Antispermatogenic, antiandrogenic activities of Albizia lebbeck (L.) Benth bark extract in male albino rats.

Abstract

Methanolic extract of Albizia lebbeck bark when administered orally at the dose level of 100 mg/rat/day to male rats of proven fertility for 60 days did not cause any significant loss in their body weights but the weights of reproductive organs, i.e. testis, epididymides, seminal vesicle and ventral prostate were decreased in a significant manner when compared to controls. Sperm motility as well as sperm density were reduced significantly which resulted in reduction of male fertility by 100%. Marked decline in the germ cell population was noticed. Population of preleptotene, pachytene, secondary spermatocytes and step-19 spermatid were declined by 60.86%, 65.81%, 71.56% and 66.55%, respectively. Cross-sectional surface area of sertoli cells as well as the cells counts were found to be depleted significantly. Leydig cells nuclear area and number of mature Leydig cells were decreased by 60.03% and 51.56%, respectively. Serum testosterone levels showed significant reduction after A. lebbeck extract feeding. Oral administration of the extract did not affect red blood cell (RBC) and white blood cell (WBC) count, haemoglobin, haematocrit and glucose in the blood and cholesterol, protein, triglyceride and phospholipid in the serum. In conclusion, A. lebbeck bark extract administration arrests spermatogenesis in male rats without noticeable side effects.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Albizia lebbeck; Antispermatogenic; Sperm motility; Sperm density; Sertoli cells; Leydig cells

Introduction

A number of plants have been identified and evaluated for fertility regulation in males. The present study is also an ongoing attempt to investigate fertility regulatory substances of plant origin. Albizia lebbeck (L.) Benth (Mimosoideae) is commonly called Indian Siris or East Indian Walnut. The tree is used in folk remedies for abdominal tumors, boils, cough, eye ailments, flu and lung ailments. The seed oil is used for leprosy and the powdered seed in scrofulous swellings. Indians use the flowers for spermatorrhea (Hartwell, 1969). It is also reported to be astringent, pectoral, rejuvenant and tonic (Hartwell, 1967-1971). The ethanolic extracts of A. lebback leaves exhibited anticonvulsant activity (Kastura et al., 2000). Saponines of A. lebbeck leaves reported to be nootropic and anxiolytic activity in albino mice (Une et al., 2001). The present investigation aims at determining the antifertility activity along with the nature of the A. lebbeck bark extract in male albino rats.

Materials and methods

Animal model

Colony-bred, healthy adult (4-5-month old) male albino rats of the Wistar strain, weighing between 150 and 200 g, were used. The animals were housed in polypropylene cages, with provision of 12h light: 12h dark regimen. The animals were fed a paletted standard rat chow supplemented with soaked gram and wheat. Water was provided ad libitum. All the experiments have been carried out under approval of institutional ethics committee.

Test material

The stem bark of A. lebbeck was collected from campus of University of Rajasthan, Jaipur. Plant material was identified (Voucher no--RUBL 19894) and authenticated from the herbarium, Department of Botany, University of Rajasthan, Jaipur-302 004, India.

Extraction of the plant material

The stem bark of A. lebbeck was shade dried and ground to powder, 3.0 kg of powdered bark was extracted with methanol for approximately (48-52 h). The obtained extract was concentrated under reduced pressure and yielded a 51 g dark brown semi-solid mass. This mass was washed with petroleum ether (40-60 [degrees]C) to remove the fatty components and then extracted with ethyl acetate. A part of this extract was fed to rats at 100 mg/rat/day and 25 g was subjected to column chromatography.

By eluting the column with petroleum ether (100%), 3-oxo-oleane-9(11)-ene was obtained. Further elution of column with benzene (100%) gave Lupenone, benzene: chloroform (1:1) gave [alpha]-amyrine, chloroform (100%) gave lupeol and [beta]-sitosterol, chloroform: acetone (1:1) gave acacic acid lactone and chloroform: acetone (3:1) gave vitalboside. The structures of these compounds were established by spectral analysis.

