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Evaluation of contraceptive activity of methanol extract of Dendrophthoe falcata stem in male albino rats
Journal of Ethnopharmacology 112 (2007) 215–218
Ethnopharmacological communication
Evaluation of contraceptive activity of methanol extract of Dendrophthoe falcata stem in male albino rats R.S. Gupta ∗ , J.B.S. Kachhawa Reproductive Physiology Section, Center of Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 23 May 2006; received in revised form 17 December 2006; accepted 23 February 2007 Available online 27 February 2007
Abstract The present study was undertaken to evaluate the contraceptive efficacy of methanol extract of Dendrophthoe falcata Ettingsh (familyLoranthaceae), stem in male albino rats as reported in folk remedies. Adult proven fertile male rats were gavaged methanol extract of D. falcata stem at 50, 100 and 200 mg/rat/day for 60 days. The activity was compared with standard drug, i.e. Lonidamine. On day 61 the animals were autopsied and the testes, epididymides, seminal vesicle and ventral prostate were dissected out and weighed. Sperm motility and density and serum testosterone level were assessed. The sperm motility and density were significantly reduced. The histoarchitecture of testes revealed degenerative changes in the seminiferous tubules, arrest of spermatogenesis at the stage of round spermatid. Serum testosterone levels were decreased significantly in all treatment groups. It is concluded that D. falcata methanol stem extract showed a significant effect on fertility in male rats as reported in folk remedies. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Dendrophthoe falcata; Contraceptive activity; Sperm dynamics; Spermatogenesis
1. Introduction Dendrophthoe falcata Ettingsh (family-Loranthaceae), is a large bushy parasitic shrub with gray bark, one of the seven species of the genus Dendrophthoe found in India (The Wealth of India, 1952). It is also known as Loranthus falcatus Linn. f. Its bark is taken as astrin, narcotic used for wounds and menstrual troubles and also as a remedy for consumption, asthma (Chopra et al., 1956). D. falcata treats kidney stones (Singh and Ali, 1992), having diuretic and antilithiatic activity (Alkutti et al., 1993). It is widely used as antibiotics (Bhakuni et al., 1998). Chemical investigations revealed that some flavonoids, i.e. quercetin, kaempeferol, quercitrin and rutin present in methanol extract of D. falcata. However, the further elution with hexane and hexane benzene (19:1) gave rise to unidentified oils and plant alcohols. With hexane–benzene elute (3:1) -amyrin acetate was isolated and identified by hydrolysis to -amyrin. In the next elute of hexane benzene (1:1) an apparently new triter-
∗
Corresponding author at: TR-03, Teacher’s Hostel, University of Rajasthan, Jaipur 302004, India. Tel.: +91 141 2711228. E-mail address: gupta [email protected] (R.S. Gupta). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.02.022
pene C33 H54 O4 was isolated. Further elution with benzene and benzene ethylacetate furnished -sitosterol, stigmasterol and oleanolic acid (Anjaneyula et al., 1977). In the present investigation contraceptive efficacy of D. falcata stem extract was investigated with the sole objective of developing a male pill of plant origin since its use in folk remedies as antifertility agent (Jain and Defillips, 1991). 2. Material and methods The fresh stems of D. falcata were collected from Nahargarh Biological Garden, Amer in the month of August 2004 and was authenticated by Department of Botany, University of Rajasthan, Jaipur, where a voucher specimen (RUBL-19905) has been deposited. The shade-dried stem (250 g) was powdered and extracted with 100% v/v methanol (b.p. 60–80 ◦ C) in a soxhlet for 36 h (WHO, 1983). The obtained extract was concentrated under reduced pressure and yielded 28 g dark brown semi-solid mass. This mass was washed with pet ether to remove the fatty components and subjected as drug, fed to rats. Fifty proven fertile male rats of Wistar strain, obtained from Jamia Hamdard, Hamdard University, New Delhi (India) were
100 (−ve) 100 (+ve) Values are in mean ± S.E.M. (n = 10). Levels of significance: * P ≤ 0.01; ** P ≤ 0.001 compared with group I (control); a P ≤ 0.01; a+ P ≤ 0.001 compared with group III; b P ≤ 0.01; b+ P ≤ 0.001 compared with group IV.
