METHOD OF EXTRACTION OF PROANTHOCYANIDINS COMPOUND FROM PINE BARK OF PINUS ROXBURGHII

Abstract

A method for synthesis of method of extraction of proanthocyanidins compound from pine barkof Pinus roxburghii comprising the steps: weighing and pulverizing bark to obtain pulverized powder, adding powder with water in steam jacketed rotary extractor to obtain an extract A, adding the residue in the extractor to obtain extract B, processing the second residue to obtain a third residue and an extract C which is further processed to obtain a third residue and extract D, mixing A, B, C, D extract to obtain liquor, subjecting vacuum concentrate and insoluble concentrate and subjecting to spray drying and vacuum drying respectively, sieving the powder followed by sterilization to obtain the Pine Bark Extract and further subjecting to HPLC analysis for proanthocyanidins content and packing the extract into fiber drums with double polybags for storage.

Claims

1. A method for extraction of proanthocyanidins compound from pine bark of Pinus roxburghii, comprising the steps: i) weighing and pulverizing dried pine bark, followed by sieving to obtain pulverized powder; ii) adding 80% ethanol and RO (Reverse Osmosis) water in said pulverized powder in a rotary extractor and carrying out extraction at a temperature in the range of 70-80? C. for a time duration in the range of 100-140 minutes, followed by filtering through a filter to obtain a first residue and an extract A; iii) adding said first residue with said 80% ethanol in said steam jacketed rotary extractor at a temperature in the range of 70-80 ? C. for a time duration in the range of 100-140 minutes, followed by filtration in a closed tank to obtain a second residue and an extract B; iv) processing said obtained second residue with said 80% ethanol in said steam jacketed rotary extractor for a time duration in the range of 100-140 minutes, followed by filtration in a closed tank to obtain a third residue and an extract C; v) processing said obtained second residue with said 80% ethanol in said steam jacketed rotary extractor for a time duration in the range of 100-140 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a fourth residue and an extract D; vi) mixing said obtained extract A, extract B, extract C, and extract D at a temperature in the range of 5-15? C. and holding for a time duration in the range of 20-28 hours in order to obtain a combined extract, followed by filtering said obtained combined extract through a centrifuge fitted with 3-5 micron Nylon bag to remove insoluble fines and obtain a liquor; vii) subjecting said obtained liquor to distillation under vacuum at a temperature in the range of 65-75? C. in a distillation kettle, making final concentration of said liquor upto 25-30% TS (Total Solids) in order to obtain a concentrate, followed by holding said concentrate at a temperature of 10-15? C. for a time duration in the range of 20-28 hours for settling and filtering said concentrate through centrifuge to separate water insoluble solid mass and collect water soluble concentrate of phenolic compounds; viii) subjecting said obtained water soluble concentrate to spray drying using a spray drier having inner chamber fixed at a temperature in the range of 185-190? ? C. and outer chamber fixed at a temperature in the range of 90-105? C., in order to obtain sterilized pine bark extract; and ix) collecting said spray dried powder, grinding, and sieving said powder through #150 sieve, passing said extract through metal detector to avoid Ferrous, Non-Ferrous & stainless-steel elements, followed by HPLC analysis for proanthocyanidins content and packing said extract into fiber drums with double polybags for storage of said extract.

2. The method as claimed in claim 1, wherein said dried pine bark is obtained by collecting fresh bark of Pinus roxburghii tree, removing outer scales of said bark, cleaning said bark from outer and inner side, and dried said cleaned bark under shade.

3. The method as claimed in claim 1, wherein estimation of proanthocyanidins in said pine bark extract is carried out by High-performance liquid chromatography (HPLC) using Maritime pine extract USP reference standard.

4. The method as claimed in claim 1, wherein said water soluble concentrate includes phenolic acids, gallic acid, protocatechuric acid, catechin, caffeic acid, taxifolin & ferulic acid.