Experimental design

Male rats of proven fertility were divided into two groups of 10 each. One group was treated with A. lebbeck bark extract (100 mg/rat/day) for 60 days. The control group received vehicle (distilled water 0.5 ml/day) for 60 days. On day 61, animals were sacrificed under ether anaesthesia testes, epididymides, seminal vesicle, ventral prostate, liver and adrenal glands were removed, cleared off fat and connective tissue and weighed.

Fertility test

The mating exposure tests on control and treated groups were performed from day 55 to day 60. The male rats were cohabited with proestrus females in a ratio of 1:3, 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 site on day 16 of pregnancy via laparotomy.

Schedule of sacrifices

On day 61, after 24 h from the last dosing for both groups, the animals were weighed and sacrificed under ether anesthesia. Testis, epididymides, seminal vesicle and ventral prostate were dissected out and weighed.

Sperm motility and density

The sperm density was assessed in cauda epididymides and testes. The tissues were mashed in physiological saline (0.9% NaCl) and the sperm were counted in a Neubauer's counting chamber. Epididymal sperm were obtained through a puncture at the cauda with a disposable hypodermic needle dispersed in saline solution and the motility was determined with a haemocytometer.

Tissue biochemistry

Tissues were kept at -20[degrees]C until assayed for protein (Lowry et al., 1951), glycogen (Montgomery, 1957), sialic acid (Warren, 1959), and fructose was estimated in seminal vesicle (Mann, 1964).

Blood and serum biochemistry

Red blood cell (RBC) and white blood cell (WBC) counts, packed cell volume and haemoglobin were recorded in the blood samples collected directly from the heart. Serum protein (Lowry et al., 1951), total cholesterol (Zlatkis et al., 1953), phospholipid (Zilversmit and Davis, 1950), triglycerides (Gottfried and Rosenberg, 1973) and HDL-cholesterol (Burstein and Scholnic, 1970) were assayed.

Hormonal assay

Serum testosterone levels were assayed from samples using radio immuno assay method (Belanger et al., 1980). The sensitivity of the assay was 10 pg/ml.

Histophathological studies

Tissues were fixed in Bouin's fluid, tissues were sectioned at the thickness of 5 [micro]m and stained with haematoxylin and eosin for the discrimination of the stages of spermatogenesis (Leblond and Clermont, 1952).

Cell population dynamics

The evaluation of cell population dynamics was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analyzed using spherically appearing sections. Abercrombie's correcting factor was introduced to correct for the better chance a big cell has to be counted (Berndtson, 1977). Interstitial cell types such as fibroblast, degenerating and mature Leydig cells were estimated applying a differential counts which were statistically verified by the binomial distribution (Dixon and Massey, 1957). Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules. Leydig cell nuclear area and Sertoli cell area were measured at x800 magnification.

Statistical analysis

All the values of body/organ weights, histometry and testicular dynamics were expressed in terms of mean[+ or -]standard error. The treated groups were compared to control using the Student's "t" test.

Results

Body and organ weights

The oral administration of A. lebbeck extract to male rats for 60 days did not cause any significant change in the body weights of treated rats. However, the weights of testis, epididymides, seminal vesicle and ventral prostate were significantly (p<0.001) reduced when compared to control values (Table 1).

Sperm density, motility and fertility

The treated rats showed significant (p<0.001) reduction in the sperm concentration of testis and cauda epididymides. The sperm motility of the cauda epididymis was also reduced significantly (p<0.001). The A. lebbeck bark extract treatment reduced, the fertility of male rats by 100% (Table 1).

Tissues biochemistry

The protein contents of testis, epididymides, seminal vesicle and ventral prostate were reduced significantly (p<0.001) in comparison with controls. Sialic acid contents of the testis, cauda epididymides, seminal vesicles and ventral prostate were depleted. Glycogen contents in the testis were decreased significantly following treatment with A. lebbeck bark extract. Seminal vesicular fructose was also decreased significantly (Table 2).

