Patent Application
20100062989
1. Field of the Invention
The invention in general relates to Emblica Officinalis Gaertn. (Amla) fruit. More specifically, the present invention relates to novel optimal biomarkers for the biological potency of amla fruits and products thereof.
2. Description of Prior Art
The fruits of E. officinalis are reported to contain hydrolysable tannins, emblicanin A and emblicanin B, along with pedunculagin and punigluconin [Ghosal, S.; Tripathi, V. K.; Chauhan, S. Active constituent of Emblica officinalis: part I—the chemistry and antioxidant effects of two new hydrolysable tannins, emblicanin A and B. Indian J. Chem. 1996, 35B, 941-948]. Important patent references in this regard include:
Amla fruits were to be considered rich sources of ascorbic acid until Ghosal et al. questioned its presence in 1996 [Ghosal, S.; Tripathi, V. K.; Chauhan, S. Active constituent of Emblica officinalis: part I—the chemistry and antioxidant effects of two new hydrolysable tannins, emblicanin A and B. Indian J. Chem. 1996, 35B, 941-948].
Similar views on ascorbic acid contents in Emblica officinalis to bring about the biological effects of amla were discussed in “A much higher concentration of Ascorbic acid is required to effect the same inhibition as Amla”—S. M. Khopde et al., Current Science, Vol. 81, No. 2, 25 Jul. 2001.
However, in 2006, Scartezzini et al. proposed a reliable HPLC-DAD for the identification and quantification of ascorbic acid and further indicated that high antioxidant activity is due to a large percentage of the presence of ascorbic acid (Scartezzini, P.; Antognoni, F.; Raggi, M. A.; Poli, F.; Sabbioni, C. Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation of Emblica officinalis Gaertn. J. Ethnopharm. 2006, 104, 113-118).
Recently, Raghu et al. compared ascorbic acid content of the fruits by conventional calorimetric estimation and specific enzymatic method and as the o-phenylene diamine derivative of dehydroascorbic acid and found contents of 34-38 mg of vitamin C equivalent to 100 g of fresh weight (Raghu, V.; Platel, K.; Srinivasan, K. Comparison of ascorbic acid content of Emblica officinalis fruits determined by different analytical methods. J. Food Compos. Anal. 2007, 20, 529-533.)
Zhang et al have reported the principal phenolic constituents of Emblica officinalis fruit juice as mucic acid 2-O-gallate, mucic acid 1,4-lactone 2-O-gallate and mucic acid 1,4-lactone 5-O-gallates. These along with 1-O-galloyl-b-D-glucose (β-glucogallin) have been suggested as possible antioxidants in the juice together with vitamin C.
The inventors of the present invention revisited the amla chemistry to identify and confirm the essential biomarkers of the fruit that confer its vital properties.
Accordingly, it is the principle object of the present invention to investigate thoroughly the chemistry of amla and unravel unknown data if any and also confirm the essential biomarkers of the fruit that confer its vital properties.
It is also another object of the present invention to develop a product standardized in terms of the most optimal biomarker molecule as identified by the chemical studies on amla fruit.
The present invention fulfills the aforesaid objectives and provides further related advantages.
The present invention relates to the most optimal biomarker for the biological potency of Emblica Officinalis Gaertn. (Amla) fruit and products standardized for 5% and above w/w of the said biomarker represented by STR#1. Further, the products described herein above contain from about 0.00010% to about 4% of free ascorbic acid depending on the raw material used. In specific embodiment of the present invention, the product standardized for 5% and above w/w of the said biomarker represented by STR#1 represents an aqueous extract from Emblica Officinalis Gaertn. fruit (Amla fruit). More specifically, the aqueous extract also comprises from about 50% to greater than 50% total gallates including mucic acid 1,4-lactone 5-O-gallate, mucic acid 2-O-gallate, mucic acid 6-Methyl ester 2-O-gallate, mucic acid 1-Methyl ester 2-O-gallate and ellagic acid.
The advantages of the present invention include
Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principle of the invention.
DESCRIPTION OF THE MOST PREFERRED EMBODIMENT (
In the most preferred embodiment, the present invention relates to the most optimal biomarker for the biological potency of Emblica Officinalis Gaertn. fruit (Amla fruit) and products standardized for 5% and above w/w of the said biomarker represented by STR#1. Further, the products described herein above contain from about 0.00010% to about 4% of free ascorbic acid depending on the raw material used.