5. The method as claimed in claim 1, wherein said proanthocyanidins content in Pine bark extract obtained from said Pinus roxburghii is ?80%, as evaluated by HPLC.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

[0022] FIG. 1 illustrates a method for extraction of Pine bark obtained from Pinus roxburghii;

[0023] FIG. 2 illustrates a graphical representation of a HPLC of standard solution: Injection 1;

[0024] FIG. 3 illustrates a graphical representation of a HPLC of standard solution: Injection;

[0025] FIG. 4 illustrates a graphical representation of a HPLC of standard solution: Injection 3;

[0026] FIG. 5 illustrates a graphical representation of a HPLC of standard solution: Injection 4;

[0027] FIG. 6 illustrates a graphical representation of a HPLC of standard solution: Injection 5;

[0028] FIG. 7 illustrates a graphical representation of a HPLC of a pine bark extract sample level 1;

[0029] FIG. 8 illustrates a graphical representation of a HPLC of a pine bark extract sample level 2; and

[0030] FIG. 9 illustrates a graphical representation of a HPLC of a pine bark extract sample level 3.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The following description includes the preferred best mode of one embodiment of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore, the present description should be seen as illustrative and not limiting. While the invention is susceptible to various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.

[0032] In any embodiment described herein, the open-ended terms comprising, comprises, and the like (which are synonymous with including, having and characterized by) may be replaced by the respective partially closed phrases consisting essentially of, consists essentially of, and the like or the respective closed phrases consisting of, consists of, the like.

[0033] As used herein, the singular forms a, an, and the designate both the singular and the plural, unless expressly stated to designate the singular only.

[0034] The present invention relates to a method for extraction of the proanthocyanidins compounds from pine bark of the Pine TreePinus roxburghii that utilizes a natural resource Pinus roxburghii pine bark for the formulation of a nutraceutical or health/dietary supplement that as an antioxidant.

[0035] According to an embodiment of the present invention, a method for the extraction of the proanthocyanidins from pine bark of the Pine TreePinus roxburghii comprises of following steps: a) weight and pulverizing a dried pine bark, followed by sieving in order to obtain pulverized powder, b) adding the powder 4 times of the 80% ethanol and RO (Reverse Osmosis) water in a jacketed rotary extractor at a temperature in the range of 70-80? C. for a time duration in the range of 100-140 minutes under circulation, followed by filtering through a filter in a clean, dry, and closed tank in to obtain a first residue and an extract A, c) adding the first residue with the 80% of ethanol in the steam jacketed rotary extractor at a temperature in the range of 70-80? C. for a time duration in the range of 100-140 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a second residue and an extract B, d) processing the obtained second residue with the 80% ethanol in the steam jacketed rotary extractor for a time duration in the range of 100-140 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a third residue and an extract C, e) processing the obtained third residue with the 80% ethanol in the steam jacketed rotary extractor for a time duration in the range of 100-140 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a third residue and an extract D.

[0036] According to an another embodiment of the present invention, the proposed method comprises of following steps which includes: f) mixing obtained extract A, extract B, extract C, and extract D at a temperature in the range of 5-15? C. and holding for a time duration in the range of 20-28 hours in order to obtain a combined extract, followed by passing the obtained combined wash centrifuge having 3-5 micron Nylon bag to remove insoluble fines and obtain a liquor, g) subjecting the obtained liquor to distillation under vacuum at a temperature in the range of 65-75? C. in a distillation kettle, followed by concentration of the liquor up to 25-30% TS (Total Solids) in order to obtain a concentrate, followed by holding the concentrate at a temperature of 10-15? C. for a time duration in the range of 20-28 hours for settling and filtering the concentrate through centrifuge to separate water insoluble solid mass & collect water soluble concentrate of phenolic compounds h) subjecting the obtained water soluble concentrate to spray drying using a spray drier having inner chamber fixed at a temperature in the range of 185-190? C. and outer chamber fixed at a temperature in the range of 90-105? C., in order to obtain sterilized pine bark extract, i) collecting the spray dried powder, grinding, and sieving with the powder through #150 sieve, passing the extract through metal detector to avoid Ferrous, Non-Ferrous & stainless steel elements, followed by HPLC analysis for proanthocyanidins content and packing the extract into fiber drums with double polybags for storage.

EXAMPLE

[0037] Referring to FIG. 1, a method for extraction of proanthocyanidins compound from pine bark extractPinus roxburghii is illustrated that comprises of following steps: a) a dried pine bark are weighed an pulverized, followed by sieving in order to obtain 10 #pulverized powder, b) 4 times of the 80% ethanol and RO (Reverse Osmosis) water are added in a jacketed rotary extractor at a temperature of 75? C. for a time duration of 120 minutes under circulation, followed by filtering through a filter in a clean, dry, and closed tank to obtain a first residue and an extract A, c) the first residue is added within the 80% of ethanol in the steam jacketed rotary extractor at a temperature of 75? C. for a time duration of 120 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a second residue and an extract B, d) the obtained second residue is processed with the 80% ethanol in the steam jacketed rotary extractor for a time duration of 120 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a third residue and an extract C, e) the obtained third residue is processed with the 80% ethanol in the steam jacketed rotary extractor for a time duration 120 minutes, followed by filtration in a separate clean, dry, and closed tank to obtain a third residue and an extract D.

