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Evaluation of contraceptive activity of a mineralo-herbal preparation in Sprague-Dawley rats
Contraception 72 (2005) 454 – 458
Original research article
Evaluation of contraceptive activity of a mineralo-herbal preparation in Sprague-Dawley rats Shobha Rani Srivastavaa, Saurabh Kesarwanib, Govind Keshria, Man Mohan Singha,T a b
Endocrinology Division, Central Drug Research Institute, Lucknow-226 001, India Department of Biomedical Sciences, Bundelkhand University, Jhansi-284 128, India Received 18 March 2005; revised 19 May 2005; accepted 26 May 2005
Abstract Objective: This study was aimed to evaluate a marketed mineralo-herbal preparation containing plants known to have potent contraceptive activity, or contraindicated for use during pregnancy in folklore/ancient Indian literature and recommended for use as an appetizer and headache, hyperacidity and chronic constipation reliever for effect on spermatogenesis and implantation-cum-early postimplantation events in adult Sprague-Dawley rats. Methods: The preparation, suspended in distilled water with the addition of sterile gum acacia, was administered at 1 g/kg daily dose (extrapolated from human dose on surface area basis) to male rats covering one spermatogenic cycle and to female rats during the entire preimplantation and early postimplantation period by oral route. Fertility performance of male rats was tested following mating with untreated fertile females. Results: Findings of this study indicate that the mineralo-herbal preparation at this dose and schedule produced no discernible effect on weight of testis, epididymis and accessory glands, spermatogenesis, vasal sperm picture or mating rate in male rats when administered during the period covering one spermatogenic cycle, but caused significant reduction in number of implantations in females mated with these male rats as well as in female rats treated during the postcoital period. Conclusions: Any adverse effect on fertility/reproductive health following administration over longer periods/at higher doses in humans habituated to continuous use of this preparation cannot be completely ruled out from this limited study. Findings also suggest caution in indiscriminate use of this and other such preparations containing varying amounts of plants/plant products reported to possess contraceptive property and available for other pharmacological indications over-the-counter in most countries. D 2005 Elsevier Inc. All rights reserved. Keywords: Mineralo-herbal preparation; Contraceptive activity; Spermatogenesis; Implantation; Early postimplantation; Fertility; Rat
1. Introduction Human health is of prime importance for a country’s development and progress. Herbal preparations/medicaments have been used since ancient times in many parts of the world including India. In recent years, their use as a popular alternative to modern medicine has increased considerably even in developed countries [1–3]. However, in view of their multiple folkloric medicinal values, a large number of plants/ plant products reported to exhibit potent contraceptive activity in animal models and certain clinical studies, or
T Corresponding author. Tel.: +91 522 2613894; fax: +91 522 2623405. E-mail address: [email protected] (M.M. Singh). 0010-7824/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2005.05.026
contraindicated for use during pregnancy, are being used in varying quantities in several marketed herbal/mineraloherbal preparations/health foods, etc., meant for other pharmacological indications [4 –7]. Interestingly, such preparations are not necessarily being sold by authorized pharmaceutical stores, but are available over-the-counter in general stores [8] in most developing countries, including India. Indiscriminate use of such preparations containing varying amounts of plants/plant products reported to possess contraceptive property might produce adverse effect on fertility/reproductive health following administration over long periods/high doses in humans habituated to their continuous use for other pharmacological indications. This study was aimed to evaluate one such marketed mineralo-herbal preparation containing leaves of Cassia
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
angustifolia (Senna), seeds of Carum copticum (Ajwain), fruits of Terminalia chebula (Himej), fruits of Embelia ribes (Vidang) and roots of Glycyrrhiza glabra (Jethimadh), and recommended for use as an appetizer and headache, hyperacidity and chronic constipation reliever for effect on fertility/reproductive potential using colony-bred adult Sprague-Dawley rats. Pertinently, while seeds of C. copticum (Ajwain) and fruits of E. ribes (Vidang) have been reported to possess antispermatogenic activity [9], embelin isolated from berries of E. ribes has also been documented to provoke significant antiimplantation/postcoital antifertility activity and induce sterility in mice, rats and dogs [10,11]. In addition, it is considered as a potential male antifertility agent as it impairs spermatogenesis and reduces sperm count to the level of infertility [10,12]. Postcoital administration of the extract of stem bark of Terminalia species is also known to cause postimplantation resorption of implantation in rat [13]. Reports of antispermatogenic/abortifacient/emmenagogue activity of Cassia alata, C. copticum and G. glabra are also available [14 –16]. 2. Materials and methods 2.1. Animals and chemicals Colony-bred adult male and female Sprague-Dawley rats (180–225 g) maintained under standard conditions (22F18C), with alternate 12-h light/dark periods and free access to regular pellet diet (Lipton India, Bangalore, India) and tap water, were used in this study. Mineralo-herbal preparation was purchased from the local market. 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 before use. The mineralo-herbal preparation was macerated with almost equal quantity of the sterile gum acacia and suspended in distilled water so as to achieve the desired concentration such that each rat received 1 mL of the suspension per 100 g body weight. The required amount of the suspension was made fresh daily.
