Background: Population explosion is worldwide problem that possess significant threat to quality of life. The only solution to this problem is availability of suitable contraceptive. Number of medicinal plants at global level has been explored in this regard. Several studies have been carried out to explore the antifertility effect of medicinal plants in male animal models. Some of these plants are recognized for their spermicidal activities, anti-spermatogenic effects, reduce sperm count, affect the mobility and viability of sperms, change testicular morphology, cause alterations in serum testosterone level and some alter antioxidant defense mechanism. More than fifty plants had been reported to possess anti-fertility agents with antispermatogenetic, anti-androgenic or sperm immobilization activities. Research for the development of male contraceptives is important for the equitability of male and female in birth control program thus sorting the issue of population growth. Objectives: Current study was designed for the comprehensive assessment of contraceptive efficacy of novel herbal plants traditionally used in male fertility regulation. The objectives of the study include: · Comparative analysis of biological activities of four selected medicinal plants. · Biological evaluation of designated methanolic leaf extracts of medicinal plants. · To determine the male contraceptive potential of selected medicinal plants using in vitro technique to target the process of oxidative stress and androgenesis in testis. · To appraise the efficacy of selected medicinal plants in vivo to inhibit spermatogenesis or disrupt epididymal and testicular function, the perturbation of which results in reversible infertility. · To analyze the processes of sperm production, its maturation and function with the aim of regulating or inhibiting specific targets that is involved in reducing the male fertility. · To study the possible effect of medicinal plants on hypothalamus- pituitary- testicular axis (HPT) through plasma Testosterone level, Luteinizing hormone and Follicle stimulating hormone concentrations. 2 Materials and Methods: Four plants belonging to different families were selected named as Chenopodium ambrosioides, Ajuga integrifolia, Rumex hastatus and Hedera nepalensis. Methanolic leaf extracts were prepared by soaking leaf powder in 99.9% methanol (leaves to solvent ratio 1:10) for seven days. After that, contents were filtered, concentrated under pressure, dried at room temperature and placed at 4ºC until further analysis. In first study, methanolic leaf extracts of selected plants were screened for their phytochemical analysis, antioxidant potential, protein kinase inhibition activity, cytotoxic activity against brine shrimp nauplii (BSLA) and phytotoxic potential against Lamna minor. In addition, gas chromatography mass spectrometry (GC-MS) study was conducted to investigate bioactive phyto-constituents present in the methanolic extracts of selected plants. In second set of experiment, testes and sperms of adult male rats were incubated with media having different concentrations (0, 1, 10, 100, 1000 μg/mL) of plant extracts at 37 ℃ in CO2 incubator under 5% CO2 and 95% air (v/v) for 2 hours. Oxidative stress in testis was determined through assessment of antioxidant enzymes activity and generation of reactive oxygen species (ROS). In addition, testicular testosterone levels were measured by Enzyme Linked Immuno Sorbant assay (ELISA) while sperm DNA damage was assessed through comet assay. Acute toxicity and in vivo studies were conducted by using findings of in vitro experiment. For this, standard solution of plant extract was formulated in methanol and further dilutions were made with normal saline. Final strength of methanol was 0.5-1% in saline. In the next series of experiments, adult male rats were given different concentrations of selected plants extracts (0, 50, 100 and 150 mg/kg/day) orally for twenty eight consecutive days. At end of experiments, fertility test was performed to check the reversibility of treatment by pairing three animals from each group with two untreated fertility proven female rats individually. Pregnancy outcome and litter size was noted. Remaining animals were decapitated, trunk blood was collected, plasma was separated and used for hormonal analysis (Testosterone, Luteinizing hormone, Follicle stimulating hormone). While testicular and epididymal tissues were used for the evaluation of alteration in sperm parameters, histological changes and determination of oxidative stress.Results: Experimental findings of phytochemical analysis showed presence of total flavonoid contents (TFC) in methanolic extracts ranging from R. hastatus (242 μg QE/mg) to H. nepalensis (216 μg QE/mg),A. integrifolia (170 μg QE/mg) and C. ambrosioides (146 μg QE/mg). Similarly, total phenolic contents (TPC) were abundant in R. hastatus (308 μg GAE/mg) followed by A. integrifolia (302 µg/mg), C. ambrosoides (291 µg/mg) and H. nepalensis (277 µg/mg). In current study, maximum antioxidant capacity was shown by methanolic extract of R. hastatus with 309 µg/mg value followed by A. integrifolia, C. ambrosoides and H. nepalensis with 302, 291 and 277 µg/mg respectively. While, A. integrifolia possess highest free radical scavenging activity as can be seen by inhibitory concentration (IC50 value) of 17μg/mL tailed followed by C. ambrosioides, R. hastatus and H. nepalensis with IC50 values of 56, 67 and 71 μg/mL respectively. In our study, methanolic extract of H. nepalensis showed high protein