[0038] The proposed method further comprises of following steps which includes: f) the obtained extract A, B, C, and D are mixed at a temperature of 10? C. and held for a time duration for 24 hours in order to obtain a combined extract, followed by passing the obtained combined wash centrifuge having 5 micron Nylon bag to remove insoluble fines and obtain a liquor, g) the obtained liquor are subjected to distillation under vacuum at a temperature of 70? C. in a distillation kettle, followed by concentration of the liquor up to 25% TS (Total Solids) in order to obtain a concentrate, followed by holding the concentrate at a temperature of 10-15? C. for a time duration of 24 hours for settling and the concentrate is filtered through centrifuge to separate water insoluble solid mass & water soluble concentrate of phenolic compounds (proanthocyanidins) are collected, h) the obtained water soluble concentrate are subjected to spray drying using a spray drier having inner chamber fixed at a temperature in the range of 185-190? C. and outer chamber fixed at a temperature in the range of 90-105? C., in order to obtain sterilized pine bark extract, i) the spray dried powder are collected, grinded, and sieved with the powder through #150 sieve, passing the extract through metal detector to avoid Ferrous, Non-Ferrous & stainless steel elements, followed by HPLC analysis for proanthocyanidins content and packing the extract into fiber drums with double polybags for storage.

[0039] The dried pine bark used herein is obtained by collecting fresh bark of Pinus roxburghii tree from Pauri Garhwal region, removing outer scales of said bark, cleaning said bark from outer and inner side, and dried said cleaned bark under shade.

[0040] The insoluble solid mass obtain after filtration contains higher molecular weight oligomers of proanthocyanidins, whereas the water soluble concentrate contains phenolic acids, gallic acid, Protocatechuric acid, catechin, caffeic acid, taxifolin & ferulic acid, which is further subjected to spray drying.

[0041] The estimation of proanthocyanidins in said pine bark extract is carried out by High-performance liquid chromatography (HPLC) using Maritime pine extract USP reference standard. Proanthocyanidins content in Pine bark extract obtained from Pinus roxburghii is ?80% by HPLC.

Characterization

[0042] The prepared extract was tested for the evaluation of physiochemical properties by executing the following testsi) Analysis of modulatory property of the pine bark extract from Pinus roxburghii (Pinorox?) against hydrogen peroxide induced toxicity in human skin keratinocyte cells, comprising H.sub.2O.sub.2 induced cytotoxicity assay and GSH modulation assay ii) In-vitro evaluation of total antioxidant capacity of pine bark extract from Pinus roxburghii (Pinorox?) against the standard. Followed by randomized, parallel group, placebo-controlled trial study.

i) Analysis of Modulatory Property of the Pine Bark Extract from Pinus roxburghii (Pinorox?) Against Hydrogen Peroxide Induced Toxicity in Human Skin Keratinocyte Cells:

[0043] The modulatory activity of pine bark extract (powder) obtained from Pinus roxburghii (Pinorox?) was analyzed in order to evaluate the in-vitro activity on GSH (Glutathione) levels in Human skin keratinocyte cells and study the treatment of Human keratinocytes with hydrogen peroxide. Post the test analysis, it was found that hydrogen peroxide considerably increased the oxidative stress, thus reducing the GSH levels. However, the test sample which is pine bark extract powder demonstrated antioxidant property by significantly increasing the GSH levels in comparison to H.sub.2O.sub.2 control.

ii) Preparation of Test Solution (Pine Bark Extract):

[0044] For conducting the analysis, 10 mg of Pine bark extract from Pinus roxburghii (Pinorox?) was dissolved in a DMSO (Dimethyl sulfoxide) solution. The volume of the test solution was made up using DMEM-HG (Dulbecco's Modified eagle Medium) and supplemented with 2% inactivated FBS (Fetal bovine serum) to acquire a solution with concentration of 10 mg/ml. The test solution was further proceeded with sterilization using syringe filtration. For carrying out the cytotoxic analysis, a two-fold serial dilution was prepared.

