Title of Invention

A PROCESS FOR THE ISOLATION OF ANTIOXIDANT AND ANTIBACTERIAL FRACTION FROM GARCINIA PEDUNCULATA

Abstract

The present invention provides a process for the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata. The preparation is a bioactive fraction which has both antioxidant and antibacterial properties. The isolation of antioxidant and antibacterial fraction from Garcinia pedunculata is not reported in the prior art. The invention provides a technology for the isolation of the bioactive fraction from the rinds of Garcinia pedunculata, which has potential application as a natural preservative.

Full Text

The present invention relates to a process for the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata. The generation of reactive oxygen species (ROS) is associated with life under aerobic conditions and produced under normal metabolism and pathophysiological conditions in addition to exogenous factors. ROS rapidly interact and damages the biomolecules such as lipids; proteins and DNA. The consequences of such extensive damages are involved in the development of various degenerative disease state like atherosclerosis, cardiovascular disease, cataracts, tissue damage in rheumatoid arthritis, decline in immune function, dysfunction of brain, and certain types of cancer. Besides the endogenous defenses, consumption of dietary antioxidants could be an important aspect of body's defense mechanism to protect against ROS and also many antioxidants are being identified as anticarcinogens. Diets rich in fruits and vegetables have been associated with lower incidence and mortality rates of cancer and low dietary intake of the same doubles the risk of cancer as compared to high intake [Ames, B.N., Shigenaga, M.K., & Hagen, T.M. Oxidants antioxidants and degenerative diseases of aging. Proc. Natl. Acad. Sci. USA, 90, 7915-7922, 1993]. It is important to note that most of the investigations regarding inhibitory effect of food component on the oxidative damages of biomolecules have been devoted to the foods of plant origin. Antioxidants are the compounds which when added to food products, specially to lipids and lipid containing foods, can increase the shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food products during the processing and storage. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytolune (BHT), have restricted use in foods as these synthetic antioxidants are suspected to be carcinogenic (Madhavi, D. L; Salunkhe, D. K. Toxicological Aspects of Food Antioxidants, In-Foocf Antioxidants, Eds.; Madavi, D. L., Deshpande, S. S. Salunkhe, D. K., Marcel Dekker Inc. New York, 1995, p.267). Therefore, the importance of search and exploitation of natural antioxidants, especially of plant origin has greatly increased in recent years (Jayaprakasha, G.K.; Jaganmohan Rao, L. Phenolic constituents from lichen Parmotrema stuppeum (Nyl.) Hale and their antioxidant activity. Zeitschrift fur Naturforschung 2000, 55c, 1018-1022). Microbial activity is a primary mode of deterioration of many foods and is often responsible for the loss of quality and safety. Concern over pathogenic and spoilage microorganisms in foods is increasing due to increase in outbreaks of foodborne disease [Tauxe RV, Dairy Food Environ Sanit 17: 788-795, 1997]. Currently there is a growing interest to use natural antibacterial compounds, like plant extracts of herbs and spices for the preservation of foods, as these possess a characteristic flavour and sometimes show antioxidant activity as well as antimicrobial activity [Smid EJ, Gorris LGM, Natural antimicrobials for food preservation, In- Shafiurr Rahman M (ed) Handbook of food preservation. Marcel Dekker, Inc, New York, pp.285-308, 1999]. Garcinia (Family: Guttiferae) is a large genus of polygamous trees or shrubs, distributed in the tropical Asia, Africa and Polynesia. It consists of 180 species, out of which about 30 species are found in India [The Wealth of India (Raw Materials), Vol.IV, CSIR, New Delhi, India, 1956, pp.99-108]. Garcinia pedunculata are found in eastern part of India. In upper Assam it is often cultivated in homesteads for its acid fruit. The tree flowers during January - March and fruits ripen in May-June. The fruits are edible, dried slices of the fruit are used in curry in place of tamarind/lemon. G. Pedunculata is one of the largest fruited species of the genus. For the last several years, small and complex molecules have been isolated from the various species of Garcinia, which include xanthones and xanthone derivatives [Bennet, G.J.; Lee, H.H. Xanthones from Guttiferae. Phytochemistry, 28, 967-998, 1989]. Literature survey revealed that, there is no report on the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata. The main object of the present invention is to provide a process for the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata which obviates the drawbacks as outlined above. Another object of the present invention to provide technology for the preparation of antioxidant and antibacterial fraction from the rinds of Garcinia pedunculata, which can be used as potential natural preservative. Yet another object of the present invention is to have an efficient process for the large-scale preparation of both antioxidant and antibacterial fraction from the dried rinds of the fruits of Garcinia pedunculata. Still another embodiment of the present invention is to prepare the natural preservative which is neutral in flavour and dark brown in colour. The process is efficient and it involves the use of simple extraction methods and solvents, which can be regenerated. Accordingly, the present invention provides the process for the isolation of antioxidant and antibacterial fraction from Garciniapedunculata, which comprises: a) Characterized in that cutting in the rinds of Garcinia pedunculata to a size ranging from 4x6 mm to 8 x 10 mm, b) extracting the above said material with hexane in a soxhlet extractor at a temperature ranging between 55 and 60°C for a period of 6-8 hr, c) distilling the above said extract to recover the solvent for recycling, d) extracting the spent obtained from step (b) with chloroform in a soxhlet extractor at a temperature ranging between 50 and 60°C for a period of 6-10 h, e) filtering the above extract using Whatman filter paper No. 1 to obtain the particle free extract, f) concentrating the particle free extract obtained in step e) at a temperature in the range of 35-40°C under vacuum at 10-25 mm Hg, g) drying the above concentrated extract at a temperature in the range of 40-50°C and 10-25 under vacuum at 10-25 mm Hg, to obtain the desired product. In an embodiment of the present invention, the fraction obtained may have a yield in the range of 4.5-6%. In another embodiment of the present invention, the fraction obtained may be of dark brown in colour and has a neutral flavour. The preparation of antioxidant and antibacterial fraction from Garcinia pedunculata was done according to following flow diagram The novelty of the process is: 1. This is the first report of preparation of antioxidant and antibacterial fraction from G. pedunculata. 