of said antistress herbal drink composition comprises water Camellia sinensis, Withania somnifera, Emblica officinalis, extracts of Withania somnifera (1.0 g), Emblica officinalis Vitis vinifera. Sucrose, Citric acid, thickening agent (stimu (1.0 g), vitis vinifera (3.0 g) and methanol extract of Ouercus leol) as per the formula given below. infectoria (0.6 g). 11. The herbal composition according to claim 1, wherein said composition is effectively used as energy health drinks. 1. Camelia Sinensis 6.0 g 12. The herbal composition according to claim 11, wherein 2. Withania somnifera 2.0 g said energy health drink composition comprises water 3. Emblica officinalis 2.0 g extracts of Camellia sinensis (6.0 g). Withania somnifera (2.0 4. Vitis vinifera 6.0 g g). Emblica officinalis (2.0 g), vitis vinifera (6.0 g) and metha 5. AXT-3 0.6 g. 6. Citric acid 3.0 g nol extract of Quercus infectoria (0.6 g). 7. Sucrose 125.0 g 13. The herbal composition according to claim 1, wherein 8. Stimuleol 0.5 g. said extract is obtained by a process comprising steps of: 9. Mixed fruit flavour 1.0 g (a) pulverizing the shade dried galls of Quercus infectoria 10. DM water (q.s.) 1.0 L to coarse powder, While this invention has been described in detail with refer (b) subjecting the resultant coarse powder of said dried ence to certain preferred embodiments, it should be appreci galls of Quercus infectoria into an extraction vessel in ated that the present invention is not limited to those precise presence of a solvent either alone or in combination embodiments. Rather, in view of the present disclosure, thereof, which describes the current best mode for practicing the (c) filtering the resultant plant extracts employing Suitable invention, many modifications and variations would present filter, themselves to those skilled in the art without departing from (d) drying the resultant solvent extracts to form a concen the scope and spirit of this invention. trate, wherein obtained extract is characterized by having anti We claim: oxidant concentration of more than about 80% and is 1. A Herbal composition comprising extract of Quercus used in the preparation of said herbal composition. infectoria having high and effective concentration of antioxi 14. A process for preparing a herbal composition compris dant and phenolic compounds, alone or in combination with ing extract of Quercus infectoria having high and effective other effective herbs and pharmaceutically acceptable excipi concentration of antioxidant and phenolic compounds, alone entS. or in combination with other effective herbs and pharmaceu 2. The composition according to claim 1, wherein said tically acceptable excipients, the process comprising: extract is obtained from all parts of the plant Quercus infec toria, preferably from galls. (a) extracting said extract by pulverizing the shade dried 3. The composition according to claim 1, wherein said galls of Quercus infectoria to coarse powder, antioxidant is preferably methyl gallate. (b) subjecting the resultant coarse powder of said dried 4. The composition according to claim 1, wherein said galls of Quercus infectoria into an extraction vessel in extract is obtained by using solvent selected from non-polar presence of a solvent either alone or in combination hydrocarbon, chlorinated solvent, an ester, ketone, an alcohol thereof, Or Water. (c) filtering the resultant plant extracts employing Suitable 5. The composition according to claim 4, wherein the Sol filter, vent is preferably selected from hexane, chloroform and (d) drying the resultant solvent extracts to form a concen dichloromethane, ethyl acetate, acetone, methanol and ethyl trate, and a thick oil portion from said concentrate, alcohol. (e) characterizing the resultant extract by analyzing the 6. The herbal composition according to claim 5, wherein concentration of antioxidants and phenolic compounds said extract is obtained by fractionation of methanol extract in the resultant and, US 2008/01 75934 A1 Jul. 24, 2008

(f) mixing the analyzed resultant extract with pharmaceu (b) subjecting the resultant coarse powder of said dried tically acceptable excipients to prepare said herbal com galls of Quercus infectoria into an extraction vessel in position. presence of a solvent either alone or in combination 15. The process according to claim 14, wherein extraction thereof, is performed employing any extraction technique. (c) filtering the resultant plant extracts employing Suitable 16. The process according to claim 14, wherein extraction filter, is performed preferably employing percolation, maceration (d) drying the resultant solvent extracts to form a concen or soxhlet method. trate. 17. The process according to claim 14, wherein the solvent 24. The composition according to claim 23, wherein the used in the process is selected from non-polar hydrocarbon, solvent is selected from non-polar hydrocarbon, chlorinated chlorinated Solvent, an ester, ketone, an alcohol or water. Solvent, an ester, ketone, an alcohol or water. 18. The process according to claim 17, wherein the solvent 25. The composition according to claim 24, wherein the used in the process is preferably selected from hexane, chlo solvent is preferably selected from hexane, chloroform and roform and dichloromethane, ethyl acetate, acetone, metha dichloromethane, ethyl acetate, acetone, methanol or ethyl nol or ethyl alcohol. alcohol. 19. The process according to claim 18, wherein said sol 26. The composition according to claim 23, wherein said vent is preferably methanol for extraction and followed by solvent is preferably methanol with hexane and acetone in 1:1 hexane and acetone (1:1) solvent mixture for fractionation. ratio. 20. The process according to claim 14, wherein obtained 27. The composition according to claim 26, wherein extract is characterized by having antioxidant concentration obtained extract is characterized by having antioxidant con of more than about 80%. centration of more than about 80%. 21. The process according to claim 14, wherein said sol 28. The composition according to claim 23, wherein said vent is preferably methanol for extraction and followed by solvent is preferably methanol with chloroform and methanol chloroform and methanol (1:1) solvent mixture for fraction in 1:1 ratio. ation. 29. The composition according to claim 23, wherein 22. The process according to claim 14, wherein obtained obtained extract is characterized by having phenolic com extract is characterized by having phenolic compounds con pounds concentration of more than about 108 w/w. centration potential of more than about 108 w/w against tan 30. The composition according to claim 23, wherein said nic acid. composition is effectively incorporated in cosmetics, phar 23. A herbal composition comprising extract of Quercus maceuticals and functional foods such as energy health drinks infectoria having high and effective concentration of antioxi to reduce oxidative stress that is associated with ageing, car dant and phenolic compounds, alone or in combination with diovascular disease, cancer, immunological disorders, other effective herbs and pharmaceutically acceptable excipi dementia, diabetes and macular degeneration and other lif ents, wherein said extract is obtained by a process comprising estyle diseases or as natural preservative or as antimicrobial steps of: agent. (a) pulverizing the shade dried galls of Quercus infectoria to coarse powder,