ORIGINAL ARTICLE
Dose-dependent effects of ethanol extract of Salvia haematodes Wall roots on reproductive function and copulatory behaviour in male rats F. W. Bansode1, S. M. Rajendran2 & R. K. Singh3 1 Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, UP, India; 2 Division of Botany, CSIR-Central Drug Research Institute, Lucknow, UP, India; 3 Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, UP, India
Keywords Organ weights—Salvia haematodes roots— sexual behaviour—sperm counts—spermatogenesis Correspondence Dr Falgun W. Bansode, Division of Endocrinology, CSIR-Central Drug Research Institute, Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India. Tel.: +91 0522 2612411 18(PABX), Extn. 4383; Fax: +91 0522 2623403/2623938; E-mail: [email protected] Accepted: January 12, 2014 doi: 10.1111/and.12255
Summary This study was aimed to investigate the dose-dependent effects of Salvia haematodes Wall roots (SHW) extract on male reproductive function and copulatory behaviour in rats. Sexually mature males were assigned to four groups: control and treated (5, 50 and 300 mg kg 1 day 1 for 30 days). At the end of treatment regimes, the reproductive activity viz. body/organ weights, testicular spermatogenesis, daily sperm production rate (DSP) and epididymal sperm counts, and sexual behaviour including mounting latency (ML), mounting frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), post-ejaculatory interval (PEI) and penile reflexes (PE) were assessed. Results showed significant increase in body weight (at 300 mg kg 1), testis/epididymis weights (at 50 and 300 mg kg 1), testicular spermatids, DSP, tubular diameter and epididymal sperm counts (at 50 and 300 mg kg 1doses) in treated compared with control rats. It also produced dose-dependant changes in sexual behaviour. The 5 mg kg 1 dose of extract increased MF and PE, whereas 50 and 300 kg 1 doses caused significant increase in MF, IF, PE, EL (but less than sildenafil citrate treatment), hit rate and seminal plug weight. It is concluded that SHW extract enhances anabolic activity, testicular function and sexual behavioural performance in a dose-dependant manner.
Introduction Sexual dysfunction is one of the most common diseases in 40- to 70-year-old men with an increasing incidence of degenerative diseases, injuries and stress. In infertile males too, the growing evidence from clinical and epidemiological studies has been shown an increasing incidence of male reproductive problems such as conjugal infertility and abnormal semen analyses (Iammarrone et al., 2003; Hassun Filho et al., 2005). Despite medical and surgical treatment modalities available for treating sexual dysfunction and infertility, plant-derived herbal remedies continue to be more popular alternative for men and women seeking to improve their sex life. Nevertheless, the efficacy of most of these herbal agents in treating sexual dysfunction remains unclear (Park et al., 2006; Kim et al., 2009). There are several aspects of male sexual behaviour, but an erection and mounting provide direct and prominent © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
measures for determining the efficacy of aphrodisiacs (Linnankoski et al., 1995; Linnankoski & Leinonen, 2010). In Unani and Ayurvedic system of medicine, the dried roots of Salvia haematodes have been used as a sex tonic. It has been mentioned to be of great value in the management of male sexual fertility regulatory activity. Previous study with S. haematodes (500 mg kg 1, orally) has shown improvement in the sexual activity that produced a significant increase in episodes of penile erection and the orientation of males towards the female by increasing anogenital investigatory behaviour, enhanced licking and grooming of the genitals and increased ejaculation latency in male rats (Islam et al., 1991). S. haematodes Wall (Family Lamiaceae), which is commonly known as ‘Lal Bahaman’, has been cultivated worldwide including India for use in folk medicine and culinary purposes (Brickell, 1996; Rustayan et al., 1999). Pharmacological studies showed significant cardio tonic and 1
Enhancement of reproductive and sexual activity by Salvia haematodes
anticonvulsant activities without any toxicity up to the dose of 5 g kg 1 in rats (Akbar et al., 1984). The different species of Salvia including S. haematodes, S. officinalis, S. leriifolia, S. triloba and S. divinorum have been reported to show various pharmacological effects as analgesic, anti-inflammatory and antipyretic (Hernandezperez et al., 1995; Al-Yousuf et al., 2002), antioxidant (Cuppett & Hall, 1998), hepatoprotective (Wasser et al., 1998), hypoglycaemic activities (Jimenez et al., 1996), anti-ischaemic (Yu, 1994), for the treatment of coronary and cerebrovascular disease, sleep disorders, hepatitis, hepatocirrhosis, chronic renal failure, dysmenorrhoea, amenorrhoea, carbuncles and ulcers (Wasser et al., 1998), sedative, hypnotic, hallucinogenic, skeletal muscle relaxant, memory enhancing, anticonvulsant, neuroprotective, antiparkinsonian activity as well as inhibition of ethanol and morphine withdrawal syndrome (Imanshahidi & Hosseinzadeh, 2006; Baricevic & Bartol, 2000). The testicular spermatogenesis, the process of sperm formation and accessory sex organs’ functions are predominantly dependent on FSH and testosterone (Steinberger & Nelson, 1995). Disruption in biosynthesis of these hormones leads to inhibition of testicular spermatogenesis, sperm formation and Leydig cells morphology and accessory sex organs’ function (Russell et al., 1992; Shan & Hardy, 1992; Kerr et al., 1999). Testosterone supplementation restores normal function of Leydig cells/ spermatogenesis and accessory sex organs (O’Donnell et al., 1994). Testicular daily sperm production rate defined as a process for semi-quantitative analysis of spermiogenesis/sperm produced in testis is a baseline to assess sexual maturity (Robb et al., 1978). It is evaluated by enumeration of elongated spermatids nuclei in steps 17– 19 of spermiogenesis in testicular homogenates. The semen analysis, including sperm count, motility and morphology, is the basis of investigations into male infertility, and number of genes participating in spermatogenesis have been reported to play an essential role in human reproductive function (Blendy et al., 1996; Nantel et al., 1996; Zhao & Garbers, 2002). Several studies have demonstrated that herbal drugs enhance sperm count and elicit androgenic effects (Saxena & Dixit, 1987; Chaturapanich et al., 2008; Sharma et al., 2009). In contrast, the plant-like Salvia fruticosa has been shown to produce adverse effects on the fertility of male and female rats (Elbetieha et al., 1998). This study was conducted to determine the potential reproductive function (viz. body/organ weights, quantitative analysis of testicular spermatogenesis, daily sperm production rate and epididymal sperm count) and sexual behavioural performance in adult male Sprague Dawley rats administered S. haematodes Wall extract in a dosedependent manner. 2
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Materials and methods Chemicals All chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO, USA). Collection of plant material The plant S. haematodes Wall (Family Lamiaceae), a garden plant used in this study, was collected locally from fields, plant nurseries and wild waste-land areas nearby Lucknow, Uttar Pradesh, India. The plant material was authenticated by one of the author, and a specimen was preserved in our Institute’s herbarium (Voucher No. 19752). Extraction and fractionation Shade-dried powdered roots of the plant, S. haematodes Wall (SHW), were extracted with 50% ethanol (3 9 1 l). The pooled filtered ethanolic extract was concentrated under reduced pressure below 50 °C. The viscous mass thus obtained was dried under high vacuum to remove last traces of the solvent. The semi-solid ethanolic extract of the collection (yield 9.11%, w/v; Islam et al., 1991) was used to test reproductive function and aphrodisiac activity in Sprague Dawley rats. Animals, treatment, tissue collection and fixation Adult male (180–200 g) and cycling female (170–180 g) rats of proven fertility (Sprague Dawley strain) used in this study were obtained from Institute’s animal house. Rats were caged in similar husbandry conditions, in environmentally controlled rooms with maintained temperature (24 1 °C) and 12/12 h light/dark cycles. Animals were fed with pelleted food (Hindustan Lever Ltd.) and water ad libitum. In the first set of experiment, male adult rats were randomly divided into four groups (Gr. I–IV) of 12 animals in each with total of 48 rats. Animals in Gr. I received distilled water (10 ml kg 1) only and served as a control. Rats in Gr. II, III and IV were administered with 50% ethanolic extract of SHW orally at 5, 50 and 300 mg kg 1 doses, respectively, for 30 days. The animals were allowed free access to food and drinking water ad labium during the entire treatment period. The physical appearances/ activities of the rats were observed daily. Body weights of rats from control and treated groups were recorded, and autopsy was performed on day 31 by anaesthetising with solvent anaesthetic ether. The reproductive organs viz. testes, epididymis and accessory sex © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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organs (e.g. prostate, seminal vesicle, vas deferens and penis) were dissected out surgically, rinsed immediately in chilled saline, freed from connective tissues, and blood clots and weights were recorded. The testes and epididymis from left sides were fixed in Bouin’s fluid (24 h) for histology purpose. These organs from right side were used for daily sperm production rate and sperm counting. The experimental protocol was approved by an Institutional Review Committee for the use of Human or Animal Subjects and the procedure is in compliance with National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23 revised 1985) and the guidelines laid down by Institute’s Animal Ethics Committee (IAEC).
Turraxâ (Janke and Kunkel, Staufen, Germany). The homogenates were further diluted with the same medium, and homogenisation-resistant spermatid nuclei in steps 17–19 of stages IV–VIII of the spermatogenic cycle were counted in triplicate using four chambers (total of 64 squares) of Neubauer’s haemocytometer under the phasecontrast BX51 Olympus trinocular microscope (Olympus) at 9100 magnification (Amann & Lambiase, 1969). Daily sperm production rate per gram testicular parenchyma was calculated by dividing the number of spermatids nuclei with the product of weight of parenchyma and time divisor of 6.3 days reported for rat (Johnson et al., 1980). The values are represented as mean SE for five animals each group.
