Male Antifertility Agent Research Articles

Effect of giving Extract Ethanol from Bitter Gourd Fruit (Momordica Charantia L) on the Quality and Quantity of Mouse Spermatozoa (Mus Musculus)

Men's participation in the Family Planning (KB) program is still very minimal due to the limited choice of contraceptive methods. Efforts need to be made to develop contraceptive methods, one of which is using natural ingredients. Bitter melon (Momordica charantia L) is known to have potential benefits as a male antifertility agent. This research used an experimental design with a sample size of 20 mice divided into 4 groups randomly. Treatment groups 1-3 were given different doses, namely 100, 80, 60 mg of bitter melon ethanol extract/100g BW/day, and the control group was given distilled water for 35 days. The mice were sacrificed the day after the last dose, then spermatozoa were collected from the epididymis and their number, motility, viability and morphology were examined. The results of this study showed a significant decrease in the number and motility of spermatozoa and was directly proportional to the size of the dose given. In terms of viability parameters, there was a significant decrease at all doses. Meanwhile, for spermatozoa morphology, only a dose of 100 mg had a significant impact. The conclusion from the results of this research is that giving bitter melon fruit extract has an effect on the number, motility, viability and morphology of spermatozoa. The most effective dose is 100 mg/100 gBB/day.

Antifertility activity of Oroxylum indicum (L.) Kurz:In vitro and in vivo study on human sperm and male wistar rats

Oroxylum indicum (L.) Kurz, commonly called as Broken bones tree or Indian trumpet flower, belonging to Fam. Bignoniaceae, is traditionally used as a contraceptive by ethnic people of Tripura, North-East India. Here, we investigated the scientific basis for use of O. indicum as male antifertility agent by folklore healers. In vitro spermicidal activity of aqueous (AEOI) and methanolic (MEOI) extracts of O. indicum stem bark were studied on human sperm. The in vivo activity was experimented on male albino rats. The treated animals were allowed to mate and the pups delivered by female rat partners were counted. Phytochemical estimation of test samples was done using HPLC. The AEOI and MEOI treatments significantly decreased human sperm motility and viability. Test extracts have increased the hypo-osmotic swelling of sperm. Both the extracts were significantly declined the weight of reproductive organ. The MEOI treated rats have shown significant decrease in sperm motility and sperm counts. AEOI and MEOI treatment significantly reduced level of testosterone, but sharply raised dihydrotestosterone and prostaglandin in rats. Results testified the traditional claim for use of O. indicum as a male contraceptive agent, where MEOI have shown reversible action on male reproductive system leading to contraception without harming the libido.

Deleterious effect of short-term gavage of an ethanol extract of cogon grass (Imperata cylindrica L.) roots on testis and epididymal sperm quality

Background and Aim:Cogon grass (Imperata cylindrica L.) (CGG) is a herbal medicine that could be developed into a male antifertility agent. The present study aims to determine the effect of an ethanol extract of CGG roots on mice testicular activity, reproductive hormone levels, and epididymal sperm quality.Materials and Methods:This study was designed as completely randomized with three different doses, such as an ethanol extract of CGG roots at 0 (control), 90, and 115 mg/kg body weight. In total, 21 male DDY mice strain were treated with the CGG extract (by gavage) for 14 days, followed by an evaluation of reproductive organs, epididymal sperm quality, testis histology, histomorphometry, and reproductive hormone assays. All quantitative data were analyzed by analysis of variance, followed by Tukey’s post hoc test at α=0.05.Results:The results showed that the administration of the CGG root ethanol extract disrupted the testis interstitial area and seminiferous tubules, resulting in decreased epididymal sperm quality as well as serum testosterone levels in a dose-dependent pattern.Conclusion:Oral administration of a CGG root ethanol extract induced testicular damage, decreased epididymal sperm quality, and impaired testosterone secretion.

