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Postcoital interceptive activity of Wrightia tinctoria in Sprague–Dawley rats: a preliminary study
Contraception 78 (2008) 266 – 270
Original research article
Postcoital interceptive activity of Wrightia tinctoria in Sprague–Dawley rats: a preliminary study☆ Govind Keshri a , Sudhir Kumar a , Dinesh Kumar Kulshreshtha b , Siron Mani Rajendran c , Man Mohan Singh a,⁎ b
a Division of Endocrinology, Central Drug Research Institute, Lucknow 226 001, India Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226 001, India c Division of Botany, Central Drug Research Institute, Lucknow 226 001, India Received 14 December 2007; revised 10 March 2008; accepted 14 March 2008
Abstract Background: This study was aimed to investigate the pregnancy-interceptive activity of the stem bark of Wrightia tinctoria R.Br. (Family Apocynaceae) administered during the preimplantation, peri-implantation and early postimplantation periods by oral route in adult female Sprague–Dawley rats. Study Design: The ethanolic extract of the stem bark and its serial fractions were administered to female rats on Days 1–7 or 1–5 postcoitum (Day 1: day of sperm-positive vaginal smear) by the oral route. At autopsy on Day 10 postcoitum, the number and status of corpora lutea and implantations were recorded. For estrogen-agonistic activity, immature rats ovariectomized 7 days earlier received the test extract or the vehicle once daily for 3 days and, at autopsy on Day 4, uterine weight, status of vaginal opening and extent of vaginal cornification were recorded. Results: The ethanolic extract of the stem bark of W. tinctoria R.Br. inhibited pregnancy in 100% of rats when administered orally at a 250mg/kg dose on Days 1–7 or 1–5 postcoitum. On fractionation, the hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions showed 100% anti-implantation effect, while n-butanol-soluble fraction intercepted pregnancy in 75% of animals when administered in the Days 1–5 postcoitum schedule. In immature rat bioassay, the active ethanolic extract and its fractions exhibited moderate to potent estrogen-agonistic activity, which might be responsible for their contraceptive action in this species. Conclusions: Findings demonstrate the antifertility activity of the ethanolic extract of the stem bark of W. tinctoria and its hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions. Studies that pursue promising natural products (to identify contraceptive agents from natural sources lacking potent estrogenic activity) towards a fruitful conclusion for development/lead generation should continue. © 2008 Elsevier Inc. All rights reserved. Keywords: Wrightia tinctoria; Stem bark; Contraceptive; Implantation; Early postimplantation; Estrogen-agonistic activity; Rat
1. Introduction Wrightia tinctoria R.Br. (Family Apocynaceae) is widely distributed throughout peninsular India, particularly in tropical areas such as Madhya Pradesh, Rajasthan and
☆ The authors thank the Ministry of Health and Family Welfare, Government of India, for financial support. S.K. thanks the CSIR, New Delhi, for the Senior Research Fellowship award, and M.M.S. thanks the Indian Council of Medical Research for appointment as Emeritus Medical Scientist. ⁎ Corresponding author. Tel.: +91 522 2612411, +91 522 2612418x4307; fax: +91 522 2623405. E-mail address: [email protected] (M.M. Singh).
0010-7824/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2008.03.016
Tamil Nadu. Ethnomedically, the bark of this plant is used: as a galactagogue, to treat abdominal pain [1,2], skin diseases and wounds [3]; as an antipyretic [4], antidysenteric, antidiarrheal and antihemorrhagic [5] agent; and as an antidote for snake poison [6]. Seeds of this plant are also used as an aphrodisiac [7]. In view of the reported severe health hazards of estrogen, such as increased risk of endometrial hyperplasia and carcinoma [8,9], breast cancer [10] and thromboembolic diseases [11], a large number of natural products showing promising antifertility activity in preliminary studies could not be pursued due to their associated estrogen-agonistic activity [12]. The present study is part of the systematic investigation of terrestrial plants and marine flora and fauna being pursued at the
G. Keshri et al. / Contraception 78 (2008) 266–270
267
Table 1 Pregnancy-interceptive activity of the ethanolic extract of the stem bark of W. tinctoria in adult female Sprague–Dawley rats following oral administration in different schedules postcoitum Treatment
Days postcoitum of treatment
Daily dose (mg/kg)
Pregnant a/treated rats
Corpora lutea a, b
Implantationsa, c
Efficacy (%)
Vehicle Ethanolic extract Vehicle Ethanolic extract
1–7 1–7 1–5 1–5
– 250 – 250
6/6 0/5 5/6 0/4
11.0±0.4 9.0±0.3 10.2±0.6 9.8±0.9
9.3±0.3 – 7.0±1.5 –
– 100 – 100
–, none. a Day 10 postcoitum. b Mean±SEM of all treated rats. c Mean±SEM of pregnant rats only.