455
on day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each rat were recorded. This procedure was repeated each day while continuing the respective treatment until each male rat of both groups had mated an untreated female. On the day following positive mating, the male rats were autopsied by cervical dislocation, and their testes, epididymides, seminal vesicles, ventral prostate and coagulating glands were carefully dissected free of adhering tissues, washed in ice-cold phosphate-buffered saline (pH 7.2), gently blotted on a wet filter paper and weighed on an electronic balance (Afcoset, Mumbai, India; Model: ER-120A). Contents of vas deferens of each rat were squeezed into a drop of phosphate-buffered saline (pH 7.2), quickly mixed with a glass rod and examined for the presence of spermatozoa and their motility under a trinocular microscope (Leica, Cambridge, England; Model: DMLB) fitted with a live Image Acquisition System (Leica; Model: IM 50). The testes of each rat were then slit and fixed in 10% neutral buffered formalin (pH 7.2). After 24 h of fixation, the tissues were cut into 3- to 4-mm pieces and refixed in fresh 10% neutral buffered formalin (pH 7.2) for another 24 h. After 48 h of fixation, the tissues were transferred to 70% isopropanol, dehydrated in ascending concentrations of isopropanol, cleared in xylene and embedded in paraffin wax. Six-micrometer-thick cross-sections were cut on a rotary microtome (Leica, Nussloch, Germany; Model: RM 2165)
2.2. Effect on spermatogenesis, accessory sex organs and fertility performance Adult male rats were randomized and treated with the mineralo-herbal preparation at a daily dose of 1 g/kg (extrapolated from human dose on surface area basis [4]) by the oral route or an equal quantity of the vehicle (gum acacia in distilled water) covering one spermatogenic cycle [17]. Fertility performance of each male rat was checked after 60 days of continuous treatment. For this purpose, each male rat of both groups was caged overnight with two female rats in proestrus stage of estrous cycle, and their vaginal smear was examined under light microscopy on the following morning. Presence of spermatozoa in the vaginal smear was considered as evidence of mating and the day of occurrence designated as day 1 postcoitum. Mated females were isolated, kept in separate cages containing fresh rice husk and autopsied
Fig. 1. Evaluation of effect of mineralo-herbal preparation administered at 1 g/kg daily oral dose covering one spermatogenic cycle on (A) body weight and (B) weight of testes, epididymides and accessory sex organs in adult male Sprague-Dawley rats. The treated male rats were autopsied on the day following mating to an untreated female. Note lack of any ) discernible effect of the treatment of the mineralo-herbal preparation ( ). with levels being almost similar to that in vehicle control rats (
456
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
Fig. 2. Hematoxylin and eosin-stained cross-sections of testis of adult male rat treated with the mineralo-herbal preparation at 1 g/kg daily oral dose covering one spermatogenic cycle. Note similar histoarchitecture of the testis with apparently normal spermatogenesis and presence of spermatozoa in the lumen of seminiferous tubules of (A and B) vehicle control and (C and D) mineralo-herbal preparation-treated rats. A and C, 100; B and D, 40.
collected on 3-aminopropyl-triethoxy silane (Sigma, St. Louis, MO, USA) and coated microslides, dewaxed, rehydrated, washed with phosphate-buffered saline, stained with hematoxylin and eosin, and photographed on an
automatic camera (Leica, Heerbrugg, Switzerland; Model: DC300) fitted to a trinocular Leica microscope. The status of spermatogenesis was evaluated by a semiquantitative scoring system [18].
Table 1 Effect of oral administration of a mineralo-herbal preparation covering one spermatogenic cycle on fertility performance of adult male Sprague-Dawley rats Male rats
Untreated female rats
Treatment
No.
Day posttreatment of mating
No.