2.03 ± 0.04**,a+,b+ 15.76 ± 1.19**,a+,b 1.51 ± 0.13**,a+,b 25.60 ± 1.24**,a+,b+ 310.01 ± 10.27*,a 450.43 ± 14.41**,a 752.40 ± 19.39**,a+,b+ 174.39 ± 5.58ns
287.79 ± 4.23**,a+,b+
100 (−ve) 100 (+ve) 2.78 ± 0.06**,a 22.47 ± 1.24**,a+ 2.06 ± 0.11**,a+ 40.50 ± 1.23**,a 487.01 ± 15.86* 897.16 ± 20.86**,a 176.72 ± 6.99ns
315.92 ± 3.45**,a
324.53 ± 11.22*,a
87.50 (−ve) 100 (+ve) 3.16 ± 0.09** 28.71 ± 1.71* 3.11 ± 0.14** 52.07 ± 2.88* 997.30 ± 23.51*
510.16 ± 14.49*
Post
172.41 ± 7.11ns
351.15 ± 9.18*
343.61 ± 10.97ns
100 (+ve) 100 (+ve) 4.27 ± 0.13 1.74 ± 0.05** 48.29 ± 4.34 17.22 ± 1.00** 4.92 ± 0.18 1.19 ± 0.10** 70.01 ± 3.46 17.88 ± 1.30** 381.92 ± 14.58 270.65 ± 8.59** 601.33 ± 17.92 465.71 ± 10.22** 1198.36 ± 34.89 683.48 ± 12.06** 168.44 ± 6.82 182.27 ± 6.49ns
Testis
Group I Control Group II Lonidamine 50 mg/rat/day Group III D. falcata stem extract 50 mg/rat/day Group I V D. falcata stem extract 100 mg/rat/day Group V D. falcata stem extract 200 mg/rat/day
435.52 ± 15.61 236.59 ± 5.09**
Fertility (%)
Pre
Serum testosterone (ng/dl) Cauda epididymides Testis
Sperm density (million/ml)
Sperm motility (%) (Cauda epididymides) Ventral prostate Seminal vesicle Epididymides
Organ weight (mg/100 g.b.wt)
The oral administration of D. falcata stem extract at all dose levels did not cause significant change in the body weight but a great decline in the weights of testes, epididymides, seminal vesicle and ventral prostate were observed in all treatment groups when compared with Group I animals, the decrease in weight was found dose dependent, however the most effective dose level was 200 mg/rat/day in comparison to standard drug (Table 1). Serum testosterone levels of treated animals were decreased by 59.25%, 25.99%, 34.89% and 52.45% in Groups II, III, IV and V, respectively, when compared to controls, i.e. Group I (Table 1). Animals treated with D. falcata methanol stem extract showed a remarkable depression of spermatogenesis which is primarily attributed by decreased testicular weight and reduced seminiferous tubular diameter, which may be due to wide spread cellular damage and androgen deprivation (Keel and Abney, 1980). Reduction in the testosterone level in the rats treated with D. falcata is due to decrease in number of Leydig cell as testosterone is produced by Leydig cells (Ramaswami and Marshall, 2003).