Histomorphometry of testis
Epididymal sperm count
Bouin’s fixed testes and epididymis from control and treated groups of rats were dehydrated in graded series (50–100%) of ethanol, cleared in xylene, infiltrated and embedded in paraffin wax (at 58 °C). Serial cross sections (5 lm) were stained with haematoxylin and eosin. Measurements of morhpometric analyses, viz. diameter of testicular tubules (100 tubules each group) and Leydig cell nuclei (100 nuclei per group) were carried out using Biovis Image Plus Software for image analysis and processing (Expert Vision Labs Pvt. Ltd., Mumbai, India) at 9100 and at 91000 (oil immersion) magnifications under Olympus Trinocular Microscope (BX51, Tokyo, Japan).
Cauda epididymidis was excised surgically, cut into small pieces in suspension medium containing 140 mmol NaCl, 0.3 mmol KCl, 0.8 mmol Na2HPO4, 0.2 mmol KH2PO4 and 1.5 mmol D-glucose (pH adjusted to 7.3 by adding 0.1 N NaOH), and spermatozoa were collected by centrifugation at 100 9 g for 2 min. The resultant precipitate was resuspended in fresh suspension medium, diluted and placed on both sides of Neubauer’s haemocytometer. The number of spermatozoa was counted in four chambers of haemocytometer in duplicate in each rat under Olympus Trinocular microscope at 9100 magnification. The values were expressed as an average of total counts of sperm number per ml of suspension (Robb et al., 1978; Srivastav et al., 2010).
Semi-quantitative analysis of spermatogenesis Semi-quantitative analysis of testicular spermatogenesis was carried out on the basis of nuclear morphology of the germ and Sertoli cells (Leblond & Clermont, 1952). The number of germ cells and Sertoli cell nuclei were counted in at least 20 round seminiferous tubules selected randomly from at least 2–3 cross sections in each rat at 9400 magnification under Olympus Trinocular microscope (BX 51; Olympus). Cell counts were corrected by Abercrombie’s formula (Abercrombie, 1946) and values expressed as an average cell counts per round seminiferous tubule in each group (Srivastav et al., 2010). Estimation of daily sperm production rate in testis Measurement of daily sperm production rate (DSP) in testicular homogenates was carried out as per method of Amann et al. (1976). Briefly, testicular parenchyma was cut into small pieces after removal of tunica, placed in 0.25 M sucrose solution (pH 7.5), weighed and homogenised in a fluid containing 150 mM NaCl l 1, 3.8 mM NaN3 l 1 and 0.05% (v/v) Triton X-100 using an Ultra © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
Copulatory behaviour test In the second set of experiment, rats were divided into five groups (Gr. I–V) of 12 animals in each with total of 60 rats. Animals in Gr. I (control group) received distilled water (10 ml kg 1) only, and in Gr. II, III and IV administered with 50% ethanol extract of S. haematodes Wall root orally at 5, 50 and 300 mg kg 1 doses, respectively, for 30 days. Rats in Gr. V (Std. Control) were given oral suspension of Sildenafil citrate (5 mg kg 1 body weight) 1 h prior to the commencement of the mating test. Copulatory test was conducted as per method described previously (Mercier et al., 1987; Lucio et al., 2001) Behaviour test consisted in placing the male in a plexiglas cylindrical arena (55 cm in diameter) for a 5min habituation period before the presentation of an ovariectomised stimulus female. Females were brought into the oestrous by subcutaneous injection of oestradiol benzoate (10 lg) and progesterone (2 mg) 48 and 4 h before the test respectively. A total of 90 females used in 3
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this study were divided into 30 groups containing three females each to permit each observation to be made using fresh females and to avoid possible effects of repeated copulation. Test session ended when the male displayed the ejaculatory pattern. Six females in each group were sacrificed, seminal plug dissected out and weights recorded. The following copulatory parameters were studied after 30 days treatment of SHW extract: mounting latency (ML), mounting frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), post-ejaculatory interval (PEI), seminal plug weight and hit rate.
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Statistical analysis Statistical analysis for significance of differences between copulatory parameters, reproductive organ weights, spermatogenic analysis, daily sperm production and epididymal sperm count between control and treated groups was performed by applying Student’s t-test followed by oneway ANOVA (one factor analysis of variance) test. Values with P < 0.05 were considered as significant and expressed as mean SEM.
Results
Potency activity test
Body and reproductive organ weights
Potency test was carried out in adult male rats as per method described previously (Hart & Haugen, 1968; Hart, 1979). Animals were divided into five groups (Gr. I–V) of six rats each. In control group (Gr. I), rats received vehicle (10.0 ml kg 1 of distilled water). Oral administration of SHW extract at 5, 50 and 300 mg kg 1 doses (Gr. II–IV) was given daily for 30 days prior to the test. Gr. V rats received oral suspension of sildenafil citrate (5 mg kg 1) 1 h prior to the commencement of the experiment. On day 31, each animal was placed on its back in a glass cylinder for partial restraint. The pre-putial sheath was pushed behind the glans penis using the thumb and index finger for a period of 30 min. During this period, a cluster of genital responses consisting of erections (PE), quick flips (QF), long flips (LF) and total Flips (TF) of the glans penis were recorded.
No significant changes were observed in the body weights of rats treated with 5 or 50 mg kg 1 dose of SHW extract but caused a significant increase (P < 0.05) in body weight at 300 mg kg 1 dose as compared to controls. The reproductive organ weights did not show any significant change in rats treated with 5 mg kg 1 extract as compared to controls. However, 50 and 300 mg kg 1 doses caused a significant (P < 0.005–0.001) increase in the testes and epididymal weights. The accessory sex organs (viz. prostate, seminal vesicle, vas deferens and penis) did not show any significant change in their weights compared with control (Table 1).
Toxicity study To determine general short-term toxicity, the control and treated animals were observed continuously for 1 h for any gross behavioural changes or death, if any.
Morphometric studies/semi-quantitative analysis of spermatogenesis In control rats, normal testicular structure was evident depicting all spermatogenic cell types viz. spermatogonia, primary spermatocytes, spermatids and spermatozoa as well as Sertoli and Leydig cells (Fig. 1a). In treated rats, the density-elongated spermatids/spermiated spermatozoa in lumen of almost all testicular tubules were
Table 1 Effect of 50% ethanol extract of Salva haematodes Wall root (SHW) on the final body weight and reproductive organ weights in adult male rats
Treatment Control SHW treated (5 mg kg 1) (50 mg kg 1) (300 mg kg 1)
Body weight (g)
Reproductive organ weights (g) Testis
Epididymis
V. Prostate
Seminal vesicles
Vas deferens
Penis
212 3.4
1.152 0.027
0.439 0.013
0.464 0.020
1.453 0.049
0.108 0.032
0.331 0.032
216 4.4NS
1.135 0.095NS
0.434 0.028NS
0.524 0.035NS
1.521 0.028NS
0.098 0.003NS
0.329 0.015NS
214 9.8NS
1.436 0.071c
0.635 0.055b
0.512 0.122NS
1.480 0.250NS
0.151 0.009NS
0.351 0.045NS
225 3.7a
1.545 0.081c
0.650 0.025c
0.552 0.130NS
1.582 0.225NS
0.161 0.013NS
0.361 0.041NS
Values are expressed as mean SEM; n = 10, number of animals for body weights and n = 6, number of animals for organ weights; Significance of difference control versus treated; NS, not significant; aP < 0.05; bP < 0.005; cP < 0.001.
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Fig. 1 Testicular cross sections from control (a) and Salvia haematodes-root-extract-treated (b–d) rats. Control rat showing normal testicular structure, displaying all germ cell types. Note the similar testicular germ cells types in treated rats with 5 mg kg 1 dose (b), but displaying increased density of round and elongated spermatids at 50 mg kg 1 (c) and 300 mg kg 1 (d) of extract. (Magnification 9400; H-E stained). A, spermatogonia; Z, zygotene; P, pachytene spermatocytes; R, round spermatids; E, elongated spermatids; SC, sertoli cells; LC, Leydig cells; L, lumen.
(a)
(b)
(c)
(d)
increased. The Sertoli cells and Leydig cells showed normalcy in treated rats (Fig. 1b–d). The testicular tubular diameter was observed to be increased (P < 0.005–0.001) at 50 and 300 mg kg 1 doses in treated rats (Table 2). Cauda epididymal tubular lumen was filled with large number of spermatozoa in treated (at 50 and 300 mg kg 1) as compared to control rats (Fig. 2). Semi-quantitative analysis of testicular spermatogenesis did not show any significant change in number of germ cells viz. spermatogonia, primary spermatocytes (nonpachytene and pachytene) and spermatids (round and elongated) in SHW-extract-treated rats at 5 mg kg 1 dose as compared to controls. But there was a significant increase in the number of round (P < 0.02) and elongated (P < 0.005–0.001) spermatids in treated rats at 50
and 300 mg kg 1 doses for 30 days. The testicular germ cell types, for example, A-spermatogonia, non-pachytene (NP) and pachytene (P) spermatocytes, Sertoli cells (SC) and Leydig cells diameter in treated rats were comparable with that in controls (Table 2). Daily sperm production rate Administration SHW extract caused a significant increase in DSP rate as evident by increased number of elongated spermatids in steps 17–19 of IV–XII stages of spermatogenic cycle in testicular homogenates, especially in 50 mg kg 1 (P < 0.05) and 300 mg kg 1 (P < 0.001) dose level. Whereas, 5 mg kg 1 dose of root extract did not produce any significant increase in DSP rate as compared to controls (Fig. 3).
