1 Introduction

A non-malignant enlargement of the prostate, or benign prostatic hyperplasia (BPH), involves the proliferation of epithelium and fibromuscular tissue, commonly seen in aged men^[1,2]. The consecutive constriction of the urethra may lead to reduction in urine flow rate, bladder outlet obstruction and irritation symptoms. Apart from surgical treatments, like transmural vaporization and prostatectomy, conventional treatments include 5-reductase inhibitors, anti-androgens, LHRH agonists, etc to reduce the symptoms of BPH, underscoring the importance of androgens in the pathogenesis of prostatic hyperplasia^[3,4]. Complications with surgical prostatectomy and side effects with the conwentional therapy exclude their use as routine treatments for BPH^[5].

Ayurveda, an ancient system of Indian medicine, cites several plants that are useful in the treatment of urogenital disorders. Prostane, an herbal formulation consists of Tribulus terrestris Linn, Areca catechu Willd, Pedalium murex Linn, Caesalpinia bonducella Fleming and Asparagus racemosus Willd.

Tribulus terrestris and Pedalium murex have been reported to be good diuretics^[6-9]. In our earlier studies, Pedalium murex and Areca catechu have shown 5-reductase inhibitory activity (paper presented at the 13th ISAS National Symposium on Analytical Techniques-2001, 1998 Nov 24-25. Bangalore, India. p 171-173). Caesalpinia bonducella has been used in the treatment of hydrocele and glandular swelling^[10]. In the present study, Prostane was evaluated for its 5-reductase inhibitory activity and -antagonistic activity in vitro, and its activity against experimental prostatic hyperplasia in rats.

2 Materials and methods

2.1 Composition of Prostane

Prostane, an herbal formulation, is a uniform mixture of pulverized Tribulus terrestris (fruit 25 %), Areca catechu (nut 25%), Pedalium murex (fruits 25%), Caesalpinia bonducella (root 15%) and Asparagus racemosus (root 10%). The constituent plants of the formulation were procured from local suppliers and were identified by Dr. R. Kannan, Botanist of The Himalaya Drug Company. A specimen of each plant was deposited in the herbarium of our R & D Centre.

2.2 In vitro study on Prostane for 5-reductase inhibition

2.2.1 Enzyme preparation

Enzyme preparation and 5-reductase inhibitory activity were done by the method described by Ashina et al^[11] with minor modification. Male Wistar rats weighing 250-275 g were anesthetized with ether, and the prostates were dissected and homogenated at 5% concentration with 100 mmol/L phosphoric acid buffer solution at pH 6.5 containing 250 mmol/L of sucrose, 1 mmol/L of dithiothreitol (DTT) and 1 mmol/L of EDTA. The homogenate was centrifuged at 10 000g for 30 min and the supernatant was taken as a source of enzyme. Testosterone was dissolved in ethanol at the concentration of 10 mmol/L. A 50 mmol/L NADPH was prepared in potassium phosphate buffer at pH 6.5.

2.2.2 Preparation of extractum

Prostane powder (10 g) was extracted with 100 mL of water in a water bath at 100 . The fluid extract was then filtered and dried; the residue (Prostane extractum) was used for the experiment. The extractive value of Prostane extractum was 10% (10 g of Prostane powder yielded 1 g of Prostane extractum).

2.2.3 Estimation of 5-reductase inhibitory activity

Various concentrations of Prostane extractum were prepared in triplicates in 2.0 mL aliquots of potassium phosphate buffer. Testosterone 100 L, NADPH 100 L and 500 L of enzyme solution were added. The mixture was incubated at 37 for 3 h and reaction terminated by the addition of 4.0 mL ethyl acetate. The mixture was vortexed for 1 min and the ethyl acetate layer was separated and was taken for the estimation of dihydro-testosterone (DHT).