Central Drug Research Institute (Lucknow, India) to identify new leads for development as effective, reversible, safe and socially acceptable contraceptives and agents for the management of age-related degenerative and lifestyle disorders for human use and welfare, and demonstrates for the first time the contraceptive and hormonal properties of this plant. 2. Materials and methods 2.1. Collection of the plant material The stem bark of this plant was collected from the Maharashtra State of India in November, identified by the Botany Division of the institute and coded (code no. 4372). A voucher (specimen no. 8499) has been prepared and kept at the institute's herbarium. 2.2. Extraction and fractionation Shade-dried powdered stem bark of this plant was extracted three times with 95% ethanol (4 L each) by cold percolation. The combined ethanolic extract was filtered, and the filtrate was concentrated under reduced pressure at below 45°C to remove the last traces of the solvent to yield a brown viscous mass. This ethanolic extract was serially fractionated into hexane-soluble, chloroform-soluble, n-butanol-soluble, water-soluble and water-insoluble fractions and evaluated biologically.
2.3. Biological evaluation Colony-bred immature (25–30 g) female and adult (180–250 g) female and male Sprague–Dawley rats maintained under standard conditions (22±1°C), with alternate 12-h light/dark periods and free access to regular pellet diet (Lipton India Ltd., Bangalore, India) and tap water, were used in this study. Postcoital contraceptive and estrogen-agonistic activities of the test extracts were assessed according to the procedures described earlier [12,13]. Briefly, for contraceptive activity evaluation, adult female rats were caged overnight with coeval males (3:1) of proven fertility, and their vaginal smears were checked on the following morning. The presence of spermatozoa in the vaginal smear was considered as evidence of mating, and the day of their occurrence was designated as Day 1 of pregnancy. Mated females were isolated, randomized (Tables 1 and 2) and treated with the test agents suspended in distilled water, with the help of an almost equal quantity of sterile gum acacia or vehicle on Days 1–7 or 1–5 postcoitum. The required amount of the suspension was made fresh daily. Gum acacia (S.D. Fine Chemicals, Mumbai, India) was extracted twice with redistilled ethanol, dried and tested for any effect on female and male fertility or estrogenicity before use. Animals of all groups were autopsied by cervical dislocation on Day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each animal were recorded.
Table 2 Pregnancy-interceptive activity of fractions of the ethanolic extract of the stem bark of W. tinctoria in adult female Sprague–Dawley rats following oral administration on Days1–5 postcoitum Treatment
Daily dose (mg/kg)
Pregnanta/treated rats
Corpora lutea a, b
Implantationsa, c (mean±SEM)
Efficacy (%)
Vehicle Hexane-soluble fraction Chloroform-soluble fraction n-Butanol-soluble fraction Water-soluble fraction Water-insoluble fraction
– 250 250 250 250 250
5/5 0/3 0/3 1/4 0/4 0/3
11.0±0.5 9.6±1.3 9.7±0.8 8.5±1.2 12.3±1.1 9.7±0.9
7.6±1.7 – – 3.0 – –
– 100 100 75 100 100
a b c
Day 10 postcoitum. Mean±SEM of all treated rats. Mean±SEM of pregnant rats only.
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For determination of estrogen-agonistic activity, immature female rats ovariectomized 7 days earlier were treated orally with the test agent or the vehicle (gum acacia in distilled water) once daily for three consecutive days; at autopsy 24 h after the last treatment, uterine fresh weight was taken, and the status of vaginal opening (open/closed) and the extent of vaginal cornification were recorded. In animals where the vagina was still closed, smear was taken by puncturing the membrane. For comparison, one group of rats was similarly treated with 17α-ethinyl estradiol (Sigma Chemical Co., St. Louis, MO, USA) dissolved in two to three drops of redistilled ethanol and diluted to the required concentration with glass-distilled water. 2.4. Statistical analysis The data were analyzed by Student's t test. 3. Results The ethanolic extract of the stem bark of W. tinctoria R. Br. inhibited pregnancy in 100% of rats when administered orally at a daily dose of 250 mg/kg on Days 1–7 postcoitum (Table 1). In addition, no implantations were observed in any of the mated female rats receiving the extract at the same dose in the Days 1–5 postcoitum schedule covering the entire preimplantation period. On fractionation, the hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions showed 100% anti-implantation activity, while the n-butanol-soluble Table 3 Estrogen-agonistic activity of the active ethanolic extract of the stem bark of W. tinctoria and its fractions in ovariectomized immature female Sprague– Dawley rats Treatment a
Daily dose Estrogen-agonistic activity (mg/kg) Vaginal opening c/ Uterine cornification d weight b
Vehicle 17α-Ethinyl estradiol Ethanolic extract of stem bark Hexane-soluble fraction Chloroform-soluble fraction n-Butanol-soluble fraction Water-soluble fraction Water-insoluble fraction
– 0.02 250
17.33±0.88 118.10±1.14a 93.66±2.18e, f
0/0 100/100 100/100
250 250
91.66±1.85e, f 100/100 75.00±4.93e, f, g 60/90
250
64.00±4.81e, f, g
–/80
250 250
65.66±3.52e, f, g 74.00±6.49e, f, g
–/50 60/80
All other relevant comparisons were statistically nonsignificant. a Once daily for three consecutive days, administered orally. Animals of all groups were autopsied 24 h after the last treatment. b Mean±SEM. c Percentage of treated rats with open vagina. d Percentage of cornified cells in vaginal smears. e Versus the vehicle control group (pb.01). f Versus the corresponding ethinyl-estradiol-treated group (pb.01). g Versus the corresponding ethanolic-extract-treated group and hexanesoluble fraction treatment group (pb.001).
