Shakti N. Upadhyay

IMMUNOMODULATORY EFFECTS OF NEEM (AZADIRACHTA INDICA) OIL

Immunomodulatory effects of neem oil were studied in mice. The animals were treated intraperitoneally (i.p.) with neem oil; control animals received the emulsifying agent with or without peanut oil. Peritoneal lavage, collected on subsequent days, showed a maximum number of leukocytic cells on day 3 following treatment with neem oil; peritoneal macrophages exhibited enhanced phagocytic activity and expression of MHC class-II antigens. Neem oil treatment also induced the production of gamma interferon. Spleen cells of neem oil-treated animals showed a significantly higher lymphocyte proliferative response to in vitro challenge with Con A or tetanus toxoid (TT) than that of the controls. Pre-treatment with neem oil, however, did not augment the anti-TT antibody response. The results of this study indicate that neem oil acts as a non-specific immunostimulant and that it selectively activates the cell-mediated immune (CMI) mechanisms to elicit an enhanced response to subsequent mitogenic or antigenic challenge.

Synergistic spermicidal activity of neem seed extract, reetha saponins and quinine hydrochloride

In order to identify potent spermicidal agents which are free from the side effects of currently available agents, spermicidal activity of purified neem seeds extract (Praneem), reetha saponins and quinine hydrochloride was studied individually and in combination. Sander-Cramer test was used to assess the activity on human sperm. Under the test conditions, minimum effective spermicidal concentrations for Praneem, reetha saponins and quinine hydrochloride were 25%, 0.05% and 0.346%, respectively. At these concentrations, 100% of the sperm were immobilised within 20 seconds. A positive synergistic effect in the spermicidal activity of these components, if used in combination, was observed which implies the use of reduced concentrations of each to bring about the desired action. The selected combination formulated into a suitable dosage form is likely to offer dual benefit of a potent contraceptive and an antimicrobial preparation.

Antifertility effects of neem (Azadirachta indica) oil by single intrauterine administration: a novel method for contraception.

A novel use of neem (Azadirachta indica) oil, a traditional plant product, for long-term and reversible blocking of fertility after a single intrauterine is described. Female Wistar rats of proven fertility were given a single dose (100 μl) of neem oil by intrauterine route; control animals received the same volume of peanut oil. Whereas all control animals became pregnant and delivered normal litters, the rats treated with neem oil remained infertile for variable periods ranging from 107 to 180 days even after repeated matings with males of proven fertility. The block in fertility was, however, reversible as half of the animals regained fertility and delivered normal litters by five months after treatment, without any apparent teratogenic effects. Unilateral administration of neem oil in the uterus blocked pregnancy only on the side of application whereas the contralateral uterine horn treated with peanut oil had normally developing foetuses; no sign of implantation or foetal resorption was noted in the neem-oil-treated horn. The ovaries on both sides had 4—6 corpora lutea indicating no effect of treatment on ovarian functions. The animals treated with neem oil showed a significant leukocytic infiltration in the uterine epithelium between days 3 and 5 post coitum, i. e. during the pre-implantation period. Intrauterine application of neem oil appears to induce a pre-implantation block in fertility; the possible mechanisms of the antifertility action are discussed.

Studies on the contraceptive efficacy of Praneem polyherbal cream.

Praneem polyherbal cream, a spermicidal formulation, has been developed at the National Institute of Immunology, which makes use of Praneem, a purified extract from the dried seeds of an ancient Indian plant Azadirachta indica (Neem), extract from the pericarp of fruits of Sapindus species and quinine hydrochloride. These ingredients have a synergistic spermicidal activity and an optimised formula was derived. The components were made into a water-soluble cream base prepared by using pharmaceutically acceptable base and stabilised by addition of IP grade antioxidant and preservatives. The cream is devoid of irritation and sensitization potential, as seen with standard Draize test on normal and abraded skin of rabbits and by 21-day cumulative skin sensitivity in human volunteers. The formulation was found to be safe under subacute toxicity studies in monkeys. The formulation has shown high contraceptive efficacy in rabbits and in monkeys after intravaginal application. The shelf-life of the cream at room temperature is estimated to be 18 months by accelerated stability studies.

Immunocontraceptive activity guided fractionation and characterization of active constituents of neem (Azadirachta indica) seed extracts

A novel approach for immunocontraception by intervention of local cell mediated immunity in the reproductive system by using single intrauterine application of neem oil has been described earlier. The reversible block in fertility was reported to last for 107-180 days in female Wistar rats (Upadhyay et al., 1990. Antifertility effects of neem oil by single intrauterine administration: A novel method of contraception. Proceedings Of The Royal Society Of London B 242, 175-180) and 7-11 months in monkeys (Upadhyay et al., 1994. Long term contraceptive effects of intrauterine neem treatment (IUNT) in bonnet monkeys: An alternative to intrauterine contraceptive devices. Contraception 49, 161-167). The present study, describes the identification and characterization of the biologically active fraction from neem seeds (Azadirachta indica A. Juss. Family Meliaceae), responsible for the above activity in adult female Wistar rats. Initial studies with the mechanically extracted oil and solvent extracts of neem seeds have revealed that the antifertility activity was present in constituents of low to intermediate polarity. A hexane extract of neem seeds was reported to be biologically active (Garg et al., 1994. Comparison of extraction procedures on the immunocontraceptive activity of neem seed extracts. Journal of Ethnopharmacology 22, 87-92). Subsequently, hexane extract was sequentially fractionated through the last active fraction using various separation techniques and tested for antifertility activity at each step. Preparative HPLC was used for isolating individual components of the active fraction in quantities, sufficient for characterization. An analytical HPLC method was developed for standardization of the fraction. The active fraction was identified to be a mixture of six components, which comprises of saturated, mono and di-unsaturated free fatty acids and their methyl esters. Dose response study was performed with the last active fractions. The antifertility activity with the active fraction was reversible in nature and it was completely active until 5% concentration. There was no systemic toxic effect following the administration of the active fraction. This study, for the first time, proposes an active fraction from neem seeds, responsible for long term and reversible blocking of fertility after a single intrauterine administration with high efficacy.