Body weighta (g)
Corporaa lutea
Implantationsa
Vehicle (gum acacia in distilled water)
1 2 3 4 5 6 6/6b 1 2 3 4 5 6 7 7/7b
60 62 60 66 65 65
1 2 3 4 5 6 6/6c 1 2 3 4 5 6 7 7/6c
220 200 230 220 220 225 219F4.1d 220 220 190 225 190 200 210 208F2.1d
13 12 15 14 17 14 14.2F0.7d 13 13 13 15 9 13 13 12.7F0.7d
12 9 12 12 14 11 11.7F0.7e 10 10 6 0 8 10 2 7.7F1.3e,f
Mineralo-herbal preparation (1 g/kg/day, po)
62 60 65 69 65 66 66
All other relevant comparisons were statistically nonsignificant. a At autopsy on day 10 postcoitum. b Adult male rats: treated/mated to fertile females. c Adult female rats: mated/pregnant on day 10 postcoitum. d MeanFSEM of all treated rats in each group. e MeanFSEM of only the pregnant rats in each group. f p b .05, vs. corresponding vehicle control group.
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
2.3. Effect on implantation and early postimplantation pregnancy Adult female Sprague-Dawley rats were caged overnight with coeval males of proven fertility (3:1), and their vaginal smears were checked on the following morning. The day of presence of spermatozoa was taken as day 1 of pregnancy. Mated females were isolated, randomized and treated with the mineralo-herbal preparation (1 g/kg) or an equal quantity of the vehicle (gum acacia in distilled water) on days 1–7 postcoitum. The rats were autopsied on day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each rat were recorded [19]. 2.4. Statistical analysis The data were analyzed by Students’ t test.
457
of the treatment on early postimplantation events as all the implantations appeared normal without any sign of resorption (Table 1). 3.2. Effect on implantation and early postimplantation pregnancy A significant (p b.05, vs. vehicle control group) reduction in number of implantations was evident in the female rats treated with 1 g/kg daily dose of the mineralo-herbal preparation covering the entire preimplantation and early postimplantation period. No apparent adverse effect of the treatment on early postimplantation events was, however, observed, and all implantations in the two groups appeared normal without any sign of resorption (Fig. 3). 4. Discussion
3. Results 3.1. Effect on spermatogenesis, accessory sex organs and fertility performance Oral administration of the mineralo-herbal preparation to colony-bred adult male Sprague-Dawley rats at a daily dose of 1 g/kg covering the entire period of spermatogenesis did not produce any effect on body weight or weight of testes, epididymides and accessory glands (Fig. 1A, B). There was also no apparent difference in motility of vasal spermatozoa examined immediately after autopsy in the two groups of animals. Histologically, the testis showed normal spermatogenesis with presence of spermatozoa in the lumen of seminiferous tubules of animals of both treatment groups (Fig. 2). While there was no effect of the mineralo-herbal preparation on mating rate and all male rats of the two groups mated when co-caged with fertile females, a significant reduction (p b.05, vs. vehicle control group) in implantation number in females becoming pregnant following mating with males treated with the mineralo-herbal preparation was observed. There was, however, no apparent adverse effect
Fig. 3. Effect of treatment of mineralo-herbal preparation at 1 g/kg daily oral dose covering entire preimplantation and early postimplantation period in adult female Sprague-Dawley rats. Note significant (p b .01) reduction in mean number of implantations in females treated with the preparation ) as compared to that of vehicle control group ( ). There was, ( however, no apparent adverse effect of the treatment on early postimplantation events as all implantations appeared normal without any sign of resorption. Values indicate meanFSEM.
Findings of this study indicate that the mineralo-herbal preparation at the dose and schedule evaluated in this study produced no discernible effect on weight of testis, epididymis and accessory glands, spermatogenesis, vasal sperm picture or mating rate in male rats when administered during the period covering one spermatogenic cycle. It, however, caused significant reduction in number of implantations in females mated with these male rats as well as in female rats treated during the entire preimplantation and early postimplantation period. Herbal preparations/medicaments have been used since ancient times in many parts of the world, including India [20]. In recent years, their use as a popular alternative to modern medicine has increased considerably even in developed countries [1–3]. In fact, the impressive number of phytopharmaceuticals currently in use in modern therapy, representing N 30% of the pharmaceutical market, substantiates the need to explore and authenticate contraceptive as well as medicinal properties of natural products for human use and welfare. However, despite lack of sufficient information on the precise nature of constituents used and absence of properly documented experimental, clinical or pharmacological data [8], people generally continue to equate herbal with safety [21]. A need for proper characterization, standardization, safety and efficacy evaluation of the Ayurvedic/ marketed herbal formulations/medicaments/health foods, etc., is now being increasingly felt worldwide [1–3,22–27]. It also seems imperative to evolve reliable, sensitive and selective methods for quality control of raw materials as well as finished products and policy for approval of quality, efficacy and safety, including interaction with other commonly used pharmaceuticals and promotion of scientifically validated herbal products. A large number of plants such as C. angustifolia (Senna), C. copticum (Ajwain), T. chebula (Himej), E. ribes (Vidang) and G. glabra (Jethimadh) have been reported to exhibit potent contraceptive activity in animal models and certain clinical studies [9–16]. However, in view of their multiple folkloric medicinal values, some of these plants are being