2.2.4 Estimation of DHT by gas chromatography

DHT was estimated by the gas chromatography method^[12] with minor modification. A Netel Micro 9100 gas chromatograph attached with a flame ionization detector was used. The instrument was equipped with a stainless steel column packed with 3 % OV-17 (2 m length & 1/8" I.D.). The temperature of the oven, injector and detector were set at 220, 250 and 280 , respectively. Nitrogen (IOLAR-1) was used as a carrier gas at a flow rate of 30 mL/min. Twenty L of the ethyl acetate layer was injected into the gas chromatographic column and the peak area of the DHT was calculated. The formation of DHT was taken as a measure of 5-reductase activity since the enzyme reduced testosterone to DHT. The inhibitory activities of various concentrations of Prostane extractum were compared with finasteride, a 5-reductase inhibitor. The amount of DHT formed in the presence of the enzyme (control) was considered 100%, and formation of DHT in the presence of Prostane extractum was then compared with the control.

2.3 In vitro study on the -adrenergic antagonistic activity

Male Wistar rats weighing 275-300 g were used. The animals were anesthetized with ether. The vas deferens was then dissected free from extraneous tissue and suspended in an organ bath containing Tyrode solution gassed with 95% O₂ and 5% CO₂ mixture at 37. Contraction of the tissue was recorded isotonically using a lever transducer attached to the Polyrite (Medicare model 207, Ambala, India).

Following an equilibration period of 30 min, contractions were induced by administration of norepinephrine (NE) at various concentrations, viz. 0.5, 1.0, 2.0 and 4.0 g/mL with or without test drug (Prostane extract) and the contractions were recorded.

2.4 In vivo study on the activity against experimental prostatic hyperplasia

Forty male Wistar rats weighing 275-300 g were selected and randomized into five groups of 8 animals each. The animals were housed in standard laboratory conditions under a temperature of (223), relative humidity 50%-55%, and 12/12-h light/dark cycle. Drinking water and synthetic pelleted diet (Lipton India Ltd., Mumbai) were supplied ad libitum throughout the study period. The animals received the following treatments:
Group I: Olive oil 1 mLkg^-1d^-1, sc for 21 days as vehicle control.
Group II: Testosterone undecanoate (TU) 3 mgkg^-1d^-1, sc in olive oil for 21 days^[13].
Group III: Prostane powder 250 mgkg^-1d^-1, po and TU 3 mgkg^-1d^-1, sc in olive oil, both for 21 days. 
Group IV: Prostane powder 500 mgkg^-1d^-1, po and TU 3 mgkg^-1d^-1, sc in olive oil, both for 21 days.
Group V: Prostane powder 750 mgkg^-1d^-1, po and TU 3 mgkg^-1d^-1, sc in olive oil, both for 21 days.

On the 22nd day, the rats were euthanised under diethyl ether anesthesia. The prostate glands were collected, and the weight of ventral, dorsal and total prostates were recorded. After being weighed, the prostates were fixed in 10% neutral buffered formalin (NBF). The formalin fixed tissues were then processed by paraffin technique, and sections of 5 m thickness cut and stained by the routine H & E method.

2.5 Statistical analysis

3 Results

Prostane extractum showed 5-reductase inhibitory activity in a dose dependent manner (Table 1). It also revealed -adrenergic antagonistic activity and shifted the dose-response curve of NE towards the right (Figure 1). Testosterone administration at a dose of 3 mg/kg to rats for 21 days resulted in a significant increase in the weights of the total prostate as well as dorsal and ventral prostates as compared to the normal control. Treatment with Prostane powder inhibited prostate enlargement dose dependently. A significant prevention in prostatic enlargement was observed at 500 and 750 mg/kg dose levels (Table 2). Sections of prostate in Group I showed secretory luminal cells lined with a single layer of low columnar epithelium and the acini were filled with pale eosinophilic material (Figure 2). In Group II the epithelial cells showed an increase in height and number (hyperplasia) and fibrovascular stromal proliferation (Figure 3). These epithelial cells showed regular arrangement, but were sometimes thrown up into papillary folds. In Groups IV and V (Prostane 500 and 750 mg/kg), a reduction in epithelial cell height and restriction of stromal proliferation was noticed (Figure 4).

Table 1. Effect of Prostane on 5-reductase activity in vitro. meanSEM. n=3. ^cP<0.001 as compared to control.