Antispermatogenic Effects of Parkinsonia aculeata. Stembark in Male Rats

Abstract

To evaluate the effect of Parkinsonia aculeata. Linn. (Caesalpiniaceae) on male reproductive function and fertility, the ethanol crude extract of its stembark was administered orally to male rats at the dose levels of 50, 100 and 200 mg/rat per day for 60 days. A dose-dependent response was found after this treatment. The body weights were not affected, whereas the weights of reproductive organ decreased significantly after this treatment. Significant (p < 0.001) suppression of cauda epididymal sperm count and motility was observed. Fertility was decreased in this treatment by 100% in Parkinsonia aculeata.–treated rats. The testicular cell population, that is, primary (preleptotene, pachytene) and secondary spermatocytes, were reduced significantly. A significant decline was also noticed in seminiferous tubular diameter and differential count of Leydig cells. Oral administration of this drug at all the dose levels did not alter the blood and serum profiles, whereas testosterone level of serum was declined. The concentration of testicular cholesterol was significantly elevated, whereas protein, sialic acid, glycogen, and fructose content were reduced significantly (p < 0.01). It is concluded that Parkinsonia aculeata. treatment has an antispermatogenic effect in male rats.

Keywords:

• Cell population dynamics
• Leydig cells
• Parkinsonia aculeata
• Sertoli cell
• sialic acid
• sperm motility

Introduction

Parkinsonia aculeata. Linn. (Caesalpiniaceae), also known as Jerusalem thorn and Mexican palo verde, is a thorny shrub or small tree native to the tropical Americas. It is distributed throughout the Indian plains and is also available in Jaipur, Rajasthan (India). In India, it is commonly known as “vilayti babul” in Hindi and “balati kikar” in Bengali (Harborne et al., 1971). The plant is available in Jaipur, Rajasthan (India). Compounds reported from Parkinsonia aculeata. are C-glycosides-epiorientin, parkinsonia A, and parkinsonia B. They are characterized as 8C-luteolin glycoside, 5-O.-methylluteolin-8C-β.-D-glucoside, and 5,7-di-O.-methyllutoline-8C-β.-D-glucoside, respectively (Bhatia et al., 1966). β.-Amyrin acetate, β.-amyrone, β.-sistosterol, and vitexine have been isolated from the bark. Only C-glycosylflavones present in leaves, which were identified as orientin, isorientin, vitexin, and isovitexin (Besson et al., 1980). A new flavone with an epoxy-iso-pentyl moiety, named parkintin, has been isolated from the methanol soluble part of Parkinsonia aculeata. (Ali et al., 2005).

Although Parkinsonia aculeata. is regarded as one of the worst weeds, the alcohol extract of its bark exhibited CNS-depressant activity in mice, whereas aqueous extract showed cholinomimetic activity (Rao et al., 1979). Vitexine and α-amyrin acetate isolated from Parkinsonia aculeata. stembark are reported for their antifertility activities (Bhargava, 1986; Gupta et al., 2004), and β.-sistosterol is reported for its spermicidal potential (Rawat et al., 1988). The above literature on Parkinsonia aculeata. encouraged us to test its effects on male reproductive function and fertility.

Materials and Methods

Collection and identification of plant material

The stembark of Parkinsonia aculeata. was collected in the months of July–August, from the Sikar district near Jaipur (India). Plant material was identified (voucher no. RUBL/19899) at the Herbarium, Department of Botany, University of Rajasthan (Jaipur, India).

Extraction and isolation of compounds

Shade-dried stembark (approximately 2 kg) was powdered and extracted with ethanol on a steam bath for 72 h. The extract was concentrated under reduced pressure. The syrupy mass so obtained was treated with acetonitrile for the removal of fats. The fat-free extract was extracted with benzene; the solvent was removed under reduced pressure. A part of this final extraction about (8%) was employed for the antifertility activity in male albino rats. The rest (40 g) of the benzene extract of Parkinsonia aculeata. was used for the isolation of constituent compounds by means of chromatographic technique over Si-gel column and eluted with solvents of increasing polarity. The following compounds were isolated: α-amyrin acetate (250 mg), β.-amyrin acetate (275 mg), 6-hydroxypentacosylpentanoate ethynoma decanoate (172 mg), and 6-hyrdoxytritriacontan-3-one (189 mg).

