Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2005; 7 (4): 405-410 |
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This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Complementary Medicine - Antispermatogenic activity of Morinda lucida extract in male rats Yinusa Raji1, Olumide S. Akinsomisoye2, Toyin M. Salman1 1Department of Physiology, College of Medicine, University of Ibadan,Ibadan, Nigeria Abstract Aim: To investigate the effect of Morinda lucida Benth (Rubiaceae) on the reproductive activity of male albino rats. Methods: Two groups of rats were treated with 400 mg/(kg·d) of Morinda lucida leaf extract for 4 and 13 weeks, respectively. The control rats received the vehicle. All the treated rats had corresponding recovery groups. At the end of each experimental period, animals were killed and organ weights, sperm characteristics, serum testosterone levels, histology of the testes and fertility were assessed. Results: Morinda lucida leaf extract did not cause any changes in body and somatic organ weights, but significantly increased the testis weight (P < 0.05). The sperm motility and viability, and the epididymal sperm counts of rats treated for 13 weeks were significantly reduced (P < 0.05). Sperm morphological abnormalities and serum testosterone levels were significantly increased (P < 0.05). There were various degrees of damage to the seminiferous tubules. The extract reduced the fertility of the treated rats by reducing the litter size. Reversal of these changes, however, occurred after a period of time. Conclusion: The extract of Morinda lucida has reversible antispermatogenic properties. (Asian J Androl 2005 Dec; 7: 405-410) Keywords: Morinda lucida; sperm; fertility; testosterone; antispermatogenic agents Correspondence to: Dr Yinusa Raji, Department of Physiology,
College of Medicine, University of Ibadan, Ibadan, Nigeria.
1 Introduction
Morinda lucida Benth (Rubiaceae) is a medium-sized tree about 15 m tall and is widely used as a medicinal plant in West Africa, especially in Nigeria. The leaves are used as an ingredient of "fever teas", which are usually taken for the traditional treatment of malaria. The plant is also used as a general febrifuge, analgesic and laxative. A weak decoction of the stem bark is used for the treatment of severe jaundice [1]. The major constituents of Morinda lucida extract are various types of alkaloids-anthraquinones and anthraquinols. Adewunmi and Adesogan [2] isolated and characterized two compounds , oruwalol and oruwal, and ten anthraquinones from the stem of the plant. Anthraquinones isolated from Morinda lucida have shown to possess potent in vitro activity against Plasmodium falciparum [3]. Trypanocidal activity of the methanol extract of the leaf of this plant was reported by Asuzu and Chineme [4]. Obih et al. [5] reported the antimalarial activity of Morinda lucida against Plasmodium berghei berghei in mice. Many antimalarial agents have been demonstrated to have various degrees of antifertility activities [6, 7]. The use of Morinda lucida extracts in the treatment of various ailments is increasing, but its impacts on the reproductive system have not been reported. The present study was to investigate the effects of Morinda lucida leaf extract on male rat reproductive functions, such as sperm characteristics, fertility and testosterone secretion.
2 Materials and methods
2.1 Plant material and extraction procedure The leaves of Morinda lucida were obtained in March 2002 from a tree beside the Pharmacognosy Laboratory, University of Ibadan. The plant specimen was authenticated at the herbarium of the Forestry Research Institute of Nigeria. The leaves were air-dried and then grounded into powder that was subjected to Soxhlet cold extraction using methanol as the solvent.
2.2 Animals Adult male Wistar strain albino rats (200 g-300 g), purchased from the Central Animal House, College of Medicine, University of Ibadan, were used for the study. The animals were housed in wire mesh cages under standard environmental conditions with the provision of 12 h light and 12 h darkness. Rat cubes (Ladokun feeds, Nig. Ltd., Ibadan, Nigeria) and water were provided ad libitum.
2.3 Experimental protocol A total of 30 male albino rats were divided into six groups of five rats each with two treated, two recovery and two control groups. The two treated groups received intragastric (i.g.) administration of 400 mg/(kg·d) for 4 and 13 weeks, respectively. The corresponding recovery groups were also treated for 4 and 13 weeks and allowed to recover for 4 and 13 weeks, respectively. The choice of duration of extract administration for 4 weeks was to mimic the use of the plant in traditional treatment of malaria while that of 13 weeks was intended to cover the spermatogenic cycles in rats. The control groups received the equivalent volume of the vehicle (Tween 80 in equivalent amount of normal saline). The animals were weighed daily throughout the duration of the study.