iii) Preparation of Cell Line and Culture Medium

[0045] The cell line of Human keratinocytes (HaCaT) was obtained from Addex Bio, USA and were cultured in DMEM-HG supplemented media with 10% inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 ?l) and amphotericin B (5 ?g/mL) in a wet atmosphere of 5% CO.sub.2 at a temperature of 37? C. until confluent. The cells were later disintegrated with TPVG (Trypsin Phosphate Versene Glucose Solution) comprising (0.2% trypsin, 0.02% EDTA (Ethylenediaminetetraacetic acid), 0.05% glucose in PBS (Phosphate buffer solution). The stock culture was grown in a culture flask with a measurement of 25 cm2. All the experiments were conducted in a microtitre plate with 96 well.

iv) Induced Cytotoxicity Assay for H.sub.2O.sub.2:

[0046] A Cell monolayer was subjected to trypsinization and the cell count was adjusted to 2.0?105 cells/ml by using respective media (DMEM-HG) comprising FBS at 10%. The Pine bark extract from the Pinus roxburghii (Pinorox?) was assayed for the modulatory activity of GSH after the treatment with H.sub.2O.sub.2. In each plate of 12 well, diluted cell suspension of 1.0 ml was added. Post 24 hours, on the formation of monolayer, the supernatant was discarded and the monolayer was subjected to washing once with the medium. The cells were treated with H.sub.2O.sub.2 (500 ?m) and was incubated for a duration of 3 hours followed by the addition of non-toxic concentrations of the test formulation (prepared in a medium with 2% FBS). Ascorbic acid was used as the positive control (standard) for the experiment at a concentration of 17.61 pg/mL.

[0047] The Pine Bark extract from Pinus roxburghii (Pinorox?) was assayed for in vitro Cytotoxicity study against HaCaT cell line by MTT assay. The cells were exposed to different concentrations of test substances (1000 ?g/ml to 7.8 pg/ml). The Pinorox? formulation was found to be safe in HaCaT cells even at the highest concentration tested (1000 ?g/mL). The Pine Bark extract from Pinus roxburghii (Pinorox?) at 500 ?g/mL showed 182.23?0.02% and at 250 ?g/mL exhibited 140.02?0.06% which is a remarkable increase in the levels of GSH compared to the standard tested i.e., Ascorbic acid which showed 95.59?0.03. The findings of the study suggest that Pine Bark extract from Pinus roxburghii (Pinorox?) could exhibit promising modulatory effect on GSH levels against hydrogen peroxide induced oxidative stress in a dose-dependent manner in Human keratinocytes.

v) Assay for GSH Modulation:

[0048] Referring to Table 1, a tabular representation of a Pinus roxburghiiPine bark extract modulatory activity on GSH in a Hacat against hydrogen peroxide promoted oxidative stress is illustrated. The cell culture supernatant was collected from different treatment wells, centrifuged for 20 min at 1000?g at a temperature of 2-8? C. The samples were analyzed to estimate the levels of GSH using Elab science GSH ELISA kit by following the manufacturer's instruction. The graph shows the % increase in GSH level over H.sub.2O.sub.2 Control. Also, the modulatory effect by pine bark extract from Pinus roxburghii (Pinorox?) on GSH in a HaCat against hydrogen peroxide promoted oxidative stress is shown below in table no 1.

TABLE-US-00001 TABLE 1 Pinus roxburghii - Pine bark extract modulatory activity on GSH in a Hacat against hydrogen peroxide promoted oxidative stress % Increase in GSH level over Tested hydrogen SI. No Samples Concentration peroxide control 1. Pinus roxburghii 500 ?g/ml 182.23 ? 0.02 (Pinorox?) 250 ?g/ml 140.02 ? 0.06 2. Ascorbic acid 17.61 ?g/ml 95.59 ? 0.03

v) Estimation of Total Antioxidant Capacity (TAC) of a Test Substance:

[0049] The assay was performed to evaluate the total antioxidant capacity of a Pinorox? i.e. Pine bark extract from Pinus roxburghii against the standard ascorbic acid for in-vitro study. Antioxidants are compounds that are capable of inhibiting the process of oxidation that occurs under the influence of atmospheric oxygen or reactive oxygen species. The antioxidants are employed for stabilizing polymeric products, petrochemicals, cosmetics and food stuff along with pharmaceuticals. Antioxidants are involved in defense mechanisms of an organism against the associated pathologies in order to attack the free radicals. The test is carried out to check the total antioxidant capacity of a pine bark extract in order to avoid nutritional loss. The total antioxidant capacity of Pinorox? i.e. Pine bark extract from Pinus roxburghii was found out to be 756.993?0.150 mM equivalent of the ascorbic acid having 1000?0.027 mM at 1000 ?g/mL.

