Process for preparation of spermicidal agent

Abstract: A process for preparation of spermicidal agent from neem oil which comprises in the step of precipitation in the presence of an aliphatic hydrocarbon solvent. The filtrate is subjected to a step of enrichment in the presence of a polar extracant to obtain a fraction. The extractant is distilled to obtain a concentrated extract enriched with spermicidal agent.

Description: FIELD OF INVENTION: This invention relates to a process for the preparation of a spermicidal agent from neem oil. Though reference is made to a spermicidal agent, it is to be understood that such an agent also contains an antifertility agent. PRIOR ART It is generally known that antifertility activity can be effected by three methods. One such method is that of a spermicidal activity by a spermicidal agent. Sperms from the male organism are killed or prevented from reaching the uterus of a female organism so as to prevent any fertilization. Yet another method is that the sperms are allowed to reach the uterus of a female organism. Such sperms are fertilized, and the fertilized ovum starts developing, and that the developing embryo travels to the wall of the uterus of a female organism and gets implanted. In such a method the implantation is prevented. The third method is to allow the implantation and the uterine milieu is altered and embryo separated and aborted. The present invention relates to a spermicidal agent so as to prevent a fertilisation. Neem oil is known to have spermicidal activity, but its shelf life is short, and the activity too low to he of practical use. thus, it is known to subject neem oil to the step of steam distillation to obtain a spermicidal agent. A disadvantage is that the process is time consuming, and steam distillation leads to thermal decomposition of the active compounds (Riar, SS et al; 1990. Volatile Fraction of neem oil as a spermicide. Contraception 42: 479-487). Australian patent no. 9468667A disclose a method of blocking fertility in a male mammal by injecting neem oil or an active compound into the vas deferens of the male. US patent no. 5196197 discloses an antifertility agent comprising neem oil and a reetha extract. S. Garg et. al: Comparison of Extraction procedures on The Immunocontraceptive Activity of Neem Seed Extracts: Journal of Ethnopharmacology, Vol 44, N°2 Oct. 1994 p. 87-92, discloses that neem oil has been reported to possess spermicidal activity and suggests several different methods of processing neem seeds and neem oil. EP-A-0 617 119 discloses a method of solvent extracting neem seeds to obtain extracts having pesticidal activity. OBJECTS OF THE INVENTION An object of this invention is to propose a process for the preparation of a spermicidal agent from neem oil and extractives. Another objects of this invention is to propose a process for the preparation of a spermicidal agent from neem oil which obviates the disadvantages associated with those of the prior art. Yet another object of this invention is to propose a process for the prepartion of a spermicidal agent from neem oil which has the required level of activity. According to this invention there is provided a process for the preparation of a spermicidal agent from neem oil obtained from neem seeds comprising in the steps of subjecting neem oil to the step of precipitation in the presence of an aliphatic hydrocarbon solvent such as n-hexane to obtain a precipitate and a filtrate, subjecting the filtrate to the step of enrichment in the presence of a polar extractant, such as a mixture of alcohol in water, to obtain a fraction comprising said extractants, and distilling the polar extractant to obtain a concentrated extract enriched with spermicidal agent. In accordance with this invention, neem oil is mixed with a solvent having a boiling point of upto 120'C. Perferably, the solvent comprises an aliphatic hydrocarbon such as n-hexane. The ratio of solvent to neem oil is 0.5:1 to 3:1. The solvent added to neem oil can be of light, middle or heavy fraction. Though all three fractions may be employed in the process of the present invention, it is preferred that the middle fraction may be employed as the loss is less and the solvent can be recycled. If the light fraction is used the boiling point is less than 60'C and, consequentially, the cost is more in comparison to that of the middle fraction. The heavy fraction has a boiling point of 110 to 120'C and, hence, greater energy is required for recovery of the solvent. It is generally known that neem oil consists of glycerides, bitter and odoriferous constituents. Certain of such odiferous constituents that