458
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
used in varying quantities in several marketed herbal/ mineralo-herbal preparations/health foods, etc., meant for other pharmacological indications. This limited study indicates that the mineralo-herbal preparation containing these plants/plant product at the dose and schedule evaluated did not exhibit any discernible effect on reproductive function in male and female rats, except significant reduction in the number of implantation sites in females mated with male rats treated with this preparation during the period covering one spermatogenic cycle, as well as in female rats treated with this preparation during the postcoital period. It, however, does not completely rule out any adverse effects of this preparation on fertility/reproductive health following administration over longer periods/at higher doses in humans habituated to their continuous use. The findings also suggest caution in indiscriminate use of this and other such preparations containing varying amounts of plants/plant products reported to possess contraceptive properties and available over-thecounter in most countries. Acknowledgments The authors thank Mr. GK Nagar and Mr. NP Mishra for technical assistance and the Ministry of Health and Family Welfare, Government of India, for financial support. One of us (SK) thanks the Director, Central Drug Research Institute, for a 3-month training program as part of the M.Sc. curriculum. CDRI communication no. 6727. References [1] Corns CM. Herbal remedies and clinical biochemistry. Ann Clin Biochem 2003;40:489 – 507. [2] Barnes J. Quality, efficacy and safety of complementary medicines: fashions, facts and the future. Part I. Regulation and quality. Br J Clin Pharmacol 2003;55:226 – 33. [3] Rousseaux CG, Schachter H. Regulatory issues concerning the safety, efficacy and quality of herbal remedies. Birth Defects Res Part B Dev Reprod Toxicol 2003;68:505 – 10. [4] Freireich EJ, Gehan EA, Rail DP, Sechmidt LH, Skipper HE. Quantitative comparison of toxicity of anti-cancer agents in mouse, rat, dog, monkey and man. Cancer Chemother Rep 1966;50:219 – 44. [5] Seetharam YN, Sujeeth H, Jyothisharan G, et al. Antifertility effect of ethanolic extract of Amalakyadi churna in male albino mice. Asian J Androl 2003;5:247 – 50. [6] Namjoshi AN. The Ayurvedic formulary of India Part-1, first edition. Govt of India Ministry of Health and Family Planning. Delhi (India)7 Health Controller Publications; 1978. p. 1 – 200. [7] Srikantha Murthy KR. Churna Kalpana. Sarngadharacharya, Sarangadhara-Samhita. 3rd ed. Varanasi (India)7 Chaukhamba Orientalia; 1993. p. 84 – 101 [quoted by Seetharam et al. Asian J Androl 2003;5: 247–50].
[8] Bond C, Hannaford P. Issues related to monitoring the safety of overthe-counter (OTC) medicines. Drug Saf 2003;26:1065 – 74. [9] Singh RH, Medikonda PK. An appraisal of the ancient Ayurvedic approach to fertility regulation. In: Chowdhury SR, Gupta CM, Kamboj VP, editors. Golden Jubilee symposium on current status in fertility regulation: indigenous and modern approaches. Lucknow (India)7 Central Drug Research Institute, Army Printing Press; 2001. p. 21 – 34. [10] Gupta S, Sanyal SN. Biodistribution of embelin, a benzoquinone of male antifertility potential. Fitoterapia 1991;62:419 – 28. [11] Purandare TV, Kholkute SD, Gurjar A, et al. Semen analysis and hormonal levels in Bonnet macaques administered Embelia ribes berries, an indigenous plant having contraceptive activity. Indian J Exp Biol 1979;17:935 – 6. [12] Gupta S, Kanwar U, Sanyal SN. Inhibition of reproductive tissue carbohydrate metabolism and reversibility of the effects of embelin, a plant benzoquinone of antifertility potential. Fitoterapia 1990;6: 133 – 43. [13] Gupta DN, Keshri G, Lakshmi V, Kapil RS. Post-coital contraceptive efficacy of Terminalia arjuna in albino rats. Fitoterapia 1989; 60:275 – 6. [14] Ali SM, Gupta UC, Jain GC. Investigation on the antifertility effects of Cassia alata Linn. in male rats. Proceedings of the National Conference on Recent Advances in Reproductive Health, Jaipur, India, 6–8 Feb; 2003. p. 99. [15] Casey RCD. Alleged anti-fertility plants of India. Indian J Med Sci 1960;14:591 – 600. [16] Saha JC, Savini EC, Kasinathan S. Ecbolic properties of Indian medicinal plants, Part I. Indian J Med Res 1961;49:130 – 51. [17] Kamboj VP, Dhawan BN. Research on plants for fertility regulation in India. J Ethnopharmacol 1982;6:191 – 226. [18] Russell LD, Ettlin RA, Sinha Hakim AP, Clegg ED. The classification and timing of spermatogenesis. Histological and histopathological evaluation of the testis, Chapter 2. Clearwater (FL)7 Cache River Press; 1990. p. 41 – 58. [19] Keshri