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Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia
Middle East Fertility Society Journal (2011) 16, 154–158
Middle East Fertility Society
Middle East Fertility Society Journal www.mefsjournal.com www.sciencedirect.com
ORIGINAL ARTICLE
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia Oshiozokhai Eboetse Yama *, Francis Ikechukwu Duru, Ademola Ayodele Oremosu, Abraham Adepoju Osinubi, Cressie Carmel Noronha, Abayomi Olugbenga Okanlawon Department of Anatomy, Faculty of Basic Medical Sciences, University of Lagos, Idi-Araba, Lagos, Nigeria Received 18 January 2011; accepted 28 February 2011 Available online 1 April 2011
KEYWORDS Momordica charantia; Sprague–Dawley; Testes; Sperm production
Abstract Introduction: Momordica charantia has been investigated for its effect on various organs and its numerous indications have been cited in literature. There are, however, scanty publications on its effect on the male reproductive system. Objective: To evaluate the effects of methanolic seed extract of M. charantia (MC) on the sperm production (sperm number and motility), testicular volume and testicular testosterone in Sprague–Dawley (S–D) rats. Materials and methods: Twenty adult male S–D rats, weighing 106–200 g allotted randomly into four main groups (A, B, C and D). Groups A, B and C received 15, 25 and 50 mg/100 g b.w/oral of MC, respectively, daily. Group IV rats (control) were fed equal volume of physiological saline. The duration of treatment for both extract and physiological saline was 56 days. The animals were sacrificed by cervical dislocation. Testicular volume, sperm count and motility and testicular testosterone estimated.
* Corresponding author. Address: Department of Anatomy, College of Medicine of the University of Lagos, P.M.B. 12003, Lagos, Nigeria. Tel.: +234 809321251. E-mail address: [email protected] (O.E. Yama). 1110-5690 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2011.02.001
Production and hosting by Elsevier
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia
155
Results: The sperm number and motility were found to be significantly decreased (p < 0.05) with increasing dose. Similarly a dose dependent decrease in the testicular testosterone concentrations and testicular volume (p < 0.05) was also recorded. Conclusion: M. charantia seed extract suppresses the sperm production in rats. Thus, it could be developed into a contraceptive agent for men. 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Tropical forest plant species have served as a source of medicines for people of the tropics for millennia. Many medical practitioners with training in pharmacology and/or pharmacognosy are well aware of the number of modern therapeutic agents that have been derived from tropical forest species. In fact, over 120 pharmaceutical products currently in use are plant-derived, and about 75% of these were discovered by examining the use of these plants in traditional medicine (1). Yet while many modern medicines are plant-derived, the origins of these pharmaceutical agents and their relationship to the knowledge of the indigenous people in the tropical forests are usually omitted. The search for drugs and dietary supplements derived from plants has accelerated in recent years; 25–50% of current pharmaceuticals are derived from them, none are used as anti-fertility agents. Traditional healers have long used plants to prevent contraception; Western medicine is trying to duplicate their successes (2). Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been used in menstrual and pregnancy disorders (3). About four decades ago, there was a strong interest in looking at plants as sources of new pharmaceutical agents. In fact, many modern pharmaceutical companies can trace their origins to products originating from plants. However, advances in molecular biology, genetic engineering, and computational chemistry in the late 1970s and 1980s and, even more recently, advances in combinatorial chemistry (4) created much promise within the pharmaceutical industry, without the need to explore nature’s chemical diversity. Some plant-derived compounds have been found to affect fertility. The use of plant products to regulate fertility is of an ancient origin. In spite of numerous studies, no plant with confirmed contraceptive efficiency but devoid of toxicity has emerged so far (5). A promising oral compound would allow metabolism by the liver and allow reduction of the dose below toxic level. Examples of some plants (herbs) reported to possess anti-fertility properties are date palm, oil palm, gossypol, Carica papaya and Momordica charantia (2,6,7). M. charantia is a monoecious climber with oblong, green coloured fruit that are extensively ribbed (8,9). The fruits are elongated and resemble a warty gourd or cucumber. They are emerald-green in colour when unripe and orange-yellow when ripe. The bitter taste increases as it ripens. It is used traditionally as both food and medicine (9). Antifertility property has been a subject of significant evaluation using animal models with interest in developing an effective oral male contraceptive. The present research was, therefore, intended at investigating the effect of various concentrations of the crude extract M. charantia seed on sperm production in Sprague–Dawley rats.
