The present invention relates to improved method for preparing Catechin (98%) isolated from Acacia catechu and more particularly the present invention relates to improved method for preparing Catechin (98%) isolated from Acacia catechu having antioxidant activity.

BACKGROUND OF THE INVENTION

Acacia catechu belongs to the family Leguminosae, and is commonly known as black catechu or cutch. The ancient Indian medicine -Ayurveda recognizes the uses of Acacia catechu in treating various conditions. Acacia catechu extract is considered as safe by US FDA and included in GRAS [Generally Recognized As Safe] list. It is officially listed in Ayurvedic pharmacopoeia of India, British Pharmacopoeia and United States Pharmacopoeia.

Acacia Catechin extract have been used in treatment of variety of ailments like fever, leucorrhoea, piles.erysipelas etc.,

The extract enriched in catechins are known to have significant antioxidant and antimicrobial effects (Naik et.al, Phytochemistry,2003,63(1)97-104).

According to Wikipedia Acacia catechu also commonly called Mimosa catechu, is a deciduous, thorny tree which grows up to 15 m (50 ft) in height.

The plant is called khair in Hindi, and kachu in Malay, hence the name was Latinized to "catechu" in Linnaean taxonomy, as the type-species from which the extracts cutch and catechu are derived. Common names for it include catechu, cachou, cutchtree, black cutch, and black catechu.

The genus name (see Acacia for further information) derives from the Greek word for its characteristic thorns, aklc (akis, thorn). The Acacia catechu is found in Asia, China, India and the Indian Ocean area.

Through derivatives of the flavanols in its extracts, the species has lent its name to the important catechins, catechols and catecholamines of chemistry and biology. The tree's seeds are a good source of protein. Kattha (catechu), an extract of its heartwood, is used as an ingredient to give red color and typical flavor to paan. Paan, from the word pan in Hindi

These known sources comprising tree of Acacia catechu are is diminishing day by day and care should be taken on the use of the same which is mainly used for treatment of variety of ailments like fever, leucorrhoea, piles.erysipelas etc

DETAILED DESCRIPTION OF THE INVENTION

It is known in the art Cell Proliferation Assays on Catechins, Antimicrobial activity of Acacia catechu and the Antioxidant studies of Total Catechin. All these studies are known and are carried out using pure catechins isolated (98%).

The applicants believe that this is the first report wherein the antimicrobial activity, antioxidant studies, Tyrosine activity and the cell proliferation assays were carried out by pure catechins (98%) isolated from Acacia catechu.

All the earlier known reported experiments were conducted on crude extracts of the species. The applications in the treatments and the related experiments were restricted to the crude extract of Acacia catechin.

The thus reported methods are more focused on pure catechin. This is to minimize the concerns arising from interfering impurities and other major & minor compounds found in crude Acacia catechu extracts. Also, the studies on pure catechin emphasizes further that the exhibited activity is purely related to the catechin and not due to plausible interference of any other active principle associated in the crude extract.

According to present invention there is provided an improved method for preparing Catechin (98%) isolated from Acacia catechu having antioxidant activity which comprises purification and re-purification of raw materials by known methods to obtain an analytically pure sample fit for the biological studies, maximizing the "active content" by diffusion, dissolving crude catechin mixture in DM water and boiling the same, filtering the suspension formed & the contents evaporated, the filtrate thus obtained was further left at RT for suitable hours viz 35-40 hrs, and the first crop of the partially purified mixture was obtained the ethanolic extract after second de¬colorization (using active charcoal) was distilled under vacuum & the residue re-dissolved in hot DM water, the contents were left to stand for overnight or maximum upto 24 hrs and the precipitate filtered and air dried to yield the product which is Catechin (98%).

Further after 2-3 more re-crystallizations from water the melting point of the product matched with the authentic sample.

Still further antioxidant activity profiling for pure catechins (98%) isolated from Acacia catechu was carried out by the use of novel DPPH free radical method and FRAP assay for pure catechins.

Still further the present invention provides for method of providing antioxidant activity comprising the use of catechin (98%) pure isolated from Acacia catechu.

Still further catechin (98%) pure isolated from Acacia catechu is used as a Tyrosinase inhibitor in a medicament.