Blood and serum biochemistry

Blood variables i.e. RBC and WBC counts, haemoglobin, haematocrit and sugar were within the normal range. Serum protein, cholesterol, phospholipid, triglycerides and HDL-cholesterol did not change significantly after A. lebbeck extract treatment to rats (Table 3).

Hormonal assay

Serum testosterone level of A. lebbeck bark extract treated animals was decreased significantly (p<0.001) in comparison to controls (Fig. 1a).

Cell population dynamics

Administration of A. lebbeck bark extract resulted in significant reduction in most of the cell types in the seminiferous tubules. Sertoli cell area, Leydig cell nuclear area and seminiferous tubular diameter were significantly reduced (Fig. 1b,c,d). Total counts of primary spermatocytes (preleptotene and pachytene), secondary spermatocyte, round spermatid, step-19 spermatid and spermatogonia were declined by 60.86%, 65.81%, 71.56%, 73.37%, 66.55% and 47.48%, respectively (Figs. 2a-d, 3a,b). Cross-sectional surface area of sertoli cell and the number of Leydig cell were also decreased (Fig. 3c,d).

Discussion

The result showed that A. lebbeck bark extract adversely affects the male reproductive functions. The possible cause of decrease in testis weight may be depletion in the number of spermatogenic elements and spermatozoa (Takihara et al., 1987). Marked decrease in the germ cell counts specially the number of secondary spermatocytes and rounded spermatid reduced the testis weight (Bone et al., 2000). Statistically significant reduction in the weight of accessory sex organs reflects interference on testosterone output and antiandrogenic nature of plant extract (Nijar et al., 1995). Reduced seminiferous tubular diameter reflects tubular shrinkage, which may occur due to sloughing of epithelial cells.

A. lebbeck bark extract might possibly inhibit the activity of adenosine triphosphate (ATP) in the spermatozoa by uncoupling of oxidative phosphorylation from the respiratory chain and prevent phosophorylation of adenosine diphosphate to ATP and thus renders the spermatozoa immotile (Kalla and Vasudev, 1981). The reduction in sperm motility in cuda epididymis is of importance with regard to fertilization (Bedford, 1983). A decrease in sperm reserve may be a responsible cause for reduction in the weight of epididymis. The significant decrease in testosterone level in the treated animals support this view (Sandhyakumary et al., 2002).

The protein synthesis, and concentration in the accessory sex organs are androgen dependent. Administration of antiandrogenic caused a significant decrease in the protein concentration of epididymis, which was restored to normal levels after testosterone therapy (Brooks and Higgins, 1980). Structural integrity of acrosomal membrane is dependent upon sialic acid and due to alteration in its content the metabolism, motility and fertilizing ability of sperms may also be affected (Riar et al., 1973). The low glycogen content in the testis after A. lebbeck administration is possibly due to the inhibition of phosphorylase activation or the depletion of certain other enzyme which could block androgen synthesis (Frayne et al., 1996). The process of fructose formation in seminal vesicle is initiated and controlled by testicular androgens (Brooks, 1979).

In the present study Sertoli cells were quantitatively and morphologically altered, which might be the results of androgens deficiency (Oko and Hrudika, 1984). The control of spermatogenic cycle is mediated by sertoli cells, which regulate cell cycle kinetics and influence the differentiation of primordial germ cells (Gasinska and Hill, 1990). 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 cell has direct bearing on spermatogenesis. Deformation of Leydig cell further indicates the inefficiency of these cells to synthesize testosterone (Watcho et al., 2001).

Conclusion

Methanolic extract of A. lebbeck bark brought about the infertile state in male rats due to interference in the testicular androgen levels altering the process of spermatogenesis.

Acknowledgments

The authors are thankful to the Head, Department of Zoology, Prof. N.K. Lohiya, Coordinator SAP, Department of Zoology, University of Rajasthan, Jaipur, for providing necessary facilities and University Grant Commission Regional office Bhopal (MP) India for financial support. One of the author (Rakesh Chaudhary) is supported by a Senior Research Fellowship from CSIR, New Delhi (India).