A Birth Control Vaccine is on the Horizon for Family Planning

Vaccines for control of fertility are likely to have an important impact on family planning methods. They are designed to act by mobilization of an internal physiological process and do not require external medication on a continuous basis. A number of birth control vaccines are at different stages of development, the most advanced being a vaccine inducing antibodies against human chorionic gonadotrophin (hCG). This vaccine consists of a heterospecies dimer (HSD, beta hCG associated with alpha-subunit of ovine luteinizing hormone, beta hCG:alpha oLH) linked to tetanus toxoid (TT) or diphtheria toxoid (DT) as carriers. The vaccine has recently passed an important milestone; it has completed the first leg of phase II efficacy trials. Women of proven fertility leading active sexual life were protected from becoming pregnant at antibody titres > or = 50 ng of hCG bioneutralization capacity per ml. This vaccine has previously been demonstrated to be reversible in its effect. It is free from any notable side-effects on endocrine, cardiovascular and other body functions. Ovulation was not disturbed and menstrual regularity was maintained. A logistic disadvantage of the present vaccine is the requirement for multiple injections. This is expected to be overcome by encapsulation of the requisite doses of the vaccine in biodegradable microspheres, which could be given at a single contact point for sustained antibody titres lasting over a year. A live recombinant vaccine has also been made that elicits high anti-hCG titres in monkeys for nearly 2 years following primary immunization and a booster at 8-9 months.

Long-term contraceptive effects of intrauterine neem treatment (IUNT) in bonnet monkeys: an alternate to intrauterine contraceptive devices (IUCD)

Antifertility effects of intrauterine neem treatment (IUNT) was studied in bonnet monkeys. A single administration of 1 ml of neem oil by an intrauterine insemination catheter blocked fertility for 7 to 12 months. The effect was, however, reversible as all the animals became pregnant subsequently and delivered normal babies. The neem oil treatment had no adverse effect on menstrual cyclicity and ovarian functions. The uterus of neem-treated animals showed normal morphology. Immunohistological studies, however, demonstrated a significant increase in the number of MHC-II antigen-positive cells in the uterine endometrium following neem treatment, indicating enhanced antigen-presenting ability of the uterus; a feature that may be related to the observed antifertility effect of neem oil. The present investigation demonstrates that an IUNT can be used for long-term, reversible contraception, without any apparent side effects, and that the method could provide an alternate to currently used intrauterine contraceptive devices (IUCD).

Clinical trial with praneem polyherbal cream in patients with abnormal vaginal discharge due to microbial infections.

EDITORIAL COMMENT: We accepted this short report of a longer manuscript which the authors agreed to summarize at our request since readers should be interested to read of a polyherbal cream that seems active against Chlamydial infection, although inactive against Candida albicans and Trichomonas vaginalis. Readers should also note the high rate of Chlamydial infection (up to 50%) in women in this community in India who present with an abnormal vaginal discharge and cervicitis.

Comparison of extraction procedures on the immunocontraceptive activity of neem seed extracts

Azadirachta indica (Neem) seed extracts are known to activate the local cell-mediated immune reactions after a single intrauterine administration, leading to a long term reversible block of fertility. In order to identify and characterize the active fraction responsible for this activity, neem seeds were extracted by both mechanical expression and solvent extraction using a range of polar to non-polar solvents which yielded 3 broad fractions. The mechanically expressed oil was fractionated using different approaches and studied for antifertility activity. The hexane extract and a corresponding column fraction showed potent and reproducible antifertility activity. Other fractions were less stable with regard to reproducibility of effects and composition. It is our conclusion that for subsequent fractionation to reach the last active fraction, the hexane extract is the most useful starting material.

Mode of long-term antifertility effect of intrauterine neem treatment (IUNT)

The mode of antifertility action of intrauterine neem treatment (IUNT) was studied. The effect of IUNT on ovarian functions and uterine responsiveness to ovarian hormones was examined in adult Wistar rats. The treated animals had normal reproductive cycles as indicated by the vaginal smears; serum progesterone levels were also in the normal range. Effect of exogenous estradiol following IUNT in ovariectomized rats showed comparable uterine weight gain as in control group; decidual cell reaction of the uterine epithelium following IUNT was also similar to that of control, indicating normal uterine responsiveness to ovarian hormones. Unilateral IUNT followed by mating resulted in degeneration of embryos on the treated side as noted between days 3-5 post coitum; normal embryos were seen on the contralateral side given peanut oil. The study shows that the mode of antifertility action of IUNT is not because of uterine unresponsiveness to the ovarian hormones but is due to impairment of embryo development. The results of this study thus confirm our earlier observations and show further that the antifertility effect of IUNT is at the pre-implantation stage, localized and without any adverse or toxic effect on the fetal development in the contralateral uterine horn of the unilaterally treated rats. The exact mechanism(s) of antifertility action of IUNT is being investigated.