Effect of Ethanolic Extract of Trigonella foenum-graecum L. Seeds on Reproductive System of Male Albino Rats

Aims: The present study was undertaken to assess the antispermatogenic and antifertility efficacy of ethanolic extract of Trigonella foenum-greacum seeds.
 Methodology: Aqueous solution of the extract (250 mg/kg b.wt/day) when administered orally for 45 days to adult male albino rat (Duckray strain).
 Results: Seeds extract caused inhibition of spermatogenesis as well as inability to mate with normal untreated female rats of proven fertility. There occurred a significant decrease in absolute and relative weights of testis and seminal vesicle and increase in epididymal weight, whereas of ventral prostate and coagulating gland remained unchanged. Sperm concentration and motility in the cauda epididymis was also decreased. Increased incidence of separation of head and tail pieces of spermatozoa was seen in the epididymal smear. Histologically, testis of experimental animals showed arrest of spermatogenesis at the secondary spermatocytes stage and there was deposition of cellular debris in semineferous tubular lumen. The spermatids were not properly developed and interstitial cells were very sparse and degenerated. However, there was no evidence of damage to the spermatogonia. Oral treatment of extract did not affect the body weight of animals. Quantitative estimation of marker testicular enzymes, e.g., sorbitol dehydrogenase showed a significant decrease whereas lactate dehydrogenase and gamma-glutamyl transpeptidase levels were significantly increased.
 In conclusion: These results suggest that T. foenum-greacum, may induce male infertility in rats, therefore, should be considered further as a potential male antifertility agent.

Does Propolis Extract Alleviate Male Reproductive Performance Through Gonadotropic Hormone Levels and Sperm Quality?

Nowadays, the exploration of natural substances as an antifertility agent is the new paradigm in controlling the production and physiological performance of male and female germ cells. Natural antifertility agents have fewer side effects compared to chemical synthetic antifertility. Among that, the propolis extract was improved the reproductive performance through increasing sperm and male gonadotropic hormone activity. However, the discrepancy remains. Even though the action of testosterone has an essential role during spermatogenesis. However, the higher level of this hormone was also suggested to correlate with infertility. Thus, the circulating testosterone levels can be used as an additional parameter for monitoring reproductive system disorders. The purpose of this study was to evaluate the effect of Propolis extract on testosterone hormone levels of Balb C mice (Mus musculus). Male Balb C strain mice (Mus musculus) with age of 10-12 weeks and weight of 25-29 g were used. There were six kinds of treatments were approved, including a control group and five serial dilutions of the propolis extract (2.5%, 5%, 7.5%, 10%, and 12.5% ). The propolis extract was given peroral for 18 days. Mice were dissected on the 19th day, and its blood was taken to count the levels of testosterone. The quantification of circulating testosterone levels was carried out by using the ELISA test. The data were analyzed by one way ANOVA and followed by LSD test. Testosterone level began to increase at 10% concentrations of propolis extract. It can be concluded that Propolis Extract can increase testosterone levels. Thus, it is expected to serve as a potential male anti-fertility agent.

Fertility Suppression by the Fruit Extract of Opuntia elatior in the Male Rat: Possible Extragonadal Action

Plant - derived male antifertility agents need to address the basic requirements of reversibility and fertility suppression without affecting libido. This study was undertaken to evaluate the male antifertility potential of the fruit extract of Opuntia elatior in the male rat and its reversibility. The methanolic extract was administered through oral route (300, 900 mg/kg bw) to male rats for 30 and 60 days, and fertility was assessed by analysing spermatogenesis, sperm count and testosterone levels. Reversibility of fertility suppression was studied by withdrawal of treatment for two weeks and mating with normally cycling virgin females. Sperm count and motility were markedly reduced in both the groups of treated rats, without commensurate decline in serum testosterone levels and testicular hydroxysteroid dehydrogenase (HSDH) activity. Fertility of the treated rats was suppressed when mated with normally cycling virgin female rats without affecting libido. Withdrawal of treatment for two weeks restored the sperm count, testicular HSDH activity, serum testosterone levels and fertility. The methanolic extract of the fruit of O. elatior produces reversible male antifertility effect without affecting the testosterone levels and libido.

Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans

Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP₃β- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

In-vitro metabolic inhibition and antifertility effect facilitated by membrane alteration: Search for novel antifertility agent using nifedipine analogues

In search of non-hormonal male contraceptives, analogues of nifedipine, which causes reversible infertility, have been synthesized and their interaction at molecular level with model membrane has been probed. Analogues act differently with respect to their antifertility action. This is achieved by altering the cell metabolism thereby directly affecting the motility which is responsible for fertility. Secondly, these drugs bind differently to the interior of the cell-membrane affecting the membrane fluidity, architecture and dynamics. Sulfasalazine and D 4 interact to a larger extent and alter the lipid bilayer phase to a hexagonal. D 1, D 2 and D 3 do not have considerable effect. D 4 is the most promising candidate as a lead compound for the development of novel non-hormonal male antifertility agents.

Reversible Antifertility Activity of Hydroalcoholic Extract of Ficus Racemosa L. in Male Mice

Objective To evaluate male antifertility activity of hydroalcoholic extract of Ficus racemosa bark. Methods Swiss male mice were orally administered hydroalcoholic extract of Ficus racemosa bark (50 mg/kg for 30 d and 100 mg/kg body weight for next 30 d), and the effect of the treatment on body weight, reproductive organs weight, sperm, biochemical profile (sialic acid in epididymis and fructose in seminal vesicle), fertility and vaginal contraceptive efficacy was investigated. Recovery studies were also performed. Results Extract reduced fertility to 70% within 60 d. Suppression of cauda epididymis sperm count, motility, viability and abnormal morphology was observed. Marked reduction was noted in the weight of reproductive organs and the level of sialic acid in epididymis and fructose in seminal vesicle. Vaginal application of bark extract exhibited 80% vaginal contraceptive efficacy. After cessation of plant extract treatment, the altered parameters recovered after 60 d. Conclusion Clinical assessment of male antifertility agents should include acceptability, safety and efficacy during and after the treatment. The above results revealed the potential, reversible male antifertility effect of hydroalcoholic extract F. racemosa bark.

Evaluation of Contraceptive Potential of Isolated Fractions from Dendrophthoe falcata Stem Methanol Extract in Male Albino Rats

Development of an ideal contraceptive for man has been the goal of several research programs during the past few decades. The advent of an effective and reversible male contraceptive method should provide a valuable alternative to the limited options of the condom, vasectomy and withdrawal that are currently available to men. The search for an effective, safe and reversible male antifertility agent with minimum side effects remains a challenge. Therefore, the present study was carried out to evaluate contraceptive efficacy of Dendrophthoe falcata since its use in folk remedies is as a antifertility agent (1). Phytochemical studies have identified several flavonoids such as Quercetin, Kaempferol, Quercitrin, Rutin, Quercetagetin, Meratin and Hyperoside (2). Quercitrin (Quercetin3Orhamnoside) was reported to be the major constituent of D. falcata. In the present study isolated fractions of chloroform:methanol solvent in different ratio of D. falcata (Ettingsh) stem methanol extract was administered at the dose level of 50mg/rat/day to evaluate the male contraceptive potential. After 60 days of treatment suppression of sperm density as well as motility in the cauda epididymis was the most pronounced effect. The protein and sialic acid content of testis and cauda epididymis was reduced significantly; however, testicular cholesterol was increased. Histological observations of testis showed altered spermatogenesis while pyknotic nuclei were seen in few of epithelial cells of epididymis. All haematological parameters remained unchanged, which suggests the nontoxic nature of the drug. Thus, the study corroborates the safe and effective male contraceptive nature of D. falcata, which affects the testicular cells and resulted in the oligospermic state in albino rats.