Still further catechin (98%) pure isolated from Acacia catechu is also used as an antimicrobial agent in the manufacture of a medicament.

According to present invention proliferation assays on catechins 98% obtained from Acacia catechu:

The in vitro cytotoxicity studies; indicate a very healthy cell proliferation. Catechins from Acacia catechu with 98.00 % purity was used throughout the experiments in the present invention. Dermal fibroblast (CRL1904) cell lines were ohtained from American Type Culture Collection (Rockville, MD). These cells were cultured in a humidified atmosphere with 5% CO2 at 37°C which is a standard method. The standard growth medium was prepared according to specific cell lines. Iscove's Dulbecco's minimum essential medium (DMEM) was used for fibroblasts. Media preparation was completed by adding 10% fetal bovine serum (FBS) and 1% 100X Fungizone (penicillin/streptomycin).

Cell Treatment for Proliferation:

After determining the cell count, fibroblast cell lines were re-suspended in 1 % fetal bovine serum (FBS) which was subsequently starved for about 24 hrs. The total cell count for proliferation assay was determined at 2,500 cells/well. The count for cytotoxicity assay was fixed at 20,000 cells/well. This is standard method adopted for determining cell count.

Experiment 1:

An aliquot of 100uL of cell suspension was placed in each well of 96 well plates.

Various concentrations of highly purified total catechin samples were used to treat the cells. In column 1, 100uL of media containing cells were added. In columns 2-12, 100uL of the different concentrations of A. catechu samples were added such that the final concentrations in the range of 0.37 to 200mg/ml_ in row 12 were achieved. Plates were incubated for 24, 48 and 72 hrs. After 72hrs of incubation, 20ml_ of the Cell Titer 96 Solution containing [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and (phenazine ethosulfate; PES) an electron coupling agent with enhanced chemical stability was added.

Assays were conducted by adding a small amount of the Cell Titter 96 reagent solution directly added to culture wells, incubating for 1- 4 hours at 37° C at humidified 5% CO2 atmosphere and then recording absorbance at 490nm with a 96-well plate reader. The quantity of formazan produced was measured at 490nm. Absorbance values being directly proportional to the number of living cells in culture.


Graph.1
(The data obtained from the proliferation assay using highly purified A. catechu on Fibroblast cells is shown in Graph 1) Data presented in this Graph. 1 indicate a strong dose-response relationship with effect of 98% total catechins, in the dose range of 0.38-100mg/mL. The peak proliferation was observed at the 10mg/ml_ dose level.

It was also demonstrated (Figure.1) that different doses of highly purified total catechins induce healthy cell proliferation.

It is noteworthy to mention here that no cytotoxicity was observed in dermal fibroblast cell lines, used in the present investigation. This becomes the first ever report of absence of cytotoxicity with catechin extracts / pure catechins.

We now give below various studies viz Cell Proliferation Assays, Antimicrobial activity of Acacia catechu, Minimum inhibitory concentration (MIC) assays, Antioxidant studies of Total Catechin (98%) ,DPPH free radical scavenging activity, Total phenolic content, Ferric reducing ability of Plasma (FRAP) Assay, Tyrosinase inhibition assay carried out in detail:-

I. Cell Proliferation Assays:-

Figure.1

(Proliferation of Dermal fibroblast cells exposed to Catechin. Cells were seeded in 96 well plates (2, 500 cells/well) This is shown in Figure 1 of the accompanying drawing) II.' Antimicrobial activity of Acacia catechu The highly purified total Catechin samples were subjected to antimicrobial study using the agar well known diffusion method of Murray et al., (Manual of Clinical Microbiology, 6th Edition. ASM Press, Washington. DC 1995, 15-18) and its modified version by Olurinola Laboratory Manual of Pharmaceutical Microbiology (Idu, Abuja, Nigeria 1996; 69-105). Nutrient agar (20 ml) was dispensed into sterile universal bottles followed by cultures (0.2 ml) mixed and inoculated. The contents were gently transferred into sterile petri dishes. After properly sterilizing by flaming a no.3 cup borer (6 mm) three to five uniform cups/wells were bored in each petri dish. A drop of molten nutrient agar was used to seal the base of each cup. The cups/wells were filled with 50ul of total Catechin samples at different concentration ranging from 50-250mg/ml and allowed to diffuse for 45 minutes. The solvents used for reconstituting the extracts were similarly analyzed. The plates were incubated at 37°c for 12hrs. The above procedure was also repeated for fungal assays using media potato dextrose agar instead of nutrient agar. For Fungal assay the incubation temperature and times was 25°C & 48 hrs respectively. The inhibition zones in both the cases were measured with antibiotic zone scale in mm followed by one more duplication. The purified total Catechin sample that exhibited antimicrobial activities were later tested to determine the Minimum inhibitory Concentration (MIC) for bacterial and fungal samples.