G, Lakshmi V, Singh MM. Pregnancy interceptive activity of Melia azedarach Linn. in adult female Sprague-Dawley rats. Contraception 1999;68:303 – 6. [20] Maurya R, Srivastava S, Kulshreshtha DK, Gupta CM. Traditional remedies for fertility regulation. Curr Med Chem 2004;11:1431 – 50. [21] De Smet PA. Health risks of herbal remedies. Drug Saf 1995;13: 81 – 93. [22] Labadie RP. Problems and possibilities in the use of traditional drugs. J Ethnopharmacol 1986;15:221 – 30. [23] Goldman P. Herbal medicines today and the roots of modern pharmacology. Ann Intern Med 2001;135:594 – 600. [24] Ghosh R, Kamboj VP, Singh MM. Interaction with anti-implantation and estrogen antagonistic activities of dl-ormeloxifene, a selective estrogen receptor modulator, by tetracycline in female SpragueDawley rats. Contraception 2001;64:261 – 9. [25] Khurana M, Lal J, Singh MM, Paliwal JK, Kamboj VP, Gupta RC. Evaluation of interaction potential of certain concurrently administered drugs with pharmacological and pharmacokinetic profile of centchroman in rats. Contraception 2002;66:47 – 56. [26] Chan K. Some aspects of toxic contaminants in herbal medicines. Chemosphere 2003;52:1361 – 71. [27] Patwardhan B, Vaidya ADB, Chorghade M. Ayurveda and natural products drug discovery. Curr Sci 2004;86:789 – 99.
Original research article
Evaluation of contraceptive activity of a mineralo-herbal preparation in Sprague-Dawley rats Shobha Rani Srivastavaa, Saurabh Kesarwanib, Govind Keshria, Man Mohan Singha,T a b
Endocrinology Division, Central Drug Research Institute, Lucknow-226 001, India Department of Biomedical Sciences, Bundelkhand University, Jhansi-284 128, India Received 18 March 2005; revised 19 May 2005; accepted 26 May 2005
Abstract Objective: This study was aimed to evaluate a marketed mineralo-herbal preparation containing plants known to have potent contraceptive activity, or contraindicated for use during pregnancy in folklore/ancient Indian literature and recommended for use as an appetizer and headache, hyperacidity and chronic constipation reliever for effect on spermatogenesis and implantation-cum-early postimplantation events in adult Sprague-Dawley rats. Methods: The preparation, suspended in distilled water with the addition of sterile gum acacia, was administered at 1 g/kg daily dose (extrapolated from human dose on surface area basis) to male rats covering one spermatogenic cycle and to female rats during the entire preimplantation and early postimplantation period by oral route. Fertility performance of male rats was tested following mating with untreated fertile females. Results: Findings of this study indicate that the mineralo-herbal preparation at this dose and schedule produced no discernible effect on weight of testis, epididymis and accessory glands, spermatogenesis, vasal sperm picture or mating rate in male rats when administered during the period covering one spermatogenic cycle, but caused significant reduction in number of implantations in females mated with these male rats as well as in female rats treated during the postcoital period. Conclusions: Any adverse effect on fertility/reproductive health following administration over longer periods/at higher doses in humans habituated to continuous use of this preparation cannot be completely ruled out from this limited study. Findings also suggest caution in indiscriminate use of this and other such preparations containing varying amounts of plants/plant products reported to possess contraceptive property and available for other pharmacological indications over-the-counter in most countries. D 2005 Elsevier Inc. All rights reserved. Keywords: Mineralo-herbal preparation; Contraceptive activity; Spermatogenesis; Implantation; Early postimplantation; Fertility; Rat
1. Introduction Human health is of prime importance for a country’s development and progress. Herbal preparations/medicaments have been used since ancient times in many parts of the world including India. In recent years, their use as a popular alternative to modern medicine has increased considerably even in developed countries [1–3]. However, in view of their multiple folkloric medicinal values, a large number of plants/ plant products reported to exhibit potent contraceptive activity in animal models and certain clinical studies, or
T Corresponding author. Tel.: +91 522 2613894; fax: +91 522 2623405. E-mail address: [email protected] (M.M. Singh). 0010-7824/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2005.05.026
contraindicated for use during pregnancy, are being used in varying quantities in several marketed herbal/mineraloherbal preparations/health foods, etc., meant for other pharmacological indications [4 –7]. Interestingly, such preparations are not necessarily being sold by authorized pharmaceutical stores, but are available over-the-counter in general stores [8] in most developing countries, including India. Indiscriminate use of such preparations containing varying amounts of plants/plant products reported to possess contraceptive property might produce adverse effect on fertility/reproductive health following administration over long periods/high doses in humans habituated to their continuous use for other pharmacological indications. This study was aimed to evaluate one such marketed mineralo-herbal preparation containing leaves of Cassia
