Background Art The notion that deleterious effects of oxidative metabolism can be ameliorated through a diet rich in antioxidants has gained credibility and the term"functional foods" (Strain, J. J.; Benzie, I. F. F. Antioxidant nutrients. In : Sadler, M. J. and Saltmarsh, M. Eds. Functional Foods: The Consumer, the Products and the Evidence. Cambridge, U. K., Royal Society of Chemistry, p. 74) has been applied to foods with these nutrients. It is important to note that most of the investigations regarding the inhibiting effects of food components on the oxidative damages of biological materials have been devoted to the foods of plant origin.

Phenolics are the dominant antioxidants found in the natural products of plant sources (Prior, R. L. and Cao, G. Variability in Dietary antioxidant related natural product supplements: The need for methods of standardization, Journal of the American Nutraceutical Association, 2,46-56, 1999).

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 butylatedhydroxy toluene (BHT), propylgallate (PG) and tert-butylhydroquinone (TBHQ) are commonly used in foods to inhibit free radical damage to lipids. However, a concern has now been expressed about the safety of certain synthetic antioxidants such as BHA and BHT, which are suspected as possible carcinogens. As a result, BHA has been removed from the GRAS (Generally Recognized As Safe) list and the use of TBHQ remains prohibited in some countries. Therefore, there has been a considerable interest by the industry and a growing trend in consumer preferences for natural antioxidants over

synthetic compounds (Madhavi, D. L.; Salunkhe, D. K. Toxicological Aspects of Food <BR> <BR> Antioxidants, In-Food Antioxidants, Eds.; Madavi, D. L. , Deshpande, S. S. Salunkhe, D.<BR> <P>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 Par7noti-ema stuppeum (Nyl.) Hale and their antioxidant activity. Zeitschrift für Naturforsc11çung 2000, SSc, 1018-1022).

Dillenia a small genus of trees found in Indo-Malaysian region extending to Tropical Australia. About six species are found in India i. e. D. indica, D. aurea, D. pentagyna, D. andamanica, D. bractea and D. scabrella. Most of the species bear showy flowers and edible fruits and yield timbers of secondary importance (The Wealth of India, 1952). For the present investigation we have used the fruits of D. indica. D. indica occurs in the moist and evergreen forests of sub-Himalayan tract, from Kumaon and Garhwal eastwards to Assam, Bengal and Orissa. A handsome evergreen tree, 30-50 ft. in height and 6 ft. in girth, with a rounded crown. Leaf oblong-lanceolate, 8-14 in. long and 2-4 in. broad, with pointed apex and toothed margin; the upper part of the leaf as well as the veins beneath are covered with hairs. Flowers are large, 5-8 in. diameter, white, fragrant and solitary. Fruits are large 3-5 in. diameter, hard, consisting of five closely fitting imbricate sepals enclosing numerous seeds embedded in a gelatinous pulp (Figure 1 and 2). Seeds are small, compressed reniform with hairy margins. The tree flowers during May-August and fruits ripen during September-February. The sepals are sour and used in curries or made into jams and jellies. The acid juice is sweetened with sugar and used as cooling drink. The fruit is said to possess tonic and laxative properties and is used in abdominal pains.

Literature survey revealed that, there is no report on the isolation of antioxidants from Dillenia indica.

Objects of the present Invention The main object of the present invention is to develop a simple and efficient one-step process for the preparation of methanolic extract from plant Dillenia indica showing anti- oxidant property whereby solvents are regenerated.

Another main object of the present invention to obtain yield of the methanolic extract is ranging between 15-35%.

Yet another main object of the present invention is to obtain the said extract showing phenolic content ranging between 25 to 35%.

Still another object of the present invention is to have the anti-oxidant activity of the extract is ranging between 55 to 85%.

Still another object of the present invention is use of methanolic extract of plant Dillenia indica as an anti-oxidant in food and pharmaceutical substances and additives.

Still another object of the present invention is to concentration of the extract in food and pharmaceutical is ranging between 0.2 to 3.0%.

Still another object of the present invention is to obtain a food and pharmaceutical preservative with no toxicity.