can be identified are 2-methyl-pentanal, ethyl propyldisulfide, 3, 4-dimethylthiophene, Di-n-propyldisulfide, 2-phenylethanol, 1,2,3-trithracycle, hexane (C 3 H 6 S 3 ), 5-methyl-2-furturylfuran, trithiacyclononane, longifolene, (7) 8, 9,10 (111)-tetrahydron, naphtho (2,1-B)-thia, naphthene, 2-methyldecalm, nona-2, 4-dienal, n-Propyl-cis-1-propenyl tetrasulfide, n-propyl-trans-1-propenyl tetrasulfide, 2-Undecanone, 1,2-Bis (isopropyl-mercapto)ethane, Hexahydrofarnesol, methylpalmitate, ethylpalmitate, palmitic acid, farnesylacetate, methyl stearate, stearic acid, and ethyloleate. The process of the present invention broadly consists in the step of precipitation and enrichment. As described hereinabove, the step of solvent partition consists in adding an aliphatic hydro-carbon solvent to neem oil in the ratio of 1:1. The addition of solvent to neem oil provides a miscible solution consisting of glycerides, odoriferous and certain of the bitter constituents. Such bitter constituents are removed by the step of precipitation, the remainder of the bitter constituents remaining in the solution. The step of precipitation is carried out at room temperature. However, lower temperatures are preferred as this enhances the number of bitter constituents that precipitate from the solution. Thus, if the step of precipitation is effected at 10°C, it is estimated that in as much as 50% of the bitter constituents are removed by precipitation. Preferably, the step of precipitation is carried out at a temperature of 0 to 15'C. The step of precipitation is carried out for a period of 15 to 60 minutes and preferably from 15 to 30 minutes. After removal of the precipitate, the filterate is subjected to the step of enrichment. For this purpose, the ratio of hydrocarbon solvent to the neem oil residue, after precipitation, is adjusted to a level so that the residue contains 0 to 75% of hydrocarbon solvent. The filtrate is either distilled off to remove excess hydrocarbon solvent or fresh excess hydrocarbon solvent is added to obtain the aforesaid condition. A polar extractant such as ethanol or methanol each containing 0 to 10% water is added to the filtrate. Such a polar extractant is added to the filterate in the ratio of 1:1 to 1:5 and preferably 1:3, and shaken to effect enrichment. The step of enrichment is carried out at a temperature of 10 to 40'C and, for example, in a separating vessel. Two layers are produced in the separating vessel. The upper layer is hydrocarbon, layer and containing most of the glycerides, which are not required in the sepermicidal agent of the present invention. The lower layer is an alcohol layer containing the odoriferous constituents and the remaining bitter constituents. The lower layer is removed and fresh polar extractant is added to the upper layer, and which step may be further repeated several times. Such a step of adding a polar solvent to the upper layer is repeated as a certain amount of odoriferous components are still retained in the upper layer. Thus, treatment with polar extractant allows a further separation of the odoriferous constituents from the upper layer. Alternatively, and in the instance where such a step of enrichment is carried out in a counter flow column, the extractant layer is recycled several times. The lower layer obtained from the step of enrichment is then subjected to the step of distillation for removal of extractant under atmospheric pressure or moderate vacuum to obtain a concentrated extract. Reference made hereinabove to a polar extractant is intended to imply a polar solvent containing 0 to 10% water. EXAMPLE-1 To 50g of neem oil, 100 ml of n-pentane was added. The liquid was swirled and the mixture chilled to 10 to 15'C for 30 minutes. The solution supernatant pentane was decanted and introduced into a separating funnel. The precipitate was dissolved in 10 ml of acetone and transferred to another flask and, wherein, acetone was distilled to leave approx. 2 g of the precipitate. 100 ml of aqueous methanol was added to the separating funnel containing the pentane solution of neem oli. After shaking, the lower layer containing aqueous methanol was drained. Such a step of separation was repeated thrice and the alcohol solution thus obtained were pooled together and passed through a bed containing 50 g. of anhydrous sodium sulphate in a funnel. The methanol was distilled off under reduced pressure of for approximately 7.99 x 10 4 Pa (600 mm Hg) to leave behind a dark brown syrupy liquid. A spermicidal activity was found to be associated with this fraction. Total Spermicidal Power 1ml of the emulsion of the spermicidal agent in saline at a concentration of 25mg/ml was able to kill the spermatozoa in 0.8ml of human semen when added in 0.1ml increment). The total spermicidal activity estimated to be 100mg of the agent for every 3ml of monkey/human semen. Spermicidal Activity of the agent When the agent was instilled into the uterine horn it did not prevent pregnancy showing that it is devoid of any anti-implantation or abortifacient activity. (Table 2). Effect of intrauterine instillation of the agent in rats NO. NO. of implants on control horn No. of implants on exptl. horn 1. 6 3 2. 6 4 3. 5 0 4. 7 4 5. 4 3 6. 7 3 Rabbit studies: The precoital application of the agent impregnated in gelatin jelly in case of rabbits showed the spermicidal effect is dose dependent (Table -3). 15mg of the agent per animal was able to inhibit pregnancy in all the animals completely, whereas 10mg of the agent per animal was able to cause 67% inhibition and 5mg was without any effect of fertility. The results show that the agent by virtue of spermicidal activity is able to inhibit pregnancy in rabbit when applied before coitus. Precoital spermicidal effect of the agent in rabbits Dose (mg) No.of animals No.of animals with implants Spermicidal effect(%) 5 6 6 0 10 6 2 67 15 6 - 100 20 6 - 100 Monkey studies: The intravaginal application of 150mg of the agent was able to prevent pregnancy in monkeys when applied precoitally in the form of gelatin jelly and mated immediately after the application of the drug. Their menstrual cycle was not affected and the estradiol and progesterone levels did not show any change when compared with the control. The studies were repeated for three cycles in all the animals and there was no pregnancy. But in the control group where 1ml of sterile jelly was applied, 60% of the females became pregnant which is the normal fertility rate in monkeys in captivity. In-vivo Precoital spermicidal actitivity of the agent: In this study it was found that 150mg of the agent in 1ml of jelly is able to kill all the sperms after mating (Table-4). This difference in the spermicidal activity from that of total spermicidal power mentioned earlier may be due to the presence of vaginal fluid which might have diluted the concentration of the agent and also due to the uneven distribution of the drug in the vagina. The above study also shows that effect of the agent is dose dependent, approximately 150mg of the agent is sufficient to kill all the spermatozoa after mating in monkey or human subjects. In vivo spermicidal activity of the agent in monkeys: Monkey No. Dose of drug (mg/ml) 1 2 3 4 5 6 50 +++ ++ ++ +++ +++ +++ 100 - - ++ ++ +++ ++ 125 - - - + + - 150 - - - - - - Key - No motility + <25%motile ++ 25 to 50% motile +++ 50 to 80%motile The agent was impregnated in 1ml of gelatin gelly and applied near the cervix.

Claims: A process for the preparation of a spermicidal agent from neem oil obtained from neem seeds comprising the steps of subjecting neem oil to precipitation in the presence of an aliphatic hydrocarbon solvent such as n-hexane to obtain a precipitate and a filtrate, subjecting the filtrate to the step of enrichment in the presence of a polar extractant, such as an aqueous alcohol solvent, to obtain a fraction comprising of said extractant and distilling the polar extractant to obtain a concentrated extract enriched with spermicidal agent. A process as claimed in claim 1 wherein the ratio of the aliphatic hydrocarbon to neem oil is 0.5:1 to 3:1 in the step of precipitation. A process as claimed in claim 2 wherein said ratio is 1:1. A process as claimed in claims 2 or 3 wherein the step of precipitation is carried out at 0 to 15'C. A process as claimed in claim 4 wherein the step of precipitation is carried out for a period of 15 to 60 minutes. A process as claimed in claim 1 wherein said extractant is selected from an alcohol such as ethanol or methanol present in 0 to 10% of water. A process as claimed in claim 6 wherein the ratio of alcohol to water is 9:1. A process as claimed in claim 5 wherein the ratio of extractant to filtrate 1:1 to 1:5 and preferably 1:3. A process as claimed in claim 8 wherein the step of enrichment is carried out at a temperature of 10 to 40'C. A process as claimed in claim 1 wherein the ratio of hydrocarbon solvant to neem oil residue left after precipitation is such that the residue contains 0 to 75% of hydrocarbon solvent.

Inventors: DEVAKUMAR CHAKRA, ILAVAZHAGAN GOVINDASWAMY, DEVAKUMAR, CHAKRA, ILAVAZHAGAN, GOVINDASWAMY
Applicants: NAT RES DEV CORPORATIONOF INDI, NATIONAL RESEARCH DEVELOPMENT CORPORATIONOF INDIA
Application year: 1998
Main classification: A61K35
IPC codes: A61K36, A01N65, A61K31