Title of Invention

ISOLATION OF SAPONIN NEW COMPOUND FROM ACACIA CONCINNA DC

Abstract

The invention relates to the antifertility activity of acetone fraction of aqueous extract of Acacia concinna. The LD50 and ED50 were observed 500 mg/kg and 50 mg/kg respectively. Female albino rats were orally fed with fraction (50 mg/kg) for 21 days throughout the study. Rats were arrested in diestrus stage and observed decrease in body weight and organs weight. Reduction in pregnancy and in implantation were observed in rats. Abortifacient activity was not observed with the treatment of fraction. Biochemical, hormonal, proteins present and pH in intrauterine fluid and histological studies were carried out. The levels of serum cholesterol and triglycerides decrease significantly. Rats showed depletion in follicle stimulating hormone, leutinizing hormone and estrogen levels. Antithyroid study showed the fraction was not similar to goiterogenic compounds. Proteins, which required for implantation were found to be absent. Decrease in the pH value of intrauterine fluid, explain the inactivity of sperms. Histological architecture of the uterus showed regressed endometrial glands. Ovary showed degeneration of developing follicle. Epithelial cells of the fallopian tube showed degeneration and accumulation in the lumen. Vagina showed marked changes in the epithelial layers. It can be concluded that the acetone fraction of aqueous extract of Acacia concinna is used as herbal contraceptive without any undesirable side effect.

Full Text

FORM 2 THE PATENT ACT 1970 (39 OF 1970) & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) TITLE OF THE INVENTION Isolation of saponin new compound from Acacia concinna DC. APPLICANT(S) Name Nationality Address Dr. Duragkar Nandakishore Jeevanrao Indian Plot No.45, Shankar Nagar, Nagpur-440 010 Dr. Gore Shubhangi Indian 7, New Sneha Nagar Near Little Flower School Nagpur-440 015 Katolkar Parimal Pradipkumar Indian Plot No.77, Kirti Nagar, Trimurty Square, New Nandanwan, Nagpur - 440009 1. PREAMBLE TO HE DESCRIPTION COMPLETE The following specification particularly describes the invention and the manner in which it is to be performed. 2. DESCRIPTION (Description shall start from next page.) 3 0 MAY 2008 DESCRIPTION Field of the invention The invention relates to the antifertility activity of Acacia concinna. Antifertility activity of Acacia concinna aimed at interception in the process of ovulation, fertilization and implantation through strategic approaches. Background of the invention ' Plant products have attracted the attention of many scientists as a primary source of naturally occurring fertility regulating agent because of their no side effects. It seems that the development of new contraceptive method for them and they may find a ready acceptance in the village population. WHO is taking an official interest in the indigenous systems of medicines, particularly plant remedies. This is because about 80% of the world population used herbal medicines and by proper development of such systems the organizations aim of making health care available to all is facilitated. Persual of literature indicates that several plant species are considered for » antifertility activity. {(1) Fransworth, N.R., Bingel, A.S., Cordell, G.A., Crrane, F.A. and Fong, H.H.S., 1975, J.P.Sc, 64(4), 535-98. (2) Fransworth, N.R., Bingel, A.S., Cordell, G.A., Crrane, F.A.a nd Fong, H.H.S., 1975, J.P.Sc, 64(5), 717-54.} Acacia concinna, DC, Shikakai (Leguminoseae) Pods freed from seeds contain alkaloid saponin 11.2 per cent, malic acid 12.75 per cent, resin 1 per cent, glucose 13.9 per cent, gum and colouring matter 21.5 per cent, crude fibre 22 and ash 3.75 per cent. The decoction of the pods and leaves is useful aperient in bilious affections. The decoction of the pods (one in forty parts of water) is used as hair wash in lieu of soap; it promotes growth of hair and remove dandruff. The tender leaves soaked into pepper water and ground up with salt, tamarind and chillies form an excellent chutney, useful in 2 bilious affections such as jaundice etc. The infusion of the leaves is useful in checking malarias fevers; it also prevents flatulence, as it acts as a mild laxative, the pods ground up and formed into an ointment make a good application in skin diseases. Object of the invention It is an object of the invention to provide herbal drug for antifertility activity without any (neurohormonal) side effects. Details of the Invention Experimental Herbal raw material used for the invention was Acacia concinna. The material