Experimental model

Sexually mature male albino rats of the Wistar strain, weighing 150–175 g, from a laboratory-bred colony were used as the experimental model. Animals were kept under standard conditions (12-h light/12-h dark; 25 ± 3°C; 35–60% relative humidity), and rat pellet diet (Ashirwad Industries Ltd., Chandigarh, India) and water were provided ad libitum..

Fertility test

The fertility test of individual males was done before the experiment and from days 55 to 60 in both control as well as in treated groups. The male rats cohabited with proestrous females in ratio of 1:2. Vaginal smears were checked for positive mating. The inseminated females were separated, and numbers of litters delivered were recorded.

Experimental protocol

The experimental protocol was approved by the animal ethics committee of the institute. Proven fertile male rats were divided into four groups of 10 rats each. Test material (i.e., crude extract of Parkinsonia aculeata.) was administered orally; during sample administration anesthetic agent was not used. Although the benzene extract was not completely soluble in water, the test material was administered in an emulsified form, taking water as medium.

• Group I: Control group, rats received vehicle only (distilled water, 0.5 mL/rat per day) for 60 days.

• Group II: Rats were treated with crude extract of Parkinsonia aculeata. (stembark) (50 mg/rat per day) for 60 days.

• Group III: Rats were treated with crude extract of Parkinsonia aculeata. (stembark) (100 mg/rat per day) for 60 days.

• Group IV: Rats were treated with crude extract of Parkinsonia aculeata. (stembark) (200 mg/rat per day) for 60 days.

Schedule of sacrifice

Rats were autopsied under light ether anesthesia on the 61st day. The reproductive organs (i.e., testes, epididymides, seminal vesicles, and ventral prostate) were dissected out and weighed. Half of the tissues were kept at − 20°C until assayed for biochemical estimation of protein (Lowry et al., 1951), sialic acid (Warren, 1959), glycogen (Montgomery, 1957), cholesterol, and fructose. Remaining tissues were fixed in Bouin's fluid.

Sperm count and motility

Sperm motility was observed in cauda epididymides, and sperm density was observed in cauda epididymides and testes (Prasad et al., 1972).

Blood and serum analysis

The blood of experimental rats was analyzed for RBC, WBC count, hemoglobin, hematocrit, and blood sugar. The serum was analyzed to estimate the total protein, cholesterol (Zlatkis et al., 1953), HDL-cholesterol (Burnstein et al., 1970), triglyceride (Gottfried & Rosenburg, 1973), and phospholipid. Serum testosterone was assessed by radioimmunoassay (Belanger et al., 1980).

Histopathologic preparations

Paraffin sections were made by fixed tissues and stained with hematoxylin and eosin for the discrimination of the stages.

Quantitative analysis

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 (Berndtson, 1977). All types of interstitial cells were counted. Diameter of Leydig cell nuclei and seminiferous tubules was determined at 800 × and 80 ×, respectively.

Statistical analysis

Data are expressed as mean ± standard error of mean (SEM), and comparisons among groups was performed using one-way ANOVA. The Student's t.-test was used to ascertain which mean was statistically significant. The level of statistical significance was set at p < 0.01 and p < 0.001.

Results

Body and reproductive organ weights

Oral administration of Parkinsonia aculeata. at all the dose levels did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicles, and ventral prostate were decreased significantly (p < 0.001) (Table 1) in a dose-dependent manner.

Table 1.. Effect of Parkinsonia aculeata. (stembark) on the body and organ weights.

		Organ weights (mg/100 g b.wt.)
Treatment	Final body weight (g)	Testes	Epididymides	Seminal vesicles	Ventral prostate
Group I (control)	220 ± 13.25	1184.20 ± 10.37	528.15 ± 10.70	656.83 ± 13.05	396.73 ± 10.58
Group II (P. aculeata. extract 50 mg/rat/day)	195 ± 7.85^ns	976.53 ± 5.82**	489.07 ± 8.20	558.78 ± 10.62	285.39 ± 5.87**
Group III (P. aculeata. extract 100 mg/rat/day)	190.05 ± 18.09^ns	670.81 ± 5.18**^aa.	436.90 ± 5.19**^a.	430.72 ± 8.57**^aa.	234.05 ± 3.51**^aa.
Group IV (P. aculeata. extract 200 mg/rat/day)	180.82 ± 20.53^ns	565.78 ± 3.76**^aa.^bb.	409.72 ± 7.38**^a.^b.	398.53 ± 6.21**^aa.^b.	210.58 ± 3.82**^a.^b.