Ill Minimum inhibitory concentration (MIC) assays:

Based on the preliminary screening, highly purified total catechin 98% samples have potent antimicrobial activity (Table-1). The Minimum Inhibitory Concentrations (MIC) of the extracts was determined according to the method of Elizabeth et. al., (Asian Jr. of Microbial. Biotech Env Sc 7,2005, No. (2) 209-212). Tween-20 0.5% v/v was used to enhance solubility. A series of two fold dilution of each extract, ranging from 0.2 to 100mg/ml, was prepared. After sterilization, the medium was inoculated with 3ul aliquots of culture containing approximately 105 CFU/ml of each organism. Slant culture incubated for 24 hrs under aseptic condition were transferred into sterile 6 inch diameter petri dishes and allowed to set at room temperatures for about 10 minutes followed by refrigeration (30 minutes).

Using a number 3-cup borer (6mm) diameter properly sterilized by flaming, the contents of the petri dish were bored to form 3-5 uniform cups/wells. A drop of molten nutrient agar was used to seal the base of each cup. Extracts ranging from 0.2 to 100 mg/ml were added to the cups/wells of each petri dish, keeping one well as control. Inhibition of growth in the plates containing test extracts was judged by comparing them with blank control plates. The MICs were determined as the lowest concentration of extracts inhibiting visible growth of each organism on the agar plate. Similarly the MICs of catechu extracts were determined against all other microorganisms. The results are tabulated below (Table -1) Table-1

IV Antioxidant studies of Total Catechin (98%)

Antioxidants are substances or nutrients in our diet which can prevent or slow down the oxidative damage to our body. When our body cells use oxygen, free radicals are generated which can cause damage. Antioxidants act as "free radical scavengers" and hence prevent and repair damage due to over oxidation. Metabolic problems such as heart disease, macular degeneration, diabetes, cancer etc are all to some or large extent caused by oxidative damage. In the present invention, the antioxidant activity profiling has been performed by novel DPPH free radical method and FRAP assay. The details of which are yet another aspect of the invention.

V DPPH free radical scavenging activity:

The antioxidant activity of purified total Catechin samples and the standard was assessed on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-free radical Braca et.al., (J Ethnopharmacol, 2002, 79: 379-381). The diluted working solutions of the test extracts were prepared in ethanol. Ascorbic acid was used as standard in 1-100 ug/ml solution. DPPH was prepared in 0.002% concentration and 1 ml of this solution was diluted with 1 ml of sample and standard solution separately. These solution mixtures were kept in dark for 30 min followed by optical density measurement at 517 nm, using Cecil-Elect Spectrophotometer. Ethanol (1ml) with DPPH solution (0.002%, 1 ml) was used as blank. The optical density was recorded and % inhibition was calculated.

Percent (%) inhibition of DPPH activity = A-B* 100 A
Where A = optical density of the blank and
B = optical density of the sample.
VI Total phenolic content:

Total phenolic content (TPC) of sample was determined using the Folin-Ciocalteu assay reported by Kahkonen et al., (J. Agric. Food Chem., 1999, 47, 3954). Samples (300ul in triplicate) were introduced into test tubes, followed by 1.5 ml of Folin-Ciocalteu's reagent (10 times dilution) and 1.2 ml of sodium carbonate (7.5% w/v). The tubes were allowed to stand for 30 min before being measured for absorbance at 765 nm. TPC was expressed as gallic acid equivalents (GAE) in mg per 100 g.

The calibration equation for gallic acid was y = 0.0111x _ 0.0148 (R2 = 0.9998).