Fig. 1. Uterine weight gain in ovariectomized immature female Sprague– Dawley rats treated with the active ethanolic extract of the stem bark of W. tinctoria and its fractions at a 250-mg/kg dose once daily for 3 days by the oral route and autopsied 24 h after the last treatment. Note the marked increase in uterine weight following treatment with the ethanolic extract, as well as its active fractions, depicting moderate to potent estrogen-agonistic activity. The effect observed after the administration of the ethanolic extract and its hexane-soluble fraction was almost comparable to that after the administration of a 0.02-mg/kg dose of 17α-ethinyl estradiol.
fraction intercepted pregnancy in 75% of animals when administered on Days 1–5 postcoitum (Table 2). There was, however, no significant difference in the number of corpora lutea (depicting the total number of ovulations) in animals treated with the ethanolic extract or its fractions in either schedules when compared with the corresponding vehicle control groups (Tables 1 and 2). In immature rat bioassay, the ethanolic extract of the stem bark, as well as all its fractions, exhibited moderate to potent estrogen-agonistic activity. The ethanolic extract induced a marked (N440%, pb.01 vs. vehicle control group) increase in uterine fresh weight, which was almost comparable to that after a 0.02-mg/kg daily dose of the orally effective estrogen 17α-ethinyl estradiol (582%, pb.01; Table 3 and Fig. 1). Moreover, both the ethanolic extract and the ethinyl estradiol induced premature opening of the vagina in 100% of rats, and all the cells in their vaginal smear were cornified. In comparison, while hexane-soluble fraction induced an almost similar estrogenic response as the ethanolic extract, showing 429% uterine weight gain and 100% premature opening of the vagina and cornification of the vaginal epithelium, the chloroform-soluble, n-butanol-soluble, water-soluble and water-insoluble fractions appeared comparatively less estrogenic with, respectively, 333%, 269%, 279% and 327% uterine weight gain (pb.01 vs. ethanolic extract and hexanesoluble fraction; Table 3 and Fig. 1). Their moderate estrogen-agonistic profile, in comparison to the ethanolic extract and the hexane-soluble fraction, was further substantiated by their lower estrogenic responses at the vaginal level. Both the chloroform-soluble and the water-insoluble fractions induced premature vaginal opening in only 60% of the treated rats, with 80–90% of cornified cells in their vaginal smear. In comparison, the n-butanol-soluble and
G. Keshri et al. / Contraception 78 (2008) 266–270
water-soluble fractions failed to induce premature opening of the vagina in any rat; their vaginal smear picture also showed a lower number (50–80%) of cornified cells (Table 3). 4. Discussion The findings of this study provide preliminary evidence of the postcoital antifertility activity of the ethanolic extract of the stem bark of W. tinctoria and its hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions. The ethanolic extract and its fractions also exhibited moderate to potent estrogen-agonistic activity. Pertinently, in most earlier studies in this laboratory, the contraceptive activity of natural products was found to be associated with potent estrogenic activity [12,14–17]. Continuation of further studies on such natural products has invariably been considered undesirable in view of the reported severe health hazards of classic estrogens. According to certain investigators, prolonged phytoestrogen therapy might pose the same health risks as the classic estrogens. This receives support from studies showing increased proliferation of MCF-7 and HeLa cells in vitro by the potent phytoestrogen Ginsenoside Rg1 [18], and of human endometrial stromal and glandular cells by certain isoflavones [19]. However, this is despite the fact that we still do not clearly understand the health risks (or benefits) that prolonged phytocontraceptive/phytoestrogen therapy might pose. While such concerns might appear justifiable from studies where the isolated pure compounds showing promising contraceptive activity also exhibited potent estrogenicity profile [20–22], efforts to pursue promising natural products (to identify contraceptive agents lacking potent estrogenic activity and having beneficial effects on other hormone-dependent and hormone-independent clinical disorders) [23–25] toward a fruitful conclusion for development/lead generation should continue. In species such as rat, mouse and gerbil that exhibit facultative delay of implantation, nidatory estrogen secreted within about 24 h of initiation of implantation is necessary for induction of endometrial receptivity to blastocyst signal (s) [26]. In comparison, in humans, the role of luteal-phase estrogen is believed to be permissive rather than obligatory [27–29], and pregnancies have been established in women without endogenous luteal estrogenic support [30]. However, the negative effect(s) of markedly elevated peak estrogen levels on implantation has been the subject of debate and controversy [31–34]. According to Ma et al. [35], estrogen within a very narrow range determines the duration of window of uterine receptivity, and while the window of uterine receptivity remains open for an extended period at lower estrogen levels, it rapidly closes at higher levels. The uterine refractoriness that follows the receptive state at high estrogen levels is accompanied by aberrant endometrial expression of receptivity/implantation-related genes. Based on this, careful regulation of estrogen levels for improvement
