The aim of this study was to investigate the effects of ethanolic extract of
The importance of sexuality in human life is well recognized in the ancient Indian system of Ayurvedic medicine. An entire category of drugs known as Vajikarana rasayan is devoted to drugs useful in treating sexual deficiencies/disorders [
Tubers of
Powdered tubers of
The dried extract was diluted in methanol/water (50 : 50) at a concentration of 1 mg/mL. Sample (5
Albino Wistar rats weighing 120–130 g and aged 3-4 months were housed in a standard laboratory condition. They were fed standard pellet diet and kept at
The experiment was carried under dim red light and the behavioral aspects were video-recorded for the duration of 60 minutes for each rat using a digital camera (Olympus, EX120). Observational and behavioral analysis was performed in a wooden chamber with a glass wall
The effect on sexual behavior of male rats was evaluated after 28 days of treatment with various doses of PT to respective groups. In brief, a male rat was placed in the observation glass chambers to acclimatize with the cage environment. After about 10 min, a sexually receptive female rat was dropped silently from one side of the chamber as a stimulus. The sexual behavior, for example, mount frequency, intromission latency, postejaculatory latency, and mount latency, was recorded. Mount latency (ML) was calculated as the time between the introduction of female and the occurrence of first mount; mount frequency (MF) was observed as total number of mounts during the period of observation; intromission latency was considered as the time for first intromission after introduction of female in the cage; postejaculation latency was calculated as the lag time between first intromission and the next within 30 min [
After 28 days of treatment as described above, the body weights of animals were taken after which animals were killed by decapitation. Testis, seminal vesicles, epididymis, and prostate glands were carefully removed and weighed. Testis and epididymis of animals were cut into small pieces and fixed in bovine’s fixative. After dehydration with varying percentage of ethanol, sections were cut (6
Epididymes of rats of each group were homogenized and taken into 5 mL of 1% sodium citrate solution and crushed thoroughly with the help of needle and forceps until a milky suspension was obtained. The solution was filtered through 80
The seminal vesicles were macerated with 3 mL of distilled water and centrifuged at 4000 rpm for 12 min. To the supernatant fluid collected after centrifugation, 0.5 mL of resorcinol and then 1.5 mL of HCl was added. The mixture was kept at 80°C for 12 min. The reaction with resorcinol developed a rosy color which was measured at 500 nm using spectrophotometer. A calibration curve was drawn using dilutions of fructose solution and measurement of the color developed with resorcinol and HCl [
After 28 days of treatment, the animals were killed by decapitation; the blood was extracted and serum was separated by centrifugation at 1000 g for 5 minutes. Isolated serum was further used for hormone level determination. Serum concentration of testosterone (EIAgen Testosterone kit, Italy, Import Lic no. NCD-164D), luteinizing, and follicle-stimulating hormones (ERBA Fertikit, Germany, Import Lic no. NCD 175/2006; lot numbers are 80425.7and 71210.10) was measured by following an immunoenzymatic method with an ELISA reader, according to the commercial protocol as defined in the kit, without any further modification.
Results are reported as mean ± SD. The treated groups were compared to control by ANOVA following Dunnet’s test. Significance level was set at
The extract of
The structure of compounds identified in
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Name | Structure |
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1 | 415 | Puerarin |
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2 | 253 | Daidzein |
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3 | 283 | Biochanin A |
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4 | 267 | Formononetin |
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LC Chromatogram of ethanolic extract of
Effect of ethanolic extract of
From the
For peak 2, the fragmentation of the deprotonated ion of m/z 253 and gave product ions of m/z 224, 223 [M–CH2O–H]−, 208, and 133. Therefore, peak 2 was identified as daidzein by comparison with the literature data.
For peak 3, the fragmentation of the deprotonated ion of m/z 283 gave product ions peaks of m/z 267, 253, and 240. The fragmentation pattern was similar to biochanin-A as reported in the literature.
For peak 5, the fragmentation of the deprotonated ion of m/z 267 and gave product ions of 252, 239, 223, and 135. The fragmentation pattern suggests that the compound could be formononetin.