2.1. Anthology of M. charantia The fresh fruits of M. charantia were procured locally in a market in Lagos State. It was authenticated in the Botany Department of University of Lagos (voucher specimen No. FHI 108422). The fruits were dried in an oven at temperatures between 30 and 40 C for about a week. The dried seeds were extracted and taken to the Pharmacognosy Department of College of Medicine University of Lagos (CMUL), where they were weighed, and the percentage yield/concentration of 230 g of M. charantia in 1000 ml of methanol prepared. 2.2. Animals Twenty adult male S–D rats were assigned randomly into four main groups: A, B, C and D. Each comprised five adult male S–D rats, weighing 106–200 g. They were obtained from the animal house, CMUL and housed in the Anatomy Department CMUL in well-ventilated metal cages under standard room conditions (temperatures 29–30 C, relative humidity 50–55%). They were exposed to a photoperiod of 12 h light, alternating with 12 h darkness; fed rat chow (Livestock feeds Plc. Ikeja, Lagos, Nigeria), and clean tap water were provided ad libitum. The animals were kept for at least 2 weeks to acclimatize to the laboratory condition before experimentation. 2.3. Experimental protocol and autopsy schedule The groups A, B and C were treated daily with 15, 25 and 50 mg/100 g body weight of MC orally, respectively. Group IV rats (control) were fed equal volume of physiological saline. The duration of treatment for both extract and physiological saline was 56 days. A metal canula was used for the gavaging and done between 13 and 16 h daily. The animals were sacrificed 24 h after the last dose. Cervical dislocation was used to induce brief anaesthesia, this followed a ventral laparotomy to deliver testes per abdomen. The harvested testes and Cauda epididymis were neatly dissected out, cleared of fat and connective tissue and weighed. Testicular weight and volume, sperm number and motility including testicular testosterone (from testicular homogenate) were all estimated. 2.4. Testicular homogenate: the supernatant processing This is done by the modified method of Buege and August (1978) (10) 0.25 g of testicular tissue sample was homogenized with a mortar and pestle, in 2.5 ml of 0.15 M KCl. The homogenate was centrifuged at 1000g and the supernatant collected. An aliquot of 2 ml of thiobabituric acid (0.375%, 1 mol/l), 15% trichloroacetic acid was added to 1 ml of the tissue supernatant and mixed vigorously, heated
156
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for 15 min in a boiling water bath (80–90 C). The samples were cooled in ice cold water and centrifuged at 1500g for 15 min. 2.5. Testicular gravimetry The testicular volume was estimated by water displacement method (Archimedes principle). The testicular weight was by electronic balance (2). 2.6. Sperm number and motility analysis Several small cuts were made in the C. epididymis which was then placed in a sterile universal specimen bottle, containing 1 ml of normal saline to allow motile sperm to swim up from the epididymis. Five microlitres of epididymal fluid was delivered onto a glass slide covered with a 22 · 22 mm cover slip (11) and examined under the light microscope at a magnification of ·400. The microscopic field was scanned systematically and each spermatozoon encountered was assessed. Motility was determined by counting the number of immotile spermatozoa and subtracting from the total count · 100%. The motility was simply classified as either motile or non-motile. The procedure was repeated and the average of the two readings taken. The sperm number was determined using the Neubauer improved haemocytometre. A dilution ratio of 1:20 from each well-mixed sample was prepared by diluting 50 ll of epididymal spermatozoa suspended in physiological saline with 950 ll diluent. The diluent was prepared by adding 50 g of sodium carbonate and 10 ml of 35% (v/v) formalin to distilled water and making up the final solution to a volume of 1000 ml (11). Both chambers of the haemocytometer were scored and the average count calculated, provided that the difference between the two counts did not exceed 1/20 of their sum (i.e., less than 10% difference). When two counts were not within 10%, they were discarded, the sample dilution re-mixed and another haemocytometer was prepared and counted. To minimize error, the count was conducted three times on each epididymis. The average of all the six counts (three from each side) from a single rat was taken and this constituted one observation for the sperm number. 2.7. Testicular testosterone assay Testosterone (T) in the homogenate supernatant was determined by the enzyme immunoassay technique based on the principle of competitive binding between T and T-horseradish peroxidase conjugate for a constant amount of rabbit anti-T