269
of female fertility in in vitro fertilization/embryo transfer programs has been suggested [35]. Similar information on phytoestrogens, when administered during the postovulatory period in any primate species, is, however, lacking. Acknowledgments The authors thank Ms. Mohini Chhabra for technical assistance, and Mr. Jagdish Prasad and Mr. B.P. Mishra for animal handling. S.M.R. was responsible for the collection and identification of plant materials. All protocols used in this study were approved by the Central Drug Research Institute's Animal Ethical Committee CDRI communication no. 7516. References [1] Reddy YSR, Venkatesh S, Ravichandran T, Subburaju T, Suresh B. Pharmacognostical studies on Wrightia tinctoria bark. Pharmaceut Biol 1999;37:291–5. [2] Joshi MC, Patel MB, Mehta PJ. Some folk medicines of drugs. Bull Med Ethnobot Res 1980;1:8–24. [3] Shah GL, Gopal GV. Ethnomedical notes from the tribal inhabitants of the north Gujarat (India). J Eco Tox Bot 1988;6:193–221. [4] Reddy MB, Reddy KR, Reddy MN. A survey of plant crude drugs of Ananthapur District, Andhra Pradesh, India. Int J Crude Drug Res 1989;27:145–55. [5] Singh VP, Sharma SK, Kare VS. Medicinal plants from Ujjain District, Madhya Pradesh: Part 2. Indian Drugs 1980;17:7–12. [6] Siddiqui MB, Hussain W. Traditional antidotes of snake poison. Fitoterapia 1990;61:41–4. [7] Chopra RN, Nayar SL, Chopra IC. Wrightia in glossary of Indian medicinal plants. New Delhi: Publications and Information Directorate, CSIR; 1956. p. 259. [8] Grady D, Grebretsadik T, Ernestwr V, Petitti D. Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol 1995;85:304–13. [9] Beresford SA, Weiss NS, Voigt LF, McKnight B. Risk of endometrial cancer in relation use of oestrogen combined with cyclic progestagen therapy in postmenopausal women. Lancet 1997;349:458–61. [10] Riggs L, Hartmann LC. Selective estrogen receptor modulators — mechanisms of action and application to clinical practice. N Engl J Med 2003;348:618–29. [11] Delmas PD. Treatment of post-menopausal osteoporosis. Lancet 2002; 359:2018–26. [12] Singh MM, Chowdhury SR, Kulshreshtha DK, Kamboj VP. Antigestagenic activity of Ixora finlaysoniana in rats. Contraception 1993;48:178–91. [13] WHO protocol of MB-70: a method for detecting estrogenicity in plant extracts administered orally in the rats, 9856F. Geneva: WHO; 1983. [14] Singh MM, Wadhwa V, Gupta DN, Pal R, Khanna NM, Kamboj VP. Post-coital contraceptive and hormonal profile of Lepidium capitatum. Planta Medica 1984;50:154–7. [15] Singh MM, Gupta DN, Wadhwa V, Jain GK, Khanna NM, Kamboj VP. Post-coital contraceptive efficacy and hormonal profile of ferujol: a new coumarin from Ferula jaeschkeana. Planta Medica 1985;51: 268–70. [16] Wadhwa V, Singh MM, Gupta DN, Singh C, Kamboj VP. Contraceptive and hormonal properties of Achyranthus aspera. Planta Medica 1986;3:231–3. [17] Keshri G, Lakshmi V, Singh MM, Kamboj VP. Post-coital contraceptive property of Ferula assafoetida Linn. in female rats. Pharmaceut Biol 1999;37:273–6.
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[18] Chan RYK, Chen WF, Dong A, Guo D, Wong MS. Estrogen-like activity of Rg1 derived from Panax notoginseng. J Clin Endocrinol Metab 2002;87:3691–5. [19] Kayisli UA, Aksu CAH, Berkkanoglu M, Arici A. Estrogenicity of isoflavones on human endometrial stromal and glandular cells. J Clin Endocrinol Metab 2002;87:5539–44. [20] Singh MM, Gupta DN, Jain GK, Khanna NM, Kamboj VP. Ferujol. Drugs Fut 1986;11:371. [21] Singh MM, Agnihotri A, Garg SN, et al. Antifertility and hormonal properties of certain carotane sesquiterpenes of Ferula jaeschkeana in rat. Planta Medica 1988;6:492–4. [22] Srivastava S, Singh MM, Kulshreshtha DK. A new alkaloid and other anti-implantation principles from Tabernaemontana hyneana. Planta Medica 2001;67:577–9. [23] Kurzer MS, Xu X. Dietary phytoestrogens. Ann Rev Nutr 1997;17: 353–81. [24] Cotterchio M, Boucher BA, Manno M, Gallinger S, Okey A, Harper P. Dietary phytoestrogen intake is associated with reduced colorectal cancer risk. J Nutr 2006;136:3046–53. [25] Bhathena SJ, Velasquez MT. Beneficial role of dietary phytoestrogens in obesity and diabetes. Amer J Clin Nutr 2002;76:1191–201. [26] Singh MM, Chauhan SC, Maitra SC, Trivedi RN, Kamboj VP. Correlation of pinopod development on uterine luminal epithelial surface with hormonal events and endometrial sensitivity in rat. Eur J Endocr 1996;135:107–17. [27] Singh MM, Booth WD. Origin of oestrogen in pre-implantation rabbit blastocysts. J Steroid Biochem 1979;11:723–8.