All values are expressed as mean ± SE. ns, non-significant.

Levels of significance: *p < 0.01;

**p < 0.001 compared with group I.

^a.p < 0.01;

^aa.p < 0.001 compared with group II.

^b.p < 0.01 compared with group III.

Sperm dynamics and fertility

The sperm motility of cauda epididymides was significantly reduced (p < 0.001) at 100 and 200 mg dose levels. Concentration of testicular and cauda epididymal spermatozoa declined by 66.26% and 78.75% at the 100 mg dose level, and 69.86% and 84.29% at the 200 mg dose level. The fertility of male rats reduced up to 100% after treatment with Parkinsonia aculeata. (stembark) (Table 2).

Table 2.. Effect of Parkinsonia aculeata. (stembark) on sperm dynamics and fertility of male rats.

		Sperm density (million/mL)
Treatment	Sperm motility (cauda epididymides) (%)	Tests	Cauda epididymides	Fertility (%)	Testosterone (ng/dL)
Group I (control)	68.58 ± 3.78	6.67 ± 1.20	48.25 ± 2.69	100	4.32 ± 0.12
Group II (P. aculeata. extract 50 mg/rat/day)	59.23 ± 6.59^ns	4.68 ± 0.33^ns	26.83 ± 2.18	48	3.82 ± 0.10
Group III (P. aculeata. extract 100 mg/rat/day)	32.44 ± 2.07**^a.	2.25 ± 0.50^a.	10.25 ± 1.05**^aa.	0	2.03 ± 0.75
Group IV (P. aculeata. extract 200 mg/rat/day)	24.89 ± 1.23**^a.^b.	2.01 ± 0.23^aa.	7.58 ± 2.30**^aa.	0	1.96 ± 0.52^a.

All values are expressed as mean ± SE. ns, non-significant.

Levels of significance: *p < 0.01;

**p < 0.001 compared with group I.

^a.p < 0.01;

^aa.p < 0.001 compared with group II.

^b.p < 0.01 compared with group III.

Biochemical observations

As shown in Table 3, the protein and sialic acid contents of testes, cauda epididymides, and seminal vesicle were reduced significantly (p < 0.001). Also, a significant (p < 0.001) decline was noticed in testicular glycogen and fructose content, whereas significant (p < 0.001) elevation was observed in testicular cholesterol.

Table 3.. Effect of Parkinsonia aculeata. (stembark) on biochemical parameters of male rats.

	Protein (mg/g)				Sialic acid (mg/g)				Glycogen (mg/g)	Cholesterol (mg/g)	Fructose (mg/g)
Treatment	Testes	Cauda epididymides	Seminal vesicles	Ventral prostate	Testes	Cauda epididymides	Seminal vesicles	Ventral prostate	Testes	Testes	Seminal vesicles
Group I (control)	240.56 ± 5.37	256.89 ± 4.69	225.43 ± 5.81	216.25 ± 2.56	5.38 ± 0.59	5.28 ± 0.18	5.20 ± 0.10	5.35 ± 0.08	2.86 ± 0.12	13.03 ± 1.05	5.87 ± 0.25
Group II (P. aculeata. extract 50 mg/ rat/day)	206.25 ± 8.12	223.47 ± 9.83	220.51 ± 5.03^ns	208.37 ± 3.81^ns	5.02 ± 0.12^ns	5.00 ± 0.32^ns	4.92 ± 0.03^ns	4.69 ± 0.21	2.25 ± 0.07	16.25 ± 2.15^ns	4.93 ± 0.46^ns
Group III (P. aculeata. extract 100 mg/ rat/day)	178.64 ± 3.00**^a.	195.00 ± 8.88**	170.45 ± 6.66**^a.	176.50 ± 6.50^a.	4.25 ± 0.06^a.	3.50 ± 0.07**^a.	4.05 ± 0.09**^aa.	3.63 ± 0.39	1.80 ± 0.03**^a.	17.99 ± 0.90	4.26 ± 0.11
Group IV (P. aculeata. extract 200 mg/ rat/day)	169.23 ± 2.58**^a.	173.85 ± 5.32**^a.	161.73 ± 4.53**^aa.	158.38 ± 2.53**^aa.	4.03 ± 0.03^a.^b.	3.01 ± 0.13**^a.^b.	3.52 ± 0.07**^aa.^b.	3.12 ± 0.54	1.51 ± 0.12**^a.	18.28 ± 2.51	3.66 ± 0.22**^a.