Table 1
2.8. Statistical analysis Results were expressed as mean ± standard deviation. Analysis was carried out using analysis of variance (ANOVA) with Scheffe’s post hoc test. The level of significance was considered at p < 0.05. All procedures involving animals in this study conformed to the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (13) and were approved by the Departmental Committee on the Use and Care of Animals in conformity with international acceptable standards. 3. Results and discussion The result of the effects of graded doses of M. charantia seed extract on sperm production ( C. epididymal sperm number and motility) reveals a dose dependent statistically significant decrease (p < 0.05) in the treated groups compared to control (Table 1). While within the groups; C treated with 50 mg/100 g significant difference (p < 0.05) compared to groups A and B treated with 15 and 25 mg/100 g (Table 1). Similarly an associated dose dependent significant decrease in testicular testosterone concentrations (p < 0.05) compared to control (Table 2) was observed. This decrease in sperm production or the cessation of spermatogenesis may be linked to the extract directly suppressing the gonadal androgens resulting in a sub-optimal testosterone levels. This supports the fact that sperm production cannot proceed to optimal completion without a continuous androgen supply (14). This is also in consonance with recent studies which showed that no human subject having a lower than normal testicular testosterone levels had a sperm count greater than 20 million/ml or motility greater than 50% (15). The extract may also have had a circumlocutory association with hormones of the extra-testicular axis (gonadotropins which are essential for initiation and maintenance of
Effects of varying doses of Momordica charantia seed extract on sperm production.
Groups (n = 20) A B C D
(12). Goat antirabbit IgG-coated wells were incubated with T standards, controls, samples (supernatants of testicular homogenates), T-horseradish peroxides conjugate reagent and rabbit anti-T reagent at 37 C for 90 min. Unbound T peroxides conjugate was removed and the wells washed. Tetramethylbenzidine was added and incubated, resulting in the development of blue colour. The colour development was stopped with the addition of 1 MS HCl, and the absorbance measured spectrophotometrically at 450 nm. A standard curve was obtained by plotting the concentration of the standard versus the absorbance and the T concentrations calculated from the standard curve.
Group details 15 mg/100 g b.w 25 mg/100 g b.w 50 mg/100 g b.w Physiological saline
All values are expressed as mean ± standard deviation; b.w = body weight. a Significant difference at p < 0.05 compared to control (group D). b Significant difference at p < 0.05 compared to groups A and B.
Sperm number (·106) a
63.5 ± 10.45 40.5 ± 10.45a 18.01 ± 21.33a,b 219.2 ± 7.52
Sperm motility (%) 70.00 ± 7.07 52.00 ± 0.01a 29.20 ± 13.25a 93.6 ± 7.89
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia Table 2
157
Effects of graded doses of Momordica charantia seed extract on testicular testosterone (TT), weight (TW) and volume (TV).
Groups (n = 20)
Treatment group
TT (mmol/l)
TW (g)
TV (ml)
A B C D
15 mg/100 g b.w 25 mg/100 g b.w 50 mg/100 g b.w Physiological saline
16.14 ± 0.78 13.50 ± 1.43a 10.30 ± 0.95a 18.01 ± 0.83
0.90 ± 0.08 0.56 ± 0.30a 0.32 ± 0.27a 1.14 ± 0.22
0.91 ± 0.08 0.57 ± 0.31a 0.33 ± 0.29a 1.14 ± 0.23
All values are expressed as mean ± standard deviation; b.w = body weight. a Significant difference at p < 0.05 compared to control (group D).
