Benign prostate hyperplasia (BPH) is a urological disorder caused by the noncancerous enlargement of the prostate as men age. As the prostate enlarges, it can constrict the urethra, inducing various symptoms including a weak urinary stream, incomplete bladder emptying, nocturia, dysuria and bladder outlet obstruction (Pais, 2010; Roehrborn, 2011). It is proliferative process of both stromal and epithelial elements of the prostate (McNeal, 1983). It is unclear what specific factors regulate the degree of hyperplasia, which ultimately dictates the size of the prostate gland, there are many consensus regarding the prostate size that qualifies for the diagnosis of benign prostatic enlargement (BPE). As men age, the caliber of the urinary stream diminishes (Girman et al., 1995). The diminution of the urinary stream was assumed to be attributable to bladder outlet obstruction (BOO) arising directly from the BPE (Lepor, 2000). Androgens may be involved in the epithelial stroma interaction. In mature prostate, androgens are known to cause several changes in prostatic epithelium through androgen receptors located in the stroma. Immunocytochemical studies have shown that prostatic smooth muscle cells are uniformly androgen receptor positive. This fact indicates that smooth muscle located in prostatic stroma may be an important target for androgen action and able to regulate the expression of prostate growth factors (Niu et al., 2003).

Although the etiology of benign prostatic hyperplasia is not completely elucidated, it involves hormonal changes in the aging man. The development and growth of prostate gland depends on androgen stimulation, mainly by dihydrotestosterone (DHT), an active metabolite formed due to enzymatic conversion of testosterone by steriod 5 α-reductase.

Production and accumulation of DHT in the prostate increases with ageing which results in encouraging cell growth and induction of hyperplasia (Carson and Ritterman, 2003; Bartsch et al., 2000). Benign prostatic hyperplasia also involves augmented adrenergic tone in prostate smooth muscles, regulated through α₁-adernoceptors (Michel et al., 1998).

Conventionally used drugs like 5 α-reductase inhibitors (finasteride and dutasteride), α-adrenoceptors antagonists (alfuzosin,doxazosin, tamsulosin, terazosin) are used to treat benign prostatic hyperplasia, but they possess various side effects like impotence, decreased libido, ejaculation disorder, gynaecomastia, dizziness, upper respiratory tract infection, headache, fatigue and chest pain (Patel and Chapple, 2008). Along with conventional therapy, some alternative therapies are also available to treat prostatic hyperplasia. Various in vitro studies have reported that fatty acids inhibit the enzymes activity (Liang and Liao, 1992). Coconut oil (Arruzabala et al., 2006), lauric and myristic acid (Veeresh Babu et al., 2010) have proven to be effective benign prostatic hyperplasia mainly due to 5 α-reductase  inhibitory activities, which is due to their high content of lauric acid and myristic acid (mainly lauric).

However, there is no evidence for efficacy combination of lauric and myristic acid on testosterone induced benign prostatic hyperplasia whether oral dose with combination of lauric/myristic acid could prevent testosterone induced hyperplasia in rats.

Animals

Male Wister rats weighing 180 to 220 g were procured from an institutional animal facility centre. They were housed individually in clean and transparent polypropylene cages maintained at room temperature with 12 h light/dark cycle and had free access to food and water. After 7 days of acclimatization, they were randomly distributed into experimental groups. All the experimental procedure was carried out in accordance with Committee for the Purpose of Control and Supervision of Experimental on Animal (CPCSEA) guidelines.

Chemicals

Lauric acid and myristic acid (obtained from Sigma-Aldrich Pvt. Ltd).

Finasteride (was obtained from FINAST, Dr.Reddy’s Lab).

Testosterone Propionate (was Courtesy of Genesis pharmaceuticals, japan).

ELISA Kit for measurement of testosterone, hydrotestosterone, and

Alanine transaminase (ALT), aspartate transaminase (AST) Kits were (purchased from Transasia Bio chemical Ltd Daman).

Experimental BPH model and drug administration

Lauric acid and myristic acid were suspended in distilled water using Tween 80 and administer orally. Testosterone propionate was diluted with distilled water using Tween 80 and injected subcutaneously. Experimental groups were divided into 5 groups. Group I (normal control group) did not receive any treatment. Testosterone treated groups were randomly divided into four groups (n = 6): Group II (positive control) testosterone propionate (TP, 3 mg/kg body weight); Group III, which received finasteride (10 mg/kg body weight) administered orally and TP (3 mg/kg body weight), Group IV: Lauric acid and myristic acid (180 and 70 mg/kg) administered orally and TP (3 mg/kg body weight) injected subcutaneously. Group V: Lauric + myristic acid (360 and 140 mg/kg) administered orally and TP (3 mg/kg body weight). All rats were treated once a day for four weeks.

