Abstract:
Disclosed is a novel herbal composition comprising extract of  Quercus infectoria  having rich and effective concentration of as antioxidants and phenolic compounds. Further the present invention provides a process for preparing said composition and use thereof in cosmeceuticals, pharmaceuticals and health drinks for treating oxidative stress and other life style diseases.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention, in general relates to a novel herbal composition comprising extracts of  Quercus infectoria . More particularly, the present invention provides a novel herbal composition comprising extract of  Quercus infectoria  having high and effective concentration of antioxidants and phenolic compounds. The invention further provides a process for preparing said composition and use thereof. 
       BACKGROUND OF THE INVENTION 
       [0002]    Reactive Oxygen species and Reactive Nitrogen species (ROS/RNS) arise in the normal course of oxidative metabolism. These reactive species (ROS/RNS) influence multiple metabolic, signaling and transcriptional processes, which are involved in the normal function of cell. 
         [0003]    Oxidative stress is a condition that occurs when there is excess and prolonged imbalance between the generation of reactive oxygen species and reactive nitrogen species and control by antioxidants. Reducing reactive oxygen species and reactive nitrogen species in the body is one of the primary target towards the protection of various diseases such as aging process, cancer, diabetes and neurodegenerative diseases. 
         [0004]    Dietary compounds such as vitamins C and E may limit oxidative damage directly but along these other dietary constituents viz. carotenoids and polyphenols may also act via indirect mechanisms such as endogenous antioxidant enzyme activity and thereby reduce the risk of a number of the age related disorders. 
         [0005]    Polyphenols occur most commonly in foods of plant origin have attracted much attention as potent antioxidants due to their ability to scavenge free radical and form inert phenoxy radical intermediates. A number of epidemiological studies have suggested that consumption of polyphenol-rich foods reduces the risk of developing chronic diseases. 
         [0006]    Although, these polyphenol antioxidants reported for the prevention of various lifestyle diseases but only a few findings show that they are active and majority of the results show that they have no clinical efficacy. The vast literature on antioxidants suggests that the failure of polyphenol antioxidant in clinical cases because it is almost impossible to eliminate many active oxygen species produced in the body by single antioxidant molecule. 
         [0007]    Polyphenol antioxidants derived from the plant origin have structurally phenol group as backbone and make it more lipid soluble. The bioavailability of these plant polyphenols is very low in GI tract and therefore most of these antioxidants have not sufficiently displayed efficacy in the body even though they have been reported as potent antioxidants in vitro. 
         [0008]    To overcome the above said difficulties in the preparation of antioxidant composition that is effective both in vitro and clinical, it is necessary to select a group of polyphenolic compounds rich composition that is selectively address the inhibition of many enzymatic pathways that produce free radicals and also the overall composition should improve the bioavailability of the antioxidant active fraction. 
       PRIOR ART 
       [0009]    U.S. Pat. No. 6,534,046 to Golz-Bemer et al. disclosed the preparation of plant derived anti-perspiration cosmetics comprising the extracts of  Equisetum arvense, Salvia officinalis, Hamamelis Virginia  and  Quercus infectoria.    
         [0010]    U.S. Pat. No. 6,264,926 to Farooqi et al. disclose a formulation useful as a natural herbal tooth powder comprising the extracts of  Quercus infectoria, Zanthoxylum amatum  and  Zingiber officinalis  etc. 
         [0011]    Kaur G et al. (J. Ethnopharmacol. 2004, 90(2-3). 285-92) have reported the anti-inflammatory activities of alcoholic extracts of galls of  Quercus infectoria.    
         [0012]    Sawangiaroen N et al (J Ethnopharmacol 2005 98(1-2) 67-72) have reported the antidiarrhoel effect of methanol extract of galls of  Quercus infectoria.    
         [0013]    United States Patent application No. 20030138509 to Pushpangadan et al. teaches the preparation of fermented herbal health drink from the plant  Andrographis paniculata.    
         [0014]    U.S. Pat. No. 4,741,915 to Farr et al. disclosed the use of purified and hydrolyzed gallotannins derived from plant materials for inhibition of oxidation in foodstuffs. 
         [0015]    U.S. Pat. No. 7,041,332 to Gaudout et al. teaches the preparation of a phenolic rich fraction obtained from the fruit of family rosaceae for use in cosmetic, dietary and nutraceutical preparation. 
         [0016]    U.S. Pat. No. 6,989,161 to Rouf et al discloses a composition comprising phytonutrients viz. lutein, lycopene, epigallocatechin gallate (EGCG), ellagic acid, hesperidin and quercetin. 
       SUMMARY OF THE INVENTION 
       [0017]    It is a principal object of the present invention to provide a herbal composition comprising extract of  Quercus infectoria  having high and effective concentration of antioxidant and phenolic compounds. 
         [0018]    Another aspect of the present invention is to obtain the extract using all parts of the plant  Quercus Infectoria , preferably from the galls. 
         [0019]    Yet another aspect of the present invention is to obtain a herbal composition comprising antioxidant rich extract of  Quercus Infectoria , wherein the antioxidant is preferably methyl gallate. 
         [0020]    Still another aspect of the present invention is to obtain the extract of  Quercus Infectoria  employing solvents selected from non-polar hydrocarbon, chlorinated solvents, an ester and ketones, an alcohol and water, preferably, selected from hexane, chloroform, ethyl acetate, acetone, methanol or ethyl alcohol. 
         [0021]    Yet another aspect of the present invention is to obtain the extract of the said herbal composition employing methanol with hexane and acetone preferably in 1:1 ratio, wherein the said extract is characterized by having antioxidant concentration of more than 80%. 
         [0022]    Still another aspect of the present invention is to obtain the extract of the said herbal composition employing methanol with chloroform and methanol preferably in 1:1 ratio, wherein said extract is characterized by having phenolic compounds concentration of more than about 108 w/w 
         [0023]    In accordance with further aspect of the present invention, there is provided an antioxidant and phenolic compounds rich herbal composition, which is obtained by a process, comprising the steps of (a) pulverizing the shade dried galls of  Quercus Infectoria  to coarse powder, (b) subjecting the resultant coarse powder of said dried galls of  Quercus Infectoria  into an extraction vessel in presence of a solvent either alone or in combination thereof, (c) filtering the resultant plant extracts employing suitable filter, (d) drying the resultant solvent extracts to form a concentrate. 
         [0024]    Further aspect of the present invention is the obtained extract is characterized by having antioxidant principle concentration of more than 80% and phenolic principle concentration of more than about 108% w/w. 
         [0025]    In yet another aspect, the present invention is endowed with a process for preparing a herbal composition comprising extract of  Quercus Infectoria  having high and effective concentration of antioxidant and phenolic principles, alone or in combination with other effective herbs and pharmaceutically acceptable excipients, wherein the said process is comprised of; (a) pulverizing the shade dried galls of  Quercus Infectoria  to coarse powder, (b) subjecting the resultant coarse powder of said dried galls of  Quercus Infectoria  into an extraction vessel in presence of a solvent either alone or in combination thereof, (c) filtering the resultant plant extracts employing suitable filter, (d) drying the resultant solvent extracts to form a concentrate, (d) characterizing the resultant extract by analyzing presence of concentration of antioxidant and phenolic compounds, (f) mixing the analyzed resultant extract with pharmaceutically acceptable excipients to prepare the said herbal composition. 