spermatogenesis) by inhibiting the latter, the testosterone production/supply is compromised, since these hormones (essentially luteinizing hormone) through specific receptors found on the surface of Leydig cells are known to control testosterone production and secretion (16–18). Although the levels of gonadotropins were not estimated in this study the observed reduction in the number of sperm number and motility may indicate lowered availability of the gonadotropins. The mean testicular weights in grams of the testes were similar to the values obtained for the testicular volume in millilitres, giving a mean testicular density of one; this follows the same pattern in all. The mean testicular weight and volume of rats fed with physiological saline were 1.14 ± 0.22 g and 1.14 ± 0.23 ml (Table 2). These values became decreased significantly with increasing concentration of the extract. Thus the extract concentrations of 15, 25 and 50 mg/100 g b.w reduced the weight of the testes to 0.90 ± 0.08, 0.56 ± 0.30 and 0.32 ± 0.27 g and the volume to 0.91 ± 0.08, 0.57 ± 0.31 and 0.33 ± 0.29 ml, respectively (Table 2). This reduced testicular weight and volume indicate a wide spread destruction (19) which could be the depleted protein elements in these testes (20,21). Similarly the testicular volume has been shown to associate positively with testosterone level, as well as testicular function (22,23). This means that the decreased testosterone concentration and reduced testicular volume and weight as indicated in our findings (Table 2) signified both an extensive testicular injury and compromised spermatogenesis and male infertility. It is concluded from these obtained data that methanolic seed extract of MC at an oral dose of 50 mg/100 g/day produced a better sterility in male rats as compared to the other doses. Although the oral ingestion of the fruits is safe as demonstrated by its’ long-term consumption in Asian cultures (9); a case of paroxysmal atrial fibrillation was reported recently with the use of the extract (24). However, the toxicity, and safety margin of the seed must be assessed in well-designed human trials. Other reported toxicities include hypoglycaemic coma and convulsions in children, a favism-like syndrome, and increases in gamma-glutamyltransferase and alkaline phosphatase levels in animals (25). Thus the future use of MC extract as a contraceptive agent would dependent on successful isolation of the active principles, toxicological evaluation and its reversibility within a predictable time frame. References (1) Fansworth NR, Bingel AS, Copdell GA, Crane FA, Fong HHS. Potential value of plants as source of new antifertility agents. Part II. J Pharmaceut Sci 1975;64:717–53.
(2) Yama OE, Osinubi AA, Noronha CC, Okanlawon AO. Effect of methanolic seed extract Momordica charantia on body weight and serum cholesterol level of male Sprague–Dawley rats. Nigerian Quart J Hosp Med 2010;20(4):209–13. (3) Technical data on Bitter melon (Momordica charantia), vol. 103; 2002. p. 3–7. (4) Biere DE, Carlson TJ, King SR. Sharma Pharmaceuticals: integrating indigenous knowledge, tropical medicinal plants, medicine, modern science and reciprocity into a novel drug discovery approach.. (5) Sharma RS, Rojalakshimi M, Anthony-Jeyaraj D. Current status of fertility control methods in India. J Biosci 2001;26:391–405. (6) Fansworth NR, Waller DP. Current status of plant products reported to inhibit sperm. Res Front Fertil Regul 1982;2:1–16. (7) Gu ZP, Mao BY, Wang YX, Zhang RA, Tan YZ, Chen ZX, et al.. Low dose gossypol for male contraception. Asian J Androl 2000;2(4):283–7. (8) Bates DM, Merrick LC, Robinson RW. Minor cucurbits. Evolution of crop plants. New York: John Wiley & Sons; 1995, p. 110. (9) Alternative Medicine Review. Description of Momordica charantia, a member medicine. Monograph, Clinical report, vol. 12(4); 2007. p. 360–3. (10) Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–10. (11) Barratt CLR. On the accuracy and clinical value of semen laboratory tests. Hum Reprod 1995;10:250–2. (12) Tietz NW. Clinical guide to laboratory tests. 3rd ed. Philadelphia: W.B. Saunders Company; 1995. (13) American Physiological Society. Guiding principles for research involving animals and human beings. Am J Physiol Regul Integr Comp Physiol 2002;283:R281–3. (14) Mohri H, Suter DA, Brown-Woodman PD, White IG, Ridley DD. Identification of the biochemical lesion produced by alphachlorohydrin in spermatozoa. Nature 1975;255(5503):75–7. (15) Osinubi AA, Adeyemi A, Banmeke A, Ajayi G. The relationship between testosterone concentration sperm count and motility in fertile Nigerian males. Afr J Endocrinol Metab 2003;1:43–5. (16) de Krester DM. Local regulation of testicular function. Int Rev Cytol 1987;10:89–112. (17) de Krester DM, Kerr JB. The cytology of the testis: the physiology of reproduction. New York: Raven Press; 1988. (18) Jarrow JP, Chen H, Rosner W, Trentacoste S, Zirkin BR. Assessment of the androgen environment within the human testis: minimally invasive method to obtain intratesticular fluid. J Androl 2001;4:640–5. (19) Abney T. The potential roles of estrogens in regulating Leydig cell development and function: a review. Steroids 1999; 64:610–7. (20) Sharma A, Verma PK, Dixit VP. Effect of semecarpus anacardium fruits, on the reproductive function of male albino rats. Asian J Androl 2003;5:121–4. (21) Lohiya NK, Manivannan B, Mishra PK, Pathak N, Sriram S, Bhande SS, Panneerdoss S. Chloroform extract of Carica papaya seeds induces long-term reversible azoospermia in langur monkey. Asian J Androl 2002;4(1):17–26.