All values are expressed as mean ± SE. ns, non-significant.

Levels of significance: *p < 0.01;

**p < 0.001 compared with group I.

^a.p < 0.01;

^aa.p < 0.001 compared with group II.

^b.p < 0.01 compared with group III.

Blood and serum profiles

Parameters of blood and serum did not show any significant change (Table 4), whereas serum testosterone level was decreased (Table 3).

Table 4.. Effect of Parkinsonia aculeata. (stembark) on some components of blood and serum of male rats.

Treatment	RBC (million/mm^3)	WBC (/mm^3)	Hemoglobin (g/dL)	Hematocrit (%)	Blood sugar (mg/dL)	Protein (mg/dL)	Cholesterol (mg/dL)	Triglyceride (mg/dL)	Phospholipid (mg/dL)	HDL-cholesterol (mg/dL)
Group I (control)	5.30 ± 0.20	8540 ± 70	14.15 ± 0.20	42.35 ± 0.35	90.98 ± 2.48	12,222.22 ± 358.3	105.51 ± 3.04	112.31 ± 5.50	105.05 ± 3.04	42.25 ± 2.30
Group II (P. aculeata. extract 50 mg/ rat/day)	5.42 ± 2.56^ns	8556 ± 80^ns	14.00 ± 0.32^ns	42.83 ± 0.83^ns	92.38 ± 1.32^ns	12,222.22 ± 375.8^ns	110.23 ± 7.82^ns	98.00 ± 8.58^ns	100.00 ± 5.36^ns	42.05 ± 1.80^ns
Group III (P. aculeata. extract 100 mg/ rat/day)	4.90 ± 0.10^ns	8350 ± 75^ns	13.10 ± 0.82^ns	36.70 ± 2.90^ns	82.80 ± 3.60^ns	12,333.32 ± 333.3^ns	118.18 ± 9.09^ns	102.00 ± 3.50^ns	96.58 ± 5.68^ns	39.80 ± 1.95^ns
Group IV (P. aculeata. extract 200 mg/ rat/day)	4.88 ± 0.30^ns	8450 ± 72^ns	14.16 ± 0.30^ns	44.50 ± 8.25^ns	86.79 ± 2.68^ns	12,222.22 ± 348.54^ns	120.25 ± 8.30^ns	95.34 ± 4.89^ns	95.52 ± 5.30^ns	37.56 ± 2.20^ns

All values are expressed as mean ± SE. ns, non-significant.

Testicular cell dynamics

Testicular cell components were decreased after oral administration of Parkinsonia aculeata.. Sertoli cells and spermatogonia were reduced markedly in this study. The preleptotene, pachytene, and secondary spermatocyte were decreased by 35.57%, 52.23%, and 54.31% at the 50 mg dose level, 61.40%, 1.68%, and 66.09% at the 100 mg dose level, and 70.02%, 83.10%, and 77.96% at the 200 mg dose level, respectively. Seminiferous tubular diameter reduced significantly (p < .001), and the nuclear area of Leydig cells was decreased only at the 200 mg dose level. The number of mature Leydig cells was reduced, whereas the number of degenerating cells increased significantly (p < 0.01) (Table 5).

Table 5.. Effect of Parkinsonia aculeata. (stembark) on testicular cell population dynamics in male rats.