         [0026]    It is another aspect of the present invention wherein; the extraction is performed employing any suitable hot or cold extraction techniques, preferably, percolation, maceration or soxhlet method. 
         [0027]    One another preferred aspect of the present invention is to effectively incorporate the said herbal composition comprising extract of galls of  Quercus Infectoria  in cosmetics, pharmaceuticals and functional foods such as energy health drinks to reduce oxidative stress that is associated with ageing, cardiovascular disease, cancer, immunological disorders, dementia, diabetes and macular degeneration and other lifestyle diseases or as natural preservative, antioxidant or as antimicrobial agent. 
         [0028]    One another aspect of the present invention is to assess the antioxidant and antimicrobial activity of methanol extract of galls of  Quercus Infectoria.    
         [0029]    It is yet another aspect of the present invention is to use the said herbal composition for preventing oxidative stress. 
         [0030]    It is yet another preferred aspect of the present invention is to evaluate the said herbal composition for antifungal activity employing fungal strains such as  Trycophyton rubrum  (MTCC296),  Candida albicans  (MTCC 741),  Tricophyton gypseum  (ATCC 8125™),  Tricophyton mentagrophytes  (ATCC 52018). 
         [0031]    It is still another preferred aspect of the present invention is to appraise the said herbal composition for antibacterial activity employing bacterial strains such as  Escherichia coli  (MTCC 443),  Pseudomonas aeruginosa  (MTCC 741),  Salmonella typhi  (MTCC 733),  Staphylococcus aureus  (MTCC 96),  Helicobacter pylori  (ATCC No-51653™),  Niesseria gonorrhoeae  (ATCC — 49226). 
         [0032]    Another aspect of the present invention is to evaluate the inhibitory activity of said herbal composition against yeast  Malassezia furfur  (MTCC-1374) causing dandruff and  Malassezia pachydermatis  causing ear infection. 
         [0033]    In one another preferred aspect of the present invention is to evaluate the antioxidant and free radical scavenging activity of the said herbal composition by employing DPPH method, Ferric reducing Power Assay Method, and determining Nitric oxide scavenging activity. 
         [0034]    Yet another aspect of the present invention is to prepare the extracts of  Quercus Infectoria  by employing percolation method and hot soxhlet method. 
         [0035]    Another aspect of the present invention is to prepare the various extracts such as methanol extract, water extract, acetone extract, ethyl acetate and acetone (1:1) extract, chloroform and methanol (8:2) extract in order to obtain 20% of the raw materials. 
         [0036]    It is another aspect of the present invention is to identify the various active fractions of the methanolic extract of  Quercus infectoria  with varying degree of activity. 
         [0037]    In one another aspect of the present invention is to estimate the percentage concentration of methyl gallate present in the various active fractions of  Quercus infectoria  by HPLC standardization. 
         [0038]    It is still another aspect of the present invention is to estimate the presence of total phenolic compounds in various solvent fractions and semi purified fractions of extract of  Quercus infectoria.    
         [0039]    One another aspect of the present invention is to employ the column chromatography for purifying the active compounds of various active fractions of  Quercus infectoria.    
         [0040]    Additional aspect of the present invention is to prepare an anti stress health drink comprising water extract of  Withania somnifera  (1.0 g),  Emblica officinalis  (1.0 g),  Vitis vinifera  (3.0 g) and methanol extract of galls of  Quercus infectoria  (AXT-3) (0.6 g). 
         [0041]    Further aspect of the present invention is to prepare the energy health drink comprising the water extracts of  Camellia sinensis  (6.0 g.),  Withania somnifera  (2.0 g),  Emblica officinalis  (2.0 g),  Vitis vinifera  (6.0 g) and methanol extract of galls of  Quercus infectoria  (AXT-3) (0.6 g.) 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0042]    Further objects of the present invention together with additional features contributing thereto and advantages accruing there from will be apparent from the description of preferred embodiments of the present invention which are shown in the accompanying drawing figures. 
           [0043]      FIG. 1 . DPPH radical scavenging activity of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-01, 02, 16, 17, 18. 
           [0044]      FIG. 2 . DPPH radical scavenging activity of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 21, 24. 
           [0045]      FIG. 3 . DPPH radical scavenging activity of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-22, 23. 
           [0046]      FIG. 4 . DPPH radical scavenging activity of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-19, 20. 
           [0047]      FIG. 5 . Reducing power assay of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-01, 02, 16, 17, 18. 
           [0048]      FIG. 6 . Reducing power assay of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, 15, 21, 24. 
           [0049]      FIG. 7 . Reducing power assay of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-22, 23. 
           [0050]      FIG. 8 . Reducing power assay of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-19, 20. 
           [0051]      FIG. 9 . Lipid peroxidation (in vitro) assay of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-01, 03, 10, 31, 33. 
           [0052]      FIG. 10 . NO (Nitric Oxide) scavenging activity of  Quercus infectoria  extracts (IC 50 -[μg/ml] values) AXT-03, 05, 07, 09, 11, 16, 17, 19, 20, 21, 23, 28, 30, 31. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0053]    The present invention involves the selection of herb and subjecting the herb for solvent extraction by various methods and screening of these solvent extracts for the antioxidant activity and antimicrobial activity. Preparation of a composition using the bioactive extract for pharmaceuticals, cosmeceuticals and health drinks as an antioxidant to treat and prevent various life style disease such as stress, ageing, cardiovascular disease, cancer, immunological disorders, dementia, diabetes and macular degeneration and also prevents microbial contamination as natural preservative. 
         [0054]      Quercus infectoria  of the family Fagaceae is a small tree or shrub and about 2-5 m. high, leaves are very rigid, 4-6 cm.long, very rigid, often glabrescent with spinous teeth, mostly distributed in the temperate regions of the northern hemisphere and extending to sub-tropical and tropical America and Asia at high altitudes. Around, 23 evergreen are found in the Himalayan region of India 
         [0055]    Gall nut is used externally for its astringent effect; it is used in ointments for the treatment of piles, and in plasters. The tannic and gallic acids extracted from the galls are often used in dysentery and diarrhoea and as a gargle. 
       EXAMPLE-1 
     Preparation of  Quercus infectoria  Extract by Percolation Method 
       [0056]    The shade dried galls of  Quercus infectoria  was pulverized to coarse powder and about 1 Kg each of powdered material placed in different percolators and the material was soaked in n-hexane, dichloromethane, chloroform, ethyl acetate, acetone, ethanol, methanol and water either alone or in combination thereof at room temperature for 24 to 48 h 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 dryness on rotatory evaporator or on steam bath at optimum temperature and under reduced pressure. 
       EXAMPLE-2 
     Preparation of  Quercus infectoria  Extract by Hot-Soxhlation Method 
       [0057]    The shade dried galls of  Quercus infectoria  was pulverized to coarse powder and about 1 Kg each of the powdered material subjected to hot-soxlation in different soxhlet apparatus using solvents n-hexane, dichloromethane, chloroform, ethyl acetate, acetone, ethanol and methanol either alone or combination thereof at optimum temperature until extraction is completed, then plant extracts were filtered through whatmann no. 1 filter paper and concentrated to dryness on rotatory evaporator or on steam bath at optimum temperature. All extracts were qualitatively similar to extracts prepared by percolation method. The yields of solvent extracts that are considerably more than 20% are summarized in the table-1. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Sl 
                   