Body weight (g)
3. Results and discussion
Treatment
used for the present study. An acclimatization period of ten days was allowed before experimentation. Water and food was given ad libitum. Ten male rats were selected randomly in the five groups and administered with 50 mg/rat/day of standard drug, i.e. Lonidamine(1-(2,4-dichlorobenzyl)-1H-indazole-3carboxylic acid) belongs to a group of indazole-carboxylic acids that were originally synthesized as anticancer drugs and were also found to be potent inhibitors of spermatogenesis (Grima et al., 2001) as well as D. falcata stem (100% MeOH) extract at the dose of 50, 100 and 200 mg/rat/day; p.o. for 60 days. The required dose was freshly dissolved in 0.5 ml of distilled water and administered to each treated animal every morning with feeding needle. The study was approved by the ethical committee of the Department of Zoology, University of Rajasthan, Jaipur (India). Indian National Science Academy, New Delhi (INSA, 2000) guidelines were followed for maintenance and use of the experimental animals. Fertility was calculated in control as well as in treated groups (WHO, 1983). Twenty-four hours after the last dose, rats were weighed and autopsied under light ether anesthesia. The reproductive organs, i.e. testes, epididymides, seminal vesicles, and ventral prostate were separated out, cleared off fats and connective tissues and weighed. Cauda epididymal sperm motility and density, and testicular sperm counts were made according to the method of Prasad et al. (1972). Serum testosterone levels were assayed from samples using radio immuno assay method (Belanger et al., 1980). One testes of each animal was fixed in Bouin’s fluid, dehydrated in graded ethanol, cleared in xylene and embedded in paraffin wax. Sections were cut at 6 m, stained with Harris hematoxylin and eosin and observed under a light microscope. Data were expressed in mean ± S.E.M. and were analyzed by using the Student’s “t”-test followed by ANOVA.
100 (+ve) 100 (−ve)
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218 Table 1 Assessment of body weight, organ weight, sperm dynamics, serum testosterone and fertility of the control, Lonidamine and Dendrophthoe falcata methanol stem extract treated groups of male rats after 60 days of treatment
216
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218
A significant decrease (P ≤ 0.01) in percent cauda epididymal sperm motility was evident in Groups II, III, IV and V animals when compared with Group I animals. After 60 days of treatment sperm motility was decreased by 74.46%, 25.62%, 42.15% and 63.43% in Group II, III, IV and V, respectively, when compared to Group I (Table 1). The sperm density of the cauda epididymis and testes were also diminished significantly in all treated groups (P ≤ 0.01). The extract reduced the fertility of male rats by 87.50% at 50 mg/rat/day whereas 100% at 100 and 200 mg/rat/day dose levels as well as with lonidamine (Table 1). D. falcata extract
217
might possibly inhibit the activity of ATP in the spermatozoa by uncoupling of oxidative phosphorylation from the respiratory chain and prevent phosphorylation of ADP to ATP and thus renders the spermatozoa immotile, which resulted in total infertility (Kalla and Vasudev, 1981). Similar findings were also observed in barleria (Gupta et al., 2000). Histological studies of control rat testis showing all successive stages of spermatogenesis, where the lumen was filled with sperm (Fig. 1A). Completely degenerating spermatogenesis could be seen with lonidamine at the dose level of 50 mg/rat/day (Fig. 1B), however histological alterations and arrest of sper-
Fig. 1. (A) (Group I) Control. Seminiferous tubules with all stages of spermatogenesis. Normal intertubular spaces with connective tissue. The lumen with healthy spermatozoa. (B) (Group II) Lonidamine 50 mg/rat/day. Shrinked seminiferous tubule with incomplete spermatogenesis. Lumen devoid of spermatozoa. (C) (Group III) D. falcata 50 mg/rat/day. Degeneration of spermatogenic element could be seen as later stages of spermatogenesis are absent. Lumen contains cellular debris. (D) (Group IV) D. falcata 100 mg/rat/day. Degeneration of secondary spermatocytes and round spermatids are visible. An increased interstitial space is seen. (E) (Group V) D. falcata 200 mg/rat/day. Seminiferous tubules exhibiting complete arrest of spermatogenesis. Vacuolization in the tubule can be seen.