Table 2 Effect of Salva haematodes Wall root (SHW) extract on testicular germ cell population dynamics, tubular diameter and nuclear diameter of Leydig cells in rats Spermatogenic germ cells number per seminiferous tubule Treatment groups Control SHW treated (5 mg kg 1) (50 mg kg 1) (300 mg kg 1)
(A)
(NP)
(P)
(R)
4.5 0.35
20.4 2.45
25.8 2.8
65.8 4.2
4.2 0.45NS 4.6 0.35NS 5.1 0.37NS
21.2 4.45NS 24.3 4.54NS 22.6 2.71NS
23.2 3.2NS 28.2 1.2NS 32.8 2.8NS
65.4 1.4NS 78.2 1.6a 76.8 4.2a
(E) 101 8.0 107.6 9.2NS 122.0 5.0b 134 4.0c
Sertoli cells/ tubule
Tubular diameter (µm)
Nuclear diameter of Leydig cells (µm)
16.1 1.4
437.2 9.7
14.9 2.5
15.9 0.6NS 17.0 2.4NS 18.1 1.5NS
438.0 8.3NS 497.7 10.5b 507.6 14.5c
12.1 1.6NS 15.3 3.5NS 16.7 3.7NS
A, spermatogonia A; NP, nonpachytene spermatocytes; P, pachytene spermatocytes; R, round spermatids; E, elongated spermatids. Values are expressed as mean SEM, n = 5, number of animals; Significance control versus treated; NS, not significant; aP < 0.02; bP < 0.005; c P < 0.001.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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Enhancement of reproductive and sexual activity by Salvia haematodes
(a)
(b)
(c)
(d)
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Fig. 2 Cauda epididymis from control (a) and Salvia haematodes-root-extract-treated (b–d) rats. Note the sperm density similar to control in treated rats at 5 mg kg 1 dose (b). In comparison, increased sperm density at 50 (c) and 300 (d) mg kg 1 doses of S. haematodes root extract can be seen. EP, epididymal epithelium; ES, epididymal stroma; S, epididymal sperms, (magnification 9400; H-E stained).
duced a significant increase in MF, IF and EL. In contrast, the sexual behavioural changes were observed to be highly increased in rats treated with 300 mg kg 1 of SHW extract. In addition, the hit rate, MF and seminal plug weight were also observed to be increased significantly at this dose compared with controls, but caused decline in PEI in treated rats. The Std. drug, sildenafil citrate (5 mg kg 1), produced a significant increase in MF, IF and EL but caused decrease in ML, IL and PEI as compared to controls (Fig. 4). Fig. 3 Effects of Salvia haematodes Wall root extract on daily sperm production rate (gm/testicular parenchyma) and cauda epididymal sperm counts (9106) per ml.
Epididymal sperm count Epididymal sperm count did not show any significant change in Gr. II (5 mg kg 1) rats as compared to Gr. I (control) rats, but caused a significant increase in epididymal sperm counts in Gr. III (50 mg kg 1, P < 0.05) and Gr. IV (300 mg kg 1, P < 0.001) rats as compared to Gr. I control (Fig. 3). Effects of SHW extract on sexual behaviour Results of copulatory behaviour test in SHW-extract-treated rats for 30 days showed dose-dependent changes (i.e. 5 mg < 50 mg < 300 mg kg 1) in the sexual behavioural performance. Oral administration of extract at lower dose (5 mg kg 1) exhibited a significant increase in the MF as compared to controls. 50 mg kg 1 dose of extract pro6
Effect of SHW extract on potency Oral administration of SHW root extract at its lower dose (5 mg kg 1) produced a significant increase PE as compared to control rats. At 50 mg kg 1 dose, a significant increase in PE and QF (P < 0.05) was observed. However, LF and TF were insignificantly increased when compared with controls. However, at 300 mg kg 1, there was a significant increase (P < 0.001) in PE as well as in the QF (P < 0.02), LF (P < 0.001) and TF (P < 0.005) compared with control as well as lower doses of extract. The results showed a dose-dependent increase in the episodes of penile reflexes as compared to control rats. The rats in Gr. V treated with Std. control showed prominent effects in penile reflexes as compared to doses of SHW extract used (Fig. 5). Toxicity study No mortality and gross behavioural changes were observed during 1–3 h of toxicity study in treated rats as compared to controls. © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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Fig. 4 Effects of Salvia haematodes Wall root extract on sexual behaviour of rats after 30 days treatment. MF, mounting frequency; IF, intromission frequency; ML, mounting latency; IL, intromission latency; EL, ejaculation latency; PEI, post-ejaculatory time interval; SPWt, seminal plug weight.
Fig. 5 Effects of Salvia haematodes Wall root extract on penile reflexes (potency test) in rat. QF, quick flips; LF, long flips; TF, total flips; PE, penile erections.
Discussion The results of the present study on determining the sexual behavioural performance of ethanol extract of SHW roots show aphrodisiac potential at different doses (5, 50 and 300 mg kg 1 body weight) in rat model. It caused significant increase in MF and IF at 50 and 300 mg kg 1 doses except at its lower dose of 5 mg kg 1 showed increase in MF only. Earlier reported studies with 500 mg kg 1 dose of SHW have also shown an increase in the orientation of males towards females, grooming and licking behaviour followed by increased number of mounts in treated rats (Islam et al., 1991). Similarly, our results exhibit an increase in ML at 300 mg kg 1 dose level, which has been demonstrated to increase MF and IF marked by increased rate and number of intromissions and considered to be the indices of libido with nutmeg (Tajuddin et al., 2003, 2005). The potency test carried out in rats showed significant increase in penile reflexes such as QF, LF, TF and PE at 300 mg kg 1 dose. In comparison, lower doses of plant extract also produced an increase in QF (at 50 mg kg 1) and PE (at 5 and 50 mg kg 1). Thus, the increased MF and IF along with © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
increased hit rate and libido may lead to increase penile erections with prolongation of coitus time. The aggregate of TF and PE was also significantly increased in test animals indicating extract-induced increase in ‘pure’ potency. In support, previous studies have demonstrated a dose-dependent, recurrent and significant increase in the episodes of penile reflexes (viz. QF, LF, TF and PE) at higher doses than lower ones of plant extracts (Islam et al., 1991; Ang et al., 2001; Ramachandran et al., 2004; Tajuddin et al., 2005; Thakur & Dixit, 2007). Further, results of the study also showed an increase in EL and seminal plug weights in treated (at 300 mg kg 1) rats. However, decreased time in PEI and MF has demonstrated that the test drug may intensify the occurrence of repeated sexual activity heavily (Tajuddin et al., 2005). There are several other aphrodisiacs that increased MF, IF, EF and penile reflexes, but decreased ML, IL and PI with herbal extracts of Allium tuberosum seeds (Guohua et al., 2009), Mucuna pruriens Linn. seeds (Suresh et al., 2009), Butea frondosa Koen ex Roxb (Ramachandran et al., 2004) and Kaempferia parviflora (Chaturapanich et al., 2008). In contrast, Catappa seeds have shown no effect on libido, sexual vigour or sexual performance at 1500 mg kg 1, and higher dose (3000 mg kg 1) reversibly inhibited all the parameters of sexual behaviour other than MF and IF and copulatory efficiency (Ratnasooriya & Dharmasiri, 2000). The results of the present study also showed increased anabolic activity that indicated an increase in the body weights (at 300 mg kg 1) and reproductive organs (viz. testis and epididymis) weights as well as testicular tubular diameters (at 50 or 300 mg kg 1), which may suggest an involvement of testosterone intervention and in improving testosterone availability to gonads in treated rats. Our results show an evidence of increased spermiogenesis marked by a characteristic increase in round and elongated spermatids (in testicular cross sections), DSP (in testicular homogenates), as well as epididymal sperm counts in rats with 50 and 300 mg kg 1 of SHW extract. These findings can be correlated with increased MF, IF, 7
Enhancement of reproductive and sexual activity by Salvia haematodes