                   
                   
                   
               
               
                 No 
                 Code No 
                 Solvents Used 
                 Source 
                 Yield (%) 
               
               
                   
               
             
             
               
                 1 
                 AXT-1 
                 Methanol 
                 Plant material 
                 82.0 
               
               
                 2 
                 AXT-2 
                 Water 
                 Plant material 
                 60.0 
               
               
                 3 
                 AXT-16 
                 Ethyl acetate:Acetone (1:1) 
                 Plant material 
                 63.3 
               
               
                 4 
                 AXT-17 
                 Chloroform:Methanol (8:2) 
                 Plant material 
                 21.5 
               
               
                 5 
                 AXT-18 
                 Acetone 
                 Plant material 
                 69.5 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-3 
     Solvent-Solvent Fractionation of Methanol Extract (AXT-1) 
       [0058]    About 1 Kg of methanol extract was macerated with different solvents alone or combination thereof as mentioned in the table-2 to obtain a purified active fraction with potent antioxidant and antimicrobial activities. The yields of active fractions obtained from the solvent-solvent fractionation are given in table-2. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Sl 
                 Code 
                   
                   
                 Yield 
               
               
                 No 
                 No 
                 Solvents Used 
                 Source 
                 (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AXT-3 
                 Hexane:acetone(1:1) 
                 Methanol extract 
                 7.0 
               
               
                 2 
                 AXT-4 
                 Hexane:acetone (6:4) 
                 Methanol extract 
                 4.0 
               
               
                 3 
                 AXT-5 
                 Chloroform:methanol (9:1) 
                 Methanol extract 
                 7.5 
               
               
                 4 
                 AXT-6 
                 Chloroform:methanol (1:1) 
                 Methanol extract 
                 70.0 
               
