Antifertility activity of Thevetia peruviana (Pers.) K. Schum leaf in female Sprague-Dawley rat.

OBJECTIVES: Thevetia peruviana (Pers.) K. Schum. (Apocynaceae) is known to possess cardioactive glycoside such as thevetin A, thevetin B, neriifolin, peruvoside, thevetoxin, and ruvoside. Traditionally, T. peruviana leaves are used as abortifacient. The aim of the present study is to evaluate antifertility potential of T. peruviana leaves. SUBJECTS AND METHODS: Cardiac glycoside freed leaves of T. peruviana were extracted with methanol using maceration method. The dried cardiac glycoside-free methanolic extract of T. peruviana leaves (TPL-Me-G) was screened for phytoconstituents and evaluated for its effect on estrogen-primed female Sprague-Dawley rat uterus model. It was further studied for effects on the estrous cycle, implantation, and effect on estrogen and progesterone. STATISTICAL ANALYSIS USED: Statistical analysis was done by ANOVA followed by Dunnett's t-test. RESULTS: Alkaloids, flavonoids, essential oils, carbohydrates, and amino acids were found to be present in the glycoside-free extract. Thin-layer chromatography (TLC) in n-butanol: acetone: water (4:1:5) revealed the presence of quercetin and kaempferol. The presence of flavonoids (quercetin 0.0326% and kaempferol 0.138% on dry weight basis) was reconfirmed by high-performance TLC analysis. The extract was able to induce uterine contractions (EC50, 0.170 mg/ml) in a dose-dependent manner. Further investigation showed significant (P < 0.001) extension of estrous cycle and anti-implantation activity of the extract by reduction of the progesterone level. CONCLUSIONS: Methanolic extract of T. peruviana leaves (TPL-Me-G) containing quercetin 0.0326% and kaempferol 0.138% possesses a significant (P < 0.001) antifertility potential by virtue of decreasing the progesterone level.

Artemisia annua as a possible contraceptive agent: a clue from mammalian rat model.

In a previous study, we evaluated the maternal and fetal safety of antimalarial herb Artemisia annua with artemisinin yield of 1.09%. Here, we attempted to ascertain the contraceptive claim of A. annua. Sexually matured female Wistar rats (180-220 g) were allotted into four study groups of six rats each. The control group received normal saline, while the A. annua-treated groups received 100, 200 and 300 mg/kg of A. annua for 2 weeks, followed by mating with proven fertile males (1:1). The rats were allowed to carry the pregnancy to term. At birth and weaning periods, selected reproductive outcome and fertility indices were determined. The results showed that A. annua significantly reduced litter size, reproductive outcome and fertility indices compared with the control (p <  0.05). These results imply that A. annua could serve as a prospective contraceptive agent in addition to its antimalarial activity.

Antifertility efficacy of Drynaria quercifolia (L.) J. Smith on female Wister albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant based traditional medicines for birth control has been in practice in rural populations of North-East India, since time immemorial. Fresh rhizome of Drynaria quercifolia (L.) J. Smith is one of the plant parts used traditionally by different ethnic communities in Tripura, India for birth control. The present investigation is aimed to justify the scientific basis in traditional use of Drynaria quercifolia rhizome as anti-fertility agent. MATERIALS AND METHODS: Ex-vivo uterotonic activity was done on uterine tissue. Acute toxicity test of the plant extract was carried out in rats of both sexes. The abortifacient and anti-implantation activities of the extract were investigated, in-vivo and estimated the level of different hormones release. RESULTS: The result revealed the effectiveness of methanol (87%) and aqueous (68%) extract of the plant on uterotonic activity. The extracts showed relatively non-toxic effect in acute toxicity study. Methanolic extract has shown higher efficacy for both abortifacient (**p<0.01) and anti-implantation performance (**p<0.01) and also effected hormone release level (**p<0.01). CONCLUSION: Methanolic extract of Drynaria quercifolia (L.) J. Smith rhizome has been proved to have significant anti-fertility activity.

Fractionation of stigmasterol derivative and study of the effects of Celsia coromandelina aerial parts petroleum ether extract on appearance of puberty and ovarian steroidogenesis in immature mice.