ChemInform Abstract: Development of Male-Fertility-Regulating Agents

Steroidal nonsteroidal plant-derived gonadotropin-related and immunological agents investigated for control of male fertility are reviewed with brief descriptions of their effects and illustrations of their structures. The physiology of the male reproductive system is presented as an introduction: an ideal male antifertility agent would inhibit spermatogenesis at the level of the Sertoli cells without affecting endogenous androgen production by Leydig cells needed for libido and potency. Androgens down-regulate their own production at physiological levels but few long-acting orally active derivatives are available. Anti-androgens with mixed androgen and progestin activity combined with a pure androgen are potentially useful. Androgen-progestin combinations are being tested by WHO as implants. Dozens of miscellaneous nonsteroidal compounds have been discovered serendipitously to have antifertility activity in men or male animals including alkylating agents antimetabolites antibiotics sulfa derivatives fungicides trichomonocides amebicides alpha blockers antimalarials coumarins and carbohydrate derivatives. Various plant alkaloids have been screened. Those of Hibiscus Vitex and Plumbago species as well as Tripterygium wilfordii glycosides which are being evaluated in combination with gossypol are mentioned here. Gossypol has been thoroughly tested in China but rejected because of its side effects particularly hypokalemic paralysis its low therapeutic index and uncertain recovery of fertility. Gonadotropin-releasing hormone (GnRH) agonists and antagonists are being researched in combination with androgens with some success. The GnRH antagonists to date have low activity or cause histamine-related side effects at higher doses; the androgens require a new route such as a long-acting implant to overcome the need for daily injections. Immunological contraception for males has not progressed beyond the research stage.

Inhibition of hyaluronidase activity of human and rat spermatozoa in vitro and antispermatogenic activity in rats in vivo by Terminalia chebula, a flavonoid rich plant

Our interest in development of hyaluronidase inhibitors as male antifertility agents led to identification of Terminalia chebula (T. chebula) plant with hyaluronidase (HAase) inhibitory activity of human spermatozoa ( approximately 93% inhibition) and rat caudal epididymal spermatozoa ( approximately 86% inhibition) in vitro at 30 mg/ml. We further demonstrated inhibition of hyaluronidase activity of testis and epididymal spermatozoa in vivo coincident with antispermatogenic activity and contraceptive efficacy of TC extract administered at 50 and 100mg/kg/day orally for 60 days in male albino rats. The significant decrease in motility, count and increase in morphological abnormalities of epididymal spermatozoa and severe reduction in fertility (-100%) of male rats treated with T. chebula fruit extract at 100mg/kg dose could be attributed to either direct effect on testis or direct or indirect interference with sperm maturation in epididymis, and/or inhibition of testicular and epididymal sperm hyaluronidase enzyme in vivo probably caused by flavonoids like tannins present in T. chebula.

Studies on the male antifertility agent--gossypol acetic acid. V. Effect of gossypol acetic acid on the fertility of male rats.

Gossypol acetic acid was given to male rats at a dose of 7.5 mg/rat/day six days a week for ten weeks. After nine weeks of gossypol treatment no implantation sites were observed in the females mated with gossypol treated males. After ten weeks of gossypol treatment all the spermatozoa in the vas deferens were non-motile. Gossypol treatment did not affect the body weight and the weights of the accessory sex organs. Plasma LH and FSH levels, hCG binding in testis and succinic dehydrogenase (SDH) and adenosine triphosphatase (ATPase) activities in liver, kidney and testis were not affected by gossypol treatment. Histological observations of the testis revealed partial damage to the seminiferous tubules. Single high doses of gossypol did not induce significant changes in the body weight and weights of testis and accessory sex organs. ATPase activity in the testis was reduced significantly after gossypol treatment, the enzyme activity in liver and kidney, was however, affected at high doses only. Gossypol treatment had no effect on the histoarchitecture of the testis. Intratesticular administration of gossypol evoked localized damage in the testis. Gossypol treatment had no effect on I125 FSH binding to the rat testis homogenate in vitro.