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
angustifolia (Senna), seeds of Carum copticum (Ajwain), fruits of Terminalia chebula (Himej), fruits of Embelia ribes (Vidang) and roots of Glycyrrhiza glabra (Jethimadh), and recommended for use as an appetizer and headache, hyperacidity and chronic constipation reliever for effect on fertility/reproductive potential using colony-bred adult Sprague-Dawley rats. Pertinently, while seeds of C. copticum (Ajwain) and fruits of E. ribes (Vidang) have been reported to possess antispermatogenic activity [9], embelin isolated from berries of E. ribes has also been documented to provoke significant antiimplantation/postcoital antifertility activity and induce sterility in mice, rats and dogs [10,11]. In addition, it is considered as a potential male antifertility agent as it impairs spermatogenesis and reduces sperm count to the level of infertility [10,12]. Postcoital administration of the extract of stem bark of Terminalia species is also known to cause postimplantation resorption of implantation in rat [13]. Reports of antispermatogenic/abortifacient/emmenagogue activity of Cassia alata, C. copticum and G. glabra are also available [14 –16]. 2. Materials and methods 2.1. Animals and chemicals Colony-bred adult male and female Sprague-Dawley rats (180–225 g) maintained under standard conditions (22F18C), with alternate 12-h light/dark periods and free access to regular pellet diet (Lipton India, Bangalore, India) and tap water, were used in this study. Mineralo-herbal preparation was purchased from the local market. 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 before use. The mineralo-herbal preparation was macerated with almost equal quantity of the sterile gum acacia and suspended in distilled water so as to achieve the desired concentration such that each rat received 1 mL of the suspension per 100 g body weight. The required amount of the suspension was made fresh daily.
455
on day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each rat were recorded. This procedure was repeated each day while continuing the respective treatment until each male rat of both groups had mated an untreated female. On the day following positive mating, the male rats were autopsied by cervical dislocation, and their testes, epididymides, seminal vesicles, ventral prostate and coagulating glands were carefully dissected free of adhering tissues, washed in ice-cold phosphate-buffered saline (pH 7.2), gently blotted on a wet filter paper and weighed on an electronic balance (Afcoset, Mumbai, India; Model: ER-120A). Contents of vas deferens of each rat were squeezed into a drop of phosphate-buffered saline (pH 7.2), quickly mixed with a glass rod and examined for the presence of spermatozoa and their motility under a trinocular microscope (Leica, Cambridge, England; Model: DMLB) fitted with a live Image Acquisition System (Leica; Model: IM 50). The testes of each rat were then slit and fixed in 10% neutral buffered formalin (pH 7.2). After 24 h of fixation, the tissues were cut into 3- to 4-mm pieces and refixed in fresh 10% neutral buffered formalin (pH 7.2) for another 24 h. After 48 h of fixation, the tissues were transferred to 70% isopropanol, dehydrated in ascending concentrations of isopropanol, cleared in xylene and embedded in paraffin wax. Six-micrometer-thick cross-sections were cut on a rotary microtome (Leica, Nussloch, Germany; Model: RM 2165)
2.2. Effect on spermatogenesis, accessory sex organs and fertility performance Adult male rats were randomized and treated with the mineralo-herbal preparation at a daily dose of 1 g/kg (extrapolated from human dose on surface area basis [4]) by the oral route or an equal quantity of the vehicle (gum acacia in distilled water) covering one spermatogenic cycle [17]. Fertility performance of each male rat was checked after 60 days of continuous treatment. For this purpose, each male rat of both groups was caged overnight with two female rats in proestrus stage of estrous cycle, and their vaginal smear was examined under light microscopy on the following morning. Presence of spermatozoa in the vaginal smear was considered as evidence of mating and the day of occurrence designated as day 1 postcoitum. Mated females were isolated, kept in separate cages containing fresh rice husk and autopsied
Fig. 1. Evaluation of effect of mineralo-herbal preparation administered at 1 g/kg daily oral dose covering one spermatogenic cycle on (A) body weight and (B) weight of testes, epididymides and accessory sex organs in adult male Sprague-Dawley rats. The treated male rats were autopsied on the day following mating to an untreated female. Note lack of any ) discernible effect of the treatment of the mineralo-herbal preparation ( ). with levels being almost similar to that in vehicle control rats (
456
S.R. Srivastava et al. / Contraception 72 (2005) 454 – 458
Fig. 2. Hematoxylin and eosin-stained cross-sections of testis of adult male rat treated with the mineralo-herbal preparation at 1 g/kg daily oral dose covering one spermatogenic cycle. Note similar histoarchitecture of the testis with apparently normal spermatogenesis and presence of spermatozoa in the lumen of seminiferous tubules of (A and B) vehicle control and (C and D) mineralo-herbal preparation-treated rats. A and C, 100; B and D, 40.