PE, ejaculations and increased seminal plug weights in treated rats. In concurrence, Chlorophytum borivilianum treatment also has shown an increase in libido, sperm count, seminal fructose content and penile erection index in male rats. The effect of extracts on inducible NO release in vitro directly correlated with the enhanced erectile function in vivo (Thakur & Dixit, 2007; Kenjale et al., 2008). It has been demonstrated that the increased testosterone level is associated with increased sexual desire, arousability, sperm counts, PE and MF (Thakur & Dixit, 2007; Sharma et al., 2009; Harding & Velotta, 2011; Yakubu & Akanji, 2011). In contrast, results with yohimbineinduced castrated rats supplemented with testosterone resulted in reduction in latencies to initial mount, intromission and ejaculation and demonstrated that testosterone is not a requisite for the enhancement of sexual motivation by yohimbine, but a2-adrenoceptors are involved in the modulation of sexual arousal (Clark et al., 1985). Results of the study did not show any significant increase in weights of androgen-dependant accessory sex organs as well as in number of androgen-sensitive primary spermatocytes and nuclear diameter of Leydig cells treated rats up to 300 mg kg 1 dose. It indicates dose and duration dependency of the test drug for enhancement of sexual behaviour and reproductive activity. Existing reports have shown that the increase in blood flow to the testis may stimulate testosterone production/ secretion, acts on the central nervous system and gonadal tissues and modulates male sexual behaviour (Larsson et al., 1975; Damber & Janson, 1978). It also causes an increase in the diameter of seminiferous tubules, seminal vesicle weight and cauda epididymal sperm density in rats. S. hypoleuca (Jasem et al., 2010) and Anacyclus pyrethrum DC (Sharma et al., 2009) have been shown to cause an increase in sperm parameters, viz. sperm count and motility and spermatogenesis and testicular weight/ accessory organ weights in rat. The extract of S. hypoleuca caused a significant increase in testicular CREM gene expression and protein level in rat (Jasem et al., 2010). It has been shown that Abutilon indicum and Withania somnifera both possessed marked aphrodisiac activity complying many facets such as enhancement in libido, increase in the sexual performance, penile erection and anabolism, increased spermatogenesis as well as sperm validity and had shown to relax penile cavernosal tissue probably by phosphodiesterase inhibition and cGMP accumulation (Ganu et al., 2010). In castrated male rats, both testosterone and dihydrotestosterone (DHT) are very effective in restoring penile erections, but testosterone is most effective in restoring mating behaviour also as compared to DHT, which is totally ineffective (Gray et al., 1980). Penile erection is a vascular event controlled by autonomous nervous system and pro-erectile events of phosphodi8
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esterase type 5 inhibitors acting in penis and melanocortin agonists acting in brain, combinedly stimulate developments (Giuliano & Rampin, 2004). There is evidence that ginseng saponin facilitated penile erection by directly inducing the vasodilatation and relaxation of penile corpus cavernosum via nitric oxide mechanism (Chen & Lee, 1995). Furthermore, the central nervous system has been shown to significantly alter the activity of hypothalamic catecholamine and nitric oxide during facilitation of copulatory behaviour and hormone secretion (Murphy & Lee, 2006). In conclusion, the 50% ethanolic extract of S. haematodes was found to be devoid of any general toxicity. In Ayurveda medicine, this plant is found to be safe sexual enhancer in male human. Results of the present study showed significant increase in body weight (at 300 mg kg 1) and reproductive activity viz. testes and epididymal weights, spermiogenesis (spermatids number), daily sperm production rate followed by epididymal sperm counts (at 50 and 300 mg kg 1) in SHW-extracttreated rats. Further, there was an enhancement in the sexual behavioural performance such as MF, IF, Hit rate, PE and EL at 50 or 300 mg kg 1 doses. These results indicate an anabolic effect, enhancement of reproductive activity and sexual behavioural performance by SHW extract. Thus, it provides a scientific rationale for traditional use of S. haematode Wall roots in the treatment of sexual disorders/erectile dysfunction in males. Acknowledgements The authors are thankful to Ms T. Firdaus, Mr Pradeep Singh and Ms Ranjeeta Maurya (a research trainee) for technical and experimental assistance. This work was supported by Ministry of Health and Family Welfare, New Delhi, India. References Abercrombie M (1946) Estimation of nuclear population from microtome sections. Anat Rec 94:238–248. Akbar S, Tariq M, Nisa M (1984) A study on CNS depressant activity of Salvia haematodes wall. Int J Crude Drug Res 22:41–44. Al-Yousuf MH, Bashir AK, Ali BH, Tanira MOM, Blunden G (2002) Some effects of Salvia aegyptiaca L. on the central nervous system in mice. J Ethnopharmacol 81:121–127. Amann RP, Lambiase JT Jr (1969) The male rabbit. III. Determination of daily sperm production by means of testicular homogenates. J Anim Sci 28:369–374. Amann RP, Johnson L, Thomson DL, Pickett BW (1976) Daily spermatozoal production, epididymal spermatozoal reserves and transit time of spermatozoa through the
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epididymis of the rhesus monkey. Biol Reprod 15:586– 592. Ang HH, Ikeda S, Gan EK (2001) Evaluation of the potency activity of aphrodisiac in Eurycoma longifolia Jack. Phytother Res 15:435–436. Baricevic D, Bartol T (2000) The biological/pharmacological activity of Salvia genus. In: SAGE The Genus Salvia. Kintzios SE (ed). Harwood Academic Publishers, Amsterdam, pp. 143–184. Blendy JA, Kaestner KH, Weinbaure GF, Nieschlag E, Schuetz G (1996) Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380:162–165. Brickell C (1996) Encyclopedia of Garden Plants. Dorling Kindersley, London. Chaturapanich G, Chaiyakul S, Verawatnapakul V, Pholpramool C (2008) Effects of Kaempferia parviflora extracts on reproductive parameters and spermatic blood flow in male rats. Reproduction 136:515–522. Chen X, Lee TJ (1995) Ginsenosides-induced nitric oxidemediated relaxation of the rabbit corpus cavernosum. Br J Pharmacol 115:15–18. Clark JT, Smith ER, Davidson JM (1985) Testosterone is not required for the enhancement of sexual motivation by yohimbine. Physiol Behav 35:517–521. Cuppett SL, Hall CA (1998) Antioxidant activity of the Labiatae. Adv Food Nutr Res 42:245–271. Damber JE, Janson OO (1978) Testicular blood flow and testosterone concentration in spermatic venous blood of anaesthetized rats. J Reprod Fertil 52:265–269. Elbetieha A, Al-Hamood MH, Alkofahi A, Bataineh H (1998) Reproductive Toxicity potentials of Salvia fruticosa (Labiatae) in rats. J Ethnopharmacol 61:67– 74. Ganu G, Nagore DH, Rangari M, Mithilesh Gupta H (2010) Pharmacological evaluation of ayurvedic plants for aphrodisiac activity in experimental animals. J Complement Integr Med 7:31. Giuliano F, Rampin O (2004) Neural control of erection. Physiol Behav 83:189–201. Gray GD, Smith ER, Davidson JM (1980) Hormonal regulation of penile erection in castrated male rats. Physiol Behav 24:463–468. Guohua H, Yanhua L, Rengang M, Dongzhi W, Zhengzhi M, Hua Z (2009) Aphrodisiac properties of Allium tuberosum seeds extract. J Ethnopharmacol 122:579–582. Harding SM, Velotta JP (2011) Comparing the relative amount of testosterone required to restore sexual arousal, motivation, and performance in male rats. Horm Behav 59:666–673. Hart BL (1979) Activation of sexual reflexes in male rats by dihydrotestosterone but not estrogen. Physiol Behav 23:107– 109. Hart BL, Haugen CM (1968) Activation of sexual reflexes in male rats by spinal implementation of testosterone. Physiol Behav 3:735–738.