In specific embodiment of the present invention, the product standardized for 5% and above w/w of the said biomarker represented by STR#1 represents an aqueous extract from Emblica Officinalis Gaertn. fruit (AmIa fruit). More specifically, the aqueous extract also comprises from about 50% to greater than 50% total gallates including mucic acid 1,4-lactone 5-O-gallate represented by STR#2, mucic acid 2-O-gallate represented by STR#3, mucic acid 6-Methyl ester 2-O-gallate represented by STR#4, mucic acid 1-Methyl ester 2-O-gallate represented by STR#5 and ellagic acid represented by STR#6.
The aforesaid most preferred embodiments are explained in detail by specific examples described herein below.
The present inventors separated the extract of Emblica officinalis into seven major fractions using Preparative Shimadzu HPLC system equipped with Class Vp software with binary gradient (LC8A) pump, UV-VIS dual wavelength detector (SPD-10AVP), system controller (SCL-10AVp) and Rheodyne injector 7725i with 5 mL sample loop. Separation was done on Phenomenex Jupiter C18 column 300A(250×50 mm, 15 μm) with mobile phase A—water (0.1% formic acid) and B—methanol. A gradient was used as 0-35 min 5% B, 35-40 min 100% B, 40-50 min. 100% B, 50-51 min. 5% B and 51-60 min. 5% B. Flow rate was 35 ml/min and monitoring at 240 and 280 nm.
Under stringent conditions of temperature and light, the present inventors isolated, freeze dried and subjected Peak 2 of
Ascorbic acid has been estimated previously by various methods. Retention time of Ascorbic acid (
I. Oxygen Radical Absorbance Capacity (ORAC)—ORAC value is a measure of inhibition of peroxyl radicals. ORAC value is represented as Trolox equivalents where the product is compared with Trolox, a standard inhibitor of peroxyl radicals. Higher the ORAC value, higher is the peroxyl radical inhibition. ORAC value is represented as Trolox equivalents per gm or liter by calculating the area under the curve formed by the inhibition of quenching of fluroscein dye induced by the peroxyl radical generator, azobis.
II. DPPH scavenging—Generation of free radicals in skin due to various stress conditions such as UV exposure, pollution, ageing etc, result in induction of melanin synthesis. The quenching of the stable free radical Diphenylpicryl hydrazyl (DPPH) in a methanolic reaction mixture by the antioxidant is an indication of the antioxidant potential.
In terms of both DPPH scavenging assay and ORAC values (
Varying concentrations of the test sample in the cell culture medium were added to the 24 hr monolayers of cells in a 96 well flat bottomed clear plates and then exposed to UV irradiation dose of 0.05 J cm-2. Each concentration was applied with ‘n’ value of 12. The plate layout was made in such a way that six of the replicates were exposed to UV and six unexposed. The unexposed region of the plate was covered by aluminum foil. After exposure, the cells were incubated in a CO2 incubator for 48 hrs and developed by the NRU staining techniques to analyze the cell viability. The percentage of UV protection was calculated with respect to the cytotoxicity in treated cells as compared to that of the untreated cells.
UV source: UV lamp of 14.7 W, 0.3 A, 55V with an intensity of 33.3 μW cm-2 was obtained from Sankyo Denki Co., Ltd, Japan.
Table C-SABERRY™: Antioxidant Profile
In-House Clinical Data on the Antioxidant Capacity of SABERRY™.
Study Design: Two healthy subjects and four unhealthy subjects (age: 40 to 50) were recruited to participate in this study. All healthy participants were in good health as determined by the following eligibility criteria: 1) no history of cardiovascular, hepatic, gastrointestinal, or renal disease; 2) no alcoholism; 3) no antibiotic or supplemental vitamin and/or mineral use for at least 4 wk before the start of the study and 4) no smoking. The unhealthy individuals were obese and frequent smokers.
A 10 mL blood sample (zero baseline samples) was obtained from fasting subjects at the start of the study, following which they were given 200 mg per day of SABERRY™ for a period of one month. Blood samples (10 mL) were collected again after one month. ORAC assay and ROS scavenging assay were conducted for all of the serum samples.
Oxygen Radical Absorbance Capacity (ORAC)
Principle: The Oxygen Radical Absorbance Capacity (ORAC) assay depends on the free radical damage to a fluorescent probe through the change in its fluorescence intensity. The change of fluorescence intensity is an index of the degree of free radical damage. In the presence of antioxidant, the inhibition of free radical damage, which is reflected in the protection against the change of probe fluorescence, is a measure of its antioxidant capacity against the free radical.