               
                 5 
                 AXT-7 
                 Dichloromethane:acetone (7:3) 
                 Methanol extract 
                 2.5 
               
               
                 6 
                 AXT-8 
                 Dichloromethane:acetone (1:1) 
                 Methanol extract 
                 46.3 
               
               
                 7 
                 AXT-9 
                 Chloroform:acetone (1:1) 
                 Methanol extract 
                 4.2 
               
               
                 8 
                 AXT-10 
                 Ethyl acetate:acetone (1:1) 
                 Methanol extract 
                 88.2 
               
               
                 9 
                 AXT-11 
                 Toluene:methanol (9:1) 
                 Methanol extract 
                 2.0 
               
               
                 10 
                 AXT-12 
                 Ethyl acetate 
                 Methanol extract 
                 69.2 
               
               
                 11 
                 AXT-13 
                 Ethyl acetate:methanol (7.5:2.5) 
                 Methanol extract 
                 94.0 
               
               
                 12 
                 AXT-14 
                 Ethyl acetate:methanol (1:1) 
                 Methanol extract 
                 96.0 
               
               
                 13 
                 AXT-15 
                 Ethyl acetate:acetone (6:4) 
                 Methanol extract 
                 80.0 
               
               
                 14 
                 AXT-21 
                 Dichloromethane:methanol (1:1) 
                 Methanol extract 
                 82.0 
               
               
                 15 
                 AXT-24 
                 Ethyl acetate:acetone (2.5:7.5) 
                 Methanol extract 
                 88.0 
               
               
                 16 
                 AXT-25 
                 Acetone 
                 Methanol extract 
                 80 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-4 
     Solvent-Solvent Fractionation of Water Extract (AXT-2) 
       [0059]    About 1 Kg each of water extract was suspended in dichloromethane:acetone (7:3) and dichloromethane:acetone (1:1) to obtain semipurified fractions AXT-22 and AXT-23 respectively. The yields of AXT-22 and AXT-23 fractions are given in table-3. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Sl 
                   
                   
                   
                 Yield 
               
               
                 No 
                 Code No 
                 Solvents Used 
                 Source 
                 (%) 
               
               
                   
               
             
             
               
                 1 
                 AXT-22 
                 Dichloromethane:acetone (7:3) 
                 Water extract 
                 5.0 
               
               
                 2 
                 AXT-23 
                 Dichloromethane:acetone (1:1) 
                 Water extract 
                 8.0 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-5 
     Solvent-Solvent Fractionation of Semipurified Ethyl Acetate Fraction (AXT-12) 
       [0060]    The ethyl acetate fraction obtained at the yield of 69.2% from the methanol extract was further fractionated 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 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                 Code 
                   
                   
                   
               
               
                 Sl No 
                 No 
                 Solvents Used 
                 Source 
                 Yield (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AXT-19 
                 Dichloromethane:acetone(7:3) 
                 Ethyl acetate 
                 9.0 
               
               
                   
                   
                   
                 fraction 
               
               
                 2 
                 AXT-20 
                 Dichloromethane:acetone(1:1) 
                 Ethyl acetate 
                 10.0 
               
               
                   
                   
                   
                 fraction 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-6 
     Column Chromatography of Methanol Extract (AXT-1) 
       [0061]    The column chromatography was performed over silica gel (60-120 mesh). About 2.5 Kg of silica gel was suspended in chloroform and packed the column. About 300 g of methanol extract was prepared as slurry using silica gel and poured on to the column. The column was then eluted with chloroform, chloroform:methanol with increasing polarity to obtain purified active fraction. The details of column fractions and their yields are summarized in the table-5. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Sl. No. 
                 Fraction No 
                 Solvent system 
                 Yield (g) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AXT-1/1 
                 Chloroform 
                 5 
               
               
                 2 
                 AXT-1/2 
                 Chloroform:methanol (95:5) 
                 7 
               
               
                 3 
                 AXT-1/3 
                 Chloroform:methanol (90:10) 
                 50 
               
               
                 4 
                 AXT-1/4 
                 Chloroform:methanol (85:15) 
                 15 
               
               
                 5 
                 AXT-1/5 
                 Chloroform:methanol (80:20) 
                 100 
               
               
                 6 
                 AXT-1/6 
                 Methanol 
                 100 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-7 
     Standardization of Extracts and Fractions by TLC and HPLC 
       [0062]    All extracts and fractions were prepared at a concentration of 100 mg in 5 mL of the respective solvent and about 50 μl of the sample solution were spotted on precoated (e-Merck) silica gel TLC plates. The TLC plates were air dried and placed in the mobile Phase: (1) Dichloromethane:Methanol (85:15) and (2) Hexane:Ethyl acetate (30:70). TLC plates were air dried and sprayed with 1% alcoholic ferric chloride solution to visualize as dark blue spots confirming the presence of phenolic compounds. 
         [0063]    All extracts and fractions were also subjected to HPLC analysis for standardization purpose taking methyl gallate as principle marker. The results are summarized in table-6. The HPLC conditions are as follows. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Column: 
                 Reverse Phase C-18 
               
               
                   
                 Mobile Phase: 
                 Solvent A:Solvent B (20:80) 
               
               
                   
                 Solvent A: 
                 0.05% phosphoric acid in ACN 
               
               
                   
                 Solvent B: 
                 0.1% phosphoric acid in water 
               
               
                   
                 Flow rate: 
                 1 ml/min 
               
               
                   