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Hassun Filho PA, Cedenho AP, Lima SB, Ortiz V, Srougi M (2005) Single nucleotide polymorphisms of the heat shock protein 90 gene in varicocele associated infertility. Int Braz J Urol 31:236–242. Hernandez-perez M, Rabanal RM, de la Torre MC, Rodriguez B (1995) Analgesic, anti-inflammatory, anti pyretic and haematological effect of aethiopinone, an o-naphthoquinone diterpeniod from Salvia anthiopis roots and two hemisynthetic derivatives. Planta Med 61:505–509. Iammarrone E, Balet R, Lower AM, Gillott C, Grudzinskas JG (2003) Male infertility. Best Pract Res Clin Obstet Gynaecol 17:211–229. Imanshahidi M, Hosseinzadeh H (2006) The harmacological effects of Salvia species on the central nervous system. Phytother Res 20:427–437. Islam MW, Tariq M, Ageel AM, Al-Said MS, Al-Yhya AM (1991) Effect of Salvia haematodes on sexual behaviour of male rats. J Ethnopharmacol 33:67–72. Jasem E, Nasim J, Gholamreza M, Naser S, Nader M, Maryam SL, Abbas N, Vahid R (2010) Evaluation of the effects of Salvia hypoleuca on the cAMP-responsive element modulator (CREM) gene expression and spermatogenesis in rat. Middle East Fertil Soc J 15:274–277. Jimenez J, Risco S, Ruiz T, Zarzuelo A (1996) Hypoglycemic activity of Salvia lavandulifolia. Planta Med b4:260–262. Johnson L, Petty CS, Neaves WB (1980) A comparative study of daily sperm production and testicular composition in humans and rats. Biol Reprod 22:1233–1243. Kenjale R, Shah R, Sathaye S (2008) Effects of Chlorophytum borivilianum on sexual behaviour and sperm count in male rats. Phytother Res 22:796–801. Kerr JB, Millar M, Maddocks S, Sharp RM (1999) Stagedependent changes in spermatogenesis and Sertoli cells in relation to the onset of spermatogenic failure following withdrawal of testosterone. Anat Rec 235:547–559. Kim TH, Jeon SH, Hahn EJ, Paek KY, Park JK, Youn NY, Lee HL (2009) Effects of tissue-cultured mountain ginseng (Panax ginseng CA Meyer) extract on male patients with erectile dysfunction. Asian J Androl 11:356–361. Larsson K, Perez-Palacios G, Moralı G, Beyer C (1975) Effects of dihydrotestosterone and estradiol benzoate pretreatment upon testosterone-induced sexual behavior in the castrated male rat. Horm Behav 6:1–8. Leblond CP, Clermont Y (1952) Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 55:548–573. Linnankoski I, Leinonen LM (2010) Compatibility of male and female sexual behaviour in Macaca arctoides. Z Tierpsychol 70:115–122. Linnankoski I, Gr€ onroos M, Carlson S, Pertovaara A (1995) Effect of cocaine on sexual behavior in male stump tail macaques (Macaca arctoides). Pharmacol Biochem Behav 52:211–216. Lucio RA, Flores-Rojas G, Aguilar F, Zempoalteca R, Pacheco P, Velazquez-Moctezuma J (2001) Effects of genitofemoral
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nerve transection on copulatory behavior and fertility in male rats. Physiol Behav 73:487–492. Mercier O, Perraud J, Stadler J (1987) A method for the routine observation of sexual behaviour in rats. Lab Anim 21:125–130. Murphy LL, Lee TJF (2006) Ginseng, sex behavior, and nitric oxide. Ann N Y Acad Sci 962:372–377. Nantel F, Monaco L, Foulkes NS, Masuilier D, Lemeur M, Henriksen K (1996) Spermiogenesis deficiency and germ cell apoptosis in CREM-mutant mice. Nature 380:159– 162. O’Donnell L, McLachlan RI, Wreford NG, Robertson DM (1994) Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 135:2608–2614. Park SW, Lee CH, Shin DH, Bang NS, Lee SM (2006) Effect of SA1, a herbal formulation, on sexual behavior and penile erection. Biol Pharm Bull 29:1383–1386. Ramachandran S, Sridhar Y, Sam SK, Saravanan M, Leonard JT, Anbalagan N, Sridhar SK (2004) Aphrodisiac activity of Butea frondosa Koen. ex Roxb. extract in male rats. Phytomedicine 11:165–168. Ratnasooriya WD, Dharmasiri MG (2000) Effects of Terminalia catappa seeds on sexual behaviour and fertility of male rats. Asian J Androl 2:213–219. Robb GW, Amann RP, Killian GJ (1978) Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J Reprod Fertil 54:103–107. Russell LD, Corbin TJ, Ren HP, Amador A, Bartke A, Ghosh S (1992) Structural changes in rat Leydig cells posthypophysectomy: a morphometric and endocrine study. Endocrinology 131:498–508. Rustayan A, Masoudi S, Monfared A, Komilizadeh H (1999) Volstile constituents of three Salvia species grown wild in Iran. Flavour Fragr J 14:267–278. Saxena S, Dixit VK (1987) Role of total alkaloids of Mucuna pruriens Baker in spermatogenesis in male rats. Indian J Nat Prod 3:3–7. Shan LX, Hardy MP (1992) Developmental changes in levels of luteinizing hormone receptor and androgen receptor in rat Leydig cells. Endocrinology 131:1107–1114.
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Sharma V, Thakur M, Chauhan NS, Dixit VK (2009) Evaluation of the anabolic, aphrodisiac and reproductive activity of Anacyclus pyrethrum DC in male rats. Sci Pharm 77:97–110. Srivastav A, Chandra A, Singh M, Jamal F, Rastogi P, Rajendran SM, Bansode FW, Lakshmi V (2010) Inhibition of hyaluronidase activity of human and rat spermatozoa in vitro and antispermatogenic activity in rats in vivo by Terminalia chebula, a flavonoid rich plant. Reprod Toxicol 29:214–224. Steinberger E, Nelson WO (1995) The effect of hypophysectomy, cryptorchidism, estrogen and androgen upon the level of hyaluronidase in the rat testis. Endocrinology 56:429–444. Suresh S, Prithiviraj E, Prakash S (2009) Dose- and timedependent effects of ethanolic extract of Mucuna pruriens Linn. seed on sexual behaviour of normal male rats. J Ethnopharmacol 122:497–501. Tajuddin SA, Latif A, Qasmi IA (2003) Aphrodisiac activity of 50% ethanolic extracts of Myristica fragrans Houtt. (nutmeg) and Syzygium aromaticum (L) Merr. & Perry. (clove) in male mice: a comparative study. BMC Complement Altern Med 3:6. Tajuddin SA, Latif A, Qasmi IA, Amin KMY (2005) An experimental study of sexual function improving effect of Myristica fragrans Houtt (nutmeg). BMC Complement Altern Med 5:16. Thakur M, Dixit VK (2007) Effect of some vajikaran herbs on pendiculation activities and in vitro sperm count in male rats. Sex Disabil 25:203–207. Wasser S, Ho JM, Ang HK, Tan CE (1998) Salvia miltiorrhiza reduce experimentally-induced hepatic fibrosis in rats. J Hepatol 29:760–771. Yakubu MT, Akanji MA (2011) Effect of aqueous extract of Massularia acuminata stem on sexual behavior of male wistar rats. ECAM 2011:1–10. Yu WG (1994) Effect of acetylsalvianolic acid A on platelet function. Yao Xue Xue Bao 29:412–416. Zhao GQ, Garbers DL (2002) Male germ cell specification and differentiation. Dev Cell 2:537–574.
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Dose-dependent effects of ethanol extract of Salvia haematodes Wall roots on reproductive function and copulatory behaviour in male rats F. W. Bansode1, S. M. Rajendran2 & R. K. Singh3 1 Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, UP, India; 2 Division of Botany, CSIR-Central Drug Research Institute, Lucknow, UP, India; 3 Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, UP, India
Keywords Organ weights—Salvia haematodes roots— sexual behaviour—sperm counts—spermatogenesis Correspondence Dr Falgun W. Bansode, Division of Endocrinology, CSIR-Central Drug Research Institute, Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India. Tel.: +91 0522 2612411 18(PABX), Extn. 4383; Fax: +91 0522 2623403/2623938; E-mail: [email protected] Accepted: January 12, 2014 doi: 10.1111/and.12255
Summary This study was aimed to investigate the dose-dependent effects of Salvia haematodes Wall roots (SHW) extract on male reproductive function and copulatory behaviour in rats. Sexually mature males were assigned to four groups: control and treated (5, 50 and 300 mg kg 1 day 1 for 30 days). At the end of treatment regimes, the reproductive activity viz. body/organ weights, testicular spermatogenesis, daily sperm production rate (DSP) and epididymal sperm counts, and sexual behaviour including mounting latency (ML), mounting frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), post-ejaculatory interval (PEI) and penile reflexes (PE) were assessed. Results showed significant increase in body weight (at 300 mg kg 1), testis/epididymis weights (at 50 and 300 mg kg 1), testicular spermatids, DSP, tubular diameter and epididymal sperm counts (at 50 and 300 mg kg 1doses) in treated compared with control rats. It also produced dose-dependant changes in sexual behaviour. The 5 mg kg 1 dose of extract increased MF and PE, whereas 50 and 300 kg 1 doses caused significant increase in MF, IF, PE, EL (but less than sildenafil citrate treatment), hit rate and seminal plug weight. It is concluded that SHW extract enhances anabolic activity, testicular function and sexual behavioural performance in a dose-dependant manner.
Introduction Sexual dysfunction is one of the most common diseases in 40- to 70-year-old men with an increasing incidence of degenerative diseases, injuries and stress. In infertile males too, the growing evidence from clinical and epidemiological studies has been shown an increasing incidence of male reproductive problems such as conjugal infertility and abnormal semen analyses (Iammarrone et al., 2003; Hassun Filho et al., 2005). Despite medical and surgical treatment modalities available for treating sexual dysfunction and infertility, plant-derived herbal remedies continue to be more popular alternative for men and women seeking to improve their sex life. Nevertheless, the efficacy of most of these herbal agents in treating sexual dysfunction remains unclear (Park et al., 2006; Kim et al., 2009). There are several aspects of male sexual behaviour, but an erection and mounting provide direct and prominent © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
measures for determining the efficacy of aphrodisiacs (Linnankoski et al., 1995; Linnankoski & Leinonen, 2010). In Unani and Ayurvedic system of medicine, the dried roots of Salvia haematodes have been used as a sex tonic. It has been mentioned to be of great value in the management of male sexual fertility regulatory activity. Previous study with S. haematodes (500 mg kg 1, orally) has shown improvement in the sexual activity that produced a significant increase in episodes of penile erection and the orientation of males towards the female by increasing anogenital investigatory behaviour, enhanced licking and grooming of the genitals and increased ejaculation latency in male rats (Islam et al., 1991). S. haematodes Wall (Family Lamiaceae), which is commonly known as ‘Lal Bahaman’, has been cultivated worldwide including India for use in folk medicine and culinary purposes (Brickell, 1996; Rustayan et al., 1999). Pharmacological studies showed significant cardio tonic and 1
Enhancement of reproductive and sexual activity by Salvia haematodes
anticonvulsant activities without any toxicity up to the dose of 5 g kg 1 in rats (Akbar et al., 1984). The different species of Salvia including S. haematodes, S. officinalis, S. leriifolia, S. triloba and S. divinorum have been reported to show various pharmacological effects as analgesic, anti-inflammatory and antipyretic (Hernandezperez et al., 1995; Al-Yousuf et al., 2002), antioxidant (Cuppett & Hall, 1998), hepatoprotective (Wasser et al., 1998), hypoglycaemic activities (Jimenez et al., 1996), anti-ischaemic (Yu, 1994), for the treatment of coronary and cerebrovascular disease, sleep disorders, hepatitis, hepatocirrhosis, chronic renal failure, dysmenorrhoea, amenorrhoea, carbuncles and ulcers (Wasser et al., 1998), sedative, hypnotic, hallucinogenic, skeletal muscle relaxant, memory enhancing, anticonvulsant, neuroprotective, antiparkinsonian activity as well as inhibition of ethanol and morphine withdrawal syndrome (Imanshahidi & Hosseinzadeh, 2006; Baricevic & Bartol, 2000). The testicular spermatogenesis, the process of sperm formation and accessory sex organs’ functions are predominantly dependent on FSH and testosterone (Steinberger & Nelson, 1995). Disruption in biosynthesis of these hormones leads to inhibition of testicular spermatogenesis, sperm formation and Leydig cells morphology and accessory sex organs’ function (Russell et al., 1992; Shan & Hardy, 1992; Kerr et al., 1999). Testosterone supplementation restores normal function of Leydig cells/ spermatogenesis and accessory sex organs (O’Donnell et al., 1994). Testicular daily sperm production rate defined as a process for semi-quantitative analysis of spermiogenesis/sperm produced in testis is a baseline to assess sexual maturity (Robb et al., 1978). It is evaluated by enumeration of elongated spermatids nuclei in steps 17– 19 of spermiogenesis in testicular homogenates. The semen analysis, including sperm count, motility and morphology, is the basis of investigations into male infertility, and number of genes participating in spermatogenesis have been reported to play an essential role in human reproductive function (Blendy et al., 1996; Nantel et al., 1996; Zhao & Garbers, 2002). Several studies have demonstrated that herbal drugs enhance sperm count and elicit androgenic effects (Saxena & Dixit, 1987; Chaturapanich et al., 2008; Sharma et al., 2009). In contrast, the plant-like Salvia fruticosa has been shown to produce adverse effects on the fertility of male and female rats (Elbetieha et al., 1998). This study was conducted to determine the potential reproductive function (viz. body/organ weights, quantitative analysis of testicular spermatogenesis, daily sperm production rate and epididymal sperm count) and sexual behavioural performance in adult male Sprague Dawley rats administered S. haematodes Wall extract in a dosedependent manner. 2
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Materials and methods Chemicals All chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO, USA). Collection of plant material The plant S. haematodes Wall (Family Lamiaceae), a garden plant used in this study, was collected locally from fields, plant nurseries and wild waste-land areas nearby Lucknow, Uttar Pradesh, India. The plant material was authenticated by one of the author, and a specimen was preserved in our Institute’s herbarium (Voucher No. 19752). Extraction and fractionation Shade-dried powdered roots of the plant, S. haematodes Wall (SHW), were extracted with 50% ethanol (3 9 1 l). The pooled filtered ethanolic extract was concentrated under reduced pressure below 50 °C. The viscous mass thus obtained was dried under high vacuum to remove last traces of the solvent. The semi-solid ethanolic extract of the collection (yield 9.11%, w/v; Islam et al., 1991) was used to test reproductive function and aphrodisiac activity in Sprague Dawley rats. Animals, treatment, tissue collection and fixation Adult male (180–200 g) and cycling female (170–180 g) rats of proven fertility (Sprague Dawley strain) used in this study were obtained from Institute’s animal house. Rats were caged in similar husbandry conditions, in environmentally controlled rooms with maintained temperature (24 1 °C) and 12/12 h light/dark cycles. Animals were fed with pelleted food (Hindustan Lever Ltd.) and water ad libitum. In the first set of experiment, male adult rats were randomly divided into four groups (Gr. I–IV) of 12 animals in each with total of 48 rats. Animals in Gr. I received distilled water (10 ml kg 1) only and served as a control. Rats in Gr. II, III and IV were administered with 50% ethanolic extract of SHW orally at 5, 50 and 300 mg kg 1 doses, respectively, for 30 days. The animals were allowed free access to food and drinking water ad labium during the entire treatment period. The physical appearances/ activities of the rats were observed daily. Body weights of rats from control and treated groups were recorded, and autopsy was performed on day 31 by anaesthetising with solvent anaesthetic ether. The reproductive organs viz. testes, epididymis and accessory sex © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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organs (e.g. prostate, seminal vesicle, vas deferens and penis) were dissected out surgically, rinsed immediately in chilled saline, freed from connective tissues, and blood clots and weights were recorded. The testes and epididymis from left sides were fixed in Bouin’s fluid (24 h) for histology purpose. These organs from right side were used for daily sperm production rate and sperm counting. The experimental protocol was approved by an Institutional Review Committee for the use of Human or Animal Subjects and the procedure is in compliance with National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23 revised 1985) and the guidelines laid down by Institute’s Animal Ethics Committee (IAEC).
Turraxâ (Janke and Kunkel, Staufen, Germany). The homogenates were further diluted with the same medium, and homogenisation-resistant spermatid nuclei in steps 17–19 of stages IV–VIII of the spermatogenic cycle were counted in triplicate using four chambers (total of 64 squares) of Neubauer’s haemocytometer under the phasecontrast BX51 Olympus trinocular microscope (Olympus) at 9100 magnification (Amann & Lambiase, 1969). Daily sperm production rate per gram testicular parenchyma was calculated by dividing the number of spermatids nuclei with the product of weight of parenchyma and time divisor of 6.3 days reported for rat (Johnson et al., 1980). The values are represented as mean SE for five animals each group.
Histomorphometry of testis
Epididymal sperm count
Bouin’s fixed testes and epididymis from control and treated groups of rats were dehydrated in graded series (50–100%) of ethanol, cleared in xylene, infiltrated and embedded in paraffin wax (at 58 °C). Serial cross sections (5 lm) were stained with haematoxylin and eosin. Measurements of morhpometric analyses, viz. diameter of testicular tubules (100 tubules each group) and Leydig cell nuclei (100 nuclei per group) were carried out using Biovis Image Plus Software for image analysis and processing (Expert Vision Labs Pvt. Ltd., Mumbai, India) at 9100 and at 91000 (oil immersion) magnifications under Olympus Trinocular Microscope (BX51, Tokyo, Japan).
Cauda epididymidis was excised surgically, cut into small pieces in suspension medium containing 140 mmol NaCl, 0.3 mmol KCl, 0.8 mmol Na2HPO4, 0.2 mmol KH2PO4 and 1.5 mmol D-glucose (pH adjusted to 7.3 by adding 0.1 N NaOH), and spermatozoa were collected by centrifugation at 100 9 g for 2 min. The resultant precipitate was resuspended in fresh suspension medium, diluted and placed on both sides of Neubauer’s haemocytometer. The number of spermatozoa was counted in four chambers of haemocytometer in duplicate in each rat under Olympus Trinocular microscope at 9100 magnification. The values were expressed as an average of total counts of sperm number per ml of suspension (Robb et al., 1978; Srivastav et al., 2010).
Semi-quantitative analysis of spermatogenesis Semi-quantitative analysis of testicular spermatogenesis was carried out on the basis of nuclear morphology of the germ and Sertoli cells (Leblond & Clermont, 1952). The number of germ cells and Sertoli cell nuclei were counted in at least 20 round seminiferous tubules selected randomly from at least 2–3 cross sections in each rat at 9400 magnification under Olympus Trinocular microscope (BX 51; Olympus). Cell counts were corrected by Abercrombie’s formula (Abercrombie, 1946) and values expressed as an average cell counts per round seminiferous tubule in each group (Srivastav et al., 2010). Estimation of daily sperm production rate in testis Measurement of daily sperm production rate (DSP) in testicular homogenates was carried out as per method of Amann et al. (1976). Briefly, testicular parenchyma was cut into small pieces after removal of tunica, placed in 0.25 M sucrose solution (pH 7.5), weighed and homogenised in a fluid containing 150 mM NaCl l 1, 3.8 mM NaN3 l 1 and 0.05% (v/v) Triton X-100 using an Ultra © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
Copulatory behaviour test In the second set of experiment, rats were divided into five groups (Gr. I–V) of 12 animals in each with total of 60 rats. Animals in Gr. I (control group) received distilled water (10 ml kg 1) only, and in Gr. II, III and IV administered with 50% ethanol extract of S. haematodes Wall root orally at 5, 50 and 300 mg kg 1 doses, respectively, for 30 days. Rats in Gr. V (Std. Control) were given oral suspension of Sildenafil citrate (5 mg kg 1 body weight) 1 h prior to the commencement of the mating test. Copulatory test was conducted as per method described previously (Mercier et al., 1987; Lucio et al., 2001) Behaviour test consisted in placing the male in a plexiglas cylindrical arena (55 cm in diameter) for a 5min habituation period before the presentation of an ovariectomised stimulus female. Females were brought into the oestrous by subcutaneous injection of oestradiol benzoate (10 lg) and progesterone (2 mg) 48 and 4 h before the test respectively. A total of 90 females used in 3
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this study were divided into 30 groups containing three females each to permit each observation to be made using fresh females and to avoid possible effects of repeated copulation. Test session ended when the male displayed the ejaculatory pattern. Six females in each group were sacrificed, seminal plug dissected out and weights recorded. The following copulatory parameters were studied after 30 days treatment of SHW extract: mounting latency (ML), mounting frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), post-ejaculatory interval (PEI), seminal plug weight and hit rate.