Body and prostate weight – ratio of prostate weight to body weight and percentage of inhibition

Animal were sacrificed after weighing by sodium pentobarbital and prostates were removed and weighted immediately.

Then, prostate weight to body weight ratio were calculated. Further percentage of inhibition was calculated as follows:

100-[(treated group - negative control) / (positive control - negative control × 100]

Blood and tissue samples

After a treatment period of 4 weeks and an overnight fast, the rats were anesthetized with sodium pentobarbital (100 mg/kg body weight, i.p.). Blood samples were collected and centrifuged at 2000 × g for 10 min. The prostate was collected from each rat and weighed. All prostatic specimens from each group were fixed with 10% buffered formalin for 24 h.

Measurement of DHT and testosterone in prostate and serum

The prostate tissue was homogenized in lysis buffer containing protease inhibitors (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.1% SDS, 1 mM EGTA, 100 μg/ml PMSF, 10 μg/ml pepstatin A, and 100 μM Na₃VO₃). The homogenates were centrifuged at 12,000 × g for 25 min at 4°C, and the protein concentrations in the supernatant fractions were determined using Bradford reagent. DHT and testosterone levels in the serum and prostates were measured with ELISA kits according to manufacturer’s instructions (Transasia Bio chemical Ltd Daman).

Group I: negative control; Group II: Positive control; Group III: Finasteride (10 mg/kg); Group IV: Lauric + myristic acid (180 and 70 mg/kg); Group V: Lauric + myristic acid (360 and 140 mg/kg). Values are expressed as mean± S.E.M. Statistical analysis is done by one way ANOVA followed by Dunnett's multiple comparisons test.

Measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum

ALT and AST levels were determined to assess liver function using commercial kits (Transasia Bio chemical Ltd Daman) and an auto analyzer

Statistical analysis

Data were expressed as mean ± standard error of mean (SEM). Statistical analysis is done by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test.

Evaluation of prostate enlargement

Prostate weights

Significant enlargement of prostate weights was found by testosterone treatment when compared with negative control. But significant reduction in elevation of prostate weights was found in group-V in testosterone treated rats. Percentage inhibition was 73.93% (Table 1).

In Group II, significant elevation in prostate weights/body weight ratio was noted when compared with Group I rats. But significant reduction in elevation of prostate weight/body weight ratio was observed by Group IV and V. Percentage inhibition was 62.12 and 78.69%, respectively when compared with Group I (Table 1).

Measurement of DHT and testosterone in serum

There was no significant difference in testosterone levels in serum found between all groups except for the group II (Table 2). DHT levels in serum, in group II, increased statistically more than Group I (8.79 ± 1.20 ng/ml). Conversely, in Group III (finasteride) (14.45 ± 2.36 ng/ml) markedly decreased the DHT levels in serum compared with the Group II. Group IV (10.56 ± 2.36 ng/ml) also significantly decreased the DHT levels in serum as well as the result of finasteride group (Table 2).

Prostate weights to body weight ratio

Measurement of DHT in prostate

As shown in Graph 1, the DHT level of group II (20.4 ± 7.87 ng/ml) significantly increased more than the normal.

Control (Group I) (10.4 ± 0.60 ng/ml). In contrast, the finasteride group (11.36 ± 6.77 ng/ml) significantly decreased the DHT level in prostate compared with the Group II. The group V (14.25 ± 9.70 ng/ml) also observed the significant reduction in the DHT level of prostate in comparison with Group II, which was similar to the result of finasteride group.

Measurement of testosterone in prostate

As shown in Graph 2, the testosterone level of group II (209.04 ± 6.87 pg/ml) significantly increased more than the normal control (Group I) (135.07 ± 0.50 pg/ml). In contrast, the finasteride Group III (172.02 ± 3.77 pg/ml) significantly increased the testosterone level in prostate compared with the Group I. The group V (174.23 ± 8.70 pg/ml) also observed the significant reduction in the testosterone level of prostate in comparison with the Group II, which was similar to the result of finasteride Group III.

Measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in serum

Treatments did not affect the activities of the serum toxicity marker enzymes, ALT and AST, indicating normal liver function (Figure 1).