vi) Test Substance and Standard Preparation:

[0050] Pine bark extract of 5.5 mg each/ascorbic acid were subjected to dissolve in 0.5 ml of DMSO (Dimethyl sulfoxide) in a separate container. 0.244 ml of 0.6 M sulphuric acid was added to a 15 ml of distilled water. 0.334 g of 28 mM sodium phosphate along with 0.074 g of 4 mM ammonium molybdate was added to the solution and was completely dissolved.

vii) Assay for Estimating Total Antioxidant Capacity of Pine Bark Extract

[0051] Referring to Table 2, a tabular representation of a total antioxidant capacity in a Pine Bark Extract is illustrated. Test solution of 0.1 ml and a standard containing a reducing species in DMSO were taken in a separate Eppendorf tube, and reagent solution of 1 ml comprising (0.6M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was added and subjected to incubation in a water bath for 90 minutes at 95? C. Post this, the samples were maintained to room temperature and 0.1 ml was pipette out in triplicates to a microtitre plate. The reagent solution is used as a blank. The absorbance was recorded at 490 mm via ELISA (enzyme-linked immunosorbent assay) reader and the values were recorded. The total activity of antioxidant was expressed as mM which is equivalent of ascorbic acid. The table no 2 representation of the total antioxidant capacity in a test substance.

TABLE-US-00002 TABLE 2 The total antioxidant capacity in a Pine Bark Extract was demonstrated as mentioned below in a tabular format Concentration SI. NO Test substance (?g/ml) TAC 1 Pine bark extract from 1000 756.993 ? 0.150 Pinus roxburghii (Pinorox?) 2 Ascorbic acid 1000 1000 ? 0.027
viii) Estimation of Proanthocyanidins by HPLC:

Chromatograpahic Condition:

[0052] Column: Luna C18, 100 ? (250 mm?4.6 mm, 5?) or equivalent [0053] Detector: UV, 280 nm [0054] Flow rate: 1.0 ml/minute [0055] Injection Volume: 10 ?L [0056] Column temperature: 400 C [0057] LC Mode: Low pressure gradient [0058] Mobile Phase Preparation A: 100% Methanol. [0059] Mobile phase preparation B: dilute 1.0 g of orthophosphoric acid in 1000 ml of water, mix well and filter through 0.45? fine pore size, degas and sonicate at least 5 minutes.

[0060] Referring to Table 3, a tabular representation of a gradient program of HPLC is illustrated.

TABLE-US-00003 TABLE 3 Gradient program of HPLC Time (min) Mobile Phase A (%) Mobile Phase B (% ) 0.01 8 92 40.00 34 66 45.00 2 98 50.00 2 98 51.00 92 8 57.00 92 8 58.00 8 92 65.00 8 92 65.01 Control Stop

Standard Solution Preparation:

[0061] Weigh accurately about 50 mg of maritime pine extract reference standard or 35.5 mg equivalent to reference standard) in 25 ml volumetric flak. Add 20 ml of Methanol & sonicate to dissolve completely. Make up the volume up to 25 ml with Methanol. Filter through 0.45? nylon syringe filter. Discard the first few ml of filtrate.

Sample Solution Preparation for Extract:

[0062] Weigh accurately about 100 mg of extract (or 71.0 mg equivalent to reference standard) in 50 mL volumetric flask. Add 30 mL of methanol and sonicate to dissolve completely. Make up the volume up to 50 mL with methanol. Filter through 0.45? nylon syringe filter. Discard the first few mL of filtrate.

Sample Preparation for Bark:

[0063] Weigh accurately about 2.0 g grinder powder of pine bark in 250 mL round bottom flask, add 50 ml of methanol and reflux in water bath for 1 Hrs., and allow to cool at room temperature, decant, and retain the solvent. Repeat until the solvent is colorless. Combine the retained solvents, filter, concentrate under vacuum to about 90 ml, transfer to a 100 ml volumetric flask, and adjust the volume with methanol. Dilute 5 ml of this solution into 50 ml volumetric flask and make up volume with methanol, before injection filter the solution through 0.45 nylon syringe filter.