was identified with the help of Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur. The part of plant used for the activity was legume. The legumes were crushed into coarse powder and charged into Soxhlet apparatus for extraction. The coarse powder was defatted with petroleum ether (60-80°C) to remove primary metabolites, fats and oil fractions. After extraction with petroleum ether, the materials were dried at loom temperature to remove the traces of petroleum ether. Again, the defatted material was macerated with water for 15 days. The water were evaporated and semisolid extract were used for the process of fractionation. The acetone fraction from semisolid aqueous extract were used for the further invention related to the antifertility activity. The acetone fraction used for study give the positive tests for saponins viz, foam test and haemolysis test. The LD50 and ED50 of acetone fraction were determined. Anti-implantation activity with various doses of acetone fraction of Acacia concinna aqueous extract, were determined to decide the dose throughout the study on female albino rats. Control group received water and food ad libitum. Each group consists often animals. After determination of LD50 and ED50 the tablets were prepared for further study. Reduction in pregnancy in female albino rats were also determined with acetone fraction. On the basis of ED50 and reduction in pregnancy, the dose of acetone fraction of aqueous extract of Acacia concinna was selected 50 mg/kg throughout the further study 3 and treatment was carried out upto 21 days. Effect of acetone fraction treatment on body weight and organ weight were determined. Effect of acetone fraction on oestrus cycle of female albino rats was determined. Early abortifacient activity of acetone fraction was determined. Acetone fraction was evaluated for biochemical parameters. Effect of acetone fraction of Acacia concinna on blood glucose of rats were determined. Effect of acetone » fraction on acid phosphate and on alkaline phosphatase of rats were determined. Cholesterol and triglycerides levels were also determined using acetone fraction of Acacia concinna in rats. Hormonal studies were also determined. Acetone fraction of Acacia concinna were used to evaluate the effect ton serum hormonal changes (oestrogen, progesterone, follicle stimulating hormone, leutinizing hormone etc) in treated female albino rats. These hormones induces regressive and degenerative changes in oriduct, uterus and vagina. Effect on T3 (Triiodothyroxin), T4 (Thyroxin) and TSH (Thyroid stimulating hormone) of acetone fraction of aqueous extracts o Acacia concinna were also evaluated. These study showed that the active compounds is not similar to the goiterogenic » compounds. Effect of acetone fraction of aqueous extract of Acacia concinna were evaluated for protein studies in inira-uterine fluid of the rats. Effect of acetone fraction on protein bands of uterine luminal fluid, which induces preimplantationary loss and abortion in rats were evaluated. Effect of acetone fraction on concentration of proteins, phosphatases, dehydrogenases and pH of luminal fluid of treated rats were evaluated. In histological studies effect of acetone fraction on the uterus, ovary, vagina, fallopian tube and liver of the rats were studied. Results and Discussion Acetone fraction of aqueous extracts of Acacia concinna treated rats showed lethal effect after administration with 500 mg/kg body weight and effective anti- 4 implantation activity found with 50 mg/kg body weight. Table no. 1 shows effect of acetone fraction of aqueous extract of Acacia concinna legumes on reduction of pregnancy in rats. Maximum present reduction or inhibition of implantation on born litters was observed in 50 mg/kg b.w. After treatment with acetone fraction, weights were reduced upto 21 days. The results are shown in table 2. Maximum reduction was observed upto 6.92%. Organ weights were recorded in all experimental as well as control rats and results are shown in Table 3. The weight of the uterus, ovary, vagina, pituitary gland and adrenal gland decreased significantly in acetone fraction of aqueous extract of Acacia concinna treated rats after 21 days. The diestrus stage is prolonged with the fraction. (Table 4) (Photo 1,2, 3 and 4). Vaginal bleeding was not recorded with acetone fraction of aqueous extracts of Acacia concinna. In rats, abortifaciertf activity was not observed in the fraction of extracts of plant. The results of biochemical parameters are shown in Table 5. The acetone fraction of aqueous extract