References

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R.S. Gupta*, J.B.S. Kachhawa, R. Chaudhary

Reproduction Physiology Section, Department of Zoology, University of Rajasthan, Jaipur 302 004, India

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (R.S. Gupta).

Table 1. Effect of A. lebbeck bark extract on the body weight, sperm
motility, density and fertility in male rats

                                 Organ weight (mg/100 g b.wt.)
Treatment       Body weight (g)  Testis           Epididymides

Control         228.30[+ or -]   1350.05[+ or -]  480.60[+ or -]
                  8.41             11.49            7.37
A. lebbeck      223.43[+ or -]    889.13[+ or -]  319.28[+ or -]
bark extract      7.92 (ns)        13.67**          11.35**
100 mg/rat/day

                                                        Sperm motility%
                     Organ weight (mg/100 g b.wt.)      (Cauda
Treatment            Seminal vesicle  Ventral prostate  epididymides)

Control              633.87[+ or -]   390.54[+ or -]    69.05[+ or -]
                       5.20             3.79              1.14
A. lebbeck           513.38[+ or -]   208.97[+ or -]    19.11[+ or -]
bark extract           18.45**          2.99**            1.61**
100 mg/rat/day

                Sperm density (million/ml)
Treatment       Testis         Cauda epididymides  Fertility%

Control         10.05[+ or -]  65.43[+ or -]       100 (+) ve
                  1.05           3.19
A. lebbeck       1.72[+ or -]   8.49[+ or -]       100 (-) ve
bark extract      0.45**         0.72**
100 mg/rat/day

Values are mean[+ or -]SEM (n = 10), ns = non-significant, **p<0.001 vs.
control.

Table 2. Effect of A. lebbeck bark extract on biochemical parameters of
male rats

                Protein (mg/g)
Treatment       Testis          Cauda epididymides  Seminal vesicle

Control         229.14[+ or -]  265.24[+ or -]      204.31[+ or -]
                  5.21            6.29                4.28
A. lebbeck      185.36[+ or -]  204.45[+ or -]      179.21[+ or -]
bark extract      3.79**          5.19**              3.49**
100 mg/rat/day

                Protein (mg/g)  Sialic acid (mg/g)
                Ventral                       Cauda
Treatment       prostate        Testis        epididymides

Control         200.04[+ or -]  5.51[+ or -]  6.18[+ or -]
                  3.05            0.03          0.04
A. lebbeck      153.52[+ or -]  3.72[+ or -]  3.99[+ or -]
bark extract      2.91**          0.02**        0.03**
100 mg/rat/day

                  Sialic acid (mg/g)
                Seminal       Ventral         Glycogen (mg/g)
Treatment       vesicle       prostate        Testis

Control         5.19[+ or -]  5.45[+ or -]    2.79[+ or -]
                  0.12          0.16            0.17
A. lebbeck      3.79[+ or -]  3.81[+ or -]    1.78[+ or -]
bark extract      0.08**        0.11**          0.07**
100 mg/rat/day

                Fructose (mg/g)
Treatment       Seminal vesicle

Control         5.63[+ or -]
                  0.24
A. lebbeck      4.13[+ or -]
bark extract      0.14**
100 mg/rat/day

Values are mean[+ or -]SEM (n = 10), **p<0.001 vs. control.

Table 3. Effect of A. lebbeck bark extract in serum and blood in rats

                Protein           Cholesterol     Triglyceride
Treatment       (mg/dl)           (mg/dl)         (mg/dl)

Control         14333.31[+ or -]  105.18[+ or -]  113.30[+ or -]
                  129.32            11.58           7.32
A. lebbeck      14111.11[+ or -]  102.43[+ or -]  109.45[+ or -]
bark extract      120.09 (ns)       9.43 (ns)       5.39 (ns)
100 mg/rat/day

                                HDL            Blood
                Phospholipids   cholesterol    sugar
Treatment       (mg/dl)         (mg/dl)        (mg/dl)