	Testicular cell counts (number/cross section)						Leydig cells
			Primary spermatocytes					Differential count (%)
Treatment	Sertoli cell	Spermatogonia	Preleptotene	Pachytene	Secondary spermatocytes	Seminiferous tubular diameter (µm)	Nuclear area (µm^2)	Fibroblast	Degenerating	Mature
Group I (control)	2.85 ± 0.25	7.05 ± 0.62	22.85 ± 2.11	35.80 ± 2.25	49.25 ± 4.05	250.00 ± 5.16	12.68 ± 2.00	23.20 ± 1.95	17.02 ± 0.52	59.18 ± 1.10
Group II (P. aculeata. extract 50 mg/ rat/day	2.61 ± 1.53^ns	5.73 ± 0.35^ns	14.72 ± 2.35	17.10 ± 3.53^ns	22.50 ± 2.56	220.50 ± 7.83	9.83 ± 1.57^ns	21.85 ± 5.23^ns	40.25 ± 2.39^ns	38.25 ± 3.65^ns
Group III (P. aculeata. extract 100 mg/ rat/day)	2.18 ± 0.15	4.72 ± 0.52	8.82 ± 0.22	8.19 ± 0.64**	16.70 ± 0.94**	148.30 ± 13.40^a.	7.56 ± 0.92^ns	19.61 ± 4.43^ns	46.67 ± 5.09**	32.32 ± 3.17**
Group IV (P. aculeata. extract 200 mg/rat/day)	2.00 ± 0.18	3.88 ± 0.10^a.^b.	6.85 ± 0.11^a.^b.	6.05 ± 0.38**^a.^b.	10.85 ± 1.80**^a.^b.	132.50 ± 4.28**^a.^b.	5.20 ± 1.03	18.50 ± 3.50^ns	52.83 ± 4.50**	22.25 ± 1.39**^a.^b.

All values are expressed as mean ± SE. ns, non-significant.

Level of significance: *p < 0.01;

**p < 0.001 compared with group I.

^a.p < 0.01;

^aa.p < 0.001 compared with group II.

^b.p < 0.01 compared with group III.

Discussion

Oral administration of Parkinsonia aculeata. caused reduction in the testosterone level and in the weights of testes and other accessory sex organs of experimental rats. The epididymis, seminal vesicle, and ventral prostate are androgen dependent, relying on testosterone for their growth and function (Klinefelter & Hess, 1998); their weight loss reflects a decline in bioavailability and production of androgen (Mathur & Chattopadhyay, 1982). Reduction in the number and diameter of Leydig cells and significant elevation in testicular cholesterol indicate decreased androgen levels.

Decreased fertility is explained by the suppression of sperm motility and density that might be due to α-amyrin acetate (Gupta et al., 2004), present in Parkinsonia aculeata. stembark. Sperm motility is one of the most important predictors of sperm fertilizing ability. Sialic acids are concerned with changing the membrane surface of maturing spermatozoa, coating of spermatozoa with certain antigens, and in the development of their fertilizing capacity (Turner et al., 1995). Low levels of sialic acid may suppress the fertilizing capacity of spermatozoa. Sperm immotility, in all the treated groups, suggests an undersupply of androgens to epididymis, resulting in impairment of epididymal functions and its weight. Motility of spermatozoa is due to the flagellar beat, which is dependent on the microtubular apparatus of the flagellum (Williamson et al., 1984) and adenosine triphosphate (ATP) (Liu et al., 1987). Low levels of fructose could inhibit the sperm motility by deficient generation of ATP (Chinoy & Bhattacharya, 1997).

Reduced sperm count in testes and epididymis may be due to inhibition of meiotic division of spermatocytes (Akbarsha et al., 2001). In Parkinsonia aculeata.–treated rats, primary and secondary spermatocytes were decreased. Similar results have been shown in Mondia whitei. Linn. (Watcho et al., 2001). Reduced protein and glycogen content could be correlated with the low sperm density (Gunaga et al., 1972; Chinoy & Bhattacharya, 1997). Seminiferous tubular diameter was decreased in this treatment, which may be due to widespread cellular damage. Alteration in the number of Sertoli cells and spermatogonia in treated rats shows androgen deprivation. Parkinsonia aculeata. treatment did not show any adverse effect on hematologic and serum parameters.

In conclusion, Parkinsonia aculeata. (stembark) treatment induced antiandrogenic effects in male rats without altering general body metabolism, possibly due to α-amyrin acetate present in its crude extract.

Acknowledgments

The authors are thankful to Dr. Jagdish Prasad for statistical help and to the Head, Department of Zoology and Chemistry; Prof. N.K. Lohiya, Coordinator, SAP, Department of Zoology, University of Rajasthan, Jaipur, India, for providing the necessary facilities.