Title of Invention

A PROCESS FOR THE ISOLATION ANTIOXIDANT RICH FRACTION FROM GRAPE SEEDS

Abstract The present invention relates to a process for the isolation of antioxidant fraction from grape seeds. Lipid peroxidation is one of the major reasons for deterioration of food products during the processing and storage. The addition of antioxidants is a method of increasing the shelf life, especially of lipids and lipid containing foods. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytolune.
Full Text The present invention relates to a process for the preparation of antioxidants from Annona squamosa peel.
Background of the invention
Animal and vegetable fats and oils, which are invariably a part of the food, cosmetic and health-care products, are prone to oxidative rancidity on storage. The oxidative deterioration is accelerated on cooking and leads to objectionable stale, rancid or "warmed-over" flavour development. Once initiated, the oxidative process proceeds at an exponential rate, which is proportional to the level of oxidative by-products present. Also, the oxidative process can occur with natural food and cosmetic colourants, such as carotenoids, which leads to objectionable aroma and flavour and loss of colour intensity. As flavour, odour and colour are the principal factors by which food quality is judged, antioxidants suitable for incorporation into food products are valued for their capacity to reduce the destructive oxidative process to provide a longer shelf life.
Presently, a number of synthetic chemical compounds are being used as antioxidants in the food and cosmetic industries. These compounds primarily consist of a group of phenolic derivatives, which include BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), propyl gallate and TBHQ (tertiary butylhydroquinone). As these antioxidants are produced by synthetic chemical processes, their inclusion in food products is severely limited by regulation in regard to both level of use and food product application. In many applications, they are completely banned. Other problems with this class of compounds include heat sensitivity, discolouration and susceptibility to loss by steam distillation.
One of the most important consumer trends in the food industry today is the demand for "all natural" food products that are free of chemical additives. This demand has focused attention on certain natural antioxidant materials obtained
from plant sources. Most work in this area has concentrated on the antioxidant activity of natural herbs and spices, such as cloves, ginger, mace, nutmeg and the Labiatae herbs rosemary, sage, thyme and oregano. Chipault et al. (Food Res. International, 17, 46, 1952, and Food Technol. 10, 209, 1956) have investigated the antioxidant properties of many ground herbs and their ethanol extracts and have shown that rosemary and sage have the highest antioxidant activity amongst them. These naturally occurring compounds have been shown to possess antioxidant activity equivalent to BHA and BHT.
The custard apple (Annona squamosa) belongs to family Annonaceae. The compound fruit is nearly round, ovoid, or conical; 6-10 cm long; its thick rind composed of knobby segments, pale-green, gray-green, bluish-green, or, in one form, dull, deep-pink externally (nearly always with a bloom); separating when the fruit is ripe and revealing the mass of conically segmented, creamy-white, glistening, delightfully fragrant, juicy, sweet, delicious flesh. Many of the segments enclose a single oblong-cylindrical, black or dark-brown seed about 1.25 cm long. There may be a total of 20 to 38, or perhaps more, in the average fruit.
The peel of Annona squamosa is non-edible and comes out as a waste from the industries involved in its processing. Literature survey revealed that there are no reports on the isolation of antioxidants from custard apple.
The present invention provides a process for preparation of an antioxidant rich fraction from Annona squamosa peel, which can be used as potential natural preservative.
The main object of the present invention is to provide a process for the preparation of antioxidants from Annona squamosa peel, which obviates the drawbacks detailed above.
Another object of the present invention is to use simple extraction protocol for antioxidants with solvents.
Still another object of the present invention is to obtain appreciable yields of antioxidant fraction, which has a brown colour and neutral flavour.Another object of the present invention is to use simple extraction protocol for antioxidants with solvents.
Still another object of the present invention is to obtain appreciable yields of antioxidant fraction, which has a brown colour and neutral flavour.
Yet another object of the present invention is to evaluate the radical scavenging activity of Methanol extract and BHA.
Accordingly, the present invention relates to a process for the isolation of antioxidant rich fraction from grape seeds, comprising the following steps:
a) drying and powdering grape seeds to get a powder,
b) extracting the resulting powder with water at temperature between 30 -60°C to
remove unwanted material,
c) extract the residue using water between temperature 120-130°C at pressure
above atmospheric pressure to extract phenolic compounds,
d) filtering the said aqueous extract,
e) concentrating said aqueous extract,
f) characterized in that extracting the concentrate with ethyl acetate at temperature
between 30-60 °C,
g) ethyl acetate extract is recovered under vacuum.
In an embodiment the yield of methanol extract is 14-15% with 34-35% phenolic content.
In an another embodiment the radical scavenging activity of methanol extract at 25 ppm concentration is in the range of 85-92% .
In yet an another embodiment the radical scavenging activity of methanol extract at 50ppm concentration is in the range of 88-94%.
In yet an another embodiment the radical scavenging activity of BHA at 25 ppm concentration is in the range of 88-92%.
In yet an another embodiment the radical scavenging activity of BHA at 50 ppm concentration is in the range of 88-94%.
In yet an another embodiment the antioxidant fraction is brown in colour with a neutral flavour.
The preparation of antioxidant fraction from Annona squamosa was done according to following flow diagram
Annona squamosa fruits
(Diagram Removed)
The novelty of the process is:
1. This is the first report of preparation of antioxidant fraction from Annona squamosa peel.
2. The invention is a three-step process to obtain the antioxidant fraction from Annona squamosa peel.
The advantage of the process is
1. The process is simple and the solvents used in this process can be recovered for further use.
The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention.
Example 1
Peel of Annona squamosa were manually separated, dried and powdered using mixer grinder to get 60 mesh size. 50 g powder was extracted using 200 ml of hexane at 60 °C for 8 h in a Soxhlet extractor. The hexane extract was concentrated under vacuum to recover the solvent. The spent was further extracted successively with 250 ml each of ethyl acetate and Methanol at 55 °C for 2 h in a Soxhlet extractor. The Methanol extract was filtered using Whatman filter paper No.1. The extract was concentrated at a temperature of 40 °C and under reduced pressure at 10 mm of mercury and dried in vacuum oven at 40 °C and 25 mm of mercury. The yield of methanol extract was 7.6g and phenolic content was found to be 33.6%.
The Methanol extract obtained by above method was screened for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method [Singh et
al., J. Agricultural and Food Chemistry, 50, 81-86, 2002]. Methanol extract and BHA showed 89.8% and 91% radical scavenging activity at 25 ppm, respectively using Diphenyl picrylhydrazyl (DPPH) method.
Example -2
The peel of Annona squamosa were manually separated, dried and powdered using mixer grinder to get 80-mesh size. 100 g powder was extracted using 500 ml of hexane at 60 °C for 6 h in a Soxhlet extractor. The extract was collected and concentrated under vacuum to recover the solvent. The spent obtained after hexane extraction was further extracted with 500 ml each of ethyl acetate and Methanol at 50 °C for 3h. The extract was filtered through Whatman filter paper no. 1. The filtrate was concentrated at a temperature of 35 °C and under a reduced pressure at 25 mm of mercury and dried in vacuum oven at a temperature of 50 °C and under vacuum of 10 mm of mercury. The yield of methanol extract was 14.9g and phenolic content was found to be 34.1%.
The methanol extract obtained by above method was screened for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method [Singh et al., J. Agricultural and Food Chemistry, 50, 81-86, 2002]. Methanol extract and BHA showed 90.7% and 91.5% radical scavenging activity at 50 ppm, respectively using Diphenyl picrylhydrazyl (DPPH) method.
Example -3
The peel of Annona squamosa were manually separated, dried and powdered using mixer grinder to get 60 mesh size. 25 g of powder was extracted with 100 ml of hexane by using Soxhlet extractor at 60 °C for 6 h. The extract was collected and concentrated under vacuum to recover the solvent. The spent obtained after hexane extraction was further extracted successively with 100 ml each of ethyl acetate and Methanol at 60 °C for 4h. The extract was filtered
through Whatman filter paper no. 1. The filtrate was concentrated at a temperature of 40 °C and under reduced pressure at 10 mm of mercury and dried in a vacuum oven at 40 °C under vacuum at 25 mm of mercury. The yield of methanol extract was 3.7g and phenolic content was found to be 34.7%.
The Methanol extract obtained by above method was screened for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) method [Singh et al., J. Agricultural and Food Chemistry, 50, 81-86, 2002]. Methanol extract and BHA showed 91.8% and 93% radical scavenging activity at 50 ppm, respectively using Diphenyl picrylhydrazyl (DPPH) method.