                 Detection: 
                 UV λ max  240 nm 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Sl No 
                 Code No 
                 (% of Methyl gallate) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AXT-1 
                 11.6 
               
               
                 2 
                 AXT-2 
                 0.2 
               
               
                 3 
                 AXT-3 
                 79.9 
               
               
                 4 
                 AXT-4 
                 70.0 
               
               
                 5 
                 AXT-5 
                 64.7 
               
               
                 6 
                 AXT-6 
                 30.4 
               
               
                 7 
                 AXT-7 
                 58.9 
               
               
                 8 
                 AXT-8 
                 37.7 
               
               
                 9 
                 AXT-9 
                 48.0 
               
               
                 10 
                 AXT-10 
                 32.4 
               
               
                 11 
                 AXT-11 
                 72.4 
               
               
                 12 
                 AXT-12 
                 27.3 
               
               
                 13 
                 AXT-13 
                 21.5 
               
               
                 14 
                 AXT-14 
                 28.8 
               
               
                 15 
                 AXT-15 
                 30.0 
               
               
                 16 
                 AXT-16 
                 7.5 
               
               
                 17 
                 AXT-17 
                 15.9 
               
               
                 18 
                 AXT-18 
                 6.0 
               
               
                 19 
                 AXT-19 
                 58.3 
               
               
                 20 
                 AXT-20 
                 36.5 
               
               
                 21 
                 AXT-21 
                 31.8 
               
               
                 22 
                 AXT-22 
                 64.6 
               
               
                 23 
                 AXT-23 
                 77.8 
               
               
                 24 
                 AXT-24 
                 30.2 
               
               
                 25 
                 AXT-25 
                 25.8 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-8 
     Quantitative Estimation of Phenolic Compounds 
       [0064]    The phenolics present in the extracts and fractions of  Quercus infectoria  was quantitatively measured using tanninc acid as standard. The greenish blue colour produced during reaction of phenolic compounds with potassium ferri cyanide and ferric chloride was measured at 720 nm. The results are summarized in the table-7. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Sl. 
                   
                 Total phenols 
               
               
                 No 
                 Code No 
                 (Tannic acid) w/w 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 AXT-1 
                 90.09 
               
