Title of Invention

"A PROCESS FOR THE EXTRACTION OF POLYPHENOLS FROM BEES WAX"

Abstract

A process for the extraction of polyphenols from bees wax which comprises in the steps of, agitating wax for 20 to 24 hours in a polar organic solvent to obtain a slurry, filtering the slurry through filler paper which retains coarse and gelatinous precipitates, respectively washing the paper, collecting the washings and pooled for precipitating the phenols, bringing the phenols to room temperature and filtering, the polar solvents in which the polyphenols are dissolved being removed under reduced pressure at a temperature of 5 to 60°C.

Full Text

FIELD OF INVENTION This invention relates to a process for the extraction of polyphenols from bees wax. BACKGROUND OF INVENTION o -3 rich oils undergo extensive deterioration on prolonged exposure to atmospheric oxygen and heat. Hydroperoxides, which are the major initial products of oxidation, decompose into a series of secondary products such as esters, aldehydes, alcohols ketones, lactones and hydrocarbons. These breakdown compounds adversely affect flavour, aroma, taste, nutritional value and overall quality of the foods. Certain oxidation products are potentially toxic at relatively low concentrations. The degree of unsaturation in lipids, availability and types of oxygen (1O2,3O2), heat, light, metal ions, radicals (O2-, ROO, OH, SO3-), phospholipids, free fatty acids, enzymes, chelators and antioxidants affect the oxidative stability of oil. The oxidized fats/oils have been reported to cause diarrhoea, liver enlargement, growth depression, etc. the products of fatty acid oxidation, especially the aldehydes are mutagenic and carcinogenic. Presently, FDA allows butylated hydroxyl anisole (BHA) for use in edible oils as a synthetic antioxidant in order to arrest the formation of lipid peroxides. At the same time, BHA is reported to possess toxicological manifestation at high levels of intake spectrum. We have developed a methodology for the extraction of polyphenols from locally available bees wax. The method relates to the preparation of low cost polyphenols, a naturally occurring phytoconstituent, having wider applications in arresting oxidative deterioration of highly unstable polyunsaturated fatty acid (PUPA) rich edible oils. The extract developed by this method can also be used for ameliorating pesticide induced oxidative stress in living systems. More specifically the present invention relates to the development of a method for extracting natural antioxidants from locally available bees wax and its utilization for arresting oxidative deterioration in fatty foods in vitro and alleviating oxidative stress related toxicity in vivo. PRIOR ART Propolis, or bee glue, a natural resinous hive product, highly rich in polyphenols, gathered by honey bees (Apis mellifera) possesses multitude of uses like antibacterial, antifungal, antiviral, hepatoprotective and anti-inflammatory. As the Asian species of Apis do not collect much of propolis, the components of the polyphenols of the wax from other species of honey bees viz., Apis dorsetta has not been put into its potential use. Polyphenols are extracted from plant foods by conventional simple methods. The methods presently available for the extraction of polyphenols are for grapes, pomegranate, apples and other fruits. Honey has been used since ancient times and has gained appreciation as the only concentrated form of sugar available world wide. Bees wax is available as a by product of bee keeping industry which is produced and used by honey bees construction of comb. The wax is produced as secretions by the wax glands of bees and results from the digestion of carbohydrates. It has been estimated that to produce 1kg of bees wax, the bees have to consume 6.8kg of honey. Therefore, it is very important to save and process every bit of this wax so that the energy spent on wax production does not go waste. The application of bees wax in folk medicine, cosmetic and pharmaceutical industries is due to its rich content of polyphenols and flavonoids. The methods/process used for the extraction of polyphenols from the wax is not available presently. Hence the local pharma and cosmetic industries use the entire wax itself in lieu of the active ingredients present in it. OBJECTS OF INVENTION An object of this invention is to propose a process for extraction of polyphenols from bee wax. Another object of this invention is to propose a process for extraction of polyphenols from bee wax which is low cost phytoconstituent with low toxicity. Yet another object of this invention is to propose a process for extraction of polyphenols from bee wax which is a natural antioxidant and capable of arresting peroxidation of  -3 rich edible oil. Still another object of this invention is to propose a process for extraction of polyphenols from bee wax which is a natural antioxidant capable of reducing lipid peroxidation and oxidative stress derived toxicity in animal system. A further object of this invention is to propose a process for extraction of polyphenols from bee wax which is a natural antioxidant as effective as BHA (a synthetic one). A still further object of this invention is to propose a process for extraction of polyphenols from bee wax which is moreefficient than propolis, with respect to its ability to reduce lipid peroxidation induced by heating the edible oil. Yet a further object of this invention is to propose a process for extraction