[28] Makker A, Singh MM. Endometrial receptivity in relation to fertility, infertility and antifertility: clinical assessment using structural, biochemical and molecular markers. Med Res Rev 2006;26:699–746. [29] Edgar DH. Oestrogen and human implantation. Hum Reprod 1995;10: 2–4. [30] Zegers-Hochschild F, Altieri E. Luteal estrogen is not required for the establishment of pregnancy in the human. J Assist Reprod Genet 1995; 12:224–8. [31] Simon C, Cano F, Valbuena D, Remohi A, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responder patients. Hum Reprod 1995;10:2432–4. [32] de Ziegler D, Fanchin R, de Moustier B, Bulleti C. The hormonal control of endometrial receptivity: estrogen (E2) and progesterone. J Reprod Immunol 1998;39:149–66. [33] Ng EHY, You WSB, Lau EYL, So WWK, Ho PC. A rapid decline in serum estradiol concentrations around the midluteal phase had no adverse effect on outcome in 763 assisted reproductive cycles. Hum Reprod 2000;15:1903–8. [34] Freidler S, Ziemerman A, Schachter M, Raziel A, Strassburger D, Ron EIR. The midluteal decline in serum estradiol levels is drastic but not deleterious for implantation after in vitro fertilization and embryo transfer in patients with normal or high responses. Fertil Steril 2005;83: 54–60. [35] Ma W-G, Song H, Das SK, Paria BC, Dey SK. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci 2003;100:2963–8.
Original research article
Postcoital interceptive activity of Wrightia tinctoria in Sprague–Dawley rats: a preliminary study☆ Govind Keshri a , Sudhir Kumar a , Dinesh Kumar Kulshreshtha b , Siron Mani Rajendran c , Man Mohan Singh a,⁎ b
a Division of Endocrinology, Central Drug Research Institute, Lucknow 226 001, India Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226 001, India c Division of Botany, Central Drug Research Institute, Lucknow 226 001, India Received 14 December 2007; revised 10 March 2008; accepted 14 March 2008
Abstract Background: This study was aimed to investigate the pregnancy-interceptive activity of the stem bark of Wrightia tinctoria R.Br. (Family Apocynaceae) administered during the preimplantation, peri-implantation and early postimplantation periods by oral route in adult female Sprague–Dawley rats. Study Design: The ethanolic extract of the stem bark and its serial fractions were administered to female rats on Days 1–7 or 1–5 postcoitum (Day 1: day of sperm-positive vaginal smear) by the oral route. At autopsy on Day 10 postcoitum, the number and status of corpora lutea and implantations were recorded. For estrogen-agonistic activity, immature rats ovariectomized 7 days earlier received the test extract or the vehicle once daily for 3 days and, at autopsy on Day 4, uterine weight, status of vaginal opening and extent of vaginal cornification were recorded. Results: The ethanolic extract of the stem bark of W. tinctoria R.Br. inhibited pregnancy in 100% of rats when administered orally at a 250mg/kg dose on Days 1–7 or 1–5 postcoitum. On fractionation, the hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions showed 100% anti-implantation effect, while n-butanol-soluble fraction intercepted pregnancy in 75% of animals when administered in the Days 1–5 postcoitum schedule. In immature rat bioassay, the active ethanolic extract and its fractions exhibited moderate to potent estrogen-agonistic activity, which might be responsible for their contraceptive action in this species. Conclusions: Findings demonstrate the antifertility activity of the ethanolic extract of the stem bark of W. tinctoria and its hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions. Studies that pursue promising natural products (to identify contraceptive agents from natural sources lacking potent estrogenic activity) towards a fruitful conclusion for development/lead generation should continue. © 2008 Elsevier Inc. All rights reserved. Keywords: Wrightia tinctoria; Stem bark; Contraceptive; Implantation; Early postimplantation; Estrogen-agonistic activity; Rat
1. Introduction Wrightia tinctoria R.Br. (Family Apocynaceae) is widely distributed throughout peninsular India, particularly in tropical areas such as Madhya Pradesh, Rajasthan and
☆ The authors thank the Ministry of Health and Family Welfare, Government of India, for financial support. S.K. thanks the CSIR, New Delhi, for the Senior Research Fellowship award, and M.M.S. thanks the Indian Council of Medical Research for appointment as Emeritus Medical Scientist. ⁎ Corresponding author. Tel.: +91 522 2612411, +91 522 2612418x4307; fax: +91 522 2623405. E-mail address: [email protected] (M.M. Singh).
0010-7824/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2008.03.016
Tamil Nadu. Ethnomedically, the bark of this plant is used: as a galactagogue, to treat abdominal pain [1,2], skin diseases and wounds [3]; as an antipyretic [4], antidysenteric, antidiarrheal and antihemorrhagic [5] agent; and as an antidote for snake poison [6]. Seeds of this plant are also used as an aphrodisiac [7]. In view of the reported severe health hazards of estrogen, such as increased risk of endometrial hyperplasia and carcinoma [8,9], breast cancer [10] and thromboembolic diseases [11], a large number of natural products showing promising antifertility activity in preliminary studies could not be pursued due to their associated estrogen-agonistic activity [12]. The present study is part of the systematic investigation of terrestrial plants and marine flora and fauna being pursued at the
G. Keshri et al. / Contraception 78 (2008) 266–270
267
Table 1 Pregnancy-interceptive activity of the ethanolic extract of the stem bark of W. tinctoria in adult female Sprague–Dawley rats following oral administration in different schedules postcoitum Treatment
Days postcoitum of treatment
Daily dose (mg/kg)
Pregnant a/treated rats
Corpora lutea a, b
Implantationsa, c
Efficacy (%)
Vehicle Ethanolic extract Vehicle Ethanolic extract
1–7 1–7 1–5 1–5
– 250 – 250
6/6 0/5 5/6 0/4
11.0±0.4 9.0±0.3 10.2±0.6 9.8±0.9
9.3±0.3 – 7.0±1.5 –
– 100 – 100
–, none. a Day 10 postcoitum. b Mean±SEM of all treated rats. c Mean±SEM of pregnant rats only.