               
                 2 
                 AXT-2 
                 74.05 
               
               
                 3 
                 AXT-3 
                 97.72 
               
               
                 4 
                 AXT-4 
                 90.45 
               
               
                 5 
                 AXT-5 
                 82.13 
               
               
                 6 
                 AXT-6 
                 108.31 
               
               
                 7 
                 AXT-7 
                 87.22 
               
               
                 8 
                 AXT-8 
                 97.02 
               
               
                 9 
                 AXT-9 
                 91.91 
               
               
                 10 
                 AXT-10 
                 83.96 
               
               
                 11 
                 AXT-11 
                 96.37 
               
               
                 12 
                 AXT-12 
                 98.53 
               
               
                 13 
                 AXT-13 
                 83.97 
               
               
                 14 
                 AXT-14 
                 80.2 
               
               
                 15 
                 AXT-15 
                 89.52 
               
               
                 16 
                 AXT-16 
                 78.22 
               
               
                 17 
                 AXT-17 
                 92.91 
               
               
                 18 
                 AXT-18 
                 92.20 
               
               
                 19 
                 AXT-19 
                 98.55 
               
               
                 20 
                 AXT-20 
                 93.65 
               
               
                 21 
                 AXT-21 
                 94.71 
               
               
                 22 
                 AXT-22 
                 96.33 
               
               
                 23 
                 AXT-23 
                 94.40 
               
               
                 24 
                 AXT-24 
                 96.32 
               
               
                 25 
                 AXT-25 
                 93.04 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-9 
     MIC Assay for Antifungal Activity by Agar Dilution Method 
       [0065]    The test organisms  Trycophyton rubrum  (MTCC 296) and  Candida albicans  (MTCC 741) were procured from IMTECH, Chandigarh, India and  Tricophyton gypseum  (ATCC 8125™),  Tricophyton mentagrophytes  (ATCC 52018), were procured from USA. These are dermatophytes causing infections of hair, nails and skin in humans. These are cosmopolitan in distribution 
         [0066]    Preparation of Test Samples (Extracts) for Screening 
         [0067]    About 100 mg of test sample (extract) was weighed in to a sterile screw capped tube and dissolved in 1 ml of sterile distilled water in case of water soluble extracts, otherwise, extracts were dissolved in 100 μL of DMSO and then subsequently in 900 μL of sterile distilled water. 
         [0068]    Media Preparation 
         [0069]    Sabouraud chloremphenicol Agar (Himedia) was used for cultivation of fungal strains. The sabouraud chloremphenicol (65 grams) was suspended in 1 Litre distilled water, heated to boil to dissolve the media completely and autoclaved at 15 lbs at 121° C. for 15 minutes. 
         [0070]    Inoculum Preparation 
         [0071]    A seven day old pure culture of the fungi was taken for the test. 
         [0072]      Candida albicans —The 24 hrs culture of  Candida albicans  was suspended from agar plate into 2 ml of sterile saline. The turbidity of the suspension was adjusted to McFarland&#39;s turbidometer standard 0.5. 
         [0073]    Procedure 
         [0074]    The required amount of the test sample was taken in the petri plate and mixed it with 4 ml of the molten sabouraud chloremphenicol Agar. (To achieve 1 mg/ml concentrations in the plate dilute 40 μL of the extract (100 mg/ml). A range of concentrations with the extracts in duplicate was prepared. A plate for negative control (media control), a plate for positive control without extract were saved and allowed to solidify. The plates were marked on the bottom for the names of the organisms viz.  Trycophyton rubrum, Tricophyton gypseum , and  Tricophyton mentagrophytes . A small amount of mycelia were picked with sterile inoculation stab and inoculated on the surface of the plate. The plates were incubated at room temp for 7 days and observed for visible growth and MIC were calculated. The results are summarized in the table-8 
       EXAMPLE-10 
     MIC Assay for Antibacterial Activity 
       [0075]    The test organisms  Escherichia coli  (MTCC 443),  Pseudomonas aeruginosa  (MTCC 741),  Salmonella typhi  (MTCC 733),  Staphylococcus aureus  (MTCC 96) were obtained from IMTECH, Chandigarh, India. 
         [0076]    Preparation of Samples for Screening 
         [0077]    About 100 mg of test sample (extract) was weighed in to a sterile screw capped tube and dissolved in 1 ml of sterile distilled water in case of water soluble extracts, otherwise, extracts were dissolved in 100 μl of DMSO and then subsequently in 900 μl of sterile distilled water. 
         [0078]    Media Preparation 
         [0079]    Mueller Hinton Agar (Himedia M173, 38 g.) was suspended in 1 Litre of distilled water and heated to dissolve the medium completely. Mueller Hinton Broth (Himedia M391) 21 g. was suspended in 1 Litre distilled water and autoclaved at 15 lbs pressure at 121° C. for 15 minutes. Miller Hinton Agar was cooled to 55° C. 
       Inoculum Preparation 
       [0080]    The overnight culture of test bacteria was suspended from the agar plate into 2 ml of sterile saline and adjusted the turbidity of the suspension to McFarlands turbidometer standard 0.5. Dilution ( 1/100 th ) of the adjusted inoculum was prepared in Muller Hinton Broth. This inoculum contained approximately 1{tilde over (×)}1 □  Organisms per ml. 
         [0081]    Procedure 
         [0082]    The required amount of the extract was taken in the petri plate and mixed it with 4 ml of the molten Mueller Hinton agar. (To achieve 1 mg/ml concentration in the plate diluted 40 μl of extract (100 mg/ml). A range of concentrations was prepared with the extracts in duplicates. A plate for negative control (media control), and a plate for positive control without extract were kept to solidify. Inoculate was spotted with 20 μl of adjusted inoculum on the surface of the agar plate. The plates were incubated for 48 hours at 37° C. Observed the plates for visual growth and MIC were calculated. The results are given in table-8. 
       EXAMPLE-11 
     Assay for Gram-Negative Rod  Helicobacter pylori  (ATCC No-51653™) 
       [0083]    Media Preparation 
         [0084]    Mueller Hinton Agar (Himedia M173) 38 g was suspended in 1 Litre of distilled water. Tryptic soya broth (Himedia) 21 g was put in 1 Litre distilled water. About 10% defibrinated sheep blood was autoclaved at 15 lbs pressure at 121° C. for 15 minutes. These were autoclaved separately. Autoclaved 10% defibrinated sheep blood was added to miller Hinton Agar. The medium was brought to 55° C. 
         [0085]    Inoculum Preparation 
         [0086]    The fully grown test bacteria (5 days) was suspended from the agar plate into 2 ml of sterile saline and adjust the turbidity of the suspension to McFarlands turbidometer standard 0.5. Prepared 1/100 th  dilution of the adjusted inoculum in Tryptic soya Broth. This inoculum contain approximately X Organisms per ml. 
         [0087]    Procedure 
         [0088]    The required amount of the extract was taken in the petriplate and mixed it with 4 ml of the molten Mueller Hinton agar+10% defibrinated sheep blood. (To achieve 1 mg/ml concentration in the plate diluted 40 micro liter of extract (100 mg/ml). A range of concentrations were prepared with the extracts in duplicates. A plate for negative control (media control), and a plate for positive control without extract were kept to solidify. Inoculate was spotted with 20 μl of adjusted inoculum on the surface of the agar plate. The plates were incubated for 5 days in anaerobic jar at 37° C. Observed the plates for visual growth and MIC were calculated. The results are given in table-8. 
       EXAMPLE-12 
     Assay for Gram-Negative Cocci  Niesseria gonorrhoeae  (ATCC 49226) 
       [0089]    Media Preparation 
         [0090]    GC Agar Base (Himedia) (7.2 g) was prepared in distilled water (100 ml) to make a double strength base. Heated to boiling to dissolve the medium completely. The same was sterilized by autoclaving at 15 lbs pressure (121° C.) for 15 minutes. And allowed to cool up to 55° C. aseptically. Separately prepared Haemoglobin (FD002 Himedia) (2%) was added. The vitamin Growth supplements (FD025 Himedia) were added to increase the selectivity of the medium. 
         [0091]    Inoculum Preparation 
         [0092]    About 48 hours incubated culture kept at 37° C. with 5% CO 2  was suspended in to 2 ml of sterile saline. The inoculum was adjusted to McFarland standard 0.5. Prepared 1/100 th  dilution of this in Trypric Soya Broth (DIFCO). 
         [0093]    Procedure 
         [0094]    The media was kept at 55° C. on hot plate to keep molten. The required amount of test sample (extract) was taken in sterile 50 mm petri plate and labeled with the name and concentration of the drug. The test sample was diluted with molten medium in duplicates (To get final concentration of the drug at 1 mg/ml dissolve 40 μL of the extract in 4 ml of the molten medium) One plate for positive control without drug and a plate for media control were kept. The plates were allowed to solidify and then were inoculated with 20 μL of the adjusted inoculum incubated for 48 hours at 37° C. in a anaerobic chamber with 5% CO 2 . [According to instructions of Gas pack (BBL)]. After incubation plates were observed for visual growth by comparing with control. Results were recorded. MIC were calculated as the lowest concentration of the drug showing no growth. The results are summarized in table-8. 
       EXAMPLE-13 
     MIC Assay for Yeast 
       [0095]    The test organisms  Malassezia furfur  (MTCC-1374) is lipophilic yeast found on skin and body surface. The organism was used to test antidandruff activity. This is procured from IMTECH, Chandigarh, India. 
         [0096]    Media Preparation 
         [0097]    Sabouraud Dextrose Agar-Emmons modified (Himedia) (23.5 g) was suspended in 500 ml distilled water. Heated to boil to dissolve the medium completely and sterilized by autoclaving at 15 lbs pressure, 121° C. for 15 minutes. After cooling by bringing to 55° C. added few drops of sterile corn oil and mixed the medium 
         [0098]    Inoculum Preparation 
         [0099]    The fresh culture of  M. furfur  (incubated for 4 or 5 days) from the Agar plate was suspended into 2 ml of sterile saline. The turbidity of the suspension was adjusted to McFarlands turbidometer standard 0.5. 
         [0100]    Procedure 
         [0101]    Sterile petriplates (50 mm) were labeled with the test sample number and percentage of the sample. The required amount of the test sample was added into petriplates and the molten medium to make up the volume to 4 ml in duplicates. A plate as negative control without inoculation of test organism, and a plate as positive control with test organism were saved. The contents were mixed gently and allowed to solidify. Adjusted inoculate 20 microlitre was spotted on the surface of the media plates. The plates were incubated for 7 days at room temp and observed for visible growth by comparing with positive control. MIC were calculated and results given in table-8. 
       EXAMPLE-14 
     MIC Assay for Yeast  Malassezia pachydermatis    
       [0102]    The test organism  Malassezia pachydermatis  is yeast causing ear infections in canines (MTCC-1369) procured from IMTECH Chandigarh, India. The organism was used to test ear infections. 
         [0103]    Media Preparation 
         [0104]    Sabouraud Dextrose Agar-Emmons modified (Himedia) (23.5 g) was suspended in 500 ml distilled water. Heated to boil to dissolve the medium completely and sterilized by autoclaving at 15 lbs pressure, 121° C. for 15 minutes. After cooling by bringing to 55° C. added few drops of sterile corn oil and mixed the medium uniformly. 
         [0105]    Inoculum Preparation 
         [0106]    The fresh culture of  M. pachydermatis  (incubated for 7 days) from the agar plate was suspended into 2 ml of sterile saline. The turbidity of the suspension was adjusted to McFarlands turbidometer standard 0.5. 
         [0107]    Procedure 
         [0108]    Sterile petriplates (50 mm) were labeled with the test sample number and percentage of the sample. The required amount of the test sample was added into petriplates and the molten medium to make up the volume to 4 ml in duplicates. A plate as negative control without inoculation of test organism, and a plate as positive control with test organism were saved. The contents were mixed gently and allowed to solidify. Adjusted inoculate 20 μL was spotted on the surface of the media plates. The plates were incubated for 7 days at room temp and observed for visible growth by comparing with positive control. MIC (mg/ml conc) were calculated and results are given in table-8. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Organisms 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Tested 
                 AXT-5 
                 AXT-7 
                 AXT-8 
                 AXT-9 
                 AXT-10 
                 AXT-11 
                 AXT-17 
                 AXT-19 
                 AXT-20 
                 AXT-21 
                 AXT-22 
                 AXT-23 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 
                   Neisseria 
                 