CONTEXT: Celsia coromandelina Vahl (Scrophulariaceae) is a shrub found throughout Bangladesh and India, and it is distributed widely in the plains of West Bengal. It is used by the tribal people to treat diarrhea, dysentery, insomnia, skin eruption, fever, syphilis, helminthes infection, and to control fertility. OBJECTIVE: The objective of this study was to fractionate stigmasterol derivative and to investigate the effects of petroleum ether extract of C. coromandelina (PECC) aerial parts on the onset of reproductive maturity and the ovarian steroidogenesis in immature female mice. MATERIALS AND METHODS: PECC was prepared by hot extraction process and one compound was isolated by preparative TLC from it. PECC was completely freed from solvent and administered in immature female mice intraperitoneally once on every alternate day for nine doses. The sexual maturity was observed by means of vaginal opening, first estrus (days), rate of body growth, changes in weight of ovary, uterus and pituitary. The content of ascorbic acid, cholesterol, Δ5-3ß-hydroxy steroid dehydrogenase (Δ5-3ß-HSD) and glucose 6-phosphate dehydrogenase (G 6-PDH) activities in ovaries and carbonic anhydrase activity in uterus were measured by means of biochemical technique in control and treated mice. The activity of PECC was compared with standard marker compound ethinyl estradiol. RESULTS: The isolated compound was characterized as stigmasterol derivative. PECC treatment caused a remarkable delay (30.27 and 18.56%, respectively, by low dose) in sexual maturity compared to vehicle control as evidenced by the age of vaginal opening and appearance of first estrus (cornified smear). PECC treatment also caused a significant fall (58.6 and 50.0%, respectively, by low dose) in Δ5-3ß-HSD and G 6-PDH activities involved in ovarian steroidogenesis compared to vehicle control. Total cholesterol and ascorbic acid content in ovaries and carbonic anhydrase activity in uterus were increased significantly (low dose by 49.3, 424.6 and 82.4%, respectively) along with a reduction in the weight of ovary, uterus and pituitary in comparison to that of control. DISCUSSION AND CONCLUSION: Overall, these results demonstrate that PECC has a good antifertility effect and is responsible for the delayed development of sexual maturity, suppression of ovarian steroidogenesis and elevation of carbonic anhydrase activity in uterus of immature mice. This supports the claim by tribal people as a potential remedy for birth control.

Simultaneous online SPE-LC-MS/MS quantification of six widely used synthetic progestins in human plasma.

Co-administration of synthetic progestin containing hormonal contraceptives (HCs) and antiepileptic drugs (AEDs) is a common clinical situation which needs specific considerations due to drug interactions. Several studies have demonstrated that lamotrigine plasma levels are significantly decreased during co-medication with HCs, and that this interaction is associated with increased seizure frequency in most of the cases. Additionally, an increase in contraceptive failure and unintended pregnancy could be observed during co-medication. Hence, monitoring of progestin plasma levels in patients with AED co-medication is of interest. A rapid and reliable online solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS) method using gradient elution in the LC domain was established and validated for the simultaneous quantitative determination of gestodene, dienogest, drospirenone, etonogestrel, cyproterone acetate, and levonorgestrel in human plasma. The online SPE-LC-MS/MS method covered a quantification concentration range of 5-100 ng/ml for dienogest, 1-100 ng/ml for etonogestrel and 2-100 ng/ml for all other analytes. Stable isotope-labeled internal standards were used for analyte quantification based on selected reaction monitoring experiments. Inter- and intra-assay precision and accuracy were determined from quality control (QC) samples at the lower limits of quantification and at low, medium, and high concentration levels within the calibration range. Inter-assay reproducibility at the QC levels was better than 10% (relative standard deviation, RSD), accuracy at these levels ranged from -3.7% to 11.3%. Total extraction efficiency, tested at three concentrations, ranged from 92.5% to 106.4%. Matrix interferences were excluded by post-column infusion experiments. To prove the applicability of the assay in clinical cohorts, a sample set (n = 298) stemming from study patients under AED/oral HC co-medication was screened for progestin plasma levels. This method has to be considered a research-use-only assay and must not be used for diagnostic or therapeutic purposes, since it did not undergo formal performance evaluation in the sense of the IVD directive (98/79/EG) of the European Community.

Extracts of Azadirachta indica and Melia azedarach seeds inhibit folliculogenesis in albino rats.

The seed oil of Azadirachta indica A. Juss (neem) is used in traditional medicine for its antidiabetic, spermicidal, antifertility, antibacterial, and wound healing properties. The present study was undertaken to investigate the quantitative aspects of follicular development in cyclic female albino rats (135 +/- 10 g; 8 groups with 6 animals in each group) after oral administration of polar (PF) and non-polar (NPF) fractions of A. indica seed extract at 3 and 6 mg kg body weight-1 day-1 and Melia azedarach Linn. (dharek) seed extract at 24 mg kg body weight-1 day-1 for 18 days. The extracts were prepared using a flash evaporator at 35 degrees C and then dissolved in olive oil to prepare doses on a per kg body weight basis. There was a significant reduction (P = 0.05) in the number of normal single layered follicles (A. indica: 0.67 +/- 0.33 and 4.67 +/- 2.03 after 3 and 6 mg/kg NPF, and 3.33 +/- 1.67 and 1.00 +/- 1.00 after 3 and 6 mg/kg PF vs control: 72.67 +/- 9.14 and M. azedarach: 0.60 +/- 0.40 and 1.80 +/- 1.2 after 24 mg/kg PF and NPF, respectively, vs control: 73.40 +/- 7.02) and follicles in various stages (I-VII) of follicular development in all treatment groups. These extracts also significantly reduced (P = 0.05) the total number of normal follicles in the neem (14.67 +/- 5.93 and 1.00 +/- 1.00 after 3 and 6 mg/kg PF and 3.67 +/- 0.88 and 5.33 +/- 2.03 after 3 and 6 mg/kg NPF) and dharek (13.00 +/- 3.58 and 14.60 +/- 2.25 after 24 mg/kg NPF and PF) treatments compared to control (216.00 +/- 15.72 and 222.20 +/- 19.52, respectively). Currently, indiscriminate use of persistent and toxic rodenticides to control rodent populations has created serious problems such as resistance and environmental contamination. Therefore, it becomes necessary to use ecologically safe and biologically active botanical substances that are metabolized and are not passed on to the next trophic level, and that interfere with the reproductive potential particularly growth and differentiation of follicles. This may help elevate the socio-economic status of the country. Thus, the present study is an attempt to investigate the effects of A. indica and M. azedarach seed extracts on reproduction of albino rats.

Extracts of Azadirachta indica and Melia azedarach seeds inhibit folliculogenesis in albino rats

The seed oil of Azadirachta indica A. Juss (neem) is used in traditional medicine for its antidiabetic, spermicidal, antifertility, antibacterial, and wound healing properties. The present study was undertaken to investigate the quantitative aspects of follicular development in cyclic female albino rats (135 ± 10 g; 8 groups with 6 animals in each group) after oral administration of polar (PF) and non-polar (NPF) fractions of A. indica seed extract at 3 and 6 mg kg body weight-1 day-1 and Melia azedarach Linn. (dharek) seed extract at 24 mg kg body weight-1 day-1 for 18 days. The extracts were prepared using a flash evaporator at 35°C and then dissolved in olive oil to prepare doses on a per kg body weight basis. There was a significant reduction (P = 0.05) in the number of normal single layered follicles (A. indica: 0.67 ± 0.33 and 4.67 ± 2.03 after 3 and 6 mg/kg NPF, and 3.33 ± 1.67 and 1.00 ± 1.00 after 3 and 6 mg/kg PF vs control: 72.67 ± 9.14 and M. azedarach: 0.60 ± 0.40 and 1.80 ± 1.2 after 24 mg/kg PF and NPF, respectively, vs control: 73.40 ± 7.02) and follicles in various stages (I-VII) of follicular development in all treatment groups. These extracts also significantly reduced (P = 0.05) the total number of normal follicles in the neem (14.67 ± 5.93 and 1.00 ± 1.00 after 3 and 6 mg/kg PF and 3.67 ± 0.88 and 5.33 ± 2.03 after 3 and 6 mg/kg NPF) and dharek (13.00 ± 3.58 and 14.60 ± 2.25 after 24 mg/kg NPF and PF) treatments compared to control (216.00 ± 15.72 and 222.20 ± 19.52, respectively). Currently, indiscriminate use of persistent and toxic rodenticides to control rodent populations has created serious problems such as resistance and environmental contamination. Therefore, it becomes necessary to use ecologically safe and biologically active botanical substances that are metabolized and are not passed on to the next trophic level, and that interfere with the reproductive potential particularly growth and differentiation of follicles. This may help elevate the socio-economic status of the country. Thus, the present study is an attempt to investigate the effects of A. indica and M. azedarach seed extracts on reproduction of albino rats.

Localization of concealed norplant implants and implant fragments by a modified mammography technique.

A modified mammography technique for localization of concealed Norplant implants and Norplant fragments was applied to four patients, ages 18, 35, 22, and 25. A dedicated mammography unit and mammography film screen system affording high resolution and high contrast was applied under the same conditions to each patient. 26 kilovoltage peak (KVp) was used, ranging from 60 to 150 milliamperes. Using both the automatic exposure control and manual techniques, specific milliampere range depended on the specific case. The technique proved successful, as concealed Norplant capsules were exquisitely visualized by films obtained with a Siemens Mammomat 3000. Applying the same modified technique, precise location of the Norplant implants was determined using a fenestrated compression plate with an alphanumeric grid in a manner similar to hookwire localization of breast lesions under mammographic guidance. The capsules were then safely retrieved. We conclude that this modified technique may provide practitioners with valuable assistance in the exact localization of the Norplant capsule(s) or fragments, thereby facilitating their removal. Application of a modified mammography technique reveals precise localization of concealed Norplant capsules facilitating their removal.

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.

The pregnane glycoside marsdekoiside A from Marsdenia koi.

A new pregnane glycoside, marsdekoiside A was isolated from the stems of Marsdenia koi (Asclepiadaceae) and its structure was elucidated from chemical and spectral data as 12-cinnamoyl-dihydrosarcostin-3-O-methyl-6-deoxy-beta-D-allopyr ano syl-(1----4)-O-beta-D-oleandropyranosyl-(1----4)-O-beta-D-++ +cymar opyranoside.

Studies on the antifertility components from Marsdenia koi.

By random screening test, Marsdenia koi was found to have antifertility activity on SD rat. From MeOH extracts of this plant two steroidal glycosides, marsdekoiside A and B, were isolated, and their structures were elucidated on the basis of spectral evidence and by comparison of the hydrolysis products with the authentic samples. Both are newly identified compounds, and marsdekoiside A has good antifertility activity.

[Structure determination of marsdekoiside C].

A pregnane glycoside ester was isolated from Marsdenia koi Tsiang by column chromatography. The structure was identified on the basis of IR, 1H-NMR, 13C-NMR and FAB-MS. It is a new compound obtained from this plant for the first time and named as marsdekoiside C.