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Statistical analysis Statistical analysis for significance of differences between copulatory parameters, reproductive organ weights, spermatogenic analysis, daily sperm production and epididymal sperm count between control and treated groups was performed by applying Student’s t-test followed by oneway ANOVA (one factor analysis of variance) test. Values with P < 0.05 were considered as significant and expressed as mean SEM.
Results
Potency activity test
Body and reproductive organ weights
Potency test was carried out in adult male rats as per method described previously (Hart & Haugen, 1968; Hart, 1979). Animals were divided into five groups (Gr. I–V) of six rats each. In control group (Gr. I), rats received vehicle (10.0 ml kg 1 of distilled water). Oral administration of SHW extract at 5, 50 and 300 mg kg 1 doses (Gr. II–IV) was given daily for 30 days prior to the test. Gr. V rats received oral suspension of sildenafil citrate (5 mg kg 1) 1 h prior to the commencement of the experiment. On day 31, each animal was placed on its back in a glass cylinder for partial restraint. The pre-putial sheath was pushed behind the glans penis using the thumb and index finger for a period of 30 min. During this period, a cluster of genital responses consisting of erections (PE), quick flips (QF), long flips (LF) and total Flips (TF) of the glans penis were recorded.
No significant changes were observed in the body weights of rats treated with 5 or 50 mg kg 1 dose of SHW extract but caused a significant increase (P < 0.05) in body weight at 300 mg kg 1 dose as compared to controls. The reproductive organ weights did not show any significant change in rats treated with 5 mg kg 1 extract as compared to controls. However, 50 and 300 mg kg 1 doses caused a significant (P < 0.005–0.001) increase in the testes and epididymal weights. The accessory sex organs (viz. prostate, seminal vesicle, vas deferens and penis) did not show any significant change in their weights compared with control (Table 1).
Toxicity study To determine general short-term toxicity, the control and treated animals were observed continuously for 1 h for any gross behavioural changes or death, if any.
Morphometric studies/semi-quantitative analysis of spermatogenesis In control rats, normal testicular structure was evident depicting all spermatogenic cell types viz. spermatogonia, primary spermatocytes, spermatids and spermatozoa as well as Sertoli and Leydig cells (Fig. 1a). In treated rats, the density-elongated spermatids/spermiated spermatozoa in lumen of almost all testicular tubules were
Table 1 Effect of 50% ethanol extract of Salva haematodes Wall root (SHW) on the final body weight and reproductive organ weights in adult male rats
Treatment Control SHW treated (5 mg kg 1) (50 mg kg 1) (300 mg kg 1)
Body weight (g)
Reproductive organ weights (g) Testis
Epididymis
V. Prostate
Seminal vesicles
Vas deferens
Penis
212 3.4
1.152 0.027
0.439 0.013
0.464 0.020
1.453 0.049
0.108 0.032
0.331 0.032
216 4.4NS
1.135 0.095NS
0.434 0.028NS
0.524 0.035NS
1.521 0.028NS
0.098 0.003NS
0.329 0.015NS
214 9.8NS
1.436 0.071c
0.635 0.055b
0.512 0.122NS
1.480 0.250NS
0.151 0.009NS
0.351 0.045NS
225 3.7a
1.545 0.081c
0.650 0.025c
0.552 0.130NS
1.582 0.225NS
0.161 0.013NS
0.361 0.041NS
Values are expressed as mean SEM; n = 10, number of animals for body weights and n = 6, number of animals for organ weights; Significance of difference control versus treated; NS, not significant; aP < 0.05; bP < 0.005; cP < 0.001.
4
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Enhancement of reproductive and sexual activity by Salvia haematodes
Fig. 1 Testicular cross sections from control (a) and Salvia haematodes-root-extract-treated (b–d) rats. Control rat showing normal testicular structure, displaying all germ cell types. Note the similar testicular germ cells types in treated rats with 5 mg kg 1 dose (b), but displaying increased density of round and elongated spermatids at 50 mg kg 1 (c) and 300 mg kg 1 (d) of extract. (Magnification 9400; H-E stained). A, spermatogonia; Z, zygotene; P, pachytene spermatocytes; R, round spermatids; E, elongated spermatids; SC, sertoli cells; LC, Leydig cells; L, lumen.
(a)
(b)
(c)
(d)
increased. The Sertoli cells and Leydig cells showed normalcy in treated rats (Fig. 1b–d). The testicular tubular diameter was observed to be increased (P < 0.005–0.001) at 50 and 300 mg kg 1 doses in treated rats (Table 2). Cauda epididymal tubular lumen was filled with large number of spermatozoa in treated (at 50 and 300 mg kg 1) as compared to control rats (Fig. 2). Semi-quantitative analysis of testicular spermatogenesis did not show any significant change in number of germ cells viz. spermatogonia, primary spermatocytes (nonpachytene and pachytene) and spermatids (round and elongated) in SHW-extract-treated rats at 5 mg kg 1 dose as compared to controls. But there was a significant increase in the number of round (P < 0.02) and elongated (P < 0.005–0.001) spermatids in treated rats at 50
and 300 mg kg 1 doses for 30 days. The testicular germ cell types, for example, A-spermatogonia, non-pachytene (NP) and pachytene (P) spermatocytes, Sertoli cells (SC) and Leydig cells diameter in treated rats were comparable with that in controls (Table 2). Daily sperm production rate Administration SHW extract caused a significant increase in DSP rate as evident by increased number of elongated spermatids in steps 17–19 of IV–XII stages of spermatogenic cycle in testicular homogenates, especially in 50 mg kg 1 (P < 0.05) and 300 mg kg 1 (P < 0.001) dose level. Whereas, 5 mg kg 1 dose of root extract did not produce any significant increase in DSP rate as compared to controls (Fig. 3).
Table 2 Effect of Salva haematodes Wall root (SHW) extract on testicular germ cell population dynamics, tubular diameter and nuclear diameter of Leydig cells in rats Spermatogenic germ cells number per seminiferous tubule Treatment groups Control SHW treated (5 mg kg 1) (50 mg kg 1) (300 mg kg 1)
(A)
(NP)
(P)
(R)
4.5 0.35
20.4 2.45
25.8 2.8
65.8 4.2
4.2 0.45NS 4.6 0.35NS 5.1 0.37NS
21.2 4.45NS 24.3 4.54NS 22.6 2.71NS
23.2 3.2NS 28.2 1.2NS 32.8 2.8NS
65.4 1.4NS 78.2 1.6a 76.8 4.2a
(E) 101 8.0 107.6 9.2NS 122.0 5.0b 134 4.0c
Sertoli cells/ tubule
Tubular diameter (µm)
Nuclear diameter of Leydig cells (µm)
16.1 1.4
437.2 9.7
14.9 2.5
15.9 0.6NS 17.0 2.4NS 18.1 1.5NS
438.0 8.3NS 497.7 10.5b 507.6 14.5c
12.1 1.6NS 15.3 3.5NS 16.7 3.7NS
A, spermatogonia A; NP, nonpachytene spermatocytes; P, pachytene spermatocytes; R, round spermatids; E, elongated spermatids. Values are expressed as mean SEM, n = 5, number of animals; Significance control versus treated; NS, not significant; aP < 0.02; bP < 0.005; c P < 0.001.
© 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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Enhancement of reproductive and sexual activity by Salvia haematodes
(a)
(b)
(c)
(d)
F. W. Bansode et al.
Fig. 2 Cauda epididymis from control (a) and Salvia haematodes-root-extract-treated (b–d) rats. Note the sperm density similar to control in treated rats at 5 mg kg 1 dose (b). In comparison, increased sperm density at 50 (c) and 300 (d) mg kg 1 doses of S. haematodes root extract can be seen. EP, epididymal epithelium; ES, epididymal stroma; S, epididymal sperms, (magnification 9400; H-E stained).
duced a significant increase in MF, IF and EL. In contrast, the sexual behavioural changes were observed to be highly increased in rats treated with 300 mg kg 1 of SHW extract. In addition, the hit rate, MF and seminal plug weight were also observed to be increased significantly at this dose compared with controls, but caused decline in PEI in treated rats. The Std. drug, sildenafil citrate (5 mg kg 1), produced a significant increase in MF, IF and EL but caused decrease in ML, IL and PEI as compared to controls (Fig. 4). Fig. 3 Effects of Salvia haematodes Wall root extract on daily sperm production rate (gm/testicular parenchyma) and cauda epididymal sperm counts (9106) per ml.