                 0.025 
                 0.050 
                 0.100 
                 0.050 
                 0.050 
                 0.050 
                 0.100 
                 0.050 
                 0.100 
                 0.100 
                 0.025 
                 0.050 
               
               
                 
                   gonorrhoeae 
                 
               
               
                 
                   Trichophyton 
                 
                 2 
                 2 
                 2 
                 2 
                 2 
                 1 
                 2 
                 2 
                 2 
                 1 
                 2 
                 2 
               
               
                 
                   rubrum 
                 
               
               
                 
                   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 
               
               
                 
                   gypseum 
                 
               
               
                 
                   Entameba 
                 
                 0.250 
                 0.250 
                 0.200 
                 0.200 
                 0.250 
                 0.250 
                 0.200 
                 0.200 
                 0.250 
                 0.250 
                 0.200 
                 0.200 
               
               
                 
                   Coli 
                 
               
               
                 
                   Staphylococcus 
                 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.500 
               
               
                 
                   aureus 
                 
               
               
                 
                   Salmonella 
                 
                 0.250 
                 0.200 
                 0.500 
                 0.500 
                 0.250 
                 0.200 
                 0.500 
                 0.500 
                 0.500 
                 0.150 
                 0.150 
                 0.150 
               
               
                 
                   Typhi 
                 
               
               
                 
                   Pseudomonas 
                 
                 2 
                 0.500 
                 1 
                 1 
                 2 
                 2 
                 2 
                 1 
                 1 
                 1 
                 2 
                 2 
               
               
                 
                   aeruginosa 
                 
               
               
                 
                   Helicobacter 
                 
                 0.500 
                 0.500 
                 0.500 
                 1 
                 1 
                 2 
                 2 
                 2 
                 0.500 
                 1 
                 1 
                 1 
               