Procedure:
25 μl of each of the serum samples was pipetted into each well followed by 150 μl of 10×10−2M (final conc) AAPH reagent (2,2′-Azobis(2-amidinopropane)dihydrochloride) made in 75 mM potassium phosphate buffer (pH 7.4). Then 150 μl of disodium fluorescein dye (final conc.) 4.8×10−7M was added and mixed before the initial reading (f0) was taken. Fluorescence reading were taken (Fluostar Optima Microplate Reader) at 485/520 nm after every 1 minute for 35 minutes (f1 . . . f35). 25 μl of phosphate buffer (75 mM) was pipetted instead of antioxidant in the blank. Trolox standard from 12.5-200 μM was also kept. The number of wells being used in the experiment should not exceed 20 to reduce the error due to time lag. Difference between duplicates also occurs due to the time lag and to avoid this, the experiment is to be repeated thrice with % CV not more than 15.
Calculation: The final ORAC values were calculated by using a quadratic regression equation (Y=a+bX+cX2) between the trolox concentration (Y) (μM) and the net area under the Fluorescence decay curve (X) and were expressed as micromoles of trolox equivalents per liter or per gram of sample (μmol TE/g or μmol TE/L).
The Area under curve AUC=(1+f1/f0+f2/f0+ . . . +f35/f0.) eq 1
Where f0 is the initial fluorescence reading at 0 min and f1 is the fluorescence reading after 1 min.
The data were analyzed by applying eq 1. The net AUC was obtained by subtracting the AUC of the blank from that of the sample. The value calculated using the net AUC of the sample and the quadratic regression equation was divided by the weight of the sample in g/L. The final value obtained is the ORAC value of the sample expressed as μmol trolox equivalents (TE)/g.
Result (Table D):
Conclusion:
The serum ORAC values for healthy individuals did not vary significantly before and after treatment with SABERRY™. However, for unhealthy individuals, the serum ORAC values increased significantly in the range of 10-25%. In conclusion, SABERRY™ is good for the maintenance of the body's antioxidant potential. The reduced levels of antioxidant potential in unhealthy conditions were raised significantly and were not increased significantly in healthy conditions.
Reactive Oxyben Species (ROS) Scavenging Assay in Swiss 3T3 Fibroblast Cell Line
Principle: A cell permeable, non-fluorescent dye, DCFH-DA (dichloro fluorescein diacetate) after intracellularly getting de-esterified is converted to dichlorofluorescein upon oxidation by the reactive oxygen species. The scavenging activity of sample is indicated by the decrease in fluorescence (wavelength 485/520 nm).
Procedure: Serum samples (0.05 ml) in 96 well black flat-bottomed tissue culture plates were incubated with 100 μl of stock solution of DCFH-DA (2 mg/ml in ethanol, diluted to 100 times in phosphate buffered saline) for 1 hr at 37° C. and the fluorescence reading was taken at wavelength 485/520 nm (Fluostar optima microplate reader).
Calculation: The optical density (OD) measured is directly proportional to the ROS generated.
Result (Table E):
Conclusion:
The serum ROS levels for healthy individuals did not vary significantly before and after treatment with SABERRY™. However, for unhealthy individuals, the serum ROS levels decreased significantly in the range of 20-25%. In conclusion, SABERRY™ is good for reducing the excess ROS levels. The increased levels of ROS in unhealthy conditions were decreased significantly and were not affected in healthy conditions.
As an additional embodiment, the present invention also relates to the use of most optimal biomarker represented by STR#1 of Emblica Officinalis Gaertn. fruit (Amla fruit) and products standardized for 5% and above w/w of the said biomarker as peroxynitrite scavengers. Further, the products described herein above contain from about 0.00010% to about 4% of free ascorbic acid depending on the raw material used. In specific embodiments of the present invention, the product standardized for 5% and above w/w of the said biomarker represented by STR#1 represents an aqueous extract from Emblica Officinalis Gaertn. fruit (Amla fruit). More specifically, the aqueous extract also comprises from about 50% to greater than 50% total gallates including mucic acid 1,4-lactone 5-O-gallate represented by STR#2, mucic acid 2-O-gallate represented by STR#3, mucic acid 6-Methyl ester 2-O-gallate represented by STR#4, mucic acid 1-Methyl ester 2-O-gallate represented by STR#5 and ellagic acid represented by STR#6. In further specific embodiments, the present invention relates use of the aforesaid product for managing peroxynitrite-mediated damage occurring in Alzheimer's disease.
While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.
This application is a continuation-in-part of application Ser. No. 12/208,024 filed on Sep. 10, 2008 at the United States Patents and Trademarks Office, the content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12208024 | Sep 2008 | US |
| Child | 12353381 | US |