Epididymal sperm count Epididymal sperm count did not show any significant change in Gr. II (5 mg kg 1) rats as compared to Gr. I (control) rats, but caused a significant increase in epididymal sperm counts in Gr. III (50 mg kg 1, P < 0.05) and Gr. IV (300 mg kg 1, P < 0.001) rats as compared to Gr. I control (Fig. 3). Effects of SHW extract on sexual behaviour Results of copulatory behaviour test in SHW-extract-treated rats for 30 days showed dose-dependent changes (i.e. 5 mg < 50 mg < 300 mg kg 1) in the sexual behavioural performance. Oral administration of extract at lower dose (5 mg kg 1) exhibited a significant increase in the MF as compared to controls. 50 mg kg 1 dose of extract pro6
Effect of SHW extract on potency Oral administration of SHW root extract at its lower dose (5 mg kg 1) produced a significant increase PE as compared to control rats. At 50 mg kg 1 dose, a significant increase in PE and QF (P < 0.05) was observed. However, LF and TF were insignificantly increased when compared with controls. However, at 300 mg kg 1, there was a significant increase (P < 0.001) in PE as well as in the QF (P < 0.02), LF (P < 0.001) and TF (P < 0.005) compared with control as well as lower doses of extract. The results showed a dose-dependent increase in the episodes of penile reflexes as compared to control rats. The rats in Gr. V treated with Std. control showed prominent effects in penile reflexes as compared to doses of SHW extract used (Fig. 5). Toxicity study No mortality and gross behavioural changes were observed during 1–3 h of toxicity study in treated rats as compared to controls. © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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Enhancement of reproductive and sexual activity by Salvia haematodes
Fig. 4 Effects of Salvia haematodes Wall root extract on sexual behaviour of rats after 30 days treatment. MF, mounting frequency; IF, intromission frequency; ML, mounting latency; IL, intromission latency; EL, ejaculation latency; PEI, post-ejaculatory time interval; SPWt, seminal plug weight.
Fig. 5 Effects of Salvia haematodes Wall root extract on penile reflexes (potency test) in rat. QF, quick flips; LF, long flips; TF, total flips; PE, penile erections.
Discussion The results of the present study on determining the sexual behavioural performance of ethanol extract of SHW roots show aphrodisiac potential at different doses (5, 50 and 300 mg kg 1 body weight) in rat model. It caused significant increase in MF and IF at 50 and 300 mg kg 1 doses except at its lower dose of 5 mg kg 1 showed increase in MF only. Earlier reported studies with 500 mg kg 1 dose of SHW have also shown an increase in the orientation of males towards females, grooming and licking behaviour followed by increased number of mounts in treated rats (Islam et al., 1991). Similarly, our results exhibit an increase in ML at 300 mg kg 1 dose level, which has been demonstrated to increase MF and IF marked by increased rate and number of intromissions and considered to be the indices of libido with nutmeg (Tajuddin et al., 2003, 2005). The potency test carried out in rats showed significant increase in penile reflexes such as QF, LF, TF and PE at 300 mg kg 1 dose. In comparison, lower doses of plant extract also produced an increase in QF (at 50 mg kg 1) and PE (at 5 and 50 mg kg 1). Thus, the increased MF and IF along with © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
increased hit rate and libido may lead to increase penile erections with prolongation of coitus time. The aggregate of TF and PE was also significantly increased in test animals indicating extract-induced increase in ‘pure’ potency. In support, previous studies have demonstrated a dose-dependent, recurrent and significant increase in the episodes of penile reflexes (viz. QF, LF, TF and PE) at higher doses than lower ones of plant extracts (Islam et al., 1991; Ang et al., 2001; Ramachandran et al., 2004; Tajuddin et al., 2005; Thakur & Dixit, 2007). Further, results of the study also showed an increase in EL and seminal plug weights in treated (at 300 mg kg 1) rats. However, decreased time in PEI and MF has demonstrated that the test drug may intensify the occurrence of repeated sexual activity heavily (Tajuddin et al., 2005). There are several other aphrodisiacs that increased MF, IF, EF and penile reflexes, but decreased ML, IL and PI with herbal extracts of Allium tuberosum seeds (Guohua et al., 2009), Mucuna pruriens Linn. seeds (Suresh et al., 2009), Butea frondosa Koen ex Roxb (Ramachandran et al., 2004) and Kaempferia parviflora (Chaturapanich et al., 2008). In contrast, Catappa seeds have shown no effect on libido, sexual vigour or sexual performance at 1500 mg kg 1, and higher dose (3000 mg kg 1) reversibly inhibited all the parameters of sexual behaviour other than MF and IF and copulatory efficiency (Ratnasooriya & Dharmasiri, 2000). The results of the present study also showed increased anabolic activity that indicated an increase in the body weights (at 300 mg kg 1) and reproductive organs (viz. testis and epididymis) weights as well as testicular tubular diameters (at 50 or 300 mg kg 1), which may suggest an involvement of testosterone intervention and in improving testosterone availability to gonads in treated rats. Our results show an evidence of increased spermiogenesis marked by a characteristic increase in round and elongated spermatids (in testicular cross sections), DSP (in testicular homogenates), as well as epididymal sperm counts in rats with 50 and 300 mg kg 1 of SHW extract. These findings can be correlated with increased MF, IF, 7
Enhancement of reproductive and sexual activity by Salvia haematodes
PE, ejaculations and increased seminal plug weights in treated rats. In concurrence, Chlorophytum borivilianum treatment also has shown an increase in libido, sperm count, seminal fructose content and penile erection index in male rats. The effect of extracts on inducible NO release in vitro directly correlated with the enhanced erectile function in vivo (Thakur & Dixit, 2007; Kenjale et al., 2008). It has been demonstrated that the increased testosterone level is associated with increased sexual desire, arousability, sperm counts, PE and MF (Thakur & Dixit, 2007; Sharma et al., 2009; Harding & Velotta, 2011; Yakubu & Akanji, 2011). In contrast, results with yohimbineinduced castrated rats supplemented with testosterone resulted in reduction in latencies to initial mount, intromission and ejaculation and demonstrated that testosterone is not a requisite for the enhancement of sexual motivation by yohimbine, but a2-adrenoceptors are involved in the modulation of sexual arousal (Clark et al., 1985). Results of the study did not show any significant increase in weights of androgen-dependant accessory sex organs as well as in number of androgen-sensitive primary spermatocytes and nuclear diameter of Leydig cells treated rats up to 300 mg kg 1 dose. It indicates dose and duration dependency of the test drug for enhancement of sexual behaviour and reproductive activity. Existing reports have shown that the increase in blood flow to the testis may stimulate testosterone production/ secretion, acts on the central nervous system and gonadal tissues and modulates male sexual behaviour (Larsson et al., 1975; Damber & Janson, 1978). It also causes an increase in the diameter of seminiferous tubules, seminal vesicle weight and cauda epididymal sperm density in rats. S. hypoleuca (Jasem et al., 2010) and Anacyclus pyrethrum DC (Sharma et al., 2009) have been shown to cause an increase in sperm parameters, viz. sperm count and motility and spermatogenesis and testicular weight/ accessory organ weights in rat. The extract of S. hypoleuca caused a significant increase in testicular CREM gene expression and protein level in rat (Jasem et al., 2010). It has been shown that Abutilon indicum and Withania somnifera both possessed marked aphrodisiac activity complying many facets such as enhancement in libido, increase in the sexual performance, penile erection and anabolism, increased spermatogenesis as well as sperm validity and had shown to relax penile cavernosal tissue probably by phosphodiesterase inhibition and cGMP accumulation (Ganu et al., 2010). In castrated male rats, both testosterone and dihydrotestosterone (DHT) are very effective in restoring penile erections, but testosterone is most effective in restoring mating behaviour also as compared to DHT, which is totally ineffective (Gray et al., 1980). Penile erection is a vascular event controlled by autonomous nervous system and pro-erectile events of phosphodi8
F. W. Bansode et al.
esterase type 5 inhibitors acting in penis and melanocortin agonists acting in brain, combinedly stimulate developments (Giuliano & Rampin, 2004). There is evidence that ginseng saponin facilitated penile erection by directly inducing the vasodilatation and relaxation of penile corpus cavernosum via nitric oxide mechanism (Chen & Lee, 1995). Furthermore, the central nervous system has been shown to significantly alter the activity of hypothalamic catecholamine and nitric oxide during facilitation of copulatory behaviour and hormone secretion (Murphy & Lee, 2006). In conclusion, the 50% ethanolic extract of S. haematodes was found to be devoid of any general toxicity. In Ayurveda medicine, this plant is found to be safe sexual enhancer in male human. Results of the present study showed significant increase in body weight (at 300 mg kg 1) and reproductive activity viz. testes and epididymal weights, spermiogenesis (spermatids number), daily sperm production rate followed by epididymal sperm counts (at 50 and 300 mg kg 1) in SHW-extracttreated rats. Further, there was an enhancement in the sexual behavioural performance such as MF, IF, Hit rate, PE and EL at 50 or 300 mg kg 1 doses. These results indicate an anabolic effect, enhancement of reproductive activity and sexual behavioural performance by SHW extract. Thus, it provides a scientific rationale for traditional use of S. haematode Wall roots in the treatment of sexual disorders/erectile dysfunction in males. Acknowledgements The authors are thankful to Ms T. Firdaus, Mr Pradeep Singh and Ms Ranjeeta Maurya (a research trainee) for technical and experimental assistance. This work was supported by Ministry of Health and Family Welfare, New Delhi, India. References Abercrombie M (1946) Estimation of nuclear population from microtome sections. Anat Rec 94:238–248. Akbar S, Tariq M, Nisa M (1984) A study on CNS depressant activity of Salvia haematodes wall. Int J Crude Drug Res 22:41–44. Al-Yousuf MH, Bashir AK, Ali BH, Tanira MOM, Blunden G (2002) Some effects of Salvia aegyptiaca L. on the central nervous system in mice. J Ethnopharmacol 81:121–127. Amann RP, Lambiase JT Jr (1969) The male rabbit. III. Determination of daily sperm production by means of testicular homogenates. J Anim Sci 28:369–374. Amann RP, Johnson L, Thomson DL, Pickett BW (1976) Daily spermatozoal production, epididymal spermatozoal reserves and transit time of spermatozoa through the
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