               
                 
                   pylori 
                 
               
               
                 
                   Candida 
                 
                 0.250 
                 0.200 
                 0.500 
                 0.500 
                 0.250 
                 0.200 
                 0.500 
                 0.500 
                 0.500 
                 0.150 
                 0.150 
                 0.150 
               
               
                 
                   albicans 
                 
               
               
                 
                   Mallethsia 
                 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.500 
               
               
                 
                   Furfur 
                 
               
               
                 
                   Mallathesia 
                 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.450 
                 0.450 
                 0.500 
                 0.450 
                 0.500 
                 0.500 
               
               
                 
                   Pachydermatis 
                 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-15 
     Antioxidant Activity of Extracts and Fractions of  Quercus Infectoria    
       [0109]    All extracts and fractions prepared from the galls of  Quercus infectoria  was screened for antioxidant activity in three in vitro models viz. DPPH methods, reducing power assay and NO scavenging activity and one ex vivo Lipid peroxidation assay methods. 
       EXAMPLE-16 
     Free Radical Scavenging Activity by DPPH Method 
       [0110]    The free radical scavenging activity of the test sample was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH. 
         [0111]    Procedure 
         [0112]    1 ml of 0.1 mM solution of DPPH in ethanol was added to 3.0 ml of test sample solution in water at different concentrations (The stock sample solutions was serially diluted with respective solvents to obtain lower dilutions). Thirty minutes later, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture was indicated higher free radical scavenging activity. Butylated hydroxy toluene (BHT) was used as standard. The capability to scavenge the DPPH radical was calculated. The antioxidant activity of the test samples was expressed as IC 50 . The IC 50  value is defined as the concentration (in μg/ml) of extracts that inhibited the formation of DPPH radicals by 50%. The results of are shown in  FIG. 1-4   
       EXAMPLE-17 
     Free Radical Scavenging Activity by Reducing Power Assay 
       [0113]    The reducing power of test sample (extract) was determined in Fe 3+ —Fe 2+  redox system according to the method of Oyaizu (1986). 
         [0114]    Procedure 
         [0115]    Various concentrations of the test samples (extracts) (The stock test sample solutions was serially diluted with respective solvents to obtain lower dilutions) in 1.0 ml of deionized water was mixed with phosphate buffer (2.5 ml, 0.2M, pH 6.6) and 1% potassium ferricyanide (2.5 ml). The mixture was incubated at 50° C. for 20 min. Aliquots of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, which was then centrifuged at 1036×g for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared FeCl 3  solution (0.5 ml, 0.1%). The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. The results are shown in  FIG. 5-8 . 
       EXAMPLE-18 
     Scavenging Activity Against Nitric Oxide 
     Nitrite Detection Method 
       [0116]    Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduce production of nitric oxide. 
         [0117]    Procedure 
         [0118]    1.0 ml Sodium nitroprusside (5 mM) in 20 mM phosphate-buffered saline (PBS) pH 7.4 was mixed with 1.0 ml of different concentrations of test samples (extracts) (The stock test sample solutions was serially diluted with respective solvents to obtain lower dilutions) and incubated at 25° C. for 150 min. The samples from the above were reacted with 1 ml of Greiss reagent. The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediamine was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite, treated in the same way with Griess reagent. The percentages of NO scavenging activity of test samples (extracts) are shown in  FIG. 10 . 
       EXAMPLE-19 
       [0119]    All herbal antistress drink were prepared using the said antioxidant composition, AXT-3 along with water extracts of  Withania somnifera, Emblica officinalis, Vitis vinifera , Sugar syrup, Citric acid, Ascorbic acid and Sodium benzoate as per the formula given below. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
               
             
             
               
                 1. 
                 
                   Withania somnifera 
                 
                 1.0 
                 g 
               
               
                 2. 
                 
                   Emblica officinalis 
                 
                 1.0 
                 g 
               
               
                 3. 
                 
                   Vitis vinifera 
                 
                 3.0 
                 g 
               
               
                 4. 
                 AXT-3 
                 0.6 
                 g 
               
               
                 5. 
                 Citric acid 
                 15.0 
                 g 
               
               
                 6. 
                 Ascorbic acid 
                 2.4 
                 g 
               
               
                 7. 
                 Sodium benzoate 
                 1.0 
                 g 
               
               
                 8. 
                 Sugar syrup 
                 1.0 
                 L 
               
               
                 9. 
                 Flavour (passion fruit) 
                 10.0 
                 g 
               
               
                 10. 
                 DM water (q.s.) 
                 2.0 
                 L 
               
               
                   
               
             
          
         
       
     
       EXAMPLE-20 
       [0120]    All herbal Energy drink was prepared using the said antioxidant composition AXT-3 along with water extracts of  Camellia sinensis, Withania somnifera, Emblica officinalis, Vitis vinifera , Sucrose, Citric acid, thickening agent (stimuleol) as per the formula given below. 
         [0000]                                            1.     Camelia sinensis     6.0   g       2.     Withania somnifera     2.0   g       3.     Emblica officinalis     2.0   g       4.     Vitis vinifera     6.0   g       5.   AXT-3   0.6   g       6.   Citric acid   3.0   g       7.   Sucrose   125.0   g       8.   Stimuleol   0.5   g       9.   Mixed fruit flavour   1.0   g       10.   DM water (q.s.)   1.0   L                    
While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those skilled in the art without departing from the scope and spirit of this invention.