System Suitability Criteria:

[0064] The % RSD of Sum of the total peak area of Gallic acid, Protocatechuic Acid, Catechin, Caffeic Acid, Taxifolin and Ferulic Acid from the Standard solution five injections should not be more than 5.0% Calculate the percentage of Oligomeric Proanthocyanidins in the portion of Pine bark extract.

Calculation:

[0065] [00001]$\mathrm{Result}\left(\%\right)=\frac{\mathrm{rV}}{\mathrm{rS}}?\frac{\mathrm{Cs}}{\mathrm{Ct}}?P$

Where, rV=Sum of the total peak areas of Gallic Acid, Protocatechuic Acid, Catechin, caffeic Acid, Taxifolin & Ferulic Acid from the sample solution.
rS=Sum of the total peak areas of Gallic Acid, Protocatechuic Acid, Catechin, caffeic Acid, Taxifolin & Ferulic Acid from the standard solution.
Cs=Concentration of standard solution.
Ct=Concentration of sample solution.
P=Purity of Maritime Pine Extract USP reference standard.

[0066] Referring to FIG. 2, a graphical representation of a HPLC of standard solution: Injection 1 is illustrated.

[0067] Referring to FIG. 3, a graphical representation of a HPLC of standard solution: Injection 2 is illustrated.

[0068] Referring to FIG. 4, a graphical representation of a HPLC of standard solution: Injection 3 is illustrated.

[0069] Referring to FIG. 5, a graphical representation of a HPLC of standard solution: Injection 4 is illustrated.

[0070] Referring to FIG. 6, a graphical representation of a HPLC of standard solution: Injection 5 is illustrated.

[0071] Referring to Table 4, a tabular representation of a component summary table name of caffeic acid is illustrated.

TABLE-US-00004 TABLE 4 Component summary table name of caffeic acid Sample % USP Plate USP Int Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Caffeic 25.873 122975 9.40 50633 1.0 BB 1308730 Solution Acid 2 Standard 2 2 Caffeic 25.882 123455 9.44 50993 1.0 BB 1308065 Solution Acid 3 Standard 2 3 Caffeic 25.897 123433 9.38 50964 1.0 BB 1316318 Solution Acid 4 Standard 2 4 Caffeic 25.947 123850 9.38 51555 1.0 BB 1319738 Solution Acid 5 Standard 2 5 Caffeic 25.969 124094 9.38 51194 1.0 BB 1322652 Solution Acid Mean 25.913 123561 1315100 % RSD 0.2 0.3 0.5

[0072] Referring to Table 5. a tabular representation of a component summary table name of Catechin is illustrated.

TABLE-US-00005 TABLE 5 Component summary table name of Catechin Sample % USP Plate USP Int Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Catechin 19.539 228711 17.48 18801 0.8 BB 1308730 Solution 2 Standard 2 2 Catechin 19.548 227832 17.42 19238 0.8 BB 1308065 Solution 3 Standard 2 3 Catechin 19.561 230252 17.49 18378 0.8 BB 1316318 Solution 4 Standard 2 4 Catechin 19.616 230286 17.45 18584 0.8 BB 1319738 Solution 5 Standard 2 5 Catechin 19.634 231311 17.49 18558 0.8 BB 1322652 Solution Mean 19.580 229678 1315100 % RSD 0.2 0.6 0.5

[0073] Referring to Table 6. a tabular representation of a component summary table name of Ferulic Acid is illustrated.

TABLE-US-00006 TABLE 6 Component summary table name of Ferulic Acid Sample % USP Plate USP Int Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Ferulic 39.009 116081 8.87 111740 1.0 BB 1308730 Solution Acid 2 Standard 2 2 Ferulic 39.017 115417 8.82 112359 1.0 BB 1308065 Solution Acid 3 Standard 2 3 Ferulic 39.053 117245 8.91 111785 1.0 BB 1316318 Solution Acid 4 Standard 2 4 Ferulic 39.097 117820 8.93 112229 1.0 BB 1319738 Solution Acid 5 Standard 2 5 Ferulic 39.134 118275 8.94 113177 1.0 BB 1322652 Solution Acid Mean 39.062 116968 1315100 % RSD 0.1 1.0 0.5

[0074] Referring to Table 7. a tabular representation of a component summary table name of Gallic Acid is illustrated.