Central Drug Research Institute (Lucknow, India) to identify new leads for development as effective, reversible, safe and socially acceptable contraceptives and agents for the management of age-related degenerative and lifestyle disorders for human use and welfare, and demonstrates for the first time the contraceptive and hormonal properties of this plant. 2. Materials and methods 2.1. Collection of the plant material The stem bark of this plant was collected from the Maharashtra State of India in November, identified by the Botany Division of the institute and coded (code no. 4372). A voucher (specimen no. 8499) has been prepared and kept at the institute's herbarium. 2.2. Extraction and fractionation Shade-dried powdered stem bark of this plant was extracted three times with 95% ethanol (4 L each) by cold percolation. The combined ethanolic extract was filtered, and the filtrate was concentrated under reduced pressure at below 45°C to remove the last traces of the solvent to yield a brown viscous mass. This ethanolic extract was serially fractionated into hexane-soluble, chloroform-soluble, n-butanol-soluble, water-soluble and water-insoluble fractions and evaluated biologically.
2.3. Biological evaluation Colony-bred immature (25–30 g) female and adult (180–250 g) female and male Sprague–Dawley rats maintained under standard conditions (22±1°C), with alternate 12-h light/dark periods and free access to regular pellet diet (Lipton India Ltd., Bangalore, India) and tap water, were used in this study. Postcoital contraceptive and estrogen-agonistic activities of the test extracts were assessed according to the procedures described earlier [12,13]. Briefly, for contraceptive activity evaluation, adult female rats were caged overnight with coeval males (3:1) of proven fertility, and their vaginal smears were checked on the following morning. The presence of spermatozoa in the vaginal smear was considered as evidence of mating, and the day of their occurrence was designated as Day 1 of pregnancy. Mated females were isolated, randomized (Tables 1 and 2) and treated with the test agents suspended in distilled water, with the help of an almost equal quantity of sterile gum acacia or vehicle on Days 1–7 or 1–5 postcoitum. The required amount of the suspension was made fresh daily. Gum acacia (S.D. Fine Chemicals, Mumbai, India) was extracted twice with redistilled ethanol, dried and tested for any effect on female and male fertility or estrogenicity before use. Animals of all groups were autopsied by cervical dislocation on Day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each animal were recorded.
Table 2 Pregnancy-interceptive activity of fractions of the ethanolic extract of the stem bark of W. tinctoria in adult female Sprague–Dawley rats following oral administration on Days1–5 postcoitum Treatment
Daily dose (mg/kg)
Pregnanta/treated rats
Corpora lutea a, b
Implantationsa, c (mean±SEM)
Efficacy (%)
Vehicle Hexane-soluble fraction Chloroform-soluble fraction n-Butanol-soluble fraction Water-soluble fraction Water-insoluble fraction
– 250 250 250 250 250
5/5 0/3 0/3 1/4 0/4 0/3
11.0±0.5 9.6±1.3 9.7±0.8 8.5±1.2 12.3±1.1 9.7±0.9
7.6±1.7 – – 3.0 – –
– 100 100 75 100 100
a b c
Day 10 postcoitum. Mean±SEM of all treated rats. Mean±SEM of pregnant rats only.
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For determination of estrogen-agonistic activity, immature female rats ovariectomized 7 days earlier were treated orally with the test agent or the vehicle (gum acacia in distilled water) once daily for three consecutive days; at autopsy 24 h after the last treatment, uterine fresh weight was taken, and the status of vaginal opening (open/closed) and the extent of vaginal cornification were recorded. In animals where the vagina was still closed, smear was taken by puncturing the membrane. For comparison, one group of rats was similarly treated with 17α-ethinyl estradiol (Sigma Chemical Co., St. Louis, MO, USA) dissolved in two to three drops of redistilled ethanol and diluted to the required concentration with glass-distilled water. 2.4. Statistical analysis The data were analyzed by Student's t test. 3. Results The ethanolic extract of the stem bark of W. tinctoria R. Br. inhibited pregnancy in 100% of rats when administered orally at a daily dose of 250 mg/kg on Days 1–7 postcoitum (Table 1). In addition, no implantations were observed in any of the mated female rats receiving the extract at the same dose in the Days 1–5 postcoitum schedule covering the entire preimplantation period. On fractionation, the hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions showed 100% anti-implantation activity, while the n-butanol-soluble Table 3 Estrogen-agonistic activity of the active ethanolic extract of the stem bark of W. tinctoria and its fractions in ovariectomized immature female Sprague– Dawley rats Treatment a
Daily dose Estrogen-agonistic activity (mg/kg) Vaginal opening c/ Uterine cornification d weight b
Vehicle 17α-Ethinyl estradiol Ethanolic extract of stem bark Hexane-soluble fraction Chloroform-soluble fraction n-Butanol-soluble fraction Water-soluble fraction Water-insoluble fraction
– 0.02 250
17.33±0.88 118.10±1.14a 93.66±2.18e, f
0/0 100/100 100/100
250 250
91.66±1.85e, f 100/100 75.00±4.93e, f, g 60/90
250
64.00±4.81e, f, g
–/80
250 250
65.66±3.52e, f, g 74.00±6.49e, f, g
–/50 60/80
All other relevant comparisons were statistically nonsignificant. a Once daily for three consecutive days, administered orally. Animals of all groups were autopsied 24 h after the last treatment. b Mean±SEM. c Percentage of treated rats with open vagina. d Percentage of cornified cells in vaginal smears. e Versus the vehicle control group (pb.01). f Versus the corresponding ethinyl-estradiol-treated group (pb.01). g Versus the corresponding ethanolic-extract-treated group and hexanesoluble fraction treatment group (pb.001).
