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Antifertility studies of the root extract of the Barleria prionitis Linn in male albino rats with special reference to testicular cell population dynamics
Journal of Ethnopharmacology 70 (2000) 111 – 117 www.elsevier.com/locate/jethpharm
Antifertility studies of the root extract of the Barleria prionitis Linn in male albino rats with special reference to testicular cell population dynamics R.S. Gupta a, Pramod Kumar a, V.P. Dixit a,*, M.P. Dobhal b a
Reproducti6e Physiology Section, Department of Zoology, Uni6ersity of Rajasthan, Jaipur 302004, India b Department of Chemistry, Uni6ersity of Rajasthan, Jaipur 302004, India Received 12 July 1999; accepted 23 August 1999
Abstract Oral administration of root extract of Barleria prionitis L. to male rats (100 mg/rat per day) for the period of 60 days did not cause body weight loss. The root extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.6% (P 50.001). No significant change was found in the population of secondary spermatocytes. However, the population of preleptotene spermatocytes were decreased by 41.9%. The extract reduced the fertility of male rats by 100%. Cross sectional surface area of Sertoli cells and mature Leydig cell numbers were significantly reduced (36.9%). The total protein, sialic acid contents of the testes, epididymides, seminal vesicle and prostate were reduced. Testicular glycogen contents were low. Antifertility effects of Barleria seemed to be mediated by disturbances in testicular somatic cells functions (Leydig and Sertoli cells) resulting in the physio-morphological events of spermatogenesis. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Barleria prionitis; Testicular cell population; Physio-morphology; Leydig cell
1. Introduction Barleria prionitis Linn (Acanthaceae) commonly known as Vajradanti, the juice of the leaf is used in cataract and fever. The dried bark is used in cough treatment and the leaves chewed to relieve toothache. The paste of the root is applied to disperse boils and glandular swellings. Little attention was paid to the examination of the action of Barleria on the reproductive system of * Corresponding author.
mammals. In the present investigation antifertility activity of B. prionitis root extract was investigated with the sole objective of developing a male pill of plant origin.
2. Material and methods
2.1. Extraction and isolation of the compounds B. prionitis roots were harvested in December 1997 from Ajmer (India) and authenticated by
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 5 0 - 6
112
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Professor T.N. Bharadwaj, Department of Botany, University of Ajmer, where a voucher specimen has been deposited. Shade-dried powdered roots (2.5 kg) were extracted with 70% MeOH to obtain a crude extract (yield 7.4%). Crude extract was Si-gel CC. Elution with CHCl3-MeOH 1:1, yield: 2.7g/10 g crude extract (Fr. I). Three compound viz. barlerin (1), acetyl barlerin (2) and apigenin-7-o-glucoside (3) were separated and characterized (Mabry et al., 1970; Taneja and Tiwari, 1975; Damtoff et al., 1982). Compounds 1 and 2 are well known iridoids which have marked physiological activity and might be resposible for the male antifertility.
2.2. Animal treatment Male rats obtained from ICMR New Delhi were used. Animals were housed in steel cages and maintained under standard conditions (12 h light/ 12 h dark cycle; 25 93°C; 35 – 60% humidity). Rat feed (Hindustan Lever) and tap water were provided ad libitum. Proven fertile male rats were taken and divided into groups of ten each. Group I-rats were given vehicle: Distilled water 0.5 ml. Group II-rats were given 100 mg/rat per day B. prionitis root extract for 60 days. On day 61 testes, epididymides, seminal vesicles, and ventral prostate were removed, fat and connective tissue cleared off and kept at − 20°C until assayed for total protein, glycogen and sialic acid (Lowry et al., 1951; Montogomery, 1957; Warren, 1959).
2.3. Histological preparations Tissues were fixed in Bouin’s fluid. Paraffin sections were made and stained with hematoxylin and eosin or periodic acid schiff reaction (PAS) to aid in the discrimination of the stages of spermatogenesis (Leblond and Clermont, 1952).