158 (22) Mahmoud AM, Goemaere S, El-Grem Y, Van-Pottelbergh I, Comhaire FH, Kaufman JM. Testicular volume, in relation to hormonal indices of gonadal function in community-dwelling elderly men. J Clin Endocr Metab 2003;88:179–84. (23) Takihara HL, Cosentino MJ, Sakatoku J, Cockett ATS. Significance of testicular size measurement in anthropology: II. Correlation of testicular size, with testicular function. J Urol 1987;137:416–9.
O.E. Yama et al. (24) Ismail E, Serkan O, Emine CE, Sabri OC. A case of atrial fibrillation due to Momordica charantia (bitter melon). Ann Saudi Med 2010;30(1):86–7. (25) Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm 2003;60(4):356–9.
Middle East Fertility Society
Middle East Fertility Society Journal www.mefsjournal.com www.sciencedirect.com
ORIGINAL ARTICLE
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia Oshiozokhai Eboetse Yama *, Francis Ikechukwu Duru, Ademola Ayodele Oremosu, Abraham Adepoju Osinubi, Cressie Carmel Noronha, Abayomi Olugbenga Okanlawon Department of Anatomy, Faculty of Basic Medical Sciences, University of Lagos, Idi-Araba, Lagos, Nigeria Received 18 January 2011; accepted 28 February 2011 Available online 1 April 2011
KEYWORDS Momordica charantia; Sprague–Dawley; Testes; Sperm production
Abstract Introduction: Momordica charantia has been investigated for its effect on various organs and its numerous indications have been cited in literature. There are, however, scanty publications on its effect on the male reproductive system. Objective: To evaluate the effects of methanolic seed extract of M. charantia (MC) on the sperm production (sperm number and motility), testicular volume and testicular testosterone in Sprague–Dawley (S–D) rats. Materials and methods: Twenty adult male S–D rats, weighing 106–200 g allotted randomly into four main groups (A, B, C and D). Groups A, B and C received 15, 25 and 50 mg/100 g b.w/oral of MC, respectively, daily. Group IV rats (control) were fed equal volume of physiological saline. The duration of treatment for both extract and physiological saline was 56 days. The animals were sacrificed by cervical dislocation. Testicular volume, sperm count and motility and testicular testosterone estimated.
* Corresponding author. Address: Department of Anatomy, College of Medicine of the University of Lagos, P.M.B. 12003, Lagos, Nigeria. Tel.: +234 809321251. E-mail address: [email protected] (O.E. Yama). 1110-5690 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Middle East Fertility Society. doi:10.1016/j.mefs.2011.02.001
Production and hosting by Elsevier
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia
155
Results: The sperm number and motility were found to be significantly decreased (p < 0.05) with increasing dose. Similarly a dose dependent decrease in the testicular testosterone concentrations and testicular volume (p < 0.05) was also recorded. Conclusion: M. charantia seed extract suppresses the sperm production in rats. Thus, it could be developed into a contraceptive agent for men. 2011 Middle East Fertility Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Tropical forest plant species have served as a source of medicines for people of the tropics for millennia. Many medical practitioners with training in pharmacology and/or pharmacognosy are well aware of the number of modern therapeutic agents that have been derived from tropical forest species. In fact, over 120 pharmaceutical products currently in use are plant-derived, and about 75% of these were discovered by examining the use of these plants in traditional medicine (1). Yet while many modern medicines are plant-derived, the origins of these pharmaceutical agents and their relationship to the knowledge of the indigenous people in the tropical forests are usually omitted. The search for drugs and dietary supplements derived from plants has accelerated in recent years; 25–50% of current pharmaceuticals are derived from them, none are used as anti-fertility agents. Traditional healers have long used plants to prevent contraception; Western medicine is trying to duplicate their successes (2). Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been used in menstrual and pregnancy disorders (3). About four decades ago, there was a strong interest in looking at plants as sources of new pharmaceutical agents. In fact, many modern pharmaceutical companies can trace their origins to products originating from plants. However, advances in molecular biology, genetic engineering, and computational chemistry in the late 1970s and 1980s and, even more recently, advances in combinatorial chemistry (4) created much promise within the pharmaceutical industry, without the need to explore nature’s chemical diversity. Some plant-derived compounds have been found to affect fertility. The use of plant products to regulate fertility is of an ancient origin. In spite of numerous studies, no plant with confirmed contraceptive efficiency but devoid of toxicity has emerged so far (5). A promising oral compound would allow metabolism by the liver and allow reduction of the dose below toxic level. Examples of some plants (herbs) reported to possess anti-fertility properties are date palm, oil palm, gossypol, Carica papaya and Momordica charantia (2,6,7). M. charantia is a monoecious climber with oblong, green coloured fruit that are extensively ribbed (8,9). The fruits are elongated and resemble a warty gourd or cucumber. They are emerald-green in colour when unripe and orange-yellow when ripe. The bitter taste increases as it ripens. It is used traditionally as both food and medicine (9). Antifertility property has been a subject of significant evaluation using animal models with interest in developing an effective oral male contraceptive. The present research was, therefore, intended at investigating the effect of various concentrations of the crude extract M. charantia seed on sperm production in Sprague–Dawley rats.
2.1. Anthology of M. charantia The fresh fruits of M. charantia were procured locally in a market in Lagos State. It was authenticated in the Botany Department of University of Lagos (voucher specimen No. FHI 108422). The fruits were dried in an oven at temperatures between 30 and 40 C for about a week. The dried seeds were extracted and taken to the Pharmacognosy Department of College of Medicine University of Lagos (CMUL), where they were weighed, and the percentage yield/concentration of 230 g of M. charantia in 1000 ml of methanol prepared. 2.2. Animals Twenty adult male S–D rats were assigned randomly into four main groups: A, B, C and D. Each comprised five adult male S–D rats, weighing 106–200 g. They were obtained from the animal house, CMUL and housed in the Anatomy Department CMUL in well-ventilated metal cages under standard room conditions (temperatures 29–30 C, relative humidity 50–55%). They were exposed to a photoperiod of 12 h light, alternating with 12 h darkness; fed rat chow (Livestock feeds Plc. Ikeja, Lagos, Nigeria), and clean tap water were provided ad libitum. The animals were kept for at least 2 weeks to acclimatize to the laboratory condition before experimentation. 2.3. Experimental protocol and autopsy schedule The groups A, B and C were treated daily with 15, 25 and 50 mg/100 g body weight of MC orally, respectively. Group IV rats (control) were fed equal volume of physiological saline. The duration of treatment for both extract and physiological saline was 56 days. A metal canula was used for the gavaging and done between 13 and 16 h daily. The animals were sacrificed 24 h after the last dose. Cervical dislocation was used to induce brief anaesthesia, this followed a ventral laparotomy to deliver testes per abdomen. The harvested testes and Cauda epididymis were neatly dissected out, cleared of fat and connective tissue and weighed. Testicular weight and volume, sperm number and motility including testicular testosterone (from testicular homogenate) were all estimated. 2.4. Testicular homogenate: the supernatant processing This is done by the modified method of Buege and August (1978) (10) 0.25 g of testicular tissue sample was homogenized with a mortar and pestle, in 2.5 ml of 0.15 M KCl. The homogenate was centrifuged at 1000g and the supernatant collected. An aliquot of 2 ml of thiobabituric acid (0.375%, 1 mol/l), 15% trichloroacetic acid was added to 1 ml of the tissue supernatant and mixed vigorously, heated
156
O.E. Yama et al.