of polyphenols from bee wax which is rich in flavonoids. DETAILED DESCRIPTION OF INVENTION According to this invention there is provided a process for the extraction of polyphenols from bees wax which comprises in the steps of: i) agitating wax for 20 to 24 hours in a polar organic solvent to obtain a slurry, ii) filtering the slurry through filler paper which retains coarse and gelatinous precipitates, iii) respectively washing the paper, iv) collecting the washings and pooled for precipitating the phenols, v) bringing the phenols to room temperature and filtering, vi) the polar solvents in which the polyphenols are dissolved being removed under reduced pressure at a temperature of 5 to 60°C. After removing honey by draining the comb, it is melted in water maintained at 80°C. The wax is accumulated on the hot water surface. The gross impurities viz. dead bees, cocoons, etc. were filtered in a wet porous cloth and allowed to solidify. The wax obtained by this method was found to be yellowish brown in colour, balsamic in taste with honey like odour and soft plastic texture. The wax prepared by the above method is subjected to the extraction of polyphenols. For this, the wax is shaken mechanically uninterrupted for 20-24 hrs in a highly polar organic solvent in the ratio of 1:2. The yellow slurry obtained was filtered through a previously wet non-fatty porous medium fast ashless qualitative filter paper, which can retain coarse and gelatinous precipitates. The paper is washed with the same solvent 3 times in three increments so that the washings do not loose the phenols7. Washings were collected and pooled immediately for precipitating the phenols at lower temperature. The maximum precipitate was found by storing the intake for 24 hrs. After the complete precipitation, the resinous substance is again brought to room temperature without warming and filtered through thick, medium speed porous paper with high retention. Brownish yellow resinous substance s obtained in solution. The polar solvents in which the polyphenols are dissolved were removed under reduced pressure at a temperature 5-6°C. The dry yellow extracts of polyphenols were obtained. The yield was found to be 2.6%. The extract was found to possess antioxidant activity at a level of 86.0% 2. The PBW was utilized for stabilizing the thermally oxidized co-3 rich.- edible oil. The synthetic antioxidants, BHA and the economically available standard propolis (.Sigma Chemicals Co., USA) were used for stabilizing the same oil for comparison. The result revealed that the % reduction of formation of Thiobarbituric acid reactive substances (TBAR5) on heating the oil coaidiiuug BHA, standard propolis and PBW at the level of 200 ppm was 21.3,16 and 34.7 respectively. The % reduction of formation of conjugated dienes on heating the oil containing the same substances was 47.7,11.4, and 40.3. It was also interesting to note that the same of hydroperoxides were 40.9, 29.0, and 40.4. The work shows that PBW prepared by the method being patented could be used as a natural antioxidant in lieu of the synthetic one, BHA for controlling lipid oxidation in co-3 rich - edible oil. At the same time, it was noted that the standard propolis, economically available in an inferior one as compared to PBW. The PBW was further evaluated for establishing its capability in reducing pesticide (hexachlcrocyclohexane) - induced lipid peroxidation in rat liver. Experimental protocol Adult male albino rats of Wistar strain (150-160g) were fed on control diet and water ad libitium and maintained under standard laboratory conditions with 12:12 h lightdark cycle. The rats were divided into'four groups of six animals each , (1) control: untreated rats (2) HCH group: rats administered with HCH (SOOmg /kg body i.p,, once, 24 h prior to sacrifice) in groundnut oil (3) PBW group: rats pretreated with polyphenols extracted from honevbee wax (200mg/kg body wt.) daily, by gavage, for 2 weeks (4) PBW+HCH group: rats pretreated with PBVV and injected with HCH , 24 h after the 14th day PBW administration and sacrificed after 24 h of HCH injection. All the rats were sacrificed under mild anesthesia (sodium pentobarbitone). Liver was quickly excised and stored in liquid nitrogen until analysis. Chemical Analysis Malondialdehyde (MDA) was assayed in liver homogenate (0.5 g) by precipitating with trichloroacetic acid (10%) and reacting with thiobarbituric acid reaction mixture (0.35%) consisting of sodium dodecyl sulphate, ferric chloride and butylated hydroxy toluene in 0.1 M of giycine-HCl buffer. After boiling and cooling, OD was taken at Ama* 532 and MDA was calculated using a molar extinction coefficient of 1.56xl05/M/cm. The lipids isolated from liver were mixed with CHCh and CHaOH mixture and cenrrifuged. The upper phase was removed by reduced pressure. The lipid residue was dissolved in cyclohexane and the amount of conjugated dienes (CD) produced were estimated. The upper layer of the same assay was utilized for the estimation of hydroperoxides (HP). For the assay of Catalase liver (0.5 g) was homogenized in phosphate buffer (5 M pH 7.4) and the homogenates were centrifuged at 700 x g. The supernatant was ascayca using hydrogen peroxide as a substrate. GKitathione reductase (GSSGR) and glutathione peroxidase (GSH-Px) activities were determined in the supernatant 01 liver homogenate prepared in phosphate buffer (0.5 M pH 7.0) using HzO2 anc NADPH as substrates. Hepatic glutathione S-transferase (GST) activity wa; determined by the procedure of Habig et al. (1974) in the supernatant from live homogenate in phosphate buffer (0.1 M pH 6.5), centrifuged at 700xg using l-chloro-2 4-dinitrobenzene as substrate. Superoxide dismutase (SOD) was measured by th inhibition of cytochrome C reduction mediated via superoxide anions generated b xanthine-xanthine oxidase and monitored at 550nm. One unit.of SOD was defined 8 the amount required for inhibiting the reduction of cytochrome C by 50%..The assa mixture for the-estimation of hepatic glucose-6-phosphate dehydrogenase (G-6-PE consisted of the enzyme source prepared in O.lmM of Tris buffer7*2mM of glucose-i . phosphate, and 0.3 rnM of NADP while AA was monitored at 340 nm. Gamma glutamyltranspeptidase (GGT) was estimated using L-Y-glutamyl-p-nitroanilide as t3 substrate in a homogenate prepared with 0.1 M of Tris-HCI buffer. Statistic analysis was carried out using one way analysis of variance (ANOVA). Table 1 presents the effect of PBW administration and the HCH injection on i liver. The MDA, CD and HP were increased significantly by the HCH injection. It known that CD and MDA are more cytotoxic and stable than reactive oxygen spec and react quickly with cellular constituents. Besides these negative effects, MI particularly is a modulator of signal transducrion pathways that disturb cellu activities. However, the PBVV exposure per se did not influence the levels of th cytotoxic products in the liver. It was interesting to note thai; the HCH-induced rise in MDA, while CD and HP levels were significantly diminished by exposing the rats to PBW. This indicated that PBW was capable of reducing the HCH-induced free radical stress. Table 1. Effects of bees wax poly phenols and HCH treatments on hepatic lipid peroxides* (n=6) (Table Removed) Mean + SD* Values bearing different superscripts in the same column are significantly different (p Table 2 presents the effect of PBW and HCH treatments on hepatic antioxidant enzymes viz. catalase, GSH-Px, GSSG-reductase, G-6-PD, SOD, and hepatic detoxifying enzyme, GGT. Hepatic catalase, SOD and G-6-PD activities were diminished significantly in rats administered with HCH which on administering PBW, there was .a significant elevation in activities hepatic catalase, GSH-Px, GSSG -reductase and G-6-PD. This observed increase in antioxidant enzymes might provide protection against HCH-induced free radical stress because it is known that peroxidation in vivo is reduced by the antioxidant enzymes. The diminished hepatic G-6-P-D activity produced by HCH could be due to inhibition by the pesticide or its metabolite and in turn, the resultant reduced availability of NADPH in the HMP shunt pathway may alter GSH turnover. However, the GSH content in liver remained constant as compared to HCH- administered rats. The significant elevation of G-6-PD in the PBW- treated rats might provide the required NADPH for the regeneration of GSH from GSSG coupled with enhanced GSSG-reductase activity. The HCH-induced reduction in the activity of these enzymes was found to be restored on PBW administration. Abnormally high levels of GGT have been observed in cells with a variety of hepatic cellular toxicity and carcinomas. The data in table 2 showed that the HCH- induced enhanced hepatic GGT activity was brought down significantly by exposing the rats to PBW. s Table 2. Effect of bees wax polyphenols and HCII treatments on hepatic antioxidant/detoxify ing enzymes in rats* (n=6) (Table Removed) Mean + SD* Value bearing different superscripts in the same column are significantly different (p @ ΔA/min/mg protein * units/mg protein ** µ moles NADP formed/min/mg protein ***µ moles NADP reduced/min/mg protein # µ moles p-mtroanilide released/min/mg protein It can be concluded from the present study that the significant antioxidant activity of the phenols extracted from bees wax could scavenge the superoxide and hydroxyl radicals generated by HCH administration and thus protect the liver from oxidative stress derived toxicity. It is to be noted that the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention, which is further set forth under the following claims:- WE CLAIM; 1. A process for the extraction of polyphenols from bees wax which comprises in the steps of: i) agitating wax for 20 to 24 hours in a polar organic solvent to obtain a slurry, ii) filtering the slurry through filler paper which retains coarse and gelatinous precipitates, iii) respectively washing the paper, iv) collecting the washings and pooled for precipitating the phenols, v) bringing the phenols to room temperature and filtering, vi) the polar solvents in which the polyphenols are dissolved being removed under reduced pressure at a temperature of 5 to 60°C. 2. A process as claimed in claim 1, wherein the ratio of wax to solvent is 1:2. 3. A process as claimed in claim 1 wherein the filtration is carried out on a wet non-fatty porous medium fast ash less filler paper.

Documents:

1795-DEL-2007-Abstract-(18-11-2011).pdf

1795-del-2007-abstract.pdf

1795-DEL-2007-Claims-(18-11-2011).pdf

1795-del-2007-claims.pdf

1795-DEL-2007-Correspondence Others-(18-11-2011).pdf

1795-del-2007-correspondence others-(30-04-2008).pdf

1795-del-2007-correspondence-others.pdf

1795-DEL-2007-Description (Complete)-(18-11-2011).pdf

1795-del-2007-description (complete).pdf

1795-del-2007-form-1.pdf

1795-del-2007-form-18-(30-04-2008).pdf

1795-del-2007-form-2.pdf

1795-DEL-2007-GPA-(18-11-2011).pdf