Studies on the male antifertility agent gossypol acetic acid. VII. Effect of motility stimulated factors on the revival of human spermatozoal motility after gossypol treatment in vitro.

Human spermatozoa were incubated with gossypol acetic acid (100 micrograms/1 X 10(6) spermatozoa/ml) at 37 degrees C for 30 min. The drug treatment inhibited the spermatozoal motility significantly. Washing of the spermatozoa, after gossypol treatment, did not effect their motility. A partial revival in the motility of the spermatozoa was observed when gossypol treated spermatozoa were incubated, after washing, with motility stimulating factors, e.g. theophylline, dibutyryl-cAMP and Kallikrein.

Development of hormone-dependent prostate cancer models for the evaluation of inhibitors of 17β-hydroxysteroid dehydrogenase Type 3

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

Effect of Methanolic Extract of Dendrophthoe falcata Stem on Reproductive Function of Male Albino Rats

In spite of the considerable development in contraceptive technology, search for male antifertility agents in plants continues to be a potential area of investigation. Many plants have been known to possess antifertility activity, but limited attempts have been made to scientifically evaluate these claims. Hence the purpose of this study was to evaluate the antifertility and reproductive toxicity potential of Dendrophthoe falcata (Loranthaceae) in male Wistar rats. An oral 70% methanolic extract of stem of D. falcata at a dose level of 100 mg/kg wt/day fed to male albino rats for 60 days did not decrease body weight, while the testes and epididymides were significantly reduced, and the seminal vesicles and ventral prostate also showed a significant reduction (P < 0.01). Treated animals showed a notable depression of spermatogenesis. As a result of 100 mg/kg extract feeding, the preleptotene spermatocytes, secondary spermatocytes, step-19 spermatids and the mature Leydig cells decreased by 74.36%, 80.03%, 79.87%, 32.37%, respectively. At this dose Leydig cell nuclear area and cytoplasmic area, as well as the cross sectional surface area of Sertoli cells, were significantly reduced (P < 0.001) when compared to controls. The reduced sperm count and motility resulted in 100% negative fertility at 100 mg/kg dose level. A significant fall in the total protein and sialic acid content in the testes, epididymides, seminal vesicle and ventral prostate, as well as in the glycogen content of testes was also observed. The level of serum protein, cholesterol, billirubin, SGOT, SGPT, blood urea, and hematological indices were unaltered. In conclusion, Dendrophthoe falcata brought about the inhibition of spermatogenesis.

Reversible contraceptive efficacy of methanol extract of aerial parts of Gynandropsis pentaphylla in male albino rats

In spite of the considerable development in contraceptive technology, search for male antifertility agents in plants continues to be a potential area of investigation. Many plants have been known to possess antifertility activity but limited attempts have been made to scientifically evaluate these claims. Hence the purpose of this study was to evaluate the antifertility and reproductive toxicity potential of Gynandropsis pentaphylla DC. (Capparidaceae) in male wistar rats. Oral administration of 100% methanol extract of aerial parts of G. pentaphylla (Capparidaceae) at the dose level of 20mg/rat/day male albino rats for 60 days did not decrease body weight, while the testes and epididymides were significantly reduced, seminal vesicles and ventral prostate showed a significant reduction (P≤0.001). Treated animals showed a notable depression of spermatogenesis. As a result of 20mg/rat/day extract feeding, the preleptotene spermatocytes, secondary spermatocytes, round spermatids and the mature Leydig cells decreased. At this dose level Leydig cell nuclear area and cytoplasmic area, as well as the cross sectional surface area of Sertoli cells, were significantly reduced (P≤0.001) when compared to controls. The reduced sperm count and motility resulted in 100% negative fertility. A significant fall in the total protein and sialic acid content in the testes, epididymides, seminal vesicle and ventral prostate, as well as in the glycogen content of testes was also observed. However a most exciting observation is that all the parameters were found in a non-significant manner after withdrawal of the drug. In conclusion methanol extract of G. pentaphylla have contraceptive activity in male rats with their reversible nature.