kinase inhibition against Streptomyces growth with 27.6 mm zone of inhibition/cytotoxicity at highest concentration, followed by A. integrifolia (20.7 mm), R. hastatus (14.7 mm) and C. ambrosioides (7.6 mm) indicating their cytotoxic potential. Results of cytotoxicity assay showed maximum percent mortality of 50, 54, 60 and 74 % at 1000 μg/mL with methanolic leaf extract of R. hastatus, A. integrifolia, C. ambrosioides and H. nepalensis respectively. However, phytotoxic activity revealed relatively moderate activity by C. ambrosioides (45%), A. integrifolia (38%) and R. hastatus (49%) at highest concentration (1000 μg/ml) while, H. nepalensis showed highest % inhibition (51%) towards Lamna minor at 1000 μg/mL. GC-MS investigation of methanolic leaves extracts indicated presence of thirteen compounds in C. ambroisoides, eleven in A. integrifolia, fifteen in R. hastatus and only three compounds were identified in H. nepalensis. In the in vitro study, significantly increased oxidative stress with reduced antioxidant activity was observed in highest dose regimen (1000 μg/mL) of methanolic extract of C. ambrosioides, A. integrifolia and H. nepalensis. However, in vitro exposure to R. hastatus caused slight increase in reactive oxygen species (ROS) production at high dose without significantly affecting antioxidant enzymes status. Increased ROS generation and lipid peroxidation lead to DNA damage in rat sperm. Similarly, decline in testicular testosterone level was noticed after two hours incubation with all 4 the doses of C. ambrosioides and A. integrifolia leaf extract, however, R. hastatus and H. nepalensis showed reduction only at higher doses regimens (100 and 1000 μg/mL). Results of in vivo experiment using C. ambrosioides and A. integrifolia extracts showed that rate of sperm motility (%) and viability (%) as well as daily sperm production (DSP) were reduced in dose dependent manner. A marked reduction in concentration of antioxidant enzymes was noted in 100mg/kg and 150mg/kg dose treated rats while increase in levels of ROS and TBARS was also quite evident only in high dose exposure. Histopathological observations of testis exposed to C. ambrosioides extract for 28 days revealed significant decrease in seminiferous tubule diameter, lumen diameter and epithelial height in treated animals. Epididymal histology showed a little alteration in tubular and luminal diameter while epithelial height in both caput and caudal epididymis was significantly declined dose dependently. A significant lowering in activities of plasma testosterone, FSH was recorded however, LH was reduced non-significantly A. integrifolia extract exposure caused histological alterations including sloughing of epithelium with wider lumen in high extract treated groups. In addition, studied plasma hormonal levels were also reduced significantly in the treated groups. Fertility test of rats exposed to C. ambrosioides and A. integrifolia extract exhibited reduced pregnancy outcome in the females paired with treated male rats. Litter size was significantly reduced in 50 mg/kg and 150 mg/kg of C. ambrosioides extract treatment, however, this reduction was not significant in A. integrifolia extract treated rats. R. hastatus extract exposure indicated no significant change in DSP, epididymal sperm number and antioxidant enzymes concentration, however, dose dependent decrease in sperm motility and viability while increase in levels of ROS and TBARS were seen only in high dose regimen. No histomorphometric changes in testis and epididymis were reported except wider lumen with slightly reduced epithelial height in maximum extract treated groups. A considerable decline in plasma levels of testosterone, FSH and LH was also recorded only in high extract treated animals. Percentage fertility and number of pups born per female paired with R. hastatus extract treated male rats were reduced non-significantly in high dose treated groups as compared to control. Exposure of adult male rats to different doses of H. nepalensis resulted in dose dependent decrease in sperm motility, viability and DSP. A marked decline in 5 antioxidant enzymes levels and rise in levels of ROS and TBARS was noted in high dose treated rats. Histopathological observations of testis revealed normal tubular arrangement with reduced epithelial height and wider lumen in 100mg/kg and 150mg/kg dose treated groups. Similarly, epididymal histology showed normally arranged epididymis with slightly narrow lumen and relatively reduced number of sperms in plant extract treated (100mg/kg and 150mg/kg) groups. A significant reduction in levels of studied reproductive hormones was observed in extract exposed groups when evaluation was made with control animals. Pregnancy outcome and litter size was considerably reduced in 150mg/kg dose treatment group, but there was no morbidity or mortality exhibited by subsequent pups. Conclusion: It is concluded that although R. hastatus affect reproduction to some extant but it is not capable of suppressing fertility in male rats. On the other hand, H. neplensis have the potential to suppress fertility either by disturbing sperm parameters or interfering with hypothalamus- pituitary- gonadal axis (HPG). C. ambrosioides and A. integrifolia caused partial male sterility by disturbing spermatogenic cycle, inducing oxidative stress and hormonal imbalance. Although, fertility was compromised, but fertility suppression was reversible after cessation of treatment and no fetal mortality or morbidity was evident by fertility test." xml:lang="en_US