Introduction

Echinops echinatus Roxb (Compositae) is a pubescent annual herb of 1–3 feet in height, being distributed throughout India [1]. Roots of this plant are sold in different Indian markets under the trade name of brahmadandi [2] and are employed in the treatment of disorders related to the reproductive system, such as abortifacients, to hasten the process of delivery, in urinary discharges, as a diuretic and in syphilis [3–6]. Experimental studies in female rats showed that the antifertility effects of E. echinatus roots and its various extracts are due to antiestrogenic activity of the plant [7]. Chaturvedi et al. [8] reported that oral administration of crude alcohol extract of E. echinatus roots to male rats at the dose of 200 mg/kg body weight resulted in reversible antifertility effects, with no adverse toxic effects. In our continuous investigations on the chemical constituents and biological activities of E. echinatus, we found the presence of terpenoids as the chief constituents in the plant; therefore, it was thought worthwhile to prepare a fraction containing terpenoids and investigate its effect on the male reproductive parameters in Wistar albino rats. The terpenoidal fraction was standardised in terms of its marker (lupeol) content using high-performance thin-layer chromatography (HPTLC).

Materials and methods

Plant material

Roots of E. echinatus were collected in the month of August 2005 from the outfield of Gulbarga City, Karnataka, India, and were authenticated in the Botany Department of The M. S. University of Baroda, Vadodara, India. A voucher specimen (no. Pharmacy/EE/05-06/01/SP) has been deposited in the Pharmacy Department of The M. S. University of Baroda, Vadodara, India.

Preparation of extract and terpenoidal fraction

A coarse powder (500 g) of the shade-dried roots was extracted with petroleum ether (60/80) in a Soxhlet apparatus. The extract was concentrated on a rotary vacuum evaporator (BUCHI Rotavapor R 200) and dried under vacuum (yield = 3.96% w/w). Vacuum-dried petroleum ether extract was subjected to saponification, and the unsaponifiable material rich in terpenoids was separated out and dried under vacuum (25% w/w) [9]. Vacuum-dried terpenoid-rich fraction of petroleum ether extract (TRFPE) was taken for further studies.

Chemical test and quantitative estimation of the marker compound by HPTLC

Obtained TRFPE was subjected to Liebermann Burchard test for terpenoids [10]. In brief, a small quantity of the TRFPE was dissolved in chloroform and treated with acetic anhydride and few drops of concentrated sulfuric acid, which gives a dark pink to red colour, indicating the presence of terpenoids, which were further separated on a thin-layer chromatography (TLC) plate and detected by treatment with anisaldehyde sulfuric acid reagent.

HPTLC conditions

The HPTLC system (CAMAG) consisting of a LINOMAT-IV applicator and TLC Scanner-III, precoated plates of silica gel G60 F254 (E-Merck) and a mobile phase containing toluene:chloroform:ethyl acetate:acetic acid (10:2:1:0.1) were used in the studies. Detection was done using antimony trichloride reagent [11] followed by scanning under ultraviolet (UV) light at 366 nm.

Preparation of sample solutions

A test solution containing 1 mg/ml of the TRFPE was prepared in chloroform and methanol (1:1), and similarly, a stock solution of the pure lupeol (obtained commercially from Sigma Chemicals, USA) was prepared by dissolving 10 mg of accurately weighed pure lupeol in 10 ml of chloroform and methanol (1:1); required concentrations were then obtained by further dilution of the stock solutions.

Calibration curve

The standard solution (20–100 ng per spot) was applied on a precoated TLC plate of uniform thickness. The plate was developed to a height of 8 cm using the proposed solvent system in a twin-trough chamber saturated previously with vapours of the mobile phase for not less than 20 min. The developed plates were treated with antimony trichloride and heated at 110°C for 10 min and then scanned densitometrically at 366 nm. The peak area was recorded and the calibration curve prepared by plotting peak area versus concentration of lupeol.