Effects of Gossypol Acetate on Proliferation and Apoptosis in Lymphoblastoid Cell Line and Primary ALL and CLL Cells.

Gossypol is a polyphenolic compound extracted from the cotton plant of Malvaceae family. It was originally demonstrated as a male antifertility agent. In the 1960's, its anticancer efficacy was identified. In the last few years, gossypol has been approved to have antiproliferative and apoptosis-inducing effects on some kinds of cancer cell lines in vitro. Very recently, it has been showed that gossypol is a potent small-molecule inhibitor of Bcl-2 and Bcl-XL proteins. It can bind to Bcl-2/Bcl-XL and block the heterodimerization of Bcl-2/Bcl-XL with proapoptotic proteins, such as Bax, Bak and Bad. Such a blocking of antiapoptotic-proapoptotic protein heterodimerization in turn may inhibit the antiapoptotic function of Bcl-2/Bcl-XL, and induce apoptosis of cancer cells. Overexpression of Bcl-2 has been observed in B-cell lymphoma, B-ALL, B-CLL and correlates closely with the occurrence, progress, recurrence and multidrug-resistance of these malignancies. In this study, we found that gossypol acetate was able to inhibit the proliferation and survival of Raji cells in vitro at concentrations of more than 5 μmol/L. The effect was dose and time dependent. IC50 were 11.9±1.2 μmol/L at 24h and 6.8±0.4 μmol/L at 72h, which were 7.1 and 9.1 folds lower than that of normal MNCs, respectively. Gossypol acetate can remarkably increased pro-apoptosis activity of dexamethasone in Raji cells. Cell cycle analysis by flow cytometry (FCM) indicated that gossypol acetate could induce G0/G1 arrest. Apoptosis was demonstrated both morphologically by Wright-Giemsa staining, Hoechst 33342 staining and by DNA ladder formation in agarose-gel electrophoresis. By determination the percentage of subdiploid amount of DNA, apoptosis was quantified. The result showed that gossypol acetate could increase the percentage of cells with fragmented DNA in time and dose dependent manners at the concentrations of more than 1μmol/L. The fraction of annexin V+/PI− cells was also increased with dose. Colorimetric assay showed that an activation of caspase-3 was seen at 12h and peaked at 24h at the concentration of 25μmol/L. Protein expression of Bcl-2 decreased 13.6±3.8% at 12h and 69.5±6.2% at 24h. Gossypol acetate also had apoptosis-inducing effects in primary leukemia cells from patients with ALL (n=7) and CLL (n=3). The concentration inducing apoptosis in CLL cells was higher than that in ALL cells. It was concluded that gossypol acetate could inhibit the proliferation and induce the apoptosis of Raji cells and primary cultured ALL and CLL cells in a dose and time dependent manner. It seems that down-regulation of Bcl-2 protein expression may play a role in the apoptosis.

Investigations on gossypol: past and present developments

Gossypol has received significant attention as a result of its potential therapeutic application as a male antifertility agent. Furthermore, recent research examining the biological activity of gossypol has revealed a number of other promising lines of enquiry. These have focused on the antitumour, antiviral and antioxidant actions of the compound in various disease states. This article provides an overview of the studies on the biological activity of gossypol, with particular attention paid to the mechanisms of its activity and its prospect as a medicinal product.

Gossypol Activates Pancreatic Polyamine Catabolism in Normal Rats and Induces Acute Pancreatitis in Transgenic Rats Over-expressing Spermidine/Spermine N 1 -Acetyltransferase

Background: The male antifertility agent gossypol has been reported to induce spermidine/spermine N 1 -acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. Methods: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma ! -amylase activity were determined after different doses of gossypol. Results: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. Conclusions: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.