VII Ferric reducing ability of Plasma (FRAP) Assay:

The total antioxidant potential of a sample was determined using the ferric ion reducing ability of plasma i.e., FRAP assay as a measure of antioxidant power Benzie etal., (Anal. Biochem. 1996, 239: 70-76). The assay was based on the reducing power of the compound (antioxidant). The latter forms a blue complex (Fe2+/TPTZ), with an enhanced absorbance at 593nm. The FRAP reagent was prepared by mixing acetate buffer (300uM, pH 3.6), a solution of 10uM TPTZ in 40uM HCI, and 20uM FeCI3 at 10:1:1 (v/v/v). The reagent (300uM) and sample solutions (10uL) were added to each well and mixed thoroughly. The absorbance measurements were calculated at 593nm after 10min. Standard curve was prepared using different concentrations of FeCI3. All solutions were used on the day of preparation. The results were corrected for dilution (e.g. 1000ml) and expressed as ascorbic acid equivalents (mmoles/ml) or FRAP units. All determinations were performed in triplicates.

The antioxidant studies prove that purified Catechin (98%) samples derived from heartwood of Acacia catechu showing powerful antioxidant properties.

VIII Tyrosinase inhibition assay

The color of mammalian skin and hair is determined by a number of factors, the most important of which is the degree and distribution of the melanin pigmentation. Melanin biosynthesis inhibitory compounds are useful not only as skin whitening agents used in cosmetics but also as a remedy for pigmentation disturbance. Tyrosinase (phenol oxidase) is known to be a key enzyme for melanin biosynthesis in plants, microorganisms and mammalian cells. Therefore, many tyrosinase inhibitors have been tested in cosmetics and pharmaceuticals as a way of inhibiting biosynthesis of melanin in epidermal layers Shimuzu etal., (Planta Medica, 2000, 66:11-15; Lida etal., Planta med. 1995, 61:425-428).

Principle of the assay is as under:
The conversion of
Tyrosinase

L-Dopa ► Dopachrome
Which is Spectrophotometrically quantified as dopachrome at475nm. IX Tyrosinase Inhibition Assay:
The active compounds catechin and a control compound (Kojic acid) were tested for their 50% enzyme (tyrosinase) activity inhibition.

Inhibition tests were carried out using aqueous solvents. Saturated stock solutions were prepared at least 48 hours before the tests were run, and were diluted appropriately upon use in order to increase the solubility in the aqueous solvents of the lipophilic test compounds.

Tests were carried out according to conventional procedure as follows. That is, 0.01 to 0.02 ml of a mushroom tyrosinase solution (1,375 U/ml, Sigma Chemical Co.), 0.98 to 0.99 ml of a 0.07M phosphate buffer solution (pH 6.8) and 1 ml of a test solution with or without a test compound were mixed. The resulting mixture was incubated at 22° C. for 8 minutes. Then, 1 ml of a 0.03% aqueous solution of l-DOPA (L-3, 4-dihydroxyphenylalanine having a purity of 99%, Sigma Chemical Co.) was added. After incubating at 22°C for 2 minutes, the amount of dopachrome in the reaction mixture was measured at 475 nm. The change in absorbance with or without different test samples was monitored every 30 seconds for the next 2.5 minutes using a Beckman DU-64 spectrophotometer (Beckman, Palo Alto, Calif.).

The % inhibition was calculated as follows:

Absorbance (control) -Absorbance (test)
% inhibition = X100
Absorbance (control)
Results:

Table 02: % Inhibition data of the tested sample in Tyrosinase inhibition assay We shall now describe the invention with reference to accompanying experiments which are being carried out given by way of illustrations but does not restrict the scope of invention:

Example 1 (given in details):-Extraction and purification methodology

General guidelines regarding the purification protocols were adopted from traditional methods [1], further re-purification methods were employed for obtaining an analytically pure sample fit for the biological studies.

A three necked 20L glass vessel equipped with a stirrer (about 50 rpm) was loaded with 10 L of DM-water and 1.0 Kg of dry chips of Acacia catechu, care was taken to immerse the chips in water.