Estimation of the lupeol in TRFPE

Next, 10 μl of the test solution (200 ng per spot) was applied in triplicate on a precoated TLC plate of uniform thickness. The plate was developed in the solvent system and the peak area of lupeol was recorded as described above for the calibration curve. The amount of lupeol present in the sample was calculated from the calibration curve of lupeol.

Animals

Adult albino Wistar rats 90 days old and weighing between 210 and 270 g were obtained from M/s Zydus Cadilla Research Centre, Ahmedabad, India, and housed at a temperature of 24–28°C with a relative humidity of 45–55% with free access to food and water ad libitum. Before the experiment began, the local committee of ethics on animal experimentation approved all experimental procedures (no. 404/01/a/CPCSEA).

Acute toxicity studies

Healthy adult albino (Wistar) rats of either sex, starved overnight, were divided into four groups (n = 6) and were fed with increasing doses (50, 100, 200 and 300 mg/kg body weight) of the TRFPE. The animals were observed up to 14 days for toxicity and mortality. Finally, 1/10th of the highest tolerable dose was taken for further studies [12].

Treatment

Male albino Wistar rats were used in the studies and randomly divided into three groups (n = 8): group 1 received the vehicle (1 ml/day of 1% tween 80 in water) orally for 8 days and served as control. Groups 2 and 3 were administered TRFPE suspended in 1% tween 80 at a dose of 30 and 60 mg/kg body weight, respectively, for a period of 8 days.

Effect on body and organ weight

The initial and final body weights of the animals were recorded, and the change in body weight was determined. The animals were sacrificed by cervical dislocation method, and the sex organs (testis, epididymis, vas deferens and seminal vesicles) were dissected out carefully, freed from adhering tissues and weighed to the nearest 0.1 mg.

Epididymal sperm count

Homogenisation-resistant epididymal sperm were counted as described by Bustos-Obregon and Gonzalez-Hormazabal [13] with some modifications. Homogenisation was performed in 5 ml of 0.9% saline, and the homogenised epididymal preparation was refrigerated at 4°C for 24 h to allow sperm to be released from the walls. Data are referred to as sperm (106) per epididymis.

Serum testosterone

Blood samples were collected simultaneously during sacrification of animals, and serum samples were separated and stored according to procedures given by the National Committee for Clinical Laboratory Standards (NCCLS) [14]. Serum levels of testosterone were assayed by a competitive immunoassay using direct chemiluminescent technology on an autoanalyser (ADIVA centaur) at Endocrine Laboratory and Invitro Allergy Testing Centre, Ahmedabad, India.

Testicular histomorphology

Testis from each group was excised quickly during the dissection of animals and fixed in 10% buffered neutral formalin. Ultrathin sections of the testicular tissue were cut and stained with hematoxylin and eosin. Histological examinations included the mean seminiferous tubular diameter (STD) and germinal epithelial cell thickness (GECT). Three slides from the upper, lower and mid portions of the testis were prepared and evaluated for each testis. The mean STD and GECT were determined in 20 seminiferous tubules of each section using a projection microscope [15], and the values were expressed in terms of micrometers (μm). Microphotographs were made using an Olympus BX 40 microscope attached to an Olympus DP12 digital camera.

Data analysis

To determine statistically significant differences among treatment groups, data were analysed by using one-way analysis of variance (ANOVA) followed by Dunnet test. Values of P < 0.05 were considered statistically significant.

Results

Preliminary chemical examination of TRFPE showed an intense pink colour with the Liebermann Burchard test, indicating rich terpenoid content in the plant, which was then detected on a TLC plate by treatment with anisaldehyde sulfuric acid reagent (Fig. 1). Under the chromatographic conditions described above, the relationship between the concentration of lupeol and the peak response was linear, with a correlation coefficient of 0.994 (Fig. 2), and the R f of lupeol was found to be 0.52. The chromatogram of TRFPE showed the presence of six peaks at R f values 0.12, 0.25, 0.36, 0.52, 0.74 and 0.99, of which lupeol was detected at R f 0.52. The content of lupeol in the TRFPE was found to be 38.3% w/w.