collected on 3-aminopropyl-triethoxy silane (Sigma, St. Louis, MO, USA) and coated microslides, dewaxed, rehydrated, washed with phosphate-buffered saline, stained with hematoxylin and eosin, and photographed on an
automatic camera (Leica, Heerbrugg, Switzerland; Model: DC300) fitted to a trinocular Leica microscope. The status of spermatogenesis was evaluated by a semiquantitative scoring system [18].
Table 1 Effect of oral administration of a mineralo-herbal preparation covering one spermatogenic cycle on fertility performance of adult male Sprague-Dawley rats Male rats
Untreated female rats
Treatment
No.
Day posttreatment of mating
No.
Body weighta (g)
Corporaa lutea
Implantationsa
Vehicle (gum acacia in distilled water)
1 2 3 4 5 6 6/6b 1 2 3 4 5 6 7 7/7b
60 62 60 66 65 65
1 2 3 4 5 6 6/6c 1 2 3 4 5 6 7 7/6c
220 200 230 220 220 225 219F4.1d 220 220 190 225 190 200 210 208F2.1d
13 12 15 14 17 14 14.2F0.7d 13 13 13 15 9 13 13 12.7F0.7d
12 9 12 12 14 11 11.7F0.7e 10 10 6 0 8 10 2 7.7F1.3e,f
Mineralo-herbal preparation (1 g/kg/day, po)
62 60 65 69 65 66 66
All other relevant comparisons were statistically nonsignificant. a At autopsy on day 10 postcoitum. b Adult male rats: treated/mated to fertile females. c Adult female rats: mated/pregnant on day 10 postcoitum. d MeanFSEM of all treated rats in each group. e MeanFSEM of only the pregnant rats in each group. f p b .05, vs. corresponding vehicle control group.
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2.3. Effect on implantation and early postimplantation pregnancy Adult female Sprague-Dawley rats were caged overnight with coeval males of proven fertility (3:1), and their vaginal smears were checked on the following morning. The day of presence of spermatozoa was taken as day 1 of pregnancy. Mated females were isolated, randomized and treated with the mineralo-herbal preparation (1 g/kg) or an equal quantity of the vehicle (gum acacia in distilled water) on days 1–7 postcoitum. The rats were autopsied on day 10 postcoitum, and the number and status of corpora lutea and implantation sites in each rat were recorded [19]. 2.4. Statistical analysis The data were analyzed by Students’ t test.
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of the treatment on early postimplantation events as all the implantations appeared normal without any sign of resorption (Table 1). 3.2. Effect on implantation and early postimplantation pregnancy A significant (p b.05, vs. vehicle control group) reduction in number of implantations was evident in the female rats treated with 1 g/kg daily dose of the mineralo-herbal preparation covering the entire preimplantation and early postimplantation period. No apparent adverse effect of the treatment on early postimplantation events was, however, observed, and all implantations in the two groups appeared normal without any sign of resorption (Fig. 3). 4. Discussion
3. Results 3.1. Effect on spermatogenesis, accessory sex organs and fertility performance Oral administration of the mineralo-herbal preparation to colony-bred adult male Sprague-Dawley rats at a daily dose of 1 g/kg covering the entire period of spermatogenesis did not produce any effect on body weight or weight of testes, epididymides and accessory glands (Fig. 1A, B). There was also no apparent difference in motility of vasal spermatozoa examined immediately after autopsy in the two groups of animals. Histologically, the testis showed normal spermatogenesis with presence of spermatozoa in the lumen of seminiferous tubules of animals of both treatment groups (Fig. 2). While there was no effect of the mineralo-herbal preparation on mating rate and all male rats of the two groups mated when co-caged with fertile females, a significant reduction (p b.05, vs. vehicle control group) in implantation number in females becoming pregnant following mating with males treated with the mineralo-herbal preparation was observed. There was, however, no apparent adverse effect
Fig. 3. Effect of treatment of mineralo-herbal preparation at 1 g/kg daily oral dose covering entire preimplantation and early postimplantation period in adult female Sprague-Dawley rats. Note significant (p b .01) reduction in mean number of implantations in females treated with the preparation ) as compared to that of vehicle control group ( ). There was, ( however, no apparent adverse effect of the treatment on early postimplantation events as all implantations appeared normal without any sign of resorption. Values indicate meanFSEM.