Biochanin-A and formononetin were first time identified in the tubers of
PT treatment resulted in an increase in the body weight of treated animals. Significant increase in weights of sexual organs like testes, prostate, seminal vesicles, and epididymis was observed in case of PT 100 and PT150, while in case of PT50 there was a nonsignificant increase. This effect was thus dose dependent in the studied doses and is described in Table
Effect of ethanolic extract of
Group | Body weight of animals on first days (gm) | Body weight of animals on 28th day (gm) | Testis weight relative to body weight (mg/100 g) | Prostate weight relative to body weight (mg/100 g) | Seminal vesicles weight relative to body weight (mg/100 g) | Epididymis weight relative to body weight (mg/100 g) |
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Control |
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PT 50 |
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PT 100 |
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PT 150 |
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7.894 | 855.41 | 2.957 | 10.752 | 12.224 |
Results are expressed as means ± SE.
One-way ANOVA followed by Dunnett’s test comparing all versus control.
PT treatment had a marked influence on sexual behavior of animals and in case of all the parameters evaluated there was a clear indication of dose dependence in improving the sexual behavior in rats. The mount latency and postejaculation latency was significantly reduced, while a three-fold increase in mount frequency was recorded with 150 mg/kg dose of PT. The results for effect of PT 50, 100, and 150 treatments are described in Table
Effect of ethanolic extract of plants on sexual behavior.
Groups | Mount frequency | Mount latency (seconds) | Intromission latency (seconds) | Postejaculatory latency (seconds) |
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Control |
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PT 50 |
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PT 100 |
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PT 150 |
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Results are expressed as means ± SE.
One-way ANOVA followed by Dunnett’s test comparing all versus control.
Transverse sections (TS) of testes of control group animals showed normal histoarchitecture. The Sertoli and Leydig cells of normal size were present. The seminiferous tubules were normal in number with bundles of spermatozoa. In the TS of PT-treated animals an increase in diameter of seminiferous tubules was observed. Extract treatment also resulted in an improved spermatogenesis in all groups as compared to control group (Figure
In the epididymides of control rats, the short columnar epithelium consisted of principal cells with elongated nucleus. Basal (cuboidal) and principal (columnar) cells of the epithelium showed a pseudostratified appearance with apical stereocilia. The nucleus of the former cell type was situated basally and that of the latter cell type more centrally. The lumen of epididymis also shows scattered spermatozoa (Figure
Effect of ethanolic extract of
The treatment with ethanolic extract influenced the fructose concentration in the seminal vesicles and a marked increase in fructose concentration was observed in treated animals. A 24.17% increase in fructose concentration was recorded with PT 150 treatment in comparison to control group animals (Table
Effect of ethanolic extract of plants on the concentration of fructose in seminal vesicle and sperm count.
S. no. | Treatment groups | Fructose contents (mg/g) | Sperms count (millions/mL) |
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1 | Control |
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2 | PT 50 | 2.40 ± 0.28 |
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3 | PT 100 |
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4 | PT 150 |
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Results are expressed as means ± SE.
One-way ANOVA followed by Dunnett’s test comparing all versus control.
The
Elevated hormone levels were recorded in animals treated with PT. The serum testosterone, luteinizing hormone, and follicular-stimulating hormone-levels were significantly increased in all the treated groups. There was a dose-dependent effect of the treatment with the following order:
Effect of ethanolic extract of
This is the first report on effect of ethanolic extract of
Recently, reports on extracts of
A number of scientific investigations have shown that phytoestrogen compounds exert biological activity via the central nervous system [
The guidelines of CPCSEA, India, the governing body for animal experimentations in India, were strictly adhered to during the whole animal experimentation protocol. The number for approval of ethical committee is 379/01/ab/CPCSEA.
Nagendra Singh Chauhan would like to thank the AICTE, New Delhi, India, for providing National Doctoral Fellowship. Alexandra Christine Helena Frankland Sawaya would like to thank CAPES for a Postdoctoral Fellowship and P. Mazzafera for the use of the UPLC-MS (Bioen-Fapesp project no. 2008/58035-6). The authors are grateful to Sung-Joon Lee, Associate Professor, Korea University, for generously providing a gift of puerarin.