218
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218
matogenesis were seen in a dose dependent manner with D. falcata treatment in which decreased number of spermatogenic elements in seminiferous tubule, cellular debris in lumen, increased interstitial space and damaged Leydig cells could be seen (Fig. 1C–E). In conclusion results of this study shows the potential contraceptive efficacy of the methanol stem extract of Dendrophthoe falcata in male albino rats. Contraceptive like activity of all three-dose levels of D. falcata was found in a dose dependent manner, however the most effective dose was 200 mg/rat/day when compared with Lonidamine. Further investigations of D. falcata isolated fractions are in progress. Acknowledgements The authors are thankful to the Head, Department of Zoology, Prof. N.K. Lohiya, Coordinator CAS, Department of Zoology, University of Rajasthan, Jaipur for providing necessary facilities, as well as U.G.C., Regional Office, Bhopal, M.P. (INDIA) for financial support. References Alkutti, N.A., Srinivasan, K.K., Gundu, R.P., Udupa, A.C., Keshavamurthy, K.R., 1993. Diuretic and antilithiatic activity of Dendrophthoe falcata. Fitoterapia 64, 325–331. Anjaneyula, A.S.R., Row, L.R., Reddy, D.S., 1977. Chemical constitutes of Loranthus falcatus Linn. f. Current Science 24 (20), 850–851. Belanger, A., Caron, S., Picard, V., 1980. Simultaneous radio-immuno assay of progestins, androgens and estrogens in rat testis. Journal of Steroidal Biochemistry 13, 185–192.
Bhakuni, D.S., Goyal, A.K., Jain, S., Mehrotra, B.N., Patnaik, G.K., Prakash, V., 1998. Screening of Indian plants for biological activity: Part XIII. Indian Journal Experimental Biology 26, 883–904. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. CSIR, New Delhi, p. 222. Grima, J., Silvestrini, B., Cheng, C.Y., 2001. Reversible inhibition of spermatogenesis in rats using a new male contraceptive, 1-(2,4dichlorobenzyl)-indazole-3-carbohydrazide. Biology of Reproduction 64, 1500–1508. Gupta, R.S., Kumar, P., Dixit, V.P., Dhobhal, M.P., 2000. Antifertility studies of root extract of Barleria prionitis Linn. in male albino rats with special reference to testicular cell population dynamics. Journal of Ethnopharmacology 70 (2), 111–117. I.N.S.A., 2000. Guidelines for Care and Use of Animals in Scientific Research. Indian National Science Academy, New Delhi. Jain, S.K., Defillips, R.A., 1991. Medicinal Plants of India, Vol. 1. Reference Publication. Inc., Michigan, p. 392. Kalla, N.R., Vasudev, M., 1981. Studies on the male antifertility agent, gossypol acetic acid. II. Effect of gossypol acetic acid on the motility and ATPase activity of human spermatozoa. Andrologia 13, 95–98. Keel, B.A., Abney, T.O., 1980. Influence of bilateral cryptorchidism in the mature rat: alternations in testicular function and serum hormone levels. Endocrinology 107, 1226–1233. Prasad, M.R.N., Chinoy, N.J., Kadam, K.M., 1972. Changes in succinate dehydrogenase level in rat epididymis under normal and altered physiologic conditions. Fertility and Sterility 23, 180–190. Ramaswami, S., Marshall, G.R., 2003. Inhibitory and stimulatory regulation of testicular inhibin B secretion by luteinizing hormone and follicle stimulating hormone, respectively in rhesus monkey. Endocrinology 144, 1175–1185. Singh, V.K., Ali, Z.A., 1992. A contribution to the ethnopharmacological study of the Udaipur forests of Rajasthan, India. Fitoterapia 63, 136–144. The Wealth of India, 1952. Raw Materials, Vol. 3. PID (CSIR), New Delhi, p. 34. WHO, 1983. Protocol CG-04, Preparation of Alcoholic Extract for Bioassay and Phytochemical Studies (APJF/IP, 1001 A). World Health Organization, Geneva.