2.4 Autopsy and organ weights At the end of the treatment and recovery periods, each rat was anesthetized with 25 % urethane at a dose of 0.6 mL/100 g (intraperitoneally, i.p.). The testes, seminal vesicles and epididymis were dissected and weighed immediately.
2.5 Blood sample collection Blood was collected from each rat via the left ventricular cardiac puncture. Serum for the estimation of testosterone levels was obtained from the blood.
2.6 Sperm characteristic analysis The testes from each rat were carefully exposed and one of them was removed together with its epididymis. The epididymis was separated and the epididymal fluid was collected from the caudal part. The progressive sperm motility, sperm count, live/dead ratio (viability) and morphology were determined [8, 9].
2.7 Testicular histology The testes of the rats were prefixed in Bouin-Hollande solution prior to the histologic studies as earlier described [6, 10].
2.8 Hormone assay Serum testosterone assay was carried out using the tube-based enzyme immunoassay (EIA) method [11]. This is a standardized method used by WHO and part of its program for human reproduction research. The EIA testosterone kits were produced by Immunometrics (London, UK) and obtained from Nzemat (Lagos, Nigeria). The procedures for the assay as contained in the manufacturer¡¯s manual were strictly followed. The within assay variation was 8.1 % and the sensitivity was 0.3 ng/mL. The optical density was read using a spectrophotometer (Jenway, 6300 spectrophotometer, UK) that was sensitive at wavelengths between 492 nm and 550 nm.
2.9 Fertility test Treated male rats were cohabited with untreated parous proestrus female albino rats at a ratio of 1:2. Cohabitation commenced on the first day of the last week of extract treatment and the first day of the last week of the recovery period. A single time point fertility test for each rat was carried out using the following formula: percentage fertility success is equal to the number of pregnant females divided by the number of mated females multiplied by 100. The litter size of the pregnant rats was also determined at the end of the gestation period. 2.10 Statistical analysis Data were expressed as mean ± SEM. Statistical significance between the various groups was determined using unpaired t-test and ANOVA [12].
3 Results
3.1 Effect on body and organ weight of male rats There were no significant changes in the body weight of treated rats compared with the controls after 4 and 13 weeks of treatment. The recovery group also showed an insignificant change in body weight compared with their control counterpart. Administration of 400 mg/(kg·d) of the extract to the rats for 4 and 13 weeks increased their testicular weights compared with the controls (Table 1). There were also no significant changes in the mean weight of the seminal vesicles in the treated groups (Table 1). 3.2 Effect on spermatozoal indices The effects on sperm motility, counts, viability and morphology were shown in Table 2.
3.2.1 Motility Oral administration of Morinda lucida leaf extract for 4 and 13 weeks, progressively and significantly reduced (P < 0.05) sperm progressive motility. There was also a significant decrease (P < 0.05) in percentage sperm motility in the recovery groups compared with the respective control groups (Table 2).
3.2.2 Epididymal sperm count Daily administration of the extract for 13 weeks significantly reduced (P < 0.01) the mean epididymal sperm counts compared with their controls and the recovery groups, which showed an insignificant increase. 3.2.3 Viability (live/dead ratio) There was a significant decrease (P < 0.05) in the mean percentage of live sperm of rats treated with the extract compared with their control counterparts. The mean percentage of live sperm of rats in the 4-week recovery group showed a significant reduction (P < 0.05).
3.2.4 Morphology The commonest morphological abnormality of sperm in rats that received the extract for 4 and 13 weeks was the "curved tail" which accounted for over 70 % of the abnormalities observed. Although the recovery groups showed a lesser occurrence of morphological abnormali-ties, the increase in abnormalities were significantly higher (P < 0.05) than those in the controls.
3.2.5 Serum testosterone levels The mean serum testosterone level of rats treated with 400 mg/(kg·d) of the extract for 4 and 13 weeks significantly increased (P < 0.01) compared with the controls (Figure 1).