Still another object of the present invention is to obtain a food and pharmaceutical preservative with no adverse effect on the taste of the said substances.

Summary of the Present invention The present invention relates to a simple and efficient one-step process for the preparation of methanolic extract of yield ranging between 15-35% from plant Dillenia indica showing anti-oxidant activity ranging between 55 to 85%, whereby solvents are regenerated, and also, use of methanolic extract of plant Dillenia indica as an anti-oxidant in food and pharmaceutical substances and additives, wherein the concentration of the extract in food and pharmaceutical is ranging between 0.2 to 3.0%.

Detailed description of the present invention Accordingly, the present invention relates to a simple and efficient one-step process for the preparation of methanolic extract of yield ranging between 15-35% from plant Dillenia indica showing anti-oxidant activity ranging between 55 to 85%, whereby solvents are regenerated, and also, use of methanolic extract of plant Dillenia indica as an anti-oxidant in food and pharmaceutical substances and additives, wherein the concentration of the extract in food and pharmaceutical is ranging between 0.2 to 3.0%.

In an embodiment of the present invention, wherein a simple and efficient one-step process for the preparation of methanolic extract from plant Dillenia indica showing anti-oxidant property, whereby solvents are regenerated, said method comprises, i). powdering the fruit rinds of Dillenia indica in a mixer grinder to mesh size of 40-80, ii). extracting the powdered material with hexane in a Soxhlet extractor at temperature ranging between 55-60 °C for the time period ranging between 6-10 h to obtain an extract and spent,

iii). distilling the extract to recover the hexane up to 90%, iv). extracting the spent of step (ii) with methanol in a Soxhlet extractor at temperature ranging between 55-60 °C for time period ranging between 6-10 h, v). filtering the above extract using Whatman filter paper No. l to obtain particle free extract, vi). Distilling the filtered extract at temperature ranging between 55-60 °C to recover 90% methanol, vii). drying the distilled extract of step iv at temperature ranging between of 40-50 °C in vacuum under reduced pressure ranging between 10-25 mm of mercury and, viii). obtaining the said extract having anti-oxidant property.

In another embodiment of the present invention, wherein the yield of the methanolic extract is ranging between 15-35%.

In yet another embodiment of the present invention, wherein said extract shows phenolic content ranging between 25 to 35%.

In still another embodiment of the present invention, wherein the anti-oxidant activity of the extract is ranging between 55 to 85%.

In still another embodiment of the present invention, wherein the anti-oxidant property of the extract is determined using bet-carotene-linoleate and DPPH method.

In another main embodiment of the present invention, wherein use of methanolic extract of plant Dillenia indica as an anti-oxidant in food and pharmaceutical substances and additives.

In still another embodiment of the present invention, wherein the anti-oxidant activity of the extract is ranging between 55 to 85%.

In still another embodiment of the present invention, wherein the concentration of the extract in food and pharmaceutical is ranging between 0.2 to 3.0%.

In still another embodiment of the present invention, wherein the said use shows no toxicity.

In still another embodiment of the present invention, wherein the said use does not affect the taste of the food and pharmaceutical substances.

Brief description of the accompanying drawings: Fig 1 shows fruit of D. indica and its longitudinal section.

Fig. 2 shows fruiting branch of D. indica.

The preparation of antioxidants from Dillenia indica was done according to following flow diagram Rinds of Dillenia indica Powdered using mixer grinder to get a mesh size of 40-80 Extraction with hexane using S hlet extractor for 6-10 h at 55-60 °C Hexane ex act Spent iE IF Distillation t recover hexane Extraction with Methanol (6-10 h 55-60 °C) Methano extract Spen Distillation tt 55-60°C ce Concentrate Solvent 1 Dried under vacuum at 40-50 °C Under 10-25 mm of mercury IF Yield of antioxidants (24-33%)

The novelty of the process is: 1. This is the first report of preparation of antioxidants from D. iiidica.

2. The invention is a one step process to obtain the antioxidants from D. indica.

The advantages of the process: 1. The process is simple and the solvents used in this process can be regenerated 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 50 g of rinds of Dillenia indica was powdered using mixer grinder to get a 60 mesh size.

The powder was extracted with hexane for 6 h in a Soxhlet extractor at 60 °C. The extract was filtered and distilled to recover the solvent. The spent was dried and further extracted with 200 ml of methanol at 60 °C for 8 h in a Soxhlet extractor. The extract was filtered using Whatman filter paper No. l. The methanol extract was distilled under vacuum to recover the solvent. The extract was dried at a temperature of 50 °C and under reduced pressure at 10 mm of mercury. The yield of methanol extract was 15.05 (w/w). The phenolic content of the above extract was 34.2%.

The extract obtained above method was screened for antioxidant activity using p-carotene- linoleate model system [Singh et al. , J. Agricultural and Food Chemistry, 50,81-86, 2002].

It showed 80.2% antioxidant activity at 100 ppm.

Example-2 100 g of Dillenia indica was powdered using mixer grinder to get a 40 mesh size. The powder was extracted with hexane for 8 h in a Soxhlet extractor at 60 °C. The extract was filtered and distilled to recover the solvent. The spent was dried and further extracted with 400 ml of methanol at 60 °C for 6 h in a Soxhlet extractor. The extract was filtered using Whatman filter paper No. l. The methanol extract was distilled under vacuum to recover the solvent. The extract was dried at a temperature of 40 °C and under reduced pressure at 25 mm of mercury. The yield of methanol extract was 24.3 g. The phenolic content of the above extract was 27.34%.

The 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 68% antioxidant activity at 100 ppm using Diphenyl picrylhydrazyl (DPPH) method.

Example-3 150 g of Dillenia indica was powdered using mixer grinder to get 80 mesh size. The powder was extracted with hexane for 10 h in a Soxhlet extractor at 55 °C. The extract was filtered and distilled to recover the solvent. The spent was dried and further extracted with 200 ml of methanol at 55 °C for 10 h in a Soxhlet extractor. The extract was filtered using Whatman filter paper No. l. The methanol extract was distilled under vacuum to recover the solvent. The extract was dried at a temperature of 40 °C and under reduced pressure at 25 mm of mercury. The yield of methanol extract was 37.05 (w/w). The phenolic content of the above extract was 29.2%.

The 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 62.5% antioxidant activity at 50 ppm using Diphenyl picrylhydrazyl (DPPH) method.

The antioxidant activities of the antioxidants from D. indica in comparison with BHA as been presented in Table 1 and 2.

Table 1 Antioxidant activity of fruit extracts of D. indica and BHA by p-carotene-linoleate model <BR> <BR> system (% inhibition of bleaching of 8-carotene). [Singh et al. , J. Agricultural and Food Chemistry, 50,81-86, 2002]; [G. IL. Jayaprakasha, R. P. Singh, K. K. Salcariah : Food Chemistry 73 (2001) 285-290]. Compond 50 ppm 100 ppm BHA 93. 3 0. 02 96. 0 0.40 Methanol Extract of 64.2 0. 40 80. 2 0.75 D. Indica Table 2 Radical scavenging activity of fruit extracts of D. indica and BHA by DPPH method.

[Andres Moure et al. Food Chemistry 72 (2001) 145-171].

Compound 25 ppm 50 ppm 100 ppm BHA 58. 580. 460 64. 261. 268 72. 2910. 809 Methanol of 56. 613. 483 62. 582. 375 68. 160. 355 D. Indica Example 4 The said methanolic extract of plant Dillenia indica is tried as an anti-oxidant in food and pharmaceutical substances and additives. Some of the food systems used tomato, apples, potato, pickles, fruit jams, soft drinks, ketchups, etc. Further, the pharmaceutical product comprises analgesics, anti-pyretic, etc.

Here, concentration of the extract in food and pharmaceutical is ranging between 0.2 to 3.0%. Further, the anti-oxidant activity of the extract is found to be ranging between 55 to 85%.

In addition, the food and pharmaceutical items with said extract as anti-oxidant with food preservation activity was found to be non-toxic. Also, the taste of the food and pharmaceutical products was not affected by adding this extract.