Fig. 1. Uterine weight gain in ovariectomized immature female Sprague– Dawley rats treated with the active ethanolic extract of the stem bark of W. tinctoria and its fractions at a 250-mg/kg dose once daily for 3 days by the oral route and autopsied 24 h after the last treatment. Note the marked increase in uterine weight following treatment with the ethanolic extract, as well as its active fractions, depicting moderate to potent estrogen-agonistic activity. The effect observed after the administration of the ethanolic extract and its hexane-soluble fraction was almost comparable to that after the administration of a 0.02-mg/kg dose of 17α-ethinyl estradiol.
fraction intercepted pregnancy in 75% of animals when administered on Days 1–5 postcoitum (Table 2). There was, however, no significant difference in the number of corpora lutea (depicting the total number of ovulations) in animals treated with the ethanolic extract or its fractions in either schedules when compared with the corresponding vehicle control groups (Tables 1 and 2). In immature rat bioassay, the ethanolic extract of the stem bark, as well as all its fractions, exhibited moderate to potent estrogen-agonistic activity. The ethanolic extract induced a marked (N440%, pb.01 vs. vehicle control group) increase in uterine fresh weight, which was almost comparable to that after a 0.02-mg/kg daily dose of the orally effective estrogen 17α-ethinyl estradiol (582%, pb.01; Table 3 and Fig. 1). Moreover, both the ethanolic extract and the ethinyl estradiol induced premature opening of the vagina in 100% of rats, and all the cells in their vaginal smear were cornified. In comparison, while hexane-soluble fraction induced an almost similar estrogenic response as the ethanolic extract, showing 429% uterine weight gain and 100% premature opening of the vagina and cornification of the vaginal epithelium, the chloroform-soluble, n-butanol-soluble, water-soluble and water-insoluble fractions appeared comparatively less estrogenic with, respectively, 333%, 269%, 279% and 327% uterine weight gain (pb.01 vs. ethanolic extract and hexanesoluble fraction; Table 3 and Fig. 1). Their moderate estrogen-agonistic profile, in comparison to the ethanolic extract and the hexane-soluble fraction, was further substantiated by their lower estrogenic responses at the vaginal level. Both the chloroform-soluble and the water-insoluble fractions induced premature vaginal opening in only 60% of the treated rats, with 80–90% of cornified cells in their vaginal smear. In comparison, the n-butanol-soluble and
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water-soluble fractions failed to induce premature opening of the vagina in any rat; their vaginal smear picture also showed a lower number (50–80%) of cornified cells (Table 3). 4. Discussion The findings of this study provide preliminary evidence of the postcoital antifertility activity of the ethanolic extract of the stem bark of W. tinctoria and its hexane-soluble, chloroform-soluble, water-soluble and water-insoluble fractions. The ethanolic extract and its fractions also exhibited moderate to potent estrogen-agonistic activity. Pertinently, in most earlier studies in this laboratory, the contraceptive activity of natural products was found to be associated with potent estrogenic activity [12,14–17]. Continuation of further studies on such natural products has invariably been considered undesirable in view of the reported severe health hazards of classic estrogens. According to certain investigators, prolonged phytoestrogen therapy might pose the same health risks as the classic estrogens. This receives support from studies showing increased proliferation of MCF-7 and HeLa cells in vitro by the potent phytoestrogen Ginsenoside Rg1 [18], and of human endometrial stromal and glandular cells by certain isoflavones [19]. However, this is despite the fact that we still do not clearly understand the health risks (or benefits) that prolonged phytocontraceptive/phytoestrogen therapy might pose. While such concerns might appear justifiable from studies where the isolated pure compounds showing promising contraceptive activity also exhibited potent estrogenicity profile [20–22], efforts to pursue promising natural products (to identify contraceptive agents lacking potent estrogenic activity and having beneficial effects on other hormone-dependent and hormone-independent clinical disorders) [23–25] toward a fruitful conclusion for development/lead generation should continue. In species such as rat, mouse and gerbil that exhibit facultative delay of implantation, nidatory estrogen secreted within about 24 h of initiation of implantation is necessary for induction of endometrial receptivity to blastocyst signal (s) [26]. In comparison, in humans, the role of luteal-phase estrogen is believed to be permissive rather than obligatory [27–29], and pregnancies have been established in women without endogenous luteal estrogenic support [30]. However, the negative effect(s) of markedly elevated peak estrogen levels on implantation has been the subject of debate and controversy [31–34]. According to Ma et al. [35], estrogen within a very narrow range determines the duration of window of uterine receptivity, and while the window of uterine receptivity remains open for an extended period at lower estrogen levels, it rapidly closes at higher levels. The uterine refractoriness that follows the receptive state at high estrogen levels is accompanied by aberrant endometrial expression of receptivity/implantation-related genes. Based on this, careful regulation of estrogen levels for improvement