for 15 min in a boiling water bath (80–90 C). The samples were cooled in ice cold water and centrifuged at 1500g for 15 min. 2.5. Testicular gravimetry The testicular volume was estimated by water displacement method (Archimedes principle). The testicular weight was by electronic balance (2). 2.6. Sperm number and motility analysis Several small cuts were made in the C. epididymis which was then placed in a sterile universal specimen bottle, containing 1 ml of normal saline to allow motile sperm to swim up from the epididymis. Five microlitres of epididymal fluid was delivered onto a glass slide covered with a 22 · 22 mm cover slip (11) and examined under the light microscope at a magnification of ·400. The microscopic field was scanned systematically and each spermatozoon encountered was assessed. Motility was determined by counting the number of immotile spermatozoa and subtracting from the total count · 100%. The motility was simply classified as either motile or non-motile. The procedure was repeated and the average of the two readings taken. The sperm number was determined using the Neubauer improved haemocytometre. A dilution ratio of 1:20 from each well-mixed sample was prepared by diluting 50 ll of epididymal spermatozoa suspended in physiological saline with 950 ll diluent. The diluent was prepared by adding 50 g of sodium carbonate and 10 ml of 35% (v/v) formalin to distilled water and making up the final solution to a volume of 1000 ml (11). Both chambers of the haemocytometer were scored and the average count calculated, provided that the difference between the two counts did not exceed 1/20 of their sum (i.e., less than 10% difference). When two counts were not within 10%, they were discarded, the sample dilution re-mixed and another haemocytometer was prepared and counted. To minimize error, the count was conducted three times on each epididymis. The average of all the six counts (three from each side) from a single rat was taken and this constituted one observation for the sperm number. 2.7. Testicular testosterone assay Testosterone (T) in the homogenate supernatant was determined by the enzyme immunoassay technique based on the principle of competitive binding between T and T-horseradish peroxidase conjugate for a constant amount of rabbit anti-T
Table 1
2.8. Statistical analysis Results were expressed as mean ± standard deviation. Analysis was carried out using analysis of variance (ANOVA) with Scheffe’s post hoc test. The level of significance was considered at p < 0.05. All procedures involving animals in this study conformed to the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (13) and were approved by the Departmental Committee on the Use and Care of Animals in conformity with international acceptable standards. 3. Results and discussion The result of the effects of graded doses of M. charantia seed extract on sperm production ( C. epididymal sperm number and motility) reveals a dose dependent statistically significant decrease (p < 0.05) in the treated groups compared to control (Table 1). While within the groups; C treated with 50 mg/100 g significant difference (p < 0.05) compared to groups A and B treated with 15 and 25 mg/100 g (Table 1). Similarly an associated dose dependent significant decrease in testicular testosterone concentrations (p < 0.05) compared to control (Table 2) was observed. This decrease in sperm production or the cessation of spermatogenesis may be linked to the extract directly suppressing the gonadal androgens resulting in a sub-optimal testosterone levels. This supports the fact that sperm production cannot proceed to optimal completion without a continuous androgen supply (14). This is also in consonance with recent studies which showed that no human subject having a lower than normal testicular testosterone levels had a sperm count greater than 20 million/ml or motility greater than 50% (15). The extract may also have had a circumlocutory association with hormones of the extra-testicular axis (gonadotropins which are essential for initiation and maintenance of
Effects of varying doses of Momordica charantia seed extract on sperm production.
Groups (n = 20) A B C D
(12). Goat antirabbit IgG-coated wells were incubated with T standards, controls, samples (supernatants of testicular homogenates), T-horseradish peroxides conjugate reagent and rabbit anti-T reagent at 37 C for 90 min. Unbound T peroxides conjugate was removed and the wells washed. Tetramethylbenzidine was added and incubated, resulting in the development of blue colour. The colour development was stopped with the addition of 1 MS HCl, and the absorbance measured spectrophotometrically at 450 nm. A standard curve was obtained by plotting the concentration of the standard versus the absorbance and the T concentrations calculated from the standard curve.
Group details 15 mg/100 g b.w 25 mg/100 g b.w 50 mg/100 g b.w Physiological saline
All values are expressed as mean ± standard deviation; b.w = body weight. a Significant difference at p < 0.05 compared to control (group D). b Significant difference at p < 0.05 compared to groups A and B.
Sperm number (·106) a
63.5 ± 10.45 40.5 ± 10.45a 18.01 ± 21.33a,b 219.2 ± 7.52
Sperm motility (%) 70.00 ± 7.07 52.00 ± 0.01a 29.20 ± 13.25a 93.6 ± 7.89
Sperm quotient in Sprague–Dawley rats fed graded doses of seed extract of Momordica charantia Table 2
157
Effects of graded doses of Momordica charantia seed extract on testicular testosterone (TT), weight (TW) and volume (TV).