2.4. Quantitati6e analysis The evaluation of cell population dynamics was based on the calculations made for each cell type
per cross tubular sections. Various cell components were quantitatively analysed using spherical tubules. Abercrombie’s correcting factor was introduced (Abercrombie, 1946; Berndtson, 1977) to correct for the better chance a big cell has to be counted. Interstitial cell type such as fibroblast, mature and degenerating Leydig cells were estimated applying a differential count. Over 200 cells of the interstitial cell population were counted and statistically verified by the binomial distribution (Dixon and Massey, 1957). Mean tubular diameters were determined by measuring and tracing an average of 100 selected seminiferous tubules. Diameters of Leydig cell nuclei were measured at × 800. The results were analysed using Student’s t-test.
2.5. Fertility test The mating exposure tests of plant extract treated male groups were performed from day 55 to day 60. They were cohabited with proestrus females in the ratio 1:3. The vaginal plug and the presence of sperm in the vaginal smear were checked for positive mating. The mated females were separated to note the implantation sites on day 16 of pregnancy through leprotomy.
3. Results Barleria root extract did not cause any significant change in the body weights of treated rats. However, the weights of testes, epididymides and ventral prostate were reduced significantly (P5 0.001) (Table 1). The weights of seminal vesicle did not show significant alterations.
3.1. Cell population dynamics The production of spermatids was reduced by 73.6% in Barleria treated rats. The population of preleptotene spermatocytes and spermatogonia were reduced by 41.9 and 44.2%, respectively. However, the population of secondary spermatocytes did not change. The total number of Sertoli cells was affected after Barleria extract feeding
Body weight (g)
Control 240 Vehicle 91.4 treatment Barleria prionitis Root extract 100 mg/rat per day oral for 60 days a
227.5 (ns) 910.9
Organ weight (mg/100g b.w.t.) Testes
Epididymides
Seminal vesicle
Ventral prostate
Normal Seminiferous tubules (%)
1345 94.7
529.5 91.2
605.7 923.1
308.5 92.02
81.6 91.02
1161**
409**
932
911
ns: non-significant. All figures9SEM. ** PB0.001.
513.1 (ns) 919.5
157.75** 98.1
10.93** 91.3
Abnormal Seminiferous tubules (%)
Seminiferous tubules diameter (mm)
Leydig cell Nuclear area (mm2)
Cytoplasmic area (mm2)
19.38 90.02
268 99.0
27.4 90.2
71.2 92.8
89.02** 91.3
255 (ns) 90.8
14.2** 91.3
Sertoli cell area (mm2)
46.6** 93.9
71.05 90.8
22.78** 90.7
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 1 Effect of B. prionitis root extract on the body weight and the organ weights together with testicular histometrya
113
114
Testicular cell counts/cross section
Leydig cell differential count
Sertoli cell
Spermatogonia Preleptotene
Pachytene
Secondary spermatocyte
Round spermatid
Mature
Control
2.819 0.02
6.87 9 0.2
29.29 90.73
48.1 90.6
34.759 0.8
82.390.26
Barleria treatment
2.069 0.04**
3.83 90.004** 11.59 90.4*
25.7 90.4*
(−) 44.2
(−) 12.01
Percent deviation a
19.95 9 1.9
(−) 41.9
ns: non-significant. All figures9SEM.Levels of significance * P50.01 ** P50.001.
44.67 (ns)91.5 (−) 7.1
Degenerated
Fibroblast
57.0 9 2.8
43.3 9 3.6
9.1690.2**
51.991.6** 91.62 9 1.5** 56.48 (ns)93.18
(−) 73.64
(−) 36.9
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 2 Testicular cell population dynamics following Barleria treatmenta
Protein
Control Barleria treatment
Sialic Acid
Testes
Cauda epididymis
178.890.5
268.7 93.6
16194.0*
157.7 910.2**
Seminal vesicle
Ventral prostate
Testes
Cauda epididymis
Seminal vesicle
186 93.4
162.4 90.18
4.64 9 0.16
5.349 0.15
4.2 90.09
107.7 9 3.5**
107.7 94.9**
3.7290.07** 3.559 0.12**
ns: non significant. Values are mean 9 SE of six determinations.Levels of significance * P50.01 ** P50.001.
a
Glycogen Testes Ventral prostate 5.290.23
3.63 (ns)90.25 3.39 9 0.29**
4.859 0.6 2.1390.2*
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 3 Effect of Barleria treatment on biochemical parameters of male rata
115
116
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
(Table 2). The cross-sectional surface area of Sertoli cells was decreased significantly (P 5 0.001) (Table 1). Approximately 89% of tubules in this study were abnormal or degenerated (Table 1). The total number of mature Leydig cells were significantly reduced by 36.9% in rats, whereas, fibroblast Leydig cells remained unaffected (Table 2). Leydig cell nuclear diameter was reduced significantly, while seminiferous tubule shrinkage was nonsignificant (Table 1).
3.2. Biochemical parameters Protein content of testes (P 50.01), epididymides, ventral prostate and seminal vesicle (P5 0.001) were reduced after Barleria treatment as compared with controls. Sialic acid contents of the testes, epididymides and auxiliary gland (SV and VP) were depleted. Glycogen content of the testes was decreased significantly (P 5 0.01) (Table 3).
3.3. Sperm concentration/motility/fertility Barleria feeding to rats significantly reduced sperm concentration of testes and epididymides (P5 0.001). The motility of the cauda epididymal sperm was also reduced significantly (P5 0.001). Barleria reduced the fertility of male rats by 100% (Table 4).
4. Discussion Oral feeding of Barleria for 60 days brought about the loss in testis weight. Many authors analysed the testis weight, which is mostly related
to the number of spermatids and spermatozoa present in the testis. The reduction of DNA-synthesizing cells is related to the testis weight loss. Low cauda epididymal sperm count, presence of non-motile spermatozoa and a significant reduction in the epididymal weight imply that Barleria induced infertility might be caused by several factors. One factor may be that Barleria interferes with enzymatic reactions including the oxidative phosphorylation uncoupling (Abou-Donia and Dieckert, 1974; Ke and Tso, 1982). The reduction in sperm density and motility in cauda epididymides is of importance with regard to fertilization (Bedford, 1983). Depletion in the number of spermatids, preleptotene and pachytene spermatocytes may affect androgen binding protein of Sertoli cells (Steinberger and Steinberger, 1974) via its action in FSH with subsequent interference in sperm maturation and release. Differentiation of primordial germ cells into spermatogonia and the consequent appearance of spermatogenic cycles are under the control of gonadotropins and of testosterone, such control being possibly mediated by Sertoli cells (Courot and Kilgour, 1984) which regulate cell cycle kinetics and influence both spermatogonia and preleptotene spermatocytes (Gasinska and Hill, 1990). Shrunken Sertoli cells in Barleria fed rat testes could be compared with cryptorchidism like activity (Jegou et al., 1983). The total number of mature Leydig cells have a direct bearing on spermatogenesis. A reduction in number and dimensions are indicative of low androgen production affecting fertility (Monet-Kuntz et al., 1984). Decreased testicular protein contents could be co-related with loss of germ cell number as well as
Table 4 Sperm motility, concentration and fertility after Barleria treatmenta Sperm Motility (%) (Cauda epididymides)
Control Barleria treatment a
74.1 92.38 49.9394.9**
All figures9SEM.Levels of significance ** P50.001.
Sperm Density (million/ml) Testes
Cauda epididymides
4.15 9 0.21 0.8 90.17**
52.2 92.66 6.13 91.2**
Fertility (%)
100%(+ve) 100%(−ve)
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
total membrane protein (Barenton et al., 1982). Levinsky et al. (1983) suggested that the reduced sialic acid contents of seminal plasma cause deteriorating effects on the structural integrity of sperm cells. Similarly a decrease in testicular glycogen is indicative of decreased number of post-meiotic germ cells (Spermatids) a site for glucose metabolism (Gunaga et al., 1972). In conclusion, inhibition of spermatogenesis followed by changes in structure activity of Sertoli cells, Leydig cells and meiotic germ cells did support the contraception like activity of Barleria root extract. Work is in progress for isolation of known compounds (Barlerin and Acetyl Barlerin) for further evalution as male fertility regulating agent.
Acknowledgements This study was supported by University Grants Commission, Central Regional Office, Bhopal (India).
References Abercrombie, M., 1946. Estimation of nuclear population from microtome sections. Anat. Rec. 94, 239–247. Abou-Donia, M.B., Dieckert, J.W., 1974. Gossypol uncoupling of respiratory chain and oxidative phosphorylation. Life Sci. 14, 1955 – 1963. Barenton, B., Blanc, M.R., Caraty, A., Hochereau-de Reviers, M.T., Perreau, C., Soumande, J., 1982. Effect of cryptorchidism in the ram: changes in the concentrations of testosterone and Estradiol and receptors for LH and FSH in the testis and its histology. Mol. Cell. Endocrinol. 28, 13– 15. Bedford, J.M., 1983. Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol. Reprod. 28, 108 – 120.
.
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Berndtson, W.E., 1977. Methods for quantifying mammalian spermatogenesis: a review. J. Anim. Sci. 44, 818 – 833. Courot, M., Kilgour, R.J., 1984. Endocrine control of mammalian testicular ontogenesis. Arch. Biol. Med. Exp. 17, 249 – 255. Damtoff, S., Jensen, S.R., Nilesen, B.J., 1982. Structural revision of barlerin and acetyl barlerin. Tetrahedron Lett. 23, 4155 – 4156. Dixon, W., Massey, F.J., 1957. Introduction to Statistical Analysis. McGraw Hill, New York, p. 228. Gasinska, A., Hill, S., 1990. The effect of hyperthermia on the mouse testis. Neoplasma 37, 357 – 366. Gunaga, K.P., Rao, M.C., Seth, A.R., Rao, S.A., 1972. The role of glycogen during the development of the rat testis and prostate. J. Reprod. Fertil. 29, 157 – 162. Jegou, P., Laws, A.O., Dekretser, D.M., 1983. The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats. J. Reprod. Fertil. 69, 137 – 145. Ke, Y.B., Tso, W.W., 1982. Variations of gossypol susceptibility in rat spermatozoa during spermatogenesis. Int. J. Fertil. 27, 42 – 46. Leblond, C.P., Clermont, Y., 1952. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci. 55, 548 – 573. Levinsky, H., Singer, R., Barnet, M., Sagiv, M., Allalouf, D., 1983. Sialic acid content of human spermatozoa and seminal plasma in relation of sperm counts. Arch. Androl. 10, 45 – 46. Lowry, O.H., Rosebrough, M.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer, Heidelberg, p. 55. Monet-Kuntz, M., Hochereau-de Reviers, T., Terqui, M., 1984. Variations in testicular androgen receptors and histology of the lamb testis from birth to puberty. J. Reprod. Fertil. 70, 203 – 210. Montogomery, R, 1957. Determination of glycogen. Arch. Biochem. Biophys. 57, 378 – 381. Steinberger, E., Steinberger, A., 1974. Hormonal control of testicular function in mammals. In: Knobil, E, Sawyer, W.H. (Eds.), Handbook of Physiology. American Physiological Society, Washington DC, p. 325. Taneja, S.C., Tiwari, H.P., 1975. Structure of two new iridoids from B. prionitis. Tetrahedron Lett. 24, 1995 – 1998. Warren, L., 1959. A thiobarbituric acid assay of sialic acid. J. Biol. Chem. 234, 1971 – 1975.
Antifertility studies of the root extract of the Barleria prionitis Linn in male albino rats with special reference to testicular cell population dynamics R.S. Gupta a, Pramod Kumar a, V.P. Dixit a,*, M.P. Dobhal b a
Reproducti6e Physiology Section, Department of Zoology, Uni6ersity of Rajasthan, Jaipur 302004, India b Department of Chemistry, Uni6ersity of Rajasthan, Jaipur 302004, India Received 12 July 1999; accepted 23 August 1999
Abstract Oral administration of root extract of Barleria prionitis L. to male rats (100 mg/rat per day) for the period of 60 days did not cause body weight loss. The root extract brought about an interference with spermatogenesis. The round spermatids were decreased by 73.6% (P 50.001). No significant change was found in the population of secondary spermatocytes. However, the population of preleptotene spermatocytes were decreased by 41.9%. The extract reduced the fertility of male rats by 100%. Cross sectional surface area of Sertoli cells and mature Leydig cell numbers were significantly reduced (36.9%). The total protein, sialic acid contents of the testes, epididymides, seminal vesicle and prostate were reduced. Testicular glycogen contents were low. Antifertility effects of Barleria seemed to be mediated by disturbances in testicular somatic cells functions (Leydig and Sertoli cells) resulting in the physio-morphological events of spermatogenesis. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Barleria prionitis; Testicular cell population; Physio-morphology; Leydig cell
1. Introduction Barleria prionitis Linn (Acanthaceae) commonly known as Vajradanti, the juice of the leaf is used in cataract and fever. The dried bark is used in cough treatment and the leaves chewed to relieve toothache. The paste of the root is applied to disperse boils and glandular swellings. Little attention was paid to the examination of the action of Barleria on the reproductive system of * Corresponding author.
mammals. In the present investigation antifertility activity of B. prionitis root extract was investigated with the sole objective of developing a male pill of plant origin.
2. Material and methods
2.1. Extraction and isolation of the compounds B. prionitis roots were harvested in December 1997 from Ajmer (India) and authenticated by
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 5 0 - 6
112
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Professor T.N. Bharadwaj, Department of Botany, University of Ajmer, where a voucher specimen has been deposited. Shade-dried powdered roots (2.5 kg) were extracted with 70% MeOH to obtain a crude extract (yield 7.4%). Crude extract was Si-gel CC. Elution with CHCl3-MeOH 1:1, yield: 2.7g/10 g crude extract (Fr. I). Three compound viz. barlerin (1), acetyl barlerin (2) and apigenin-7-o-glucoside (3) were separated and characterized (Mabry et al., 1970; Taneja and Tiwari, 1975; Damtoff et al., 1982). Compounds 1 and 2 are well known iridoids which have marked physiological activity and might be resposible for the male antifertility.
2.2. Animal treatment Male rats obtained from ICMR New Delhi were used. Animals were housed in steel cages and maintained under standard conditions (12 h light/ 12 h dark cycle; 25 93°C; 35 – 60% humidity). Rat feed (Hindustan Lever) and tap water were provided ad libitum. Proven fertile male rats were taken and divided into groups of ten each. Group I-rats were given vehicle: Distilled water 0.5 ml. Group II-rats were given 100 mg/rat per day B. prionitis root extract for 60 days. On day 61 testes, epididymides, seminal vesicles, and ventral prostate were removed, fat and connective tissue cleared off and kept at − 20°C until assayed for total protein, glycogen and sialic acid (Lowry et al., 1951; Montogomery, 1957; Warren, 1959).
2.3. Histological preparations Tissues were fixed in Bouin’s fluid. Paraffin sections were made and stained with hematoxylin and eosin or periodic acid schiff reaction (PAS) to aid in the discrimination of the stages of spermatogenesis (Leblond and Clermont, 1952).
2.4. Quantitati6e analysis The evaluation of cell population dynamics was based on the calculations made for each cell type
per cross tubular sections. Various cell components were quantitatively analysed using spherical tubules. Abercrombie’s correcting factor was introduced (Abercrombie, 1946; Berndtson, 1977) to correct for the better chance a big cell has to be counted. Interstitial cell type such as fibroblast, mature and degenerating Leydig cells were estimated applying a differential count. Over 200 cells of the interstitial cell population were counted and statistically verified by the binomial distribution (Dixon and Massey, 1957). Mean tubular diameters were determined by measuring and tracing an average of 100 selected seminiferous tubules. Diameters of Leydig cell nuclei were measured at × 800. The results were analysed using Student’s t-test.
2.5. Fertility test The mating exposure tests of plant extract treated male groups were performed from day 55 to day 60. They were cohabited with proestrus females in the ratio 1:3. The vaginal plug and the presence of sperm in the vaginal smear were checked for positive mating. The mated females were separated to note the implantation sites on day 16 of pregnancy through leprotomy.
3. Results Barleria root extract did not cause any significant change in the body weights of treated rats. However, the weights of testes, epididymides and ventral prostate were reduced significantly (P5 0.001) (Table 1). The weights of seminal vesicle did not show significant alterations.
3.1. Cell population dynamics The production of spermatids was reduced by 73.6% in Barleria treated rats. The population of preleptotene spermatocytes and spermatogonia were reduced by 41.9 and 44.2%, respectively. However, the population of secondary spermatocytes did not change. The total number of Sertoli cells was affected after Barleria extract feeding
Body weight (g)
Control 240 Vehicle 91.4 treatment Barleria prionitis Root extract 100 mg/rat per day oral for 60 days a
227.5 (ns) 910.9
Organ weight (mg/100g b.w.t.) Testes
Epididymides
Seminal vesicle
Ventral prostate
Normal Seminiferous tubules (%)
1345 94.7
529.5 91.2
605.7 923.1
308.5 92.02
81.6 91.02
1161**
409**
932
911
ns: non-significant. All figures9SEM. ** PB0.001.
513.1 (ns) 919.5
157.75** 98.1
10.93** 91.3
Abnormal Seminiferous tubules (%)
Seminiferous tubules diameter (mm)
Leydig cell Nuclear area (mm2)
Cytoplasmic area (mm2)
19.38 90.02
268 99.0
27.4 90.2
71.2 92.8
89.02** 91.3
255 (ns) 90.8
14.2** 91.3
Sertoli cell area (mm2)
46.6** 93.9
71.05 90.8
22.78** 90.7
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 1 Effect of B. prionitis root extract on the body weight and the organ weights together with testicular histometrya
113
114
Testicular cell counts/cross section
Leydig cell differential count
Sertoli cell
Spermatogonia Preleptotene
Pachytene
Secondary spermatocyte
Round spermatid
Mature
Control
2.819 0.02
6.87 9 0.2
29.29 90.73
48.1 90.6
34.759 0.8
82.390.26
Barleria treatment
2.069 0.04**
3.83 90.004** 11.59 90.4*
25.7 90.4*
(−) 44.2
(−) 12.01
Percent deviation a
19.95 9 1.9
(−) 41.9
ns: non-significant. All figures9SEM.Levels of significance * P50.01 ** P50.001.
44.67 (ns)91.5 (−) 7.1
Degenerated
Fibroblast
57.0 9 2.8
43.3 9 3.6
9.1690.2**
51.991.6** 91.62 9 1.5** 56.48 (ns)93.18
(−) 73.64
(−) 36.9
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 2 Testicular cell population dynamics following Barleria treatmenta
Protein
Control Barleria treatment
Sialic Acid
Testes
Cauda epididymis
178.890.5
268.7 93.6
16194.0*
157.7 910.2**
Seminal vesicle
Ventral prostate
Testes
Cauda epididymis
Seminal vesicle
186 93.4
162.4 90.18
4.64 9 0.16
5.349 0.15
4.2 90.09
107.7 9 3.5**
107.7 94.9**
3.7290.07** 3.559 0.12**
ns: non significant. Values are mean 9 SE of six determinations.Levels of significance * P50.01 ** P50.001.
a
Glycogen Testes Ventral prostate 5.290.23
3.63 (ns)90.25 3.39 9 0.29**
4.859 0.6 2.1390.2*
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
Table 3 Effect of Barleria treatment on biochemical parameters of male rata
115
116
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
(Table 2). The cross-sectional surface area of Sertoli cells was decreased significantly (P 5 0.001) (Table 1). Approximately 89% of tubules in this study were abnormal or degenerated (Table 1). The total number of mature Leydig cells were significantly reduced by 36.9% in rats, whereas, fibroblast Leydig cells remained unaffected (Table 2). Leydig cell nuclear diameter was reduced significantly, while seminiferous tubule shrinkage was nonsignificant (Table 1).
3.2. Biochemical parameters Protein content of testes (P 50.01), epididymides, ventral prostate and seminal vesicle (P5 0.001) were reduced after Barleria treatment as compared with controls. Sialic acid contents of the testes, epididymides and auxiliary gland (SV and VP) were depleted. Glycogen content of the testes was decreased significantly (P 5 0.01) (Table 3).
3.3. Sperm concentration/motility/fertility Barleria feeding to rats significantly reduced sperm concentration of testes and epididymides (P5 0.001). The motility of the cauda epididymal sperm was also reduced significantly (P5 0.001). Barleria reduced the fertility of male rats by 100% (Table 4).
4. Discussion Oral feeding of Barleria for 60 days brought about the loss in testis weight. Many authors analysed the testis weight, which is mostly related
to the number of spermatids and spermatozoa present in the testis. The reduction of DNA-synthesizing cells is related to the testis weight loss. Low cauda epididymal sperm count, presence of non-motile spermatozoa and a significant reduction in the epididymal weight imply that Barleria induced infertility might be caused by several factors. One factor may be that Barleria interferes with enzymatic reactions including the oxidative phosphorylation uncoupling (Abou-Donia and Dieckert, 1974; Ke and Tso, 1982). The reduction in sperm density and motility in cauda epididymides is of importance with regard to fertilization (Bedford, 1983). Depletion in the number of spermatids, preleptotene and pachytene spermatocytes may affect androgen binding protein of Sertoli cells (Steinberger and Steinberger, 1974) via its action in FSH with subsequent interference in sperm maturation and release. Differentiation of primordial germ cells into spermatogonia and the consequent appearance of spermatogenic cycles are under the control of gonadotropins and of testosterone, such control being possibly mediated by Sertoli cells (Courot and Kilgour, 1984) which regulate cell cycle kinetics and influence both spermatogonia and preleptotene spermatocytes (Gasinska and Hill, 1990). Shrunken Sertoli cells in Barleria fed rat testes could be compared with cryptorchidism like activity (Jegou et al., 1983). The total number of mature Leydig cells have a direct bearing on spermatogenesis. A reduction in number and dimensions are indicative of low androgen production affecting fertility (Monet-Kuntz et al., 1984). Decreased testicular protein contents could be co-related with loss of germ cell number as well as
Table 4 Sperm motility, concentration and fertility after Barleria treatmenta Sperm Motility (%) (Cauda epididymides)
Control Barleria treatment a
74.1 92.38 49.9394.9**
All figures9SEM.Levels of significance ** P50.001.
Sperm Density (million/ml) Testes
Cauda epididymides
4.15 9 0.21 0.8 90.17**
52.2 92.66 6.13 91.2**
Fertility (%)
100%(+ve) 100%(−ve)
R.S. Gupta et al. / Journal of Ethnopharmacology 70 (2000) 111–117
total membrane protein (Barenton et al., 1982). Levinsky et al. (1983) suggested that the reduced sialic acid contents of seminal plasma cause deteriorating effects on the structural integrity of sperm cells. Similarly a decrease in testicular glycogen is indicative of decreased number of post-meiotic germ cells (Spermatids) a site for glucose metabolism (Gunaga et al., 1972). In conclusion, inhibition of spermatogenesis followed by changes in structure activity of Sertoli cells, Leydig cells and meiotic germ cells did support the contraception like activity of Barleria root extract. Work is in progress for isolation of known compounds (Barlerin and Acetyl Barlerin) for further evalution as male fertility regulating agent.
Acknowledgements This study was supported by University Grants Commission, Central Regional Office, Bhopal (India).
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