Following 4-5 hrs of reflux under constant stirring the contents were left to soak for further period of 40 hrs, thereby maximizing the "active content" diffusion. The contents of the flask were subsequently filtered and treated with active charcoal to eliminate some brown colored impurities. The water layer was vacuum distilled (40°C, 50 mm Hg) to yield about 200 g of a crude free flowing powder.

The crude catechin mixture (200 g) was further dissolved in DM water (1L) and boiled. The fine suspension formed was filtered & the contents evaporated to about 500 ml.

The filtrate thus obtained was further left at RT for next 35-40 hrs, and the first crop of the partially purified mixture was obtained. After filtration the product cake was again re-dissolved in ethanol & filtered. The ethanolic extract after second de-colorization (using active charcoal) was distilled under vacuum & the residue re-dissolved in hot DM water (500 ml). The contents were left to stand for 24 hrs and the precipitate filtered and air dried to yield about 40.0 g of the product (mp 97°C).(HPLC purity 80%)

After 2-3 more re-crystallizations from water the melting point of the product matched with the authentic sample.

IR spectra: (KBr) showed band at 2363 (broad), 1610, 1516, 1472, 1373, 1283, 1144.8, 1111.2,1025, 974 cm-1.

Mass spectra: Exhibited M+ at 291.1. Other major fragments were obtained at 146.1 and 120. The molecular mass corresponds to 291.1.

UV spectra: Amax [methanol] exhibited maxima at 277nm and 220 nm. 1H-NMR (400 MHz, DMSO-d6): Exhibited peaks at 5TMS 2.69 [H-2, d, J(H-3.2), 3.80[H-1, E), 4.00 (H-1bs), 4.48 (H-1, dd, 5=6) 4.63(H-1; d), 4.73 (H-1, s), 4.85 [H-1, d, J=4.4) 5.71 (H-1,d,J=1.6),5.88 (H-1, d, J=1.6), 6.58(H-1, d, J=1.2) 6.69 (H-1,d, J=6), 6,72 (H-1,d, J=1.2, 6.88 (H-1.S ).

13C-NMR (400 MHz DMSO-d6): Exhibited peaks at 5TMS 27.9 (C-12), 81.1 (C-11), 94.29 (C-4), 94.29 (C-2), 99.29 (C-2), 99.23 (C-13), 114.64 (C-10), 115.28 (C-7), 118.62(C-6), 130.77 (C-5), 144.58 (C-8), 144.97 (C-9), 155.50(C-3), 155.94 (C-1), 156.6 (C-14).

We Claim:

1. An improved method for preparing Catechin (98%) isolated from Acacia catechu having antioxidant activity which comprises purification and re-purification of raw materials by known methods to obtain an analytically pure sample fit for the biological studies, maximizing the "active content" by diffusion, dissolving crude catechin mixture in DM water and boiling the same, filtering the suspension formed & the contents evaporated, the filtrate thus obtained was further left at RT for suitable hours viz 35-40 hrs, and the first crop of the partially purified mixture was obtained the ethanolic extract after second de-colorization (using active charcoal) was distilled under vacuum & the residue re-dissolved in hot DM water, the contents were left to stand for overnight or maximum upto 24 hrs and the precipitate filtered and air dried to yield the product which is Catechin (98%).

2. Process as claimed in claim 1 wherein after 2-3 more re-crystallizations from water, the melting point of the product matched with the authentic sample.

3. Process as claimed in claims 1 or 2 wherein for antioxidant activity, profiling for pure catechins (98%) isolated from Acacia catechu was carried out by the use of novel DPPH free radical method and FRAP assay for pure catechins.

4. Process as claimed in claim 3 wherein method of providing antimicrobial or antioxidant activity comprising the use of catechin (98 %) pure isolated from Acacia catechu.

5. Process as claimed in claim 4 wherein for method of providing antioxidant activity comprising the use of catechin (98%) pure isolated from Acacia catechu.

6. Process as claimed in claims 4 or 5 wherein catechin (98%) pure isolated from Acacia catechu is used as a Tyrosinase inhibitor in a medicament.

7. Process as claimed in claim 6 wherein catechin (98%) pure isolated from Acacia catechu is used as an antimicrobial agent in the manufacture of a medicament.