of Acacia concinna exhibited significance hypoglycemia after 21 days of the treatment. The level of acid phosphatase was decreased significantly with acetone fraction of aqueous extract of Acacia concinna. There was no significant change in the level of alkaline phosphatase with the treatment of same. The levels of serum cholesterol and triglycerides decreased significantly with the treatment of acetone fraction of aqueous extract of Acacia concinna. Hormonal changes were observed in acetone fraction of aqueous extract of Acacia concinna treated rats showed depletion in follicle stimulating hormone, leutinizing hormone and oestrogen levels and it was found to be elevated in progesterone and prolactine. The results are showed in Table 6. Antithyroid effect (Table 7) of drug augment antifertility action. In the present study the T3, T4 and TSH levels were appeared to be normal as compared to control. Shortly it can be said that the action of fraction is not similar to goiterogenic compounds. 5 1 Proteins which are required for implantation, were found to be absent in rats treated with acetone fraction of aqueous extract of Acacia concinna. Treatment of rats with fraction resulted in preimplantation loss, which was indicated by the absence 13000, 19000, 26000 and 75000 Da (Table 8) molecular weight proteins, play an important role in the implantation. (Photo 5). Decrease in the pH value (Table, 9) of intrauterine fluid with the acetone fraction of aqueous extract of Acacia concinna. The pH changes play an important role in activity and inactivity of sperms. The decrease of the pH alone in the treated animals could explain the inactivity of sperms. Histological architecture of the uterus showed regressed endometrial glands and when compared with the control (Photo 6) animal structure of the uteri of the experimental animals showed epithelial cells non-secretary and degenerated (Photo 7). Ovary showed degeneration at developing follicles. Degenerated follicular cells were accumulated in the antral fluid. The development of the Grafian follicles was arrested which was resulted into the distortion of follicles. (Photo 8 and 9). Epithelial cells of the fallopian tube showed degeneration and accumulation in the lumen (Photo 10 and 11). Vagina showed marked changes in the epithelial layers (Photo 12 and 13). The changes observed were similar as observed in the diestrus stage of the rat. Histological changes in liver showed (Photo 14 and 15) no significant changes after the treatment indicated that there might not be any histopathological side effects. 6 Table 1: Effect of acetone fraction of aqueous extract of Acacia concinna (AFAE) on reduction of pregnancy in rats. Sr. No. Extracts Dose mg/kg b.w. No. of rats pregnant/ treated % reduction in pregnancy No. of implants in individual rats No. of rats delivered (No. of pups) 1 Control 10/10 0 13,13,11,9, 6, 8, 9, 9, 8, 5 10(10,13, 11,9,6,8,9, 9, 8, 5) 2 AFAE 10 8/10 , 20 6, 7,6,0, 3, 3,0,3,4,5 10 (6, 7, 6, 0, 3,0,3,4,5) 3 AFAE 25 5/10 50 4, 3,0,0, 2, 0, 5, 0, 0, 3 10(4,3,0,0, 2, 0, 5, 0, 0, 3) 4 AFAE 50 0/10 100 0,0,0,0,0, 0, 0, 0, 0, 0 10 (0,0,0, 0, 0, 0,0, 0, 0, 0) 5 AFAE 100 0/10 100 0, 0, 0, 0, 0, 0,0,0,0,0 10(0,0,0,0, 0, 0,0, 0,0, 0) Table 2: Body weight changes in control as well as experimental female albino rats treated with acetone fraction of aqueous extract of Acacia concinna(AF AE) Sr. No. Duration of treatment Control Tween 80 saline AFAE 50 mg/kg 1 Initial weight 254 ± 9.06 260 ± 8.40 2 7 Days 256 ± 9.00 (+ 0.79) 253 ± 7.70* (-2.69) 3 14 Days 260 ±10.96 (+2.31) 248 ± 8.90** (-4.61) 4 21 Days 266 ±10.42 (+4.72) 242 ± 8.42 (-6.92)** Values are mean ± SEM often animals per group **p Figures in parenthesis indicate percent change over control 7 Table 3: Change in organ weight of female rats administered with acetone fraction of aqueous extract of Acacia concinna (AFAE) Sr. No. Organ Control / Experimental Weights 1 Uterus (mg) Control 2147 ±8.34 AFAE (50 mg/kg) 1282 ±8.56** (-40.97) 2 Vagina (mg) Control 300.17 ±0.74 AFAE (50 mg/kg) 284.40 ±0.47** (-5.25) 3 Ovary (mg) Control 341.57 ±0.17 AFAE (60 mg/kg) 320.17 ±0.78** (-6.26) 4 Adrenal gland (mg) Control 100.17 ±0.70 AFAE (50 mg/kg) 94.16 ±0.13* (-5.99) 5 Pituitary gland (mg) Control 2.58 ±0.34 AFAE (50 mg/kg) 1.92 ±0.16* (-25.58) 6 Kidney (mg) Control 697.47 ±6.12 AFAE (50 mg/kg) 798.56 ± 6.76* (+14.49) 7 Liver (g) Control 12.74 ±0.70 AFAE (50 mg/kg) 13.96 ±0.78* (+8.73) Values are mean ± SEM of ten animals per group refers to the average of combined weight of the right and left organ. **p Figures in parenthesis indicate percent change over control. Table 4: Effect of acetone fraction of aqueous extract of Acacia concinna on estrus cycle Sr. No. Acetone fraction of aqueous extract of Acacia concinna treatment . 