3.2.6 Histologic evaluation Administration of 400 mg/(kg·d) of the extract for 4 and 13 weeks caused visible lesions within the seminiferous tubules compared with the controls (Figure 2). There was degeneration and disorganization of the plasmalemma in the basal portion of some of the seminiferous tubules. However, regenerative changes were observed in the recovery group.
3.2.7 Fertility tests The fertility of the treated rats in terms of percentage mated female rats was unaffected. However, the fertility of these rats was significantly affected by the extract treatments in terms of the number of litters born by the cohabited female rats. Female rats cohabited with treated male rats bore significantly reduced (P < 0.05) number of litters: 6.20 ± 0.32 and 4.00 ± 0.27 for 4 and 13 weeks, respectively (control: 9.40 ± 0.44).
4 Discussion
Chronic administration of Morinda lucida leaf extract could impair reproductive activities in male albino rats. The extract caused an increase in the weight of the testes, which was accompanied by an increase in the serum levels of testosterone. Similar changes have been reported with the extract of Zingiber officinale and Pentadiplendra brazzeana in rats [13, 14]. Others have reported testicular weight reduction accompanied by decreased serum testosterone levels in male rats treated with the extracts of Quassia amara [7], Azadirachta indica [6] and gossypol, a phenolic compound extracted from the cotton seed [15]. Both Quassia amara and Azadirachta indica have been demonstrated to possess potent antimalarial properties [6, 7]. Since all organs of male reproduction are androgen dependent, they serve as indicators of the Leydig cell function or androgen action [16]. Testosterone in association with follicle stimulating hormone normally acts on the seminiferous tubules to initiate and maintain spermatogenesis [16]. However, a significant decrease in the epididymal sperm count of rats treated with the extract for 13 weeks was recorded despite the high serum testosterone level. This was supported by the various degrees of degeneration in the histologic sections of the testes, suggesting that Morinda lucida extract administration for a long period was capable of permeating the blood-testis barrier [17]. This probably led to tubular fluid retention, hence non-restoration of testicular weights during recovery. The appreciable increase in the epididymal sperm counts of rats in the recovery groups suggests that the extract could be responsible for the observed decrease in the first instance. This was further corroborated by the fact that no primary abnormalities were recorded in sperm morphology, which normally occurs during the process of spermatogenesis. Reduction in the progressive epididymal sperm motility of the treated rats could be responsible for the decrease in the average number of litters born by the female rats cohabited with the extract treated male rats leading to the suspicion for the presence of a spermatoxic agent on maturing or mature spermatozoa [18] in the extract. Sperm motility depends on the coordinated propagated flagella wave under acetyl cholinesterase control [19]. Fructose utilization and glucose oxidation are important means by which spermatozoa derive energy for their motility. Morinda lucida has been shown to possess hypoglycemic and anti-hyperglycemic activities [20]. The reduction in motility recorded in this study could be due to the acetylcholinesterase inhibition and glucose lowering properties of this plant. The progressive motility improved in the recovery groups, suggesting that the deleterious effect was reversible. Phytochemical analyses revealed that Morinda lucida contains various types of anthraquinones and anthranquinols [2]. Koumaglo et al. [3] and Sittie et al. [21] reported the strong activity of anthraquinones isolated from Morinda lucida against the growth of Plasmodium falciparum. Furthermore anthraquinones isolated from Morinda lucida extract have been reported to possess potent activities against the growth of the chloroquine resistant Plasmodium falciparum. Since several studies have reported antifertility effects of antimalarial agents including chloroquine, the antifertility activities exhibited by Morinda lucida in this study could be associated with anthraquinones presented in the extract. Further studies aimed at elucidating the activities of Morinda lucida extract would be worthwhile.
Acknowledgment
This work was partly supported by the University of Ibadan Senate Research grants SRG/COM/2000/11A to Yinusa Raji and we would, therefore, like to express our gratitude to the University. References 1 Oliver-Bever B. Medicinal Plants in Tropical West Africa. Cambridge: Cambridge University Press; 1986. p89-90. 2 Adewunmi CO, Adesogan EK. Anthraquinones and oruwacin from Morinda lucida as possible agents in fasciolasis and schistosomiasis control. Fitoterapia 1984; 55: 259-63. 3 Koumaglo K, Gbeassor M, Nikabu O, de Souza C, Werner W. Effects of three compounds extracted from Morinda lucida on Plasmodium falciparum. Planta Med 1992; 58: 533-4. 4 Asuzu IU, Chineme CN. Effects of Morinda lucida leaf extract on Trypanosoma brucei brucei infection in mice. J Ethnopharmacol 1990; 30: 307-31. 5 Obih PO, Makinde JM, Laoye JO: Investigations of various extracts of Morinda lucida for antimalaraial actions on Plasmodium berghei berghei in mice. Afr J Med Med Sci 1985; 14: 45-9. 6 Raji Y, Udoh US, Mewoyeka OO, Ononye FC, Bolarinwa AF. Implication of reproductive endocrine malfunction in male antifertility efficacy of Azadirachta indica extract in rats. Afr J Med Med Sci 2003; 32: 159-65. 7 Raji Y, Bolarinwa AF. Antifertility activity of Quassia amara in male rats - in vivo study. Life Sci 1997; 64: 1067-74. 8 World Health Organization. WHO Laboratory Manual for Examination of Human Semen and Semen-Cervical Mucus Interaction, 2nd ed. London: Cambridge University Press; 1987. p1-10. 9 Blom E. The ultrastructure of some characteristic sperm defects and a proposal for a new classification of the bull spermiogram. Mord Vet Med 1973; 25: 283-5. 10 Akpantah AO, Oremosu AA, Ajala MO, Noronha CC, Okanlawon AO. The effects of crude extract of Garcinia kola seed on the histology and hormonal milieu of male Sprague-Dawley rats' reproductive organs. Nig J Health & Biomed Sci 2003; 2: 40-6. 11 Raji Y, Ifabunmi SO, Akinsomisoye OS, Morakinyo AO, Oloyo AK. Gonadal responses to antipsychotic drugs: Chlopromazine and thioridazine reversibly suppress testicular functions in albino rats. Intl J Pharmacol 2005; 1: 287-92. 12 Snedecor GW, Cochran WG. Statistical Method, 7th edn. Iowa: Iowa State University Press; 1980. p215. 13 Kamtchouing P, Mbongue Fandio GY, Dimo T, Jatsa HB. Evaluation of androgenic activity of Zingiber officinale and Pentadiplandra brazzeana in male rats. Asian J Androl 2002; 4: 299-301. 14 Watcho P, Kamtchouing P, Sokeng SD, Moundipa PF, Tantchou J, Essame JL, Koueta N. Androgenic effect of Modia whitei roots in male rats. Asian J Androl 2004; 6: 269-72. 15 Qian SZ, Wang ZG. Gossypol: a potential antifertility agent for males. Annu Rev Pharmacol Toxicol 1984;24: 329-60. 16 Rommerts FF. How much androgen is required for maintenance of spermatogenesis? J Endocrinol 1988; 116: 7-9. 17 Baldessarini RJ. Drugs and the treatment of psychiatric disorders: the pharmacological basis of psychiatric disorders. In: Goodman LS, Limbird LE, Milinoff PB, Ruddon RW, Gilman AG, editors. Alfred Goodman and Gilman, The Pharmacological Basis of Therapeutics. 6th ed. New York: Mac-Millian; 1980. p301-417. 18 Verma RJ, Chinoy NJ. Effect of papaya seed extract on microenvironment of cauda epididymis. Asian J Androl 2001; 3: 143-6. 19 Nelson L. Quantitative evaluation of sperm motility control mechanisms. Biol Reprod 1972; 6: 319-24. 20 Olajide OA, Awe SO, Makinde JM, Morebise O. Evaluation of the anti-diabetic property of Morinda lucida leaves in streptozotocin-diabetic rats. J Pharm Pharmacol 1999; 51: 1321-4. 21 Sittie AA, Lemmich E, Hviid L, Kharazmi A, Nkrumah FK, Christensen SB. Structure-activity studies: in vitro antilei-shmanial and antimalarial activities of anthraquinones from Morinda lucida. Planta Med 1999; 65: 259-61. |
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