Findings of this study indicate that the mineralo-herbal preparation at the dose and schedule evaluated in this study produced no discernible effect on weight of testis, epididymis and accessory glands, spermatogenesis, vasal sperm picture or mating rate in male rats when administered during the period covering one spermatogenic cycle. It, however, caused significant reduction in number of implantations in females mated with these male rats as well as in female rats treated during the entire preimplantation and early postimplantation period. Herbal preparations/medicaments have been used since ancient times in many parts of the world, including India [20]. In recent years, their use as a popular alternative to modern medicine has increased considerably even in developed countries [1–3]. In fact, the impressive number of phytopharmaceuticals currently in use in modern therapy, representing N 30% of the pharmaceutical market, substantiates the need to explore and authenticate contraceptive as well as medicinal properties of natural products for human use and welfare. However, despite lack of sufficient information on the precise nature of constituents used and absence of properly documented experimental, clinical or pharmacological data [8], people generally continue to equate herbal with safety [21]. A need for proper characterization, standardization, safety and efficacy evaluation of the Ayurvedic/ marketed herbal formulations/medicaments/health foods, etc., is now being increasingly felt worldwide [1–3,22–27]. It also seems imperative to evolve reliable, sensitive and selective methods for quality control of raw materials as well as finished products and policy for approval of quality, efficacy and safety, including interaction with other commonly used pharmaceuticals and promotion of scientifically validated herbal products. A large number of plants such as C. angustifolia (Senna), C. copticum (Ajwain), T. chebula (Himej), E. ribes (Vidang) and G. glabra (Jethimadh) have been reported to exhibit potent contraceptive activity in animal models and certain clinical studies [9–16]. However, in view of their multiple folkloric medicinal values, some of these plants are being
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used in varying quantities in several marketed herbal/ mineralo-herbal preparations/health foods, etc., meant for other pharmacological indications. This limited study indicates that the mineralo-herbal preparation containing these plants/plant product at the dose and schedule evaluated did not exhibit any discernible effect on reproductive function in male and female rats, except significant reduction in the number of implantation sites in females mated with male rats treated with this preparation during the period covering one spermatogenic cycle, as well as in female rats treated with this preparation during the postcoital period. It, however, does not completely rule out any adverse effects of this preparation on fertility/reproductive health following administration over longer periods/at higher doses in humans habituated to their continuous use. The findings also suggest caution in indiscriminate use of this and other such preparations containing varying amounts of plants/plant products reported to possess contraceptive properties and available over-thecounter in most countries. Acknowledgments The authors thank Mr. GK Nagar and Mr. NP Mishra for technical assistance and the Ministry of Health and Family Welfare, Government of India, for financial support. One of us (SK) thanks the Director, Central Drug Research Institute, for a 3-month training program as part of the M.Sc. curriculum. CDRI communication no. 6727. References [1] Corns CM. Herbal remedies and clinical biochemistry. Ann Clin Biochem 2003;40:489 – 507. [2] Barnes J. Quality, efficacy and safety of complementary medicines: fashions, facts and the future. Part I. Regulation and quality. Br J Clin Pharmacol 2003;55:226 – 33. [3] Rousseaux CG, Schachter H. Regulatory issues concerning the safety, efficacy and quality of herbal remedies. Birth Defects Res Part B Dev Reprod Toxicol 2003;68:505 – 10. [4] Freireich EJ, Gehan EA, Rail DP, Sechmidt LH, Skipper HE. Quantitative comparison of toxicity of anti-cancer agents in mouse, rat, dog, monkey and man. Cancer Chemother Rep 1966;50:219 – 44. [5] Seetharam YN, Sujeeth H, Jyothisharan G, et al. Antifertility effect of ethanolic extract of Amalakyadi churna in male albino mice. Asian J Androl 2003;5:247 – 50. [6] Namjoshi AN. The Ayurvedic formulary of India Part-1, first edition. Govt of India Ministry of Health and Family Planning. Delhi (India)7 Health Controller Publications; 1978. p. 1 – 200. [7] Srikantha Murthy KR. Churna Kalpana. Sarngadharacharya, Sarangadhara-Samhita. 3rd ed. Varanasi (India)7 Chaukhamba Orientalia; 1993. p. 84 – 101 [quoted by Seetharam et al. Asian J Androl 2003;5: 247–50].
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