1 ; 1 Stages of estrus cycle observed on post treatment days 1 3 6 12 15 18 21 1 Control E M E LE P M E 2 50 mg/kg E M E LE D D D PHProestrus, E=Estrus, LE=Late estrus, M=Metaestrus, D=Diestrus 8 Table 5: Effect of acetone fraction of aqueous extract of Acacia concinna (AFAE)on biochemical levels of female albino rats. Sr. No. Biochemical parameters Control / Experimental Levels 1 Cholesterol (mg/l00ml) Control 133.00 ±5.972 AFAE ' (50 mg/kg) 58.10 ±3.590*** 2 Triglycerides (mg/l00ml) Control 106.00 ±6.481 AFAE (50 mg/kg) 58.50 ±3.167*** 3 Blood glucose (mg/l00ml) Control 87.00 ±2.241 AFAE (50 mg/kg) 64.3 ±1.350*** 4 Acid phosphatase (GG units/100 ml) Control 7.44 ± 0.2544 AFAE (50 mg/kg) 5.20 ±0.2422 5 Alkaline phosphatase (KA units) Control 13.1 ±0.7951 AFAE (50 mg/kg) 11.5 ±0.9339 Values are mean ± SEM often animals per group. ***p Table 6: Effect of acetone fraction of aqueous extract of Acacia concinna on hormonal levels of female albino rats. Sr. No. Hormonal parameters Control/ Experimental Levels 1 Follicle stimulating hormone (mlU/ml) Control 4.96 ± 0.2802 AFAE (50 mg/kg) 3.49 ±0.1326 2 Leutinizing hormone (mlU/ml) Control ' 4.908 ±0.1242 AFAE (50 mg/kg) 3.134± 0.1506 3 Estrogen (pg/ml) Control 37.992 ±1.069 AFAE (50 mg/kg) 19.852 ±0.9053 4 Progesterone (ng/ml) Control 6.49 ± 0.4494 AFAE (50 mg/kg) 8.953 ±0.1843 5 Prolactine (ng/ml) Control 3.914 ±0.1431 AFAE (50 mg/kg) 8.552 ±0.2834 Values are mean ± SEM of ten animals per group. Table 7: Changes observed in T3 (Triiodothyroxine), T4 (Thyroxine) and TSH (Thyroid stimulating hormone) with acetone fraction of aqueous extract of Acacia concinna (AFAE) Sr. No. Hormones Control / Experimental Levels 1 Triiodothyroxine (ng/dl) Control 89.41 ± 3.023 AFAE , (50 mg/kg) 105.8 ±2.417 2 Thyroxine Control 7.731 ±0.2222 AFAE (50 mg/kg) 6.553 ±0.2899 3 Thyroid stimulating hormone µIU/ml) Control ' 1.117-± 0.1165 AFAE (50 mg/kg) 1.61 ±0.2395 Values are mean ± SEM often animals per group. Table 8: Effect of acetone fraction of aqueous extract of Acacia concinna (AFAE) on proteins of uterine luminal fluid, which induced pre-implantationary loss and abortion in rats. Sr. No. Treatment Molecular weight of proteins Preimplantation loss in % Abortions in % 1 Control (LMl) 19000 Da (LM2) 26000 Da (LM3) 35000 Da (LM4) 44000 Da (LM5) 65000 Da (HMl) 75000 Da 0% 0% 2 AFAE (50 mg/kg) (LMl) 55000 Da (HMl) 91000 Da 100% 100% LM-Low molecular weight, HM-High molecular weight. 10 Table 9: Effect of acetone fraction of aqueous extracts of Acacia concinna (AFAE) on concentration of proteins, phosphatases, dehydrogenases and pH of luminal fluid of the rats. Sr. No. Control/ Experi¬mental Proteins (µg/ml) Acid phosphatase (U/L) Alkaline phosphatase (U/L) Lactate dehydragenase (U/L) pH observed 1 Control 578.2 ± 5.89 6.3 ± 3.43 86.33 ± 3.54 134.15 ±7.45 8.4 ±0.78 2 AFAE (50 mg/kg) 550.7±4.34* (-4-75) 4.00 ±3.43** (-37.19) 89.45 ± 4.85 (+ 4.02) 129.67 ±7.89* (-3.21) 6.2 ±0.34* (-26.19) Values are mean ± SEM often animals per group **p i / WE CLAIM 1. A process of isolation of saponin from Acacia concinna, comprising a. The legumes of Acacia concinna are crushed into coarse powder and charged into soxhlet apparatus for extraction for defatting with petroleum ether (60-80°C ) to remove the primary metabolites, fats and oil fractions, b. After extraction with petroleum ether, the coarse powder are dried at room temperature to remove the traces of petroleum ether, c. The defatted material are macerated with water for 15 days, the water is evaporated and semisolid extract are used for the process of fractionation with acetone at a temperature in the range of 20-30°C, d. Testing of the acetone fraction obtained from semisolid aqueous extract for saponins viz; foam test and haemolysis test, e. From the acetone fraction of semisolid aqueous extract get pure powder of saponins by air drying below 50 C. 2. The process has claimed in claim 1, wherein the water used is distilled, deionised and acetone and petroleum ether is also distilled. 3. The process as in claimed in claim 1, wherein the maceration is carried out for 15 days with occasional shaking in the morning and in the evening, daily. 4. The process as claimed in claim 1, wherein the fractionation is carried out with vigorous shaking with acetone, of aqueous semisolid extract.

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