Fig. 1
figure 1

Detection of terpenoids on thin-layer chromatography (TLC) using anisaldehyde sulfuric acid reagent

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Fig. 2
figure 2

Linearity of the calibration curve for quantification of lupeol in terpenoid-rich fraction of petroleum ether extract (TRFPE)

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Results of acute toxicity studies showed that TRFPE, when administered orally at the maximum dose of 300 mg/kg body weight, did not produce any evident sign of toxicity or mortality. The final body weights of animals in both groups were increased compared with their respective initial weights; however, the increase was found to be greater in the control group compared with the treated groups. On the other hand, a decrease in relative weights of testis, cauda epididymides and seminal vesicles were observed in the treated group compared with the control group (Table 1).

Table 1 Change in the body weight and weight of reproductive organs after terpenoid-rich fraction of petroleum ether extract (TRFPE) treatment in male rats
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Treated animals exhibited a significant decrease in serum testosterone levels and cauda epididymal sperm concentration compared with animals in the control group (Table 2). Histology of the testis in the control group showed normal features with successive stages of transformation of the seminiferous epithelium into spermatozoa, whereas histological examination of the testis after acute treatment with two different doses of TRFPE at 30 and 60 mg/kg body weight showed the inhibitory effects on the growth of seminiferous epithelium and on spermatogenesis (Fig. 3), with a significant reduction in the STD and GECT (Fig. 4; Table 2).

Table 2 Effect of oral administration of terpenoid-rich fraction of petroleum ether extract (TRFPE) on testicular histomorphology and epididymal sperm concentration
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Fig. 3
figure 3

Histology of the testis in different groups. a Control. b Terpenoid-rich fraction of petroleum ether extract (TRFPE) 30 mg/kg. c TRFPE 60 mg/kg showing successive stages of transformation of the seminiferous epithelium into spermatozoa (×40)

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Fig. 4
figure 4

Effect of terpenoid-rich fraction of petroleum ether extract (TRFPE) on seminiferous tubular diameter and germinal epithelial cell thickness of different groups. a Control. b Treated with TRFPE 30 mg/kg. c TRFPE 60 mg/kg (×10)

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Discussion

Increase in the final body weight of the animals suggests that the treatment of rats with TRFPE did not induce any overtoxicity to the animals. A significant decrease in testicular weight was observed in the animals treated with TRFPE, which is known to be related to the number of spermatids and spermatozoa in the tissue [16]. Observation of the testicular histomorphology showed that the extract inhibits the growth of seminiferous epithelium and consequently a decrease in seminiferous tubular diameter. It is probable that the observed effects on testis induced by the E. echinatus extract were due to the significant decrease in testosterone level, which is responsible for the diminished spermatogenesis, and hence is reduction in sperm counts [17, 18]. Circulating levels of testosterone are required for the maintenance of normal structure and function of the accessory sex organs, though the threshold levels may vary with organs. Reduced epididymal sperm concentration and weight of the accessory sex organs further supports the suppressed concentration of testosterone in the circulation [19, 20].

The results are further supported by the fact that plants containing triterpenoids, especially those of the lupane group including pure lupeol acetate exhibit antifertility effects in male rats [21, 22]. Preliminary phytochemical screening revealed the presence of terpenoids as the chief constituents in the TRFPE. During the phytochemical investigations, we detected the presence of lupeol in the roots of E. echinatus, and it has also been reported to contain taraxasterol acetate [23], a pentacyclic triterpenoid of the lupane skeleton. The observed activity may thus be due to a rich content of terpenoids, mainly those of the lupane type, in the roots of the plant E. echinatus.

Conclusions

In conclusion, acute treatment with TRFPE of the E. echinatus roots to male rats was found to exhibit antifertility properties by interfering with the reproductive function of the testis, which may be due to the major class of chemical constituents (viz., triterpenoids of the lupane group) in the extract. Further investigations on characterisation of chemical constituents and more clear mechanisms may result in justifying this plant as a safe and effective therapeutic agent for regulation of male fertility.