2. The invention is a two step process to obtain the bioactive fraction from G. pedunculata. 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 Fifty grams of rinds of Garcinia pedunculata was manually cut into small pieces and 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 with 250 ml chloroform at 60 °C for 6 h in a Soxhlet extractor. The chloroform 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 chloroform extract was 2.4 g. The antibacterial assay for the chloroform extract of G. pedunculata was test by pour plate method against different bacteria namely, Bacillus cereus, B. coagulans, B. subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus by the method of Negi et al. [J. Agricultural and Food Chemistry 47(10), 4297-4300, 1999]. To flasks containing 20 ml melted nutrient agar, different concentration of test material in propylene glycol were added. Equivalent amounts of propylene glycol were used as controls. One hundred ^ (about 1Q3 cfu/ml) of each bacterium to be tested was inoculated into the flasks under aseptic conditions. The media was then poured into sterilized petri plates in quadruplet and incubated at 37 °C for 20-24 h. The colonies developed after incubation were counted and expressed as colony forming units per ml of culture (cfu/ml). All the cultures were tested as in pour plate method and the minimum inhibitory concentration (MIC) was reported as the lowest concentration of the compound capable of inhibiting the complete growth of the bacterium being tested. The MIC values of Garcinia pedunculate extract ranged from 400 to 1250 ppm. Example 2 The dried rinds (100 g) of Garcinia pedunculata were extracted with 400 ml of hexane by using Soxhlet extractor at 55 °C for 8 h. The extract was collected and concentrated under vacuum to recover the solvent. The spent obtained after hexane extraction was further extracted with 500 ml chloroform at 50 °C for 10 h. 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 chloroform extract was The chloroform extract obtained above method was screened for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) [Singh et al., J. Agricultural and Food Chemistry, 50, 81-86, 2002]. It showed 65% antioxidant activity at 500 ppm using Diphenyl picrylhydrazyl (DPPH) method. Example 3 Rinds (150 g) of Garcinia pedunculate were extracted with 600 ml of hexane by using Soxhlet extractor at 60 °C for 8 h. The extract was collected and concentrated under vacuum to recover the solvent. The spent obtained after hexane extraction was further extracted with 750 ml chloroform at 60 °C for 10 h. 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 chloroform extract was 8.2 g. The chloroform extract obtained above method was screened for antioxidant activity using p-carotene-linoleate model systems. The extract of G. pedunculata showed 68% antioxidant activity at 500 ppm using p-carotenelinoleate method [Jayaprakasha G.K. and Jaganmohan Rao L. Phenolic constituents from lichen Parmotrema stuppeum (Nyl.) Hale and their antioxidant activity, ZeitschriftfurNaturforschung, 55c, 1018-1022, 2000]. The advantages of the process is 1. The process is simple and the solvents used in this process can be regenerated for further use. 2. The left over spent can be further used for the extraction of (-)- Hydroxycitric acid. We Claim: 1. A process for the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata, which comprises: a) Characterized in that cutting in the rinds of Garcinia pedunculata to a size ranging from 4x6 mm to 8 x 10 mm, b) extracting the above said material with hexane in a soxhlet extractor at a temperature ranging between 55 and 60°C for a period of 6-8 hr, c) distilling the above said extract to recover the solvent for recycling, d) extracting the spent obtained from step (b) with chloroform in a soxhlet extractor at a temperature ranging between 50 and 60°C for a period of 6-10 h, e) filtering the above extract using Whatman filter paper No. 1 to obtain the particle free extract, f) concentrating the particle free extract obtained in step e) at a temperature in the range of 35-40°C under vacuum at 10-25 mm Hg, g) drying the above concentrated extract at a temperature in the range of 40-50°C and 10-25 under vacuum at 10-25 mm Hg, to obtain the desired product. 2. A process as claimed in claim 1, wherein the fraction obtained has a yield in the range of 4.5-6.0%. 3. A process as claimed in claims 1 & 2, wherein the fraction obtained is dark brown in colour and has a neutral flavour. 4. A process for the isolation of antioxidant and antibacterial fraction from Garcinia pedunculata substantially as herein described with reference to the examples.

Documents:

358-DEL-2002-Abstract-(27-10-2008).pdf

358-del-2002-abstract.pdf

358-DEL-2002-Claims-(25-02-2009).pdf

358-DEL-2002-Claims-(27-10-2008).pdf

358-del-2002-claims.pdf

358-del-2002-complete specification (granted).pdf

358-DEL-2002-Correspondence-Others-(25-02-2009).pdf

358-DEL-2002-Correspondence-Others-(27-10-2008).pdf

358-del-2002-correspondence-others.pdf

358-del-2002-correspondence-po.pdf

358-del-2002-description (complete)-(25-02-2008).pdf

358-DEL-2002-Description (Complete)-(27-10-2008).pdf

358-del-2002-description (complete).pdf

358-del-2002-form-1.pdf

358-del-2002-form-18.pdf

358-DEL-2002-Form-2-(27-10-2008).pdf

358-del-2002-form-2.pdf

358-DEL-2002-Form-3-(27-10-2008).pdf

358-del-2002-form-3.pdf