TABLE-US-00007 TABLE 7 Component summary table name of Gallic Acid Sample % USP Plate USP Int Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Gallic 7.369 87230 6.67 4680 0.7 BB 1308730 Solution Acid 2 Standard 2 2 Gallic 7.370 87495 6.69 4547 0.7 BB 1308065 Solution Acid 3 Standard 2 3 Gallic 7.370 88029 6.69 4771 0.7 BB 1316318 Solution Acid 4 Standard 2 4 Gallic 7.393 87255 6.61 4734 0.7 BB 1319738 Solution Acid 5 Standard 2 5 Gallic 7.398 87619 6.62 4529 0.7 BB 1322652 Solution Acid Mean 7.380 87526 1315100 % RSD 0.2 0.4 0.5

[0075] Referring to Table 8. a tabular representation of a component summary table name of Protocatechuic Acid is illustrated.

TABLE-US-00008 TABLE 8 Component summary table name of Protocatechuic Acid Sample % USP Plate USP Int Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Protocatechuic 13.072 71463 5.46 16687 0.9 BB 1308730 Solution Acid 2 Standard 2 2 Protocatechuic 13.080 71631 5.48 17154 0.9 BB 1308065 Solution Acid 3 Standard 2 3 Protocatechuic 13.083 71457 5.43 16832 0.9 BB 1316318 Solution Acid 4 Standard 2 4 Protocatechuic 13.118 72019 5.46 16682 0.9 BB 1319738 Solution Acid 5 Standard 2 5 Protocatechuic 13.130 73002 5.52 16726 0.9 BB 1322652 Solution Acid Mean 13.096 71915 1315100 % RSD 0.2 0.9 0.5

[0076] Referring to Table 9. a tabular representation of a component summary table name of Taxifolin is illustrated.

TABLE-US-00009 TABLE 9 Component summary table name of Taxifolin Sample % USP Plate USP In Total Name Vial Inj Name RT Area Area Count Tailing Type Area 1 Standard 2 1 Taxifolin 38.072 682268 52.13 111483 1.0 BB 1308730 Solution 2 Standard 2 2 Taxifolin 38.080 682234 52.16 111910 1.0 BB 1308065 Solution 3 Standard 2 3 Taxifolin 38.115 685902 52.11 112266 1.0 BB 1316318 Solution 4 Standard 2 4 Taxifolin 38.161 688508 52.17 112124 1.0 BB 1319738 Solution 5 Standard 2 5 Taxifolin 38.198 688351 52.04 112814 1.0 BB 1322652 Solution Mean 38.125 685453 1315100 % RSD 0.1 0.5 0.5

[0077] Referring to FIG. 7, a graphical representation of a HPLC of a pine bark extract sample level 1 is illustrated.

[0078] Referring to Table 10, a tabular representation of a HPLC of level 1 of a pine bark extract is illustrated.

TABLE-US-00010 TABLE 10 HPLC of level 1 of a pine bark extract Height Int USP Plate USP Peak Name RT (?V) Area Type Count Tailing 1 Total Area 94549 1780835 Group 2 Gallic Acid 7.399 102 1447 BB 6244 1.3 3 Protocatechuic 13.410 3364 50494 BB 17311 0.9 Acid 4 Catechin 19.985 17764 365544 BB 18085 0.8 5 Caffeic Acid 26.127 Missing 6 Taxifolin 38.600 73318 1363349 BB 99634 0.9 7 Ferulic Acid 39.500 Missing

[0079] Referring to FIG. 8, a graphical representation of a HPLC of a pine bark extract sample level 2 is illustrated.

[0080] Referring to Table 11, a tabular representation of a HPLC of level 2 of a pine bark extract is illustrated.

TABLE-US-00011 TABLE 11 HPLC of level 2 of a pine bark extract Height Int USP Plate USP Peak Name RT (?V) Area Type Count Tailing 1 Total Area 93809 1770563 Group 2 Gallic Acid 7.417 103 1408 BB 7231 1.3 3 Protocatechuic 13.431 3351 50963 BB 17092 0.9 Acid 4 Catechin 20.020 17673 362789 BB 18237 0.8 5 Caffeic Acid 26.127 Missing 6 Taxifolin 38.635 72682 1355403 BB 99144 0.9 7 Ferulic Acid 39.500 Missing

[0081] Referring to FIG. 9, a graphical representation of a HPLC of a pine bark extract sample level 3 is illustrated.

[0082] Referring to Table 12, a graphical representation of a HPLC of a pine bark extract sample level 3 is illustrated.