We Claim:
1. A process for the isolation of antioxidant rich fraction from grape seeds, comprising the
following steps:
a) drying and powdering grape seeds to get a powder,
b) extracting the resulting powder with water at temperature between 30 -60°C to remove
unwanted material,
c) extract the residue using water between temperature 120-130°C at pressure above
atmospheric pressure to extract phenolic compounds,
d) filtering the said aqueous extract,
e) concentrating said aqueous extract,
f) characterized in that extracting the concentrate with ethyl acetate at temperature
between 30-60 °C,
g) ethyl acetate extract is recovered under vacuum.
2. A process for the isolation of antioxidant rich fraction from grape seeds substantially as herein
described with reference to the examples.

Documents:

485-DEL-2004-Abstract-(19-04-2010).pdf

485-del-2004-abstract.pdf

485-DEL-2004-Claims-(19-04-2010).pdf

485-del-2004-claims.pdf

485-DEL-2004-Correspondence-Others-(19-04-2010).pdf

485-del-2004-correspondence-others.pdf

485-del-2004-correspondence-po.pdf

485-DEL-2004-Description (Complete)-(19-04-2010).pdf

485-del-2004-description (complete).pdf

485-DEL-2004-Form-1-(19-04-2010).pdf

485-del-2004-form-1.pdf

485-del-2004-form-18.pdf

485-DEL-2004-Form-2-(19-04-2010).pdf

485-del-2004-form-2.pdf

485-DEL-2004-Form-3-(19-04-2010).pdf

485-del-2004-form-3.pdf

485-del-2004-form-5.pdf


Patent Number 242890
Indian Patent Application Number 485/DEL/2004
PG Journal Number 39/2010
Publication Date 24-Sep-2010
Grant Date 17-Sep-2010
Date of Filing 16-Mar-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 GUDDADARANGAVVANHALLY KRISHNAREDDY JAYAPRAKASHA CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
2 RAVEENDRA PRATAP SINGH CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA