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Effects of Plumieride, an iridoid on spermatogenesis in male albino rats
Phytomedicine 11: 169–174, 2004 http://www.elsevier-deutschland.de/phymed
Effects of Plumieride, an iridoid on spermatogenesis in male albino rats R.S. Gupta1, A.K. Bhatnager2, Y. C. Joshi3, R. Sharma1, and A. Sharma1 1
Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur, India Department of Chemistry, S.S. Jain Subodh P.G. College, Jaipur, India 3 Department of Chemistry, University of Rajasthan, Jaipur, India 2
Summary Oral feeding of male rats with plumieride (15 mg/rat/day) for the period of 60 days did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced when compared to control values. The production of step-19 spermatids was reduced by 87.26% in plumieride treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 64.26% and 55.13% respectively. Spermatogonia and sertoli cell population was also affected. Plumieride treatment resulted in an arrest of spermatogenesis without any systemic side effect. Sperm motility as well as sperm density was reduced significantly. The number of mature Leydig cells was decreased and complete suppression of fertility was observed. A significant fall in the protein and sialic acid contents of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes was also noticed. Fructose in seminal vesicle was lowered whereas testicular cholesterol was elevated. There was no significant change in RBC and WBC count, haemoglobin, haematocrit and sugar in the whole blood and total protein, cholesterol, phospholipid and triglycerides in the serum. Conclusion: Plumieride administration arrests spermatogenesis in male rats without noticeable side effects. For the clinical use more experiments should be carried out in a phased programme. Key words: Plumieride, testicular cell population, sperm motility, sialic acid, Sertoli cell, Leydig cells
Introduction Plumeria bicolor, a plant of the Apocynaceae family is commonly known as “Champa” in India. Plumeria is grown as an ornamental plant in India, Indonesia, Philippines and South Africa. Various species of this plant are used as medicine for the cure of many diseases and used as antipsychotic, diuretic or antitumour agents. Some iridoids and triterpenoids obtained from this plant are reported to have algicidal (Coppen and Cobb, 1983; Coppen, 1983), antibacterial (Sandujza et al. 1985) and cytotoxic activity (Anderson et al. 1988; Adam et al. 1979). Plumieride, an iridoid has also been reported to show the antidermantophytic activity (Tiwari et al. 2002). The plant has also been re-
ported to have antifertility and anticancerous activity. Some species of this plant are used for the cure of rheumatism, diarrhoea, blennorhea, venereal disease and leprosy (Perry and Metzer, 1980; Watt and Brreyer, 1962; Kardono et al. 1990). Major constituent of chloroform extract of bark is plumieride, an iridoid. Two reasons, availability of plumieride approximately 10% of extract and reported antifertility activity of plumeria species, prompted us to evaluate the active principle i.e. antifertility activity of plumieride. Since very less attention has been given to male antifertility, we started work with male rats. 0944-7113/04/11/02–03-169 $ 30.00/0
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R. S. Gupta et al.
Plant and Material
cauda epididymis by the method of Prasad (Prasad et al. 1972).
The plant was collected from the campus of University of Rajasthan, Jaipur and identified by the Department of Botany, University of Rajasthan, Jaipur.
Blood and serum analysis
Isolation of Plumieride
The bark of plant was shade dried and grinded to powder. 4 kg. of powdered bark was extracted by methanol extensively for approximately 72 hrs. The extract was distilled and brown mass obtained was treated with acetonitrile to remove fatty parts and acetonitrile was removed under reduced pressure. The brown mass obtained was approximately 100 gm which was re-extracted by chloroform. Approximately 40 gm extract was obtained on removal of chloroform by distillation process. This chloroform extract was subjected to column chromatography for separation and isolation of pure compounds. For this purpose a column of height 1.2 meter with diameter 5 cm filled with 900 gm of Si gel was taken. The column was chromatographed with various solvents and mixture of solvents in the order of increasing polarity. When column was eluted with chloroform and ethyl acetate in ratio of 1:1, plumieride was isolated. Plumieride (I) was recrystallised by methanol to obtain pure plumieride, which was used in the experiment of the antifertility activity. Its mp was 225–228 °C. The structure was established by HNMR and 13C NMR data (Abe et al. 1984). Animals
Male albino rats of wistar strain were used in the present investigation. Rats weighing between 150–170 g were housed in polypropylene cages under controlled environmental conditions. They were fed with rat feed pellets and free access to water. Study Protocol
Male rats of proven fertility were divided into 2 groups of 10 each. Group – 1: Rats receiving vehicle orally (distilled water 0.5 ml/day ) for 60 days. Group – 2: Rats treated with Plumieride orally (15 mg/ rat/day) for 60 days. After 24 hrs. of last dosing of 60 days duration, the animals were autopsied under ether anaesthesia. The testes, epididymides, seminal vesicles, ventral prostate and liver were removed, cleared off fat and connective tissue, weighed and kept at –20 °C until assayed for protein (Lowry et al. 1951), glycogen (Montogomery, 1957), sialic acid (Warren, 1959) and cholesterol (Oser, 1965).
Whole blood was analysed for RBC, WBC count, haemoglobin (Crossby et al. 1954) haematocrit and sugar (Astoor and King, 1954) and serum was analysed to estimate total protein, cholesterol (Zlatkis et al. 1953), triglyceride (Gottfried and Rosenberg, 1973) and phospholipids (Zelversmit et al. 1950). Testicular cell dynamics
Tissues were fixed in Bouin’s fluid, Paraffin sections were made at the thickness of 5 µm and stained with hematoxylin and eosin. The evaluation of cell population was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analysed using spherically appearing sections. These counts were designated as crude counts and these crude counts were corrected by Abercrombie’s formula (Berndtson, 1977). Fibroblast, mature and degenerating Leydig cells were counted. Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules. Diameters of Leydig cell nuclei were measured at 800×. Statistical analysis
All the values of body/organ weight, biochemical estimation, histometry and testicular dynamics were expressed in terms of mean ± standard error. The treated groups were compared to control using the Student’s t-test (Ipstein and Poly, 1970).
Results The oral administration of plumieride to male rats for 60 days did not cause any significant change in the body weight of treated rats. However the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly (p < 0.001) reduced when compared to control values (Table 1).
Sperm motility and Sperm density
The motility of cauda epididymal spermatozoa was recorded. Sperm density was assessed in testes and
Fig. 1. Plumieride (I).
1345 ± 4.7 1009.99** ± 20.5
529.5 ± 11.2 312.1** ± 18
605 ± 9.5 312.1** ± 15
Values are mean ± SEM (n = 10); ns: non significant. ** p < 0.001 vs control.
245 ± 20 230 ± 18ns
6.87 ± 0.02 2.81 ± 0.02 2.783** ± 0.368 1.99* ± 0.18) (–59.49) (–29.18)
19.95 ± 1.9 29.29 ± 0.73 –7.13** ± 0.9 13.14** ± 1.02 (–64.26) (–55.13)
48.1 ± 0.6 19.8** ± 3.7 (–58.83)
a
178 ± 0.5 113.4** ± 4.4
268 ± 3.6 119.9** ± 4.5
186 ± 3.4 122.1** ±3
162 ± 0.18 131** ± 5.7
35.1 ± 1.07 4.47** ± 1.86 (–87.26)
4.62 ± 0.16 5.3 ± 0.15 3.11** 3.84** ± 0.03 ± 0.15
4.2 ± 0.09 2.99** ± 0.03
268 ± 9 182.4** ± 0.28
11.1 ± 0.02 5.6** ± 0.168
Seminiferous Leydig cell Tubule diameter nuclear diameter –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– µm µm
5.2 ± 0.23 3.78** ± 0.1
7.3 ± 0.2 14.99** ± 0.5
Testes
Testes 4.85 ± 0.6 2.8* ± 0.05
Cholesterol (mg/g)
99.7 ± 0.26 63.47* ± 7.83 (–36.33)
Glycogen (mg/g)
43.3 ± 3.6 59.903ns ± 9.70
4.52± 0.19 3.11** ± 0.11
Seminal vesicle
Fructose (mg/g)
57.0 ± 2.8 66.61ns ± 3.16
Leydig cell differential count –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Fibroblast Mature Degenerated
Sialic acid (mg/g) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda Seminal Ventral epididymis vesicle prostate
Values are mean ± SEM (n = 10); ns: non significant * p < 0.01; ** p < 0.001 vs control.
Control Plumieride 15 mg/rat/day
Protein (mg/g) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda Seminal Ventral epididymis vesicle prostate
Table 3. Effect of Plumieride on tissue biochemistry in rats.a
a Values are mean ± SEM (n = 10); ns: non significant * p < 0.01; ** p < 0.001 vs control. Percent variations vs control in parentheses.
Control Plumieride
Testicular cell counts (number/10 cross section) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Spermatogonia Sertoli cell Preleptotene Pachytene Secondary Step-19 spermatocytes spermatid
308.5 ± 2.02 177.10** ± 10.8
Organ weight (mg/100 g body wt.) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Epididymis Seminal vesicle Ventral Prostate
Table 2. Effect of Plumieride on testicular cell population in rats.a
a
Control Plumieride 15 mg/rat/day
Body Weight (g)
Table 1. Effect of Plumieride on body weight, organ weights together with Seminiferous tubule and Leydig cell nuclear diameter.a
Effects of Plumieride, an iridoid on spermatogenesis 171
R. S. Gupta et al. Triglyceride mg/dl
82.9 ± 3.87
94.4 6.67
150.05 ± 10.4
170.5ns 6.945
The number of step-19 spermatids was reduced by 87.26%; spermatogonia and preleptotene spermatocytes were decreased by 59.49 and 64.26% respectively. Pachytene spermatocytes reduced by 55.1%. The number of sertoli cells were reduced by 29.18%. The number of mature Leydig cells was decreased significantly (p < 0.001, Table 2). The seminiferous tubule diameter and Leydig cell nuclear diameter were reduced significantly (p < 0.001, Table 1).
The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly (p < 0.001) in comparison with controls (Table 3). Sialic acid contents of the testes, cauda epididymides, seminal vesicle and ventral prostate were depleted (Table 3). Glycogen contents in the testes were decreased significantly following treatment with plumieride. Seminal vesicular fructose was decreased significantly, whereas, testicular cholesterol was elevated significantly (p < 0.001). Sperm concentration/ motility/ fertility
Values are mean ± SEM (n = 10); ns: non significant vs control.
As shown in Table 4, the treated rats showed significant (p < 0.001) reduction in the sperm concentration of testes and cauda epididymides. The sperm motility of the cauda epididymides was also reduced significantly (p < 0.001). The plumieride treatment reduced the fertility of male rats by 100%.
a
106 ± 5.2 12160 ± 407
12333.32 111.11
89.1 ± 2.5
99.99ns ± 4.165
39.7 ± 2.0
32.46ns ± 2.11
14.4 ± 0.2
13.8ns ± 0.2
8516 ± 201 5.1 ± 0.16 Control
Plumieride 5.62ns 15 mg/rat/day ± 0.085
8176ns ± 100
Total protein mg/dl Haemoglobin gm%
Blood sugar mg/dl
122.22ns 11.11
Biochemical parameters
Haematocrit (%)
Total cholesterol mg/dl
Phospholipid mg/dl
Cell population dynamics
WBC’s no/mm3 RBC’s million/mm3
All Figures ± SEM; level of significance ** p < 0.001 a
Table 5. Effect of Plumieride on some components in whole blood and serum of rats.a
100% (±ve) 100% (–ve) 4.2 ± 0.06 1.225** ± 0.075 70.25 ± 0.53 17.59** ± 1.72 Control Plumieride treatment
50.04 ± 0.59 5.55** ± 0.35
Sperm density (million/ml) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda epididymides Sperm motility (%) Cauda epididymides
Table 4. Effect of Plumieride on Sperm motility concentration and fertility in rats.a
Fertility
172
Blood and serum analysis
Blood variables i.e. RBC and WBC counts, haemoglobin, haematocrit and sugar were within the normal range. Protein, cholesterol, phospholipid and triglycerides did not change significantly after plumieride treatment to rats (Table 5).
Discussion Oral administration of plumieride to male rats for 60 days brought about a significant loss in testes weight, which is known to be mostly related to the number of spermatids and spermatozoa present in the tissue. The reduced testicular weights and shrunken seminiferous tubular dimensions indicate wide spread damage (Keel and Abney, 1980). The treatment with plumieride exerted strong inhibiting effect on epididymides, seminal vesicle and prostate gland as evinced by decrease in their weight. Reduction in the weights of accessory re-
Effects of Plumieride, an iridoid on spermatogenesis productive organs directly supports the reduced availability of androgens (Mukerjee et al. 1992). Russell and Clermont (1977) found that the regression of seminiferous tubule epithelium after depletion of pituitary hormone was due to discriminate degeneration of mid pachytene spermatocytes and step-19 spermatids in stage VII. In the mammalian spermatogenic cycle, normally step -19 spermatids are found in stage VII and VIII and particularly androgen dependent (Ritzen et al. 1981; Parvinen and Ruokonen, 1982). The number of sertoli cells are reduced by 29.18% which adversely affects the spermatogenesis since sertoli cells provide all or most of nutritional and physical support for the developing germ cells. Sperm count shows statistically significant correlation with antifertility (Bostofte et al. 1984). In the present study, treatment with plumieride caused a significant reduction in sperm density of cauda epididymis as well as testes. The motility of sperms in cauda epididymides was reduced significantly (p < 0.001) after the administration of plumieride. Complete inhibition of fertility may be due to suppressed sperm motility and decreased sperm count, since fast and progressive sperms in a sufficient number are necessary for fertilization (Aitken et al. 1984). The number of mature Leydig cells was decreased significantly (p < 0.001). The impairment of Leydig cell function was evinced by its reduced nuclear area and lower number of mature Leydig cells. The number of mature Leydig cells has a direct bearing on spermatogenesis (Gupta et al. 2000). The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly. It is evident that testicular function would be altered by reduced protein content. The principal cells of epididymis synthesize proteins which have important role for maturation of spermatozoa (Kasturi et al. 1995). Sialic acid acts as ‘lubricant’ to facilitate the downward movement of sperms and to reduce friction among spermatozoa (Robaine and Hermo, 1988). Mukherjee et al. (1992) studied the effects of flutamide on testes and accessory sex organs of male rats and also found reduction in sialic acid contents. Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells. Increased level of testicular cholesterol may be due to decreased androgen production, which resulted in accumulation of cholesterol in testes, hence impaired spermatogenesis (Bedwal et al. 1994). Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells (Gunaga, 1972). Seminal vesicular fructose was decreased significantly, the reduction might be due to decreased secretory activity. It has been observed that the blood and serum parameters were within the normal range indicating nontoxicity of plumieride on general body metabolism.
173
Conclusion: Our results reflect anti spermatogenic effect of plumieride in male albino rats. Acknowledgements
The authors are thankful to the Head, Dept. of Zoology and Chemistry; Prof. N. K. Lohiya, Coordinator, SAP, Dept. of Zoology, University of Rajasthan and Principal, S. S. Jain Subodh P.G. College, Jaipur (India) for providing necessary facilities and University Grants Commission, Regional Office, Bhopal (MP) India for financial support.
References Abe F, Mori T, Yamauchi T (1984) Iridoids of Apocynaceae III. Minor Iridoids from Allamanda neriifolia. Chem Pharm Bull 32: 2947–2956 Adam G, Khoi NH, Bergner C, Lien NT (1979) Plant growth inhibiting properties of Plumieride from Plumeria obtusifolia. Phytochemistry 18: 1399 Aitken RJ, Best FSM, Warner P, Templeton A (1984) A prospective study of the relationship between semen quality and fertility in cases of unexplained infertility. J Androl 5: 297–303 Anderson JE, Chang CJ, Mclaughlein JL (1988) Bioactive components of Allamanda schottii. J Nat Prod 51: 307 Astoor A, King EJ (1954) Simplified colorimetric blood sugar method. Biochem J 56: XLIV Bedwal RS, Edwards MS, Katoch M, Bahuguna A, Dewan R (1994) Histological and biochemical changes in testes of Zinc deficient BALB/C strain of mice. Ind J Exp Biol 32: 243–247 Berndtson WE (1977) Methods for quantifying mammalian spermatogenesis: a review. J Anim Sci 44: 818–833 Bostofte E, Jorgen S, Rebbe H (1984) Interrelations among the characteristics of human semen and a new system for classification of male infertility. Fertil Steril 41: 95 –102 Coppen JJ, Cobb AL (1983) The occurrence of iridoids in Plumeria and Allamanda. Phytochemistry 22: 165 Coppen JJW (1983) Iridoids with algicidal properties from Allamanda. Phytochemistry 22: 179 Crossby WH, Munn JI, Furth FW (1954) Standardizing method for clinical hemoglobinometry. US Armed Force Med J 5: 695–703 Gottfried SP, Rosenberg B (1973) Improved manual spectrophotometric procedure for determination of serum triglycerides. Clin Chem 19: 1077–1078 Gunaga KP, Rao MC, Seth AR, Rao SA (1972) The role of glycogen during the development of the rat testes and prostate. J Reprod Fert 29: 157–162 Gupta RS, Kumar P, Dixit VP, Dobhal MP (2000) Antifertility studies of the root extract of the Barleria prionitis Linn. in male albino rats with special reference to testicular cell population dynamics. J Ethnopharmacol 70: 111–117 Ipstein J, Poly F (1970) In: Banchroft’s introduction to biostatistics 2nd ed p 44–64, Harper International, New York Kardono LBS, Palmawinata K, Kinghorin AD (1990) A flavon-3-ol glycoside from bark of Plumeria rubra. Phytochemistry 29: 2995
174
R. S. Gupta et al.
Kasturi M, Manivannan B, Ahmad RN, Shaikh PD, Pathan KM (1995) Ind. Changes in epididymal structure and function of albino rats treated with Azardirachta indica leaves. J Exp Biol 33: 725–729 Keel AB, Abney TO (1980) Influence of bilateral cryptorchidism in the mature rat: Alteration in testicular function and serum hormonal levels. Endocrinol 107: 1226–1233 Lowry OH, Rosebrough MJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193: 265–275 Montogomery R (1957) Determination of glycogen. Arch. Biochem. Biophys 57: 378–381 Mukherjee M, Chattopadhyay S, Mathur PP (1992) Effect of Flutamide on the physiological status of epididymis and epididymal sperms. Andrologia 24: 113–116 Oser BL (1965) Hawk’s Physiological chemistry. 14th ed. p.246. Mc Graw Hill, New York Parvinen M, Ruokonen A (1982) Endogenous steroids in the rat seminiferous tubules. Comparison of the stages of the epithelial cycles isolated by transillumination assisted microdissection. J Androl 3: 211–220 Perry LM, Metzer J (1980) Medicinal Plants of East and South Asia. p 29. MIT, Cambridge Prasad MRN, Chinoy NJ, Kadam KM (1972) Changes in succinate dehydrogenase levels in the rat epididymis under normal and altered physiological conditions. Fertil Steril 23: 186–190 Ritzen EM, Boitani C, Parvinen M (1981) Cyclic secretion of protein by the rat seminiferous tubules depending on the stage of spermatogenesis. Int J Androl (suppl.) 3: 57–58 Robaine B, Hermo L (1988) Efferent ducts, Epididymis and Vas deferens. Structure, function and their regulation In:
the Physiology of Reproduction, Knobil E, Neil JD (eds) p 999–1080, Raven Press New York Russell LD, Clermont Y (1977) Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. Anat Rec 187: 347–366 Sandujza R, Weinheimer AJ, Enler KL, Adam M (1985) Unusual occurrence of flavoplumarine, an antibacterial pigment in the mollusk Nerita albicilla. J Nat Prod 48: 335 Tiwari TN, Pandey VB, Dubey NK (2002) Plumieride from Allamanda cathartica as an antidermantophytic agent. Phytother Res 16 (4): 393–394 Warren L (1959) A thiobarbituric acid assay of Sialic acid. J Biol Chem 234: 1971–1975 Watt JM, Brreyer–Brranduijik MG (1962) Medicinal and Poisonous Plants of Southern and Eastern Africa p. 94 Zelversmit DB, Davis AK, Hamphestern MT (1950) Microdetermination of plasma phospholipid by trichloro-acetic acid precipitation method. J Lab Clin Invest 35: 155–160 Zlatkis A, Zak B, Boyle AJ (1953) A new method for the direct determination of serum cholesterol. J Lab Clin Med 41: 486–492
Address R. S. Gupta, Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur 302004, India Tel.: +91-141-701137; Fax: +91-141-2395-453, 2711-158; e-mail: [email protected]
Effects of Plumieride, an iridoid on spermatogenesis in male albino rats R.S. Gupta1, A.K. Bhatnager2, Y. C. Joshi3, R. Sharma1, and A. Sharma1 1
Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur, India Department of Chemistry, S.S. Jain Subodh P.G. College, Jaipur, India 3 Department of Chemistry, University of Rajasthan, Jaipur, India 2
Summary Oral feeding of male rats with plumieride (15 mg/rat/day) for the period of 60 days did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced when compared to control values. The production of step-19 spermatids was reduced by 87.26% in plumieride treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 64.26% and 55.13% respectively. Spermatogonia and sertoli cell population was also affected. Plumieride treatment resulted in an arrest of spermatogenesis without any systemic side effect. Sperm motility as well as sperm density was reduced significantly. The number of mature Leydig cells was decreased and complete suppression of fertility was observed. A significant fall in the protein and sialic acid contents of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes was also noticed. Fructose in seminal vesicle was lowered whereas testicular cholesterol was elevated. There was no significant change in RBC and WBC count, haemoglobin, haematocrit and sugar in the whole blood and total protein, cholesterol, phospholipid and triglycerides in the serum. Conclusion: Plumieride administration arrests spermatogenesis in male rats without noticeable side effects. For the clinical use more experiments should be carried out in a phased programme. Key words: Plumieride, testicular cell population, sperm motility, sialic acid, Sertoli cell, Leydig cells
Introduction Plumeria bicolor, a plant of the Apocynaceae family is commonly known as “Champa” in India. Plumeria is grown as an ornamental plant in India, Indonesia, Philippines and South Africa. Various species of this plant are used as medicine for the cure of many diseases and used as antipsychotic, diuretic or antitumour agents. Some iridoids and triterpenoids obtained from this plant are reported to have algicidal (Coppen and Cobb, 1983; Coppen, 1983), antibacterial (Sandujza et al. 1985) and cytotoxic activity (Anderson et al. 1988; Adam et al. 1979). Plumieride, an iridoid has also been reported to show the antidermantophytic activity (Tiwari et al. 2002). The plant has also been re-
ported to have antifertility and anticancerous activity. Some species of this plant are used for the cure of rheumatism, diarrhoea, blennorhea, venereal disease and leprosy (Perry and Metzer, 1980; Watt and Brreyer, 1962; Kardono et al. 1990). Major constituent of chloroform extract of bark is plumieride, an iridoid. Two reasons, availability of plumieride approximately 10% of extract and reported antifertility activity of plumeria species, prompted us to evaluate the active principle i.e. antifertility activity of plumieride. Since very less attention has been given to male antifertility, we started work with male rats. 0944-7113/04/11/02–03-169 $ 30.00/0
170
R. S. Gupta et al.
Plant and Material
cauda epididymis by the method of Prasad (Prasad et al. 1972).
The plant was collected from the campus of University of Rajasthan, Jaipur and identified by the Department of Botany, University of Rajasthan, Jaipur.
Blood and serum analysis
Isolation of Plumieride
The bark of plant was shade dried and grinded to powder. 4 kg. of powdered bark was extracted by methanol extensively for approximately 72 hrs. The extract was distilled and brown mass obtained was treated with acetonitrile to remove fatty parts and acetonitrile was removed under reduced pressure. The brown mass obtained was approximately 100 gm which was re-extracted by chloroform. Approximately 40 gm extract was obtained on removal of chloroform by distillation process. This chloroform extract was subjected to column chromatography for separation and isolation of pure compounds. For this purpose a column of height 1.2 meter with diameter 5 cm filled with 900 gm of Si gel was taken. The column was chromatographed with various solvents and mixture of solvents in the order of increasing polarity. When column was eluted with chloroform and ethyl acetate in ratio of 1:1, plumieride was isolated. Plumieride (I) was recrystallised by methanol to obtain pure plumieride, which was used in the experiment of the antifertility activity. Its mp was 225–228 °C. The structure was established by HNMR and 13C NMR data (Abe et al. 1984). Animals
Male albino rats of wistar strain were used in the present investigation. Rats weighing between 150–170 g were housed in polypropylene cages under controlled environmental conditions. They were fed with rat feed pellets and free access to water. Study Protocol
Male rats of proven fertility were divided into 2 groups of 10 each. Group – 1: Rats receiving vehicle orally (distilled water 0.5 ml/day ) for 60 days. Group – 2: Rats treated with Plumieride orally (15 mg/ rat/day) for 60 days. After 24 hrs. of last dosing of 60 days duration, the animals were autopsied under ether anaesthesia. The testes, epididymides, seminal vesicles, ventral prostate and liver were removed, cleared off fat and connective tissue, weighed and kept at –20 °C until assayed for protein (Lowry et al. 1951), glycogen (Montogomery, 1957), sialic acid (Warren, 1959) and cholesterol (Oser, 1965).
Whole blood was analysed for RBC, WBC count, haemoglobin (Crossby et al. 1954) haematocrit and sugar (Astoor and King, 1954) and serum was analysed to estimate total protein, cholesterol (Zlatkis et al. 1953), triglyceride (Gottfried and Rosenberg, 1973) and phospholipids (Zelversmit et al. 1950). Testicular cell dynamics
Tissues were fixed in Bouin’s fluid, Paraffin sections were made at the thickness of 5 µm and stained with hematoxylin and eosin. The evaluation of cell population was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analysed using spherically appearing sections. These counts were designated as crude counts and these crude counts were corrected by Abercrombie’s formula (Berndtson, 1977). Fibroblast, mature and degenerating Leydig cells were counted. Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules. Diameters of Leydig cell nuclei were measured at 800×. Statistical analysis
All the values of body/organ weight, biochemical estimation, histometry and testicular dynamics were expressed in terms of mean ± standard error. The treated groups were compared to control using the Student’s t-test (Ipstein and Poly, 1970).
Results The oral administration of plumieride to male rats for 60 days did not cause any significant change in the body weight of treated rats. However the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly (p < 0.001) reduced when compared to control values (Table 1).
Sperm motility and Sperm density
The motility of cauda epididymal spermatozoa was recorded. Sperm density was assessed in testes and
Fig. 1. Plumieride (I).
1345 ± 4.7 1009.99** ± 20.5
529.5 ± 11.2 312.1** ± 18
605 ± 9.5 312.1** ± 15
Values are mean ± SEM (n = 10); ns: non significant. ** p < 0.001 vs control.
245 ± 20 230 ± 18ns
6.87 ± 0.02 2.81 ± 0.02 2.783** ± 0.368 1.99* ± 0.18) (–59.49) (–29.18)
19.95 ± 1.9 29.29 ± 0.73 –7.13** ± 0.9 13.14** ± 1.02 (–64.26) (–55.13)
48.1 ± 0.6 19.8** ± 3.7 (–58.83)
a
178 ± 0.5 113.4** ± 4.4
268 ± 3.6 119.9** ± 4.5
186 ± 3.4 122.1** ±3
162 ± 0.18 131** ± 5.7
35.1 ± 1.07 4.47** ± 1.86 (–87.26)
4.62 ± 0.16 5.3 ± 0.15 3.11** 3.84** ± 0.03 ± 0.15
4.2 ± 0.09 2.99** ± 0.03
268 ± 9 182.4** ± 0.28
11.1 ± 0.02 5.6** ± 0.168
Seminiferous Leydig cell Tubule diameter nuclear diameter –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– µm µm
5.2 ± 0.23 3.78** ± 0.1
7.3 ± 0.2 14.99** ± 0.5
Testes
Testes 4.85 ± 0.6 2.8* ± 0.05
Cholesterol (mg/g)
99.7 ± 0.26 63.47* ± 7.83 (–36.33)
Glycogen (mg/g)
43.3 ± 3.6 59.903ns ± 9.70
4.52± 0.19 3.11** ± 0.11
Seminal vesicle
Fructose (mg/g)
57.0 ± 2.8 66.61ns ± 3.16
Leydig cell differential count –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Fibroblast Mature Degenerated
Sialic acid (mg/g) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda Seminal Ventral epididymis vesicle prostate
Values are mean ± SEM (n = 10); ns: non significant * p < 0.01; ** p < 0.001 vs control.
Control Plumieride 15 mg/rat/day
Protein (mg/g) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda Seminal Ventral epididymis vesicle prostate
Table 3. Effect of Plumieride on tissue biochemistry in rats.a
a Values are mean ± SEM (n = 10); ns: non significant * p < 0.01; ** p < 0.001 vs control. Percent variations vs control in parentheses.
Control Plumieride
Testicular cell counts (number/10 cross section) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Spermatogonia Sertoli cell Preleptotene Pachytene Secondary Step-19 spermatocytes spermatid
308.5 ± 2.02 177.10** ± 10.8
Organ weight (mg/100 g body wt.) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Epididymis Seminal vesicle Ventral Prostate
Table 2. Effect of Plumieride on testicular cell population in rats.a
a
Control Plumieride 15 mg/rat/day
Body Weight (g)
Table 1. Effect of Plumieride on body weight, organ weights together with Seminiferous tubule and Leydig cell nuclear diameter.a
Effects of Plumieride, an iridoid on spermatogenesis 171
R. S. Gupta et al. Triglyceride mg/dl
82.9 ± 3.87
94.4 6.67
150.05 ± 10.4
170.5ns 6.945
The number of step-19 spermatids was reduced by 87.26%; spermatogonia and preleptotene spermatocytes were decreased by 59.49 and 64.26% respectively. Pachytene spermatocytes reduced by 55.1%. The number of sertoli cells were reduced by 29.18%. The number of mature Leydig cells was decreased significantly (p < 0.001, Table 2). The seminiferous tubule diameter and Leydig cell nuclear diameter were reduced significantly (p < 0.001, Table 1).
The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly (p < 0.001) in comparison with controls (Table 3). Sialic acid contents of the testes, cauda epididymides, seminal vesicle and ventral prostate were depleted (Table 3). Glycogen contents in the testes were decreased significantly following treatment with plumieride. Seminal vesicular fructose was decreased significantly, whereas, testicular cholesterol was elevated significantly (p < 0.001). Sperm concentration/ motility/ fertility
Values are mean ± SEM (n = 10); ns: non significant vs control.
As shown in Table 4, the treated rats showed significant (p < 0.001) reduction in the sperm concentration of testes and cauda epididymides. The sperm motility of the cauda epididymides was also reduced significantly (p < 0.001). The plumieride treatment reduced the fertility of male rats by 100%.
a
106 ± 5.2 12160 ± 407
12333.32 111.11
89.1 ± 2.5
99.99ns ± 4.165
39.7 ± 2.0
32.46ns ± 2.11
14.4 ± 0.2
13.8ns ± 0.2
8516 ± 201 5.1 ± 0.16 Control
Plumieride 5.62ns 15 mg/rat/day ± 0.085
8176ns ± 100
Total protein mg/dl Haemoglobin gm%
Blood sugar mg/dl
122.22ns 11.11
Biochemical parameters
Haematocrit (%)
Total cholesterol mg/dl
Phospholipid mg/dl
Cell population dynamics
WBC’s no/mm3 RBC’s million/mm3
All Figures ± SEM; level of significance ** p < 0.001 a
Table 5. Effect of Plumieride on some components in whole blood and serum of rats.a
100% (±ve) 100% (–ve) 4.2 ± 0.06 1.225** ± 0.075 70.25 ± 0.53 17.59** ± 1.72 Control Plumieride treatment
50.04 ± 0.59 5.55** ± 0.35
Sperm density (million/ml) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Testes Cauda epididymides Sperm motility (%) Cauda epididymides
Table 4. Effect of Plumieride on Sperm motility concentration and fertility in rats.a
Fertility
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Blood and serum analysis
Blood variables i.e. RBC and WBC counts, haemoglobin, haematocrit and sugar were within the normal range. Protein, cholesterol, phospholipid and triglycerides did not change significantly after plumieride treatment to rats (Table 5).
Discussion Oral administration of plumieride to male rats for 60 days brought about a significant loss in testes weight, which is known to be mostly related to the number of spermatids and spermatozoa present in the tissue. The reduced testicular weights and shrunken seminiferous tubular dimensions indicate wide spread damage (Keel and Abney, 1980). The treatment with plumieride exerted strong inhibiting effect on epididymides, seminal vesicle and prostate gland as evinced by decrease in their weight. Reduction in the weights of accessory re-
Effects of Plumieride, an iridoid on spermatogenesis productive organs directly supports the reduced availability of androgens (Mukerjee et al. 1992). Russell and Clermont (1977) found that the regression of seminiferous tubule epithelium after depletion of pituitary hormone was due to discriminate degeneration of mid pachytene spermatocytes and step-19 spermatids in stage VII. In the mammalian spermatogenic cycle, normally step -19 spermatids are found in stage VII and VIII and particularly androgen dependent (Ritzen et al. 1981; Parvinen and Ruokonen, 1982). The number of sertoli cells are reduced by 29.18% which adversely affects the spermatogenesis since sertoli cells provide all or most of nutritional and physical support for the developing germ cells. Sperm count shows statistically significant correlation with antifertility (Bostofte et al. 1984). In the present study, treatment with plumieride caused a significant reduction in sperm density of cauda epididymis as well as testes. The motility of sperms in cauda epididymides was reduced significantly (p < 0.001) after the administration of plumieride. Complete inhibition of fertility may be due to suppressed sperm motility and decreased sperm count, since fast and progressive sperms in a sufficient number are necessary for fertilization (Aitken et al. 1984). The number of mature Leydig cells was decreased significantly (p < 0.001). The impairment of Leydig cell function was evinced by its reduced nuclear area and lower number of mature Leydig cells. The number of mature Leydig cells has a direct bearing on spermatogenesis (Gupta et al. 2000). The protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly. It is evident that testicular function would be altered by reduced protein content. The principal cells of epididymis synthesize proteins which have important role for maturation of spermatozoa (Kasturi et al. 1995). Sialic acid acts as ‘lubricant’ to facilitate the downward movement of sperms and to reduce friction among spermatozoa (Robaine and Hermo, 1988). Mukherjee et al. (1992) studied the effects of flutamide on testes and accessory sex organs of male rats and also found reduction in sialic acid contents. Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells. Increased level of testicular cholesterol may be due to decreased androgen production, which resulted in accumulation of cholesterol in testes, hence impaired spermatogenesis (Bedwal et al. 1994). Depletion in testicular glycogen possibly attributed to decreased number of post meiotic germ cells (Gunaga, 1972). Seminal vesicular fructose was decreased significantly, the reduction might be due to decreased secretory activity. It has been observed that the blood and serum parameters were within the normal range indicating nontoxicity of plumieride on general body metabolism.
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Conclusion: Our results reflect anti spermatogenic effect of plumieride in male albino rats. Acknowledgements
The authors are thankful to the Head, Dept. of Zoology and Chemistry; Prof. N. K. Lohiya, Coordinator, SAP, Dept. of Zoology, University of Rajasthan and Principal, S. S. Jain Subodh P.G. College, Jaipur (India) for providing necessary facilities and University Grants Commission, Regional Office, Bhopal (MP) India for financial support.
References Abe F, Mori T, Yamauchi T (1984) Iridoids of Apocynaceae III. Minor Iridoids from Allamanda neriifolia. Chem Pharm Bull 32: 2947–2956 Adam G, Khoi NH, Bergner C, Lien NT (1979) Plant growth inhibiting properties of Plumieride from Plumeria obtusifolia. Phytochemistry 18: 1399 Aitken RJ, Best FSM, Warner P, Templeton A (1984) A prospective study of the relationship between semen quality and fertility in cases of unexplained infertility. J Androl 5: 297–303 Anderson JE, Chang CJ, Mclaughlein JL (1988) Bioactive components of Allamanda schottii. J Nat Prod 51: 307 Astoor A, King EJ (1954) Simplified colorimetric blood sugar method. Biochem J 56: XLIV Bedwal RS, Edwards MS, Katoch M, Bahuguna A, Dewan R (1994) Histological and biochemical changes in testes of Zinc deficient BALB/C strain of mice. Ind J Exp Biol 32: 243–247 Berndtson WE (1977) Methods for quantifying mammalian spermatogenesis: a review. J Anim Sci 44: 818–833 Bostofte E, Jorgen S, Rebbe H (1984) Interrelations among the characteristics of human semen and a new system for classification of male infertility. Fertil Steril 41: 95 –102 Coppen JJ, Cobb AL (1983) The occurrence of iridoids in Plumeria and Allamanda. Phytochemistry 22: 165 Coppen JJW (1983) Iridoids with algicidal properties from Allamanda. Phytochemistry 22: 179 Crossby WH, Munn JI, Furth FW (1954) Standardizing method for clinical hemoglobinometry. US Armed Force Med J 5: 695–703 Gottfried SP, Rosenberg B (1973) Improved manual spectrophotometric procedure for determination of serum triglycerides. Clin Chem 19: 1077–1078 Gunaga KP, Rao MC, Seth AR, Rao SA (1972) The role of glycogen during the development of the rat testes and prostate. J Reprod Fert 29: 157–162 Gupta RS, Kumar P, Dixit VP, Dobhal MP (2000) Antifertility studies of the root extract of the Barleria prionitis Linn. in male albino rats with special reference to testicular cell population dynamics. J Ethnopharmacol 70: 111–117 Ipstein J, Poly F (1970) In: Banchroft’s introduction to biostatistics 2nd ed p 44–64, Harper International, New York Kardono LBS, Palmawinata K, Kinghorin AD (1990) A flavon-3-ol glycoside from bark of Plumeria rubra. Phytochemistry 29: 2995
174
R. S. Gupta et al.
Kasturi M, Manivannan B, Ahmad RN, Shaikh PD, Pathan KM (1995) Ind. Changes in epididymal structure and function of albino rats treated with Azardirachta indica leaves. J Exp Biol 33: 725–729 Keel AB, Abney TO (1980) Influence of bilateral cryptorchidism in the mature rat: Alteration in testicular function and serum hormonal levels. Endocrinol 107: 1226–1233 Lowry OH, Rosebrough MJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193: 265–275 Montogomery R (1957) Determination of glycogen. Arch. Biochem. Biophys 57: 378–381 Mukherjee M, Chattopadhyay S, Mathur PP (1992) Effect of Flutamide on the physiological status of epididymis and epididymal sperms. Andrologia 24: 113–116 Oser BL (1965) Hawk’s Physiological chemistry. 14th ed. p.246. Mc Graw Hill, New York Parvinen M, Ruokonen A (1982) Endogenous steroids in the rat seminiferous tubules. Comparison of the stages of the epithelial cycles isolated by transillumination assisted microdissection. J Androl 3: 211–220 Perry LM, Metzer J (1980) Medicinal Plants of East and South Asia. p 29. MIT, Cambridge Prasad MRN, Chinoy NJ, Kadam KM (1972) Changes in succinate dehydrogenase levels in the rat epididymis under normal and altered physiological conditions. Fertil Steril 23: 186–190 Ritzen EM, Boitani C, Parvinen M (1981) Cyclic secretion of protein by the rat seminiferous tubules depending on the stage of spermatogenesis. Int J Androl (suppl.) 3: 57–58 Robaine B, Hermo L (1988) Efferent ducts, Epididymis and Vas deferens. Structure, function and their regulation In:
the Physiology of Reproduction, Knobil E, Neil JD (eds) p 999–1080, Raven Press New York Russell LD, Clermont Y (1977) Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. Anat Rec 187: 347–366 Sandujza R, Weinheimer AJ, Enler KL, Adam M (1985) Unusual occurrence of flavoplumarine, an antibacterial pigment in the mollusk Nerita albicilla. J Nat Prod 48: 335 Tiwari TN, Pandey VB, Dubey NK (2002) Plumieride from Allamanda cathartica as an antidermantophytic agent. Phytother Res 16 (4): 393–394 Warren L (1959) A thiobarbituric acid assay of Sialic acid. J Biol Chem 234: 1971–1975 Watt JM, Brreyer–Brranduijik MG (1962) Medicinal and Poisonous Plants of Southern and Eastern Africa p. 94 Zelversmit DB, Davis AK, Hamphestern MT (1950) Microdetermination of plasma phospholipid by trichloro-acetic acid precipitation method. J Lab Clin Invest 35: 155–160 Zlatkis A, Zak B, Boyle AJ (1953) A new method for the direct determination of serum cholesterol. J Lab Clin Med 41: 486–492
Address R. S. Gupta, Reproduction Physiology section, Department of Zoology, University of Rajasthan, Jaipur 302004, India Tel.: +91-141-701137; Fax: +91-141-2395-453, 2711-158; e-mail: [email protected]