TABLE-US-00012 TABLE 12 HPLC of a pine bark extract sample level 3 Height Int USP Plate USP Peak Name RT (?V) Area Type Count Tailing 1 Total Area 95346 1790637 Group 2 Gallic Acid 7.451 99 1414 BB 6401 1.3 3 Protocatechuic 13.478 3383 51081 BB 17443 0.9 Acid 4 Catechin 20.077 17993 367648 BB 18797 0.8 5 Caffeic Acid 26.127 Missing 6 Taxifolin 38.717 73870 1370494 BB 100587 0.9 7 Ferulic Acid 39.500 Missing
viii) Evaluation of Clinical Trial to Study the Safety and Efficacy of Pine Bark Extract:

[0083] A potential, double blind, placebo comparative, interventional clinical study was conducted to evaluate the efficacy and safety of Pinorox? for improving physical and athletic performance in normal and sports subjects. The study parameters are mentioned below.

Volunteer Health Condition and the Problem Studied (Placebo Study)

[0084] Healthy sedentary volunteers of age group 18 years to 40 years following (Daily routine with no or little exercises) and sports subjects (Average in more than 3 sessions per week). A comparator agent namely placebo each capsule containing 200 mg of Maltodextrin Route: (Oral) was given daily once after a meal or in between the meals for a period of 90 days. The precaution to be followed while studying the volunteers was to avoid the intake of medicine on empty stomach. The intervention acquired from the study was that each capsule contains 200 mg pine bark extract that is Pinorox? given to the volunteers once in a day daily after meal or between the meals for 90 days. The precaution to be followed while studying the volunteers was to avoid the intake of medicine on empty stomach.

Brief Overview of the Placebo Study (Details Pertaining to the Volunteers)

[0085] Volunteers from age group comprising of 18-40 years with BMI between 18.0-24.9 kg/m.sup.2 were selected for conducting the study. The sedentary subjects were mentally, physically and legally eligible to give their informed consent for participating in a study trial. The healthy sedentary consists of both male and female subjects following (Daily routine with no or little exercises) and sports subjects following the routine as same as healthy sedentary subjects with (Average in more than 3 sessions per week) change. The patient voluntarily agreed to comply with all trial requirement. The selected patents who were chosen for the trial had not participated in a similar investigation in the past last three months. During the trial the subject was in good health and appropriate fit for exercise. The subject agreed to not use any dietary or herbal supplements and maintain their normal dietary intake until the study completion and also agreed to avoid consumption of anti-oxidant rich food. In addition to this, the subjects were instructed to refrain from exercise, 48 hours prior to the experiment and had to fast two hours before. The subject should have normal haematology and biochemistry ranges.

Computer Generated Randomization Study Trial

[0086] In this clinical study trial, the participant and investigator were blinded. The total sample size for studying the trial was 50. The primary outcome of the trial states that a change was reported in baseline in the rate of oxygen consumption VO.sub.2 max (endurance) in comparison to the placebo study. The baseline time point for the study was 30, 60 and 90 days. The outcome was assessed on the basis of anaerobic power and rate of fatigue by the use of Wingate test. The Wingate test measures the anaerobic power peak achieved during the commencement of the test.

[0087] Basically, the test talks about the anaerobic capacity produced over the entire 30-second period including the fatigue index in the test. The secondary outcome drawn from the trial enlists the change in assessment values by the use of 1-RM test and the baseline along with the day was 90. Secondary outcome also comprises of a questionnaire assessment on quality of life using SF-36. SF-36 is a questionnaire that measures eight main parameters comprising vitality (VT), bodily pain (BP), general health (GH), physical functioning (PF), role physical (RP), social functioning (SF), role emotional (RE), and mental health (MH). The last outcome or the tertiary outcome of the trial talks about the laboratory tests including CBC (Complete Blood count) and the level of serum creatinine, SGOT serum glutamic-oxaloacetic transaminase test is a type of blood test that targets on profiling the functionality of liver and SGPT test that stands for serum glutamic Pyruvic Transaminase. The test is executed to measure the glutamate pyruvate transaminase (GPT) amount in the blood serum.

[0088] Therefore, the pine bark extract obtained from Pinus roxburghii having an antioxidant property, which can be used in the nutraceutical or health/dietary supplement having functional application as an antioxidant.

[0089] Although the field of the invention has been described herein with limited reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention.