Title of Invention	"A PROCESS FOR THE PREPARATION OF ANTIOXIDANT CONSERVE FROM MAMMEA IONGIFOLIA"
Abstract	A process for the preparation of antioxidant conserves from Nagkesar (Mammea longofolia), which comprises: pulverising & extracting with apolar solvent & with a small chain fatty alcohol, fractioning the alcohol extract by chromatography or precipitation method.

Full Text

This invention relates to the process for the preparation of antioxidant conserve from Mammea longifolia. Antioxidants play a crucial role in preventing or delaying autoxidation and have attracted a lot of attention as food additives. The deterioration of food with time, results from its biological nature largely and is inevitable. During the production, processing, distribution and storage prior to actual consumption, food undergoes various modes of deterioration that involve biological changes by microbes as well as chemical changes. The latter is ascribed to enzymatic and non-enzymatic oxidation of lipids and phenolic substance, which cause undesirable changes in flavour, appearance, physical character, nutritional value and toxicity. Deoxygenation, airtight packing, and other techniques have solved some of these problems to a certain extent, but the role of antioxidants as either food constituents or as additives cannot be overlooked. Both synthetic and natural antioxidants are widely used in many food products. The subject of natural antioxidants has been developing since past decade, mainly because of the increasing limitations on the use of synthetic antioxidants and enhanced public awareness of health issues. In general, consumer prefers natural antioxidants because these are considered safe and environmental friendly. Commercial antioxidants are generally synthetic compounds. There has been a growing interest in replacing them with natural ingredients due to possible toxicity of synthetic antioxidants ( Z Naturforsch, 55C, 1018, 2000) as shown by clinical studies (Toxicological aspects of antioxidants used as food additives, In Food antioxidants; Hudson,B.J.F., Ed.; Elsevier; London, 1990; p253). Further the use of some common synthetic antioxidants such as Butylated hydroxy anisole (BHA) and Butylated hydroxy toluene (BHT) has become controversial issue because of adverse toxicological data (Food antioxidants, edited by Madhavi, Despande and Salunke, Marcel Dekker, Inc. Newyork 1995, p.267). The use of BHT is already banned in India. The use of natural antioxidants in food is limited due to the lack compounds in the raw materials and the availability of relevant toxicological data. Hence, evaluation of the antioxidative activity of naturally occurring substances has been of interest in recent years (Z Naturforsch, 55C, 1018, 2000). Currently, the use of some naturally occurring antioxidants in preventive and therapeutic medicine is gaining importance. Literature survey revealed that there is no report on the antioxidant activity of extracts from Indian minor spice Nagkesar (buds of Mammea longifolia). Buds of Mammea longifolia Planch & Triana syn (Guttiferae) are well known in India as Nagkesar (Hindi) and used as a minor spice. M. longifolia is a large tree growing to a height of 12-18 m and found in south western India from Khandla south wards to malabar and coibatore region. It bears flowers once a year in the month of March and the flower buds are globose, white or pink in colour. The flower buds are stimulant, carminative and astringent, used in dyspepsia and haemerrhoids (Ambasta, S.R. Ed., The useful Plants of India, NISCOM, CSIR, New-Delhi, 2000; pp353). Its dried flower buds resemble the enlarged clove buds and are extensively used in culinary preparations especially in spice blends and Garam Masala powders. The buds are also used as substitute to cloves in making 'Pan Masala', which is a chewing product in India to improve digestion. The main object of the present invention is to provide a process for the preparation of antioxidant conserve from the Indian minor spice Nagkesar. Another object of the invention is to provide a n antioxidant more activ ethan standard drug rutin respectively. Accordingly the present invention provides to a process for the preparation of antioxidant conserves from Nagkesar (Mammea longifolia), which comprises : (i) pulverising the buds of minor spice Nagkesar to get a particle size of 0.1mm to 1mm, (ii) extracting the pulverised material obtained from the step (i) using an apolar solvent and with a small chain fatty alcohol successively. (iii) fractionating the above alcohol extract obtained in step (ii) by chromatography or precipitaion method to get antioxidant fraction In an embodiment of the present invention, the adsorbent used in step (iii) may be silica gel, and/ sephadex gel. In another embodiment of the present invention, the yield of the antioxidnat conserve (AC-1, Scheme-1) in step (iii) may be 0.20 - 0.40% by chromatography method with respect to weight of nagkesar buds. In yet another embodiment of the present invention, the yield of the antioxidnat conserve (AC-2, Scheme-2) in step (iv) may be 0.60 - 0.85% by solvent partition method with respect to weight of nagkesar buds. In still another embodiment of the present invention the antioxidant activities of the AC-1 and AC-2 are 1.4 -2.0 times more than the standard compound rutin . The present invention provides a process for the preparation of antioxidant conserves from the Indian minor spice Nagkesar, which comprises, (i) pulverising the minor spice Nagkesar using mechanical device to get a particle size of 0.1mm to 1mm. (ii) extracting the pulverised material obtained from the step (i) using an apolar solvent selected from pentane, hexane, heptane, petroleum ether and their mixtures and with a small chain fatty alcohol successively, (iii) fractionating of the above alcohol extract in step (ii) by chromatography using adsorbent as stationary phase and solvent mixtures as eluting phase containing halogenated solvent like chloroform, methanol and water, (iv) fractionating of the above alcohol extract in step (ii) by precipitation method using solvents selected from lower fatty alchol, lower ketone and water., (v) testing of antioxidant activity The process for the preparation of antioxidant conserves from the Indian minor spice Nagkesar was carried out according to following flow diagram. SCHEME-1 Chromatographic method Nagkesar powder 1. Extraction with an apolar solvent followed by 2. Extraction with alcohol Alcohol Extract Chromatography over Silica gel Other solvent elutes Alcohol elute Concentration Residue Alcohol + Water Insoluble Soluble Chromatography over Sephadex M+W=l:l A+W=7:3 A+W=l:l M = = methanol W = = water A = : Acetone Antioxidant conserve (AC-1) SCHEME-2 Solvent partition method Nagkesar powder 1. Extraction with an apolar solvent followed by 2. Extraction with alcohol Alcohol Extract Re-dissolved in alcohol Insoluble Soluble Acetone addition and kept over night Precipitate Filtrate Concentration and kept over night Precipitate Filtrate Concentration and acetone addition Filtrate Precipitate water Fiterate Precipitate [Antioxidant conserve (AC-2)] Antioxidant activity assay methods: 1. Chemiluminescence assay: 0.5M Tris-HCl buffer solution. Tris(hydroxymethyl)amino methane (15. lg) is dissolved in 250 ml of EDTA (lOOuM). (HC1 was used to regulate pH - 7.8). Reagent A (Substrate solution): Xanthine 1.52 mg is dissolved in 0.5 M Tris-HCl buffer solution (10ml). Reagent B (coloring agent): Reagent A (1.5ml) is mixed with 0.5M Tris-HCl buffer (13.5 ml) and 3 mM MCLA in H2O (5mul). [MCLA: 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[ 1,2-a] pyrazin-3-one hydrochloride] Reagent C (Enzyme): 5 units/ml suspension of XOD (Xanthine Oxidase) (1.5 ul) is mixed with 0.5M Tris-HCl buffer (2 ml) and diluted to 20ml with water. Preparation of extract solutions: Extracts (lmg/ml in MeOH or DMSO) were prepared and 300 mul of this solution taken in the first column of wells of microplate and 168.6 mul of the solution is transferred to the next column of wells and made up to 300 mul using the solvent. These solutions were diluted subsequently and serially. Finally, each well contains 131.4 mul of extract solution with serial dilution. Measurement of chemiluminescence: 10 (mul each of the above solutions were transferred to three other microplates for chemiluminiscence measurement. Reagent B (l00mul) and C (l00mul) were added and chemiluminescence is measured after 19-20 sec. 2. DPPH (l,l-diphenyl-2-picrylhydrazyl radical )Assay: DPPH reagent solution: l00muM of DPPH in EtOH:H2O(l:l) Measurement of absorbance: 15 mul each of the above solutions were transferred to three other microplates for absorbance measurement. DPPH reagent (185mul) were added and O.D. is measured at 550 nm after 5 min. The advantages of the process are: This is the first report of a process for the isolation of antioxidant conserves from the minor spice nagkesar. The methanol extract of buds and its isolated fractions could be used as natural antioxidant / radical scavenging conserve in food systems Nagkesar is a source of natural antioxidant. This process will help in the proper utilisation of minor spice nagkesar This process will add to the economics of producer of nagkesar. The novelty of the process is isolation of antioxidant conserve from nagkesar. Separation of antioxidant conserves using simple chromatography techniques. Separation of antioxidant conserves using solvent mixtures at unique ratios. The following examples are given by way of illustrations of the present invention and therefore should not be construed to limit the scope of the present invention. Example-1 Separation of antioxidant conserve: Buds (250g) of Mammea longifolia were powdered and extracted with hexane followed by methanol. Methanol extract (22.30g) was chromatographed on a column of silica gel (225g; 10 times of extract W/W). The methanol elutes (7.2g) fraction from silica gel CC has showed strong radical scavenging activity. It was dried and dissolved in methanol (M) - water (W) (1:1). Insoluble (2.28g; yield in weight 40%) was separated, and soluble (4.32g; 60%) was subjected to Sephadex-LH20 column (3cm X 15cm). The fractions were eluted as follows: Fr 1- methanol (M) - water (W) (1:1, 600ml, 3.48g; yield in weight, 80.6%); Fr 2- Acetone (A)-Water (W) (7:3, 200ml, 0.57g; 13.2%); Fr 3 - A:W (1:1, 200ml, 0.004g; 0.1%). Solvent was removed by evaporation at 35°C under vacuum, and water was removed by freeze drying. Residue from fraction-2 showed highest antioxidant activity and labeled as antioxidant conserve-1 (AC-1). Evaluation antioxidant activity of AC-1 using chemiluminescence and DPPH assays showed 1.5 and 1.7 times more active than standard rutin respectively. Example-2 Isolation of antioxidant conserve by precipitation method: Buds (250g) of Mammea longifolia were powdered and extracted with hexane followed by methanol. Methanol extract (22.3g) was dissolved of methanol (17.5 times W/V). Insoluble material (2.0 lg; 9%) was separated by filtration. Equal amount of (0.78g; 3.5%) was filtered. The filtrate was concentrated and kept over night. Separated solid (3.35g; 15%) was filtered, and the filtrate was concentrated. Acetone (9 times) was added 1o the filtrate, and kept over night in the cold room. Precipitate (5.13g; 23%) was filtered and was dissolved in water (55 times W/V) by stirring. Insoluble material (2.10g; 41%) was filtered, showed the highest antioxidant activity and labeled as antioxidant conserve-2 (AC-2). Filtrate is subjected to freeze drying to obtain remaining (3.03g; 59%) residue. Evaluation antioxidant activity of AC-2 using chemiluminescence and DPPH assays showed 1.7 and 1.5 times more active than standard drug rutin respectively.

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