Ethnopharmacological communication
Evaluation of contraceptive activity of methanol extract of Dendrophthoe falcata stem in male albino rats R.S. Gupta ∗ , J.B.S. Kachhawa Reproductive Physiology Section, Center of Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 23 May 2006; received in revised form 17 December 2006; accepted 23 February 2007 Available online 27 February 2007
Abstract The present study was undertaken to evaluate the contraceptive efficacy of methanol extract of Dendrophthoe falcata Ettingsh (familyLoranthaceae), stem in male albino rats as reported in folk remedies. Adult proven fertile male rats were gavaged methanol extract of D. falcata stem at 50, 100 and 200 mg/rat/day for 60 days. The activity was compared with standard drug, i.e. Lonidamine. On day 61 the animals were autopsied and the testes, epididymides, seminal vesicle and ventral prostate were dissected out and weighed. Sperm motility and density and serum testosterone level were assessed. The sperm motility and density were significantly reduced. The histoarchitecture of testes revealed degenerative changes in the seminiferous tubules, arrest of spermatogenesis at the stage of round spermatid. Serum testosterone levels were decreased significantly in all treatment groups. It is concluded that D. falcata methanol stem extract showed a significant effect on fertility in male rats as reported in folk remedies. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Dendrophthoe falcata; Contraceptive activity; Sperm dynamics; Spermatogenesis
1. Introduction Dendrophthoe falcata Ettingsh (family-Loranthaceae), is a large bushy parasitic shrub with gray bark, one of the seven species of the genus Dendrophthoe found in India (The Wealth of India, 1952). It is also known as Loranthus falcatus Linn. f. Its bark is taken as astrin, narcotic used for wounds and menstrual troubles and also as a remedy for consumption, asthma (Chopra et al., 1956). D. falcata treats kidney stones (Singh and Ali, 1992), having diuretic and antilithiatic activity (Alkutti et al., 1993). It is widely used as antibiotics (Bhakuni et al., 1998). Chemical investigations revealed that some flavonoids, i.e. quercetin, kaempeferol, quercitrin and rutin present in methanol extract of D. falcata. However, the further elution with hexane and hexane benzene (19:1) gave rise to unidentified oils and plant alcohols. With hexane–benzene elute (3:1) -amyrin acetate was isolated and identified by hydrolysis to -amyrin. In the next elute of hexane benzene (1:1) an apparently new triter-
∗
Corresponding author at: TR-03, Teacher’s Hostel, University of Rajasthan, Jaipur 302004, India. Tel.: +91 141 2711228. E-mail address: gupta [email protected] (R.S. Gupta). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.02.022
pene C33 H54 O4 was isolated. Further elution with benzene and benzene ethylacetate furnished -sitosterol, stigmasterol and oleanolic acid (Anjaneyula et al., 1977). In the present investigation contraceptive efficacy of D. falcata stem extract was investigated with the sole objective of developing a male pill of plant origin since its use in folk remedies as antifertility agent (Jain and Defillips, 1991). 2. Material and methods The fresh stems of D. falcata were collected from Nahargarh Biological Garden, Amer in the month of August 2004 and was authenticated by Department of Botany, University of Rajasthan, Jaipur, where a voucher specimen (RUBL-19905) has been deposited. The shade-dried stem (250 g) was powdered and extracted with 100% v/v methanol (b.p. 60–80 ◦ C) in a soxhlet for 36 h (WHO, 1983). The obtained extract was concentrated under reduced pressure and yielded 28 g dark brown semi-solid mass. This mass was washed with pet ether to remove the fatty components and subjected as drug, fed to rats. Fifty proven fertile male rats of Wistar strain, obtained from Jamia Hamdard, Hamdard University, New Delhi (India) were
100 (−ve) 100 (+ve) Values are in mean ± S.E.M. (n = 10). Levels of significance: * P ≤ 0.01; ** P ≤ 0.001 compared with group I (control); a P ≤ 0.01; a+ P ≤ 0.001 compared with group III; b P ≤ 0.01; b+ P ≤ 0.001 compared with group IV.