Control         108.57[+ or -]  51.32[+ or -]  94.36[+ or -]
                  5.62            2.21           3.46
A. lebbeck      111.12[+ or -]  49.45[+ or -]  89.43[+ or -]
bark extract      6.01 (ns)       2.14 (ns)      3.92 (ns)
100 mg/rat/day

                RBC
                (million/
Treatment       [mm.sup.3])   WBC ([mm.sup.-3])  Haematocrit (%)

Control         5.19[+ or -]  7290[+ or -]       37.38[+ or -]
                  0.17          55.03              1.59
A.lebbeck       5.02[+ or -]  7142[+ or -]       35.39[+ or -]
bark extract      0.08 (ns)     51.21 (ns)         1.72 (ns)
100 mg/rat/day

Treatment       Haemoglobin (g%)

Control         14.07[+ or -]
                  0.40
A.lebbeck       14.20[+ or -]
bark extract      0.50 (ns)
100 mg/rat/day

Values are mean[+ or -]SEM (n = 10), ns = non-significant.

(a)

                              Serum Testosterone
                              (ng/ml)

Control                       4.31 [+ or -] 0.09
Albizia lebbeck bark extract  2.33 [+ or -] 0.07**

(b)

                              Sertoli cell Area
                              [[micro]g.sup.2]

Control                       46.81 [+ or -] 1.89
Albizia lebbeck bark extract  18.40 [+ or -] 0.93**

(c)

                              Leydig cell nuclear area
                              [[micro]m.sup.2]

Control                       17.89 [+ or -] 0.88
Albizia lebbeck bark extract   7.15 [+ or -] 0.52**

(d)

                              Seminiferous tubules diameter
                              [micro]m

Control                       262.28 [+ or -] 4.57
Albizia lebbeck bark extract  204.41 [+ or -] 1.27**

Values are mean [+ or -] SEM (n = 10); ** P<0.001 vs Control.

Fig. 1. Effect of Albizia lebbeck bark extract on serum testosterone
level and testicular histometry in male rats.

Note: Table made from bar graph.

(a)

                              Preleptotene
                              number/cross-section

Control                       22.82 [+ or -] 2.11
Albizia lebbeck bark extract   8.93 [+ or -] 0.64**

(b)

                              Pachytene
                              number/cross-section

Control                       35.40 [+ or -] 3.37
Albizia lebbeck bark extract  12.10 [+ or -] 0.88**

(c)

                              Secondary spermatocytes
                              number/cross-section

Control                       67.87 [+ or -] 5.86
Albizia lebbeck bark extract  19.30 [+ or -] 2.08**

(d)

                              Rounded spermatides
                              number/cross-section

Control                       30.83 [+ or -] 2.18
Albizia lebbeck bark extract   8.09 [+ or -] 0.57**

Values are mean [+ or -] SEM (n = 10); ** P<0.001 vs Control.

Fig. 2. Effect of Albizia lebbeck bark extract treatment on testicular
cell population dynamics in male rats.

Note: Table made from bar graph.

(a)

                              Step-19 spermatides
                              number/cross-section

Control                       30.83 [+ or -] 2.13
Albizia lebbeck bark extract  10.09 [+ or -] 0.54**

(b)

                              Spermatogonia
                              number/cross-section

Control                       6.97 [+ or -] 0.77
Albizia lebbeck bark extract  3.66 [+ or -] 0.54**

(c)

                              Sertoli cells
                              number/cross-section

Control                       2.81 [+ or -] 0.05
Albizia lebbeck bark extract  2.02 [+ or -] 0.07**

(d)

                         Leydig cells (differential counts)
                         Percent (%)

Control                  21.12 [+ or -]  24.05 [+ or -]  54.33 [+ or -]
                           1.14            2.11**          3.02
Albizia lebbeck bark     40.02 [+ or -]  33.50 [+ or -]  26.30 [+ or -]
  extract                  1.51**          2.24**          2.02**

Values are mean [+ or -] SEM (n = 10); ** P<0.001 vs Control.

Fig. 3. Effect of Albizia lebbeck bark extract treatment on testicular
cell population dynamics and testicular histometry in male rats.

Note: Table made from bar graph.