Groups (n = 20)
Treatment group
TT (mmol/l)
TW (g)
TV (ml)
A B C D
15 mg/100 g b.w 25 mg/100 g b.w 50 mg/100 g b.w Physiological saline
16.14 ± 0.78 13.50 ± 1.43a 10.30 ± 0.95a 18.01 ± 0.83
0.90 ± 0.08 0.56 ± 0.30a 0.32 ± 0.27a 1.14 ± 0.22
0.91 ± 0.08 0.57 ± 0.31a 0.33 ± 0.29a 1.14 ± 0.23
All values are expressed as mean ± standard deviation; b.w = body weight. a Significant difference at p < 0.05 compared to control (group D).
spermatogenesis) by inhibiting the latter, the testosterone production/supply is compromised, since these hormones (essentially luteinizing hormone) through specific receptors found on the surface of Leydig cells are known to control testosterone production and secretion (16–18). Although the levels of gonadotropins were not estimated in this study the observed reduction in the number of sperm number and motility may indicate lowered availability of the gonadotropins. The mean testicular weights in grams of the testes were similar to the values obtained for the testicular volume in millilitres, giving a mean testicular density of one; this follows the same pattern in all. The mean testicular weight and volume of rats fed with physiological saline were 1.14 ± 0.22 g and 1.14 ± 0.23 ml (Table 2). These values became decreased significantly with increasing concentration of the extract. Thus the extract concentrations of 15, 25 and 50 mg/100 g b.w reduced the weight of the testes to 0.90 ± 0.08, 0.56 ± 0.30 and 0.32 ± 0.27 g and the volume to 0.91 ± 0.08, 0.57 ± 0.31 and 0.33 ± 0.29 ml, respectively (Table 2). This reduced testicular weight and volume indicate a wide spread destruction (19) which could be the depleted protein elements in these testes (20,21). Similarly the testicular volume has been shown to associate positively with testosterone level, as well as testicular function (22,23). This means that the decreased testosterone concentration and reduced testicular volume and weight as indicated in our findings (Table 2) signified both an extensive testicular injury and compromised spermatogenesis and male infertility. It is concluded from these obtained data that methanolic seed extract of MC at an oral dose of 50 mg/100 g/day produced a better sterility in male rats as compared to the other doses. Although the oral ingestion of the fruits is safe as demonstrated by its’ long-term consumption in Asian cultures (9); a case of paroxysmal atrial fibrillation was reported recently with the use of the extract (24). However, the toxicity, and safety margin of the seed must be assessed in well-designed human trials. Other reported toxicities include hypoglycaemic coma and convulsions in children, a favism-like syndrome, and increases in gamma-glutamyltransferase and alkaline phosphatase levels in animals (25). Thus the future use of MC extract as a contraceptive agent would dependent on successful isolation of the active principles, toxicological evaluation and its reversibility within a predictable time frame. References (1) Fansworth NR, Bingel AS, Copdell GA, Crane FA, Fong HHS. Potential value of plants as source of new antifertility agents. Part II. J Pharmaceut Sci 1975;64:717–53.
(2) Yama OE, Osinubi AA, Noronha CC, Okanlawon AO. Effect of methanolic seed extract Momordica charantia on body weight and serum cholesterol level of male Sprague–Dawley rats. Nigerian Quart J Hosp Med 2010;20(4):209–13. (3) Technical data on Bitter melon (Momordica charantia), vol. 103; 2002. p. 3–7. (4) Biere DE, Carlson TJ, King SR. Sharma Pharmaceuticals: integrating indigenous knowledge, tropical medicinal plants, medicine, modern science and reciprocity into a novel drug discovery approach.
158 (22) Mahmoud AM, Goemaere S, El-Grem Y, Van-Pottelbergh I, Comhaire FH, Kaufman JM. Testicular volume, in relation to hormonal indices of gonadal function in community-dwelling elderly men. J Clin Endocr Metab 2003;88:179–84. (23) Takihara HL, Cosentino MJ, Sakatoku J, Cockett ATS. Significance of testicular size measurement in anthropology: II. Correlation of testicular size, with testicular function. J Urol 1987;137:416–9.
O.E. Yama et al. (24) Ismail E, Serkan O, Emine CE, Sabri OC. A case of atrial fibrillation due to Momordica charantia (bitter melon). Ann Saudi Med 2010;30(1):86–7. (25) Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm 2003;60(4):356–9.