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of female fertility in in vitro fertilization/embryo transfer programs has been suggested [35]. Similar information on phytoestrogens, when administered during the postovulatory period in any primate species, is, however, lacking. Acknowledgments The authors thank Ms. Mohini Chhabra for technical assistance, and Mr. Jagdish Prasad and Mr. B.P. Mishra for animal handling. S.M.R. was responsible for the collection and identification of plant materials. All protocols used in this study were approved by the Central Drug Research Institute's Animal Ethical Committee CDRI communication no. 7516. References [1] Reddy YSR, Venkatesh S, Ravichandran T, Subburaju T, Suresh B. Pharmacognostical studies on Wrightia tinctoria bark. Pharmaceut Biol 1999;37:291–5. [2] Joshi MC, Patel MB, Mehta PJ. Some folk medicines of drugs. Bull Med Ethnobot Res 1980;1:8–24. [3] Shah GL, Gopal GV. Ethnomedical notes from the tribal inhabitants of the north Gujarat (India). J Eco Tox Bot 1988;6:193–221. [4] Reddy MB, Reddy KR, Reddy MN. A survey of plant crude drugs of Ananthapur District, Andhra Pradesh, India. Int J Crude Drug Res 1989;27:145–55. [5] Singh VP, Sharma SK, Kare VS. Medicinal plants from Ujjain District, Madhya Pradesh: Part 2. Indian Drugs 1980;17:7–12. [6] Siddiqui MB, Hussain W. Traditional antidotes of snake poison. Fitoterapia 1990;61:41–4. [7] Chopra RN, Nayar SL, Chopra IC. Wrightia in glossary of Indian medicinal plants. New Delhi: Publications and Information Directorate, CSIR; 1956. p. 259. [8] Grady D, Grebretsadik T, Ernestwr V, Petitti D. Hormone replacement therapy and endometrial cancer risk: a meta-analysis. Obstet Gynecol 1995;85:304–13. [9] Beresford SA, Weiss NS, Voigt LF, McKnight B. Risk of endometrial cancer in relation use of oestrogen combined with cyclic progestagen therapy in postmenopausal women. Lancet 1997;349:458–61. [10] Riggs L, Hartmann LC. Selective estrogen receptor modulators — mechanisms of action and application to clinical practice. N Engl J Med 2003;348:618–29. [11] Delmas PD. Treatment of post-menopausal osteoporosis. Lancet 2002; 359:2018–26. [12] Singh MM, Chowdhury SR, Kulshreshtha DK, Kamboj VP. Antigestagenic activity of Ixora finlaysoniana in rats. Contraception 1993;48:178–91. [13] WHO protocol of MB-70: a method for detecting estrogenicity in plant extracts administered orally in the rats, 9856F. Geneva: WHO; 1983. [14] Singh MM, Wadhwa V, Gupta DN, Pal R, Khanna NM, Kamboj VP. Post-coital contraceptive and hormonal profile of Lepidium capitatum. Planta Medica 1984;50:154–7. [15] Singh MM, Gupta DN, Wadhwa V, Jain GK, Khanna NM, Kamboj VP. Post-coital contraceptive efficacy and hormonal profile of ferujol: a new coumarin from Ferula jaeschkeana. Planta Medica 1985;51: 268–70. [16] Wadhwa V, Singh MM, Gupta DN, Singh C, Kamboj VP. Contraceptive and hormonal properties of Achyranthus aspera. Planta Medica 1986;3:231–3. [17] Keshri G, Lakshmi V, Singh MM, Kamboj VP. Post-coital contraceptive property of Ferula assafoetida Linn. in female rats. Pharmaceut Biol 1999;37:273–6.
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[28] Makker A, Singh MM. Endometrial receptivity in relation to fertility, infertility and antifertility: clinical assessment using structural, biochemical and molecular markers. Med Res Rev 2006;26:699–746. [29] Edgar DH. Oestrogen and human implantation. Hum Reprod 1995;10: 2–4. [30] Zegers-Hochschild F, Altieri E. Luteal estrogen is not required for the establishment of pregnancy in the human. J Assist Reprod Genet 1995; 12:224–8. [31] Simon C, Cano F, Valbuena D, Remohi A, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responder patients. Hum Reprod 1995;10:2432–4. [32] de Ziegler D, Fanchin R, de Moustier B, Bulleti C. The hormonal control of endometrial receptivity: estrogen (E2) and progesterone. J Reprod Immunol 1998;39:149–66. [33] Ng EHY, You WSB, Lau EYL, So WWK, Ho PC. A rapid decline in serum estradiol concentrations around the midluteal phase had no adverse effect on outcome in 763 assisted reproductive cycles. Hum Reprod 2000;15:1903–8. [34] Freidler S, Ziemerman A, Schachter M, Raziel A, Strassburger D, Ron EIR. The midluteal decline in serum estradiol levels is drastic but not deleterious for implantation after in vitro fertilization and embryo transfer in patients with normal or high responses. Fertil Steril 2005;83: 54–60. [35] Ma W-G, Song H, Das SK, Paria BC, Dey SK. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci 2003;100:2963–8.