2.03 ± 0.04**,a+,b+ 15.76 ± 1.19**,a+,b 1.51 ± 0.13**,a+,b 25.60 ± 1.24**,a+,b+ 310.01 ± 10.27*,a 450.43 ± 14.41**,a 752.40 ± 19.39**,a+,b+ 174.39 ± 5.58ns
287.79 ± 4.23**,a+,b+
100 (−ve) 100 (+ve) 2.78 ± 0.06**,a 22.47 ± 1.24**,a+ 2.06 ± 0.11**,a+ 40.50 ± 1.23**,a 487.01 ± 15.86* 897.16 ± 20.86**,a 176.72 ± 6.99ns
315.92 ± 3.45**,a
324.53 ± 11.22*,a
87.50 (−ve) 100 (+ve) 3.16 ± 0.09** 28.71 ± 1.71* 3.11 ± 0.14** 52.07 ± 2.88* 997.30 ± 23.51*
510.16 ± 14.49*
Post
172.41 ± 7.11ns
351.15 ± 9.18*
343.61 ± 10.97ns
100 (+ve) 100 (+ve) 4.27 ± 0.13 1.74 ± 0.05** 48.29 ± 4.34 17.22 ± 1.00** 4.92 ± 0.18 1.19 ± 0.10** 70.01 ± 3.46 17.88 ± 1.30** 381.92 ± 14.58 270.65 ± 8.59** 601.33 ± 17.92 465.71 ± 10.22** 1198.36 ± 34.89 683.48 ± 12.06** 168.44 ± 6.82 182.27 ± 6.49ns
Testis
Group I Control Group II Lonidamine 50 mg/rat/day Group III D. falcata stem extract 50 mg/rat/day Group I V D. falcata stem extract 100 mg/rat/day Group V D. falcata stem extract 200 mg/rat/day
435.52 ± 15.61 236.59 ± 5.09**
Fertility (%)
Pre
Serum testosterone (ng/dl) Cauda epididymides Testis
Sperm density (million/ml)
Sperm motility (%) (Cauda epididymides) Ventral prostate Seminal vesicle Epididymides
Organ weight (mg/100 g.b.wt)
The oral administration of D. falcata stem extract at all dose levels did not cause significant change in the body weight but a great decline in the weights of testes, epididymides, seminal vesicle and ventral prostate were observed in all treatment groups when compared with Group I animals, the decrease in weight was found dose dependent, however the most effective dose level was 200 mg/rat/day in comparison to standard drug (Table 1). Serum testosterone levels of treated animals were decreased by 59.25%, 25.99%, 34.89% and 52.45% in Groups II, III, IV and V, respectively, when compared to controls, i.e. Group I (Table 1). Animals treated with D. falcata methanol stem extract showed a remarkable depression of spermatogenesis which is primarily attributed by decreased testicular weight and reduced seminiferous tubular diameter, which may be due to wide spread cellular damage and androgen deprivation (Keel and Abney, 1980). Reduction in the testosterone level in the rats treated with D. falcata is due to decrease in number of Leydig cell as testosterone is produced by Leydig cells (Ramaswami and Marshall, 2003).
Body weight (g)
3. Results and discussion
Treatment
used for the present study. An acclimatization period of ten days was allowed before experimentation. Water and food was given ad libitum. Ten male rats were selected randomly in the five groups and administered with 50 mg/rat/day of standard drug, i.e. Lonidamine(1-(2,4-dichlorobenzyl)-1H-indazole-3carboxylic acid) belongs to a group of indazole-carboxylic acids that were originally synthesized as anticancer drugs and were also found to be potent inhibitors of spermatogenesis (Grima et al., 2001) as well as D. falcata stem (100% MeOH) extract at the dose of 50, 100 and 200 mg/rat/day; p.o. for 60 days. The required dose was freshly dissolved in 0.5 ml of distilled water and administered to each treated animal every morning with feeding needle. The study was approved by the ethical committee of the Department of Zoology, University of Rajasthan, Jaipur (India). Indian National Science Academy, New Delhi (INSA, 2000) guidelines were followed for maintenance and use of the experimental animals. Fertility was calculated in control as well as in treated groups (WHO, 1983). Twenty-four hours after the last dose, rats were weighed and autopsied under light ether anesthesia. The reproductive organs, i.e. testes, epididymides, seminal vesicles, and ventral prostate were separated out, cleared off fats and connective tissues and weighed. Cauda epididymal sperm motility and density, and testicular sperm counts were made according to the method of Prasad et al. (1972). Serum testosterone levels were assayed from samples using radio immuno assay method (Belanger et al., 1980). One testes of each animal was fixed in Bouin’s fluid, dehydrated in graded ethanol, cleared in xylene and embedded in paraffin wax. Sections were cut at 6 m, stained with Harris hematoxylin and eosin and observed under a light microscope. Data were expressed in mean ± S.E.M. and were analyzed by using the Student’s “t”-test followed by ANOVA.
100 (+ve) 100 (−ve)
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218 Table 1 Assessment of body weight, organ weight, sperm dynamics, serum testosterone and fertility of the control, Lonidamine and Dendrophthoe falcata methanol stem extract treated groups of male rats after 60 days of treatment
216
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218
A significant decrease (P ≤ 0.01) in percent cauda epididymal sperm motility was evident in Groups II, III, IV and V animals when compared with Group I animals. After 60 days of treatment sperm motility was decreased by 74.46%, 25.62%, 42.15% and 63.43% in Group II, III, IV and V, respectively, when compared to Group I (Table 1). The sperm density of the cauda epididymis and testes were also diminished significantly in all treated groups (P ≤ 0.01). The extract reduced the fertility of male rats by 87.50% at 50 mg/rat/day whereas 100% at 100 and 200 mg/rat/day dose levels as well as with lonidamine (Table 1). D. falcata extract
217
might possibly inhibit the activity of ATP in the spermatozoa by uncoupling of oxidative phosphorylation from the respiratory chain and prevent phosphorylation of ADP to ATP and thus renders the spermatozoa immotile, which resulted in total infertility (Kalla and Vasudev, 1981). Similar findings were also observed in barleria (Gupta et al., 2000). Histological studies of control rat testis showing all successive stages of spermatogenesis, where the lumen was filled with sperm (Fig. 1A). Completely degenerating spermatogenesis could be seen with lonidamine at the dose level of 50 mg/rat/day (Fig. 1B), however histological alterations and arrest of sper-
Fig. 1. (A) (Group I) Control. Seminiferous tubules with all stages of spermatogenesis. Normal intertubular spaces with connective tissue. The lumen with healthy spermatozoa. (B) (Group II) Lonidamine 50 mg/rat/day. Shrinked seminiferous tubule with incomplete spermatogenesis. Lumen devoid of spermatozoa. (C) (Group III) D. falcata 50 mg/rat/day. Degeneration of spermatogenic element could be seen as later stages of spermatogenesis are absent. Lumen contains cellular debris. (D) (Group IV) D. falcata 100 mg/rat/day. Degeneration of secondary spermatocytes and round spermatids are visible. An increased interstitial space is seen. (E) (Group V) D. falcata 200 mg/rat/day. Seminiferous tubules exhibiting complete arrest of spermatogenesis. Vacuolization in the tubule can be seen.
218
R.S. Gupta, J.B.S. Kachhawa / Journal of Ethnopharmacology 112 (2007) 215–218
matogenesis were seen in a dose dependent manner with D. falcata treatment in which decreased number of spermatogenic elements in seminiferous tubule, cellular debris in lumen, increased interstitial space and damaged Leydig cells could be seen (Fig. 1C–E). In conclusion results of this study shows the potential contraceptive efficacy of the methanol stem extract of Dendrophthoe falcata in male albino rats. Contraceptive like activity of all three-dose levels of D. falcata was found in a dose dependent manner, however the most effective dose was 200 mg/rat/day when compared with Lonidamine. Further investigations of D. falcata isolated fractions are in progress. Acknowledgements The authors are thankful to the Head, Department of Zoology, Prof. N.K. Lohiya, Coordinator CAS, Department of Zoology, University of Rajasthan, Jaipur for providing necessary facilities, as well as U.G.C., Regional Office, Bhopal, M.P. (INDIA) for financial support. References Alkutti, N.A., Srinivasan, K.K., Gundu, R.P., Udupa, A.C., Keshavamurthy, K.R., 1993. Diuretic and antilithiatic activity of Dendrophthoe falcata. Fitoterapia 64, 325–331. Anjaneyula, A.S.R., Row, L.R., Reddy, D.S., 1977. Chemical constitutes of Loranthus falcatus Linn. f. Current Science 24 (20), 850–851. Belanger, A., Caron, S., Picard, V., 1980. Simultaneous radio-immuno assay of progestins, androgens and estrogens in rat testis. Journal of Steroidal Biochemistry 13, 185–192.
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