Immobilization effect of Ruta graveolens L. on human sperm: A new hope for male contraception

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Journal of Ethnopharmacology 115 (2008) 36–41

Immobilization effect of Ruta graveolens L. on human sperm: A new hope for male contraception Zhila Naghibi Harat a , Mohammad Reza Sadeghi b,∗ , Hamid Reza Sadeghipour a , Mohammad Kamalinejad c , Mohammad Reza Eshraghian d b

a Department of Physiology, School of of Medicine, Medical Sciences/University of Tehran, Tehran, Iran Department of Endocrinology and Embryology, Reproductive Biotechnology Research Center, Avesina Research Institute, Tehran, Iran c Department of Pharmacognosy, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Department of Epidemiology and Biostatics, School of Public Health, Medical Sciences/University of Tehran, Tehran, Iran

Received 12 March 2007; received in revised form 2 September 2007; accepted 4 September 2007 Available online 14 September 2007

Abstract Aim of the study: Contraceptive plants which were introduced by folk in traditional remedies are investigated worldwide. In this study, the contraceptive effects of Ruta graveolens L., which has been mentioned for male contraceptive in Iranian traditional folk medicine, was experimented on human sperm. Materials and Methods: Different doses of lyophilized aqueous extract of Ruta graveolens L. were added to an amount of fresh semen, containing 106 cells in a 1:1 volumic ratio. Motility and viability of cells, DNA status, mitochondrial activity and sperm revival tests were carried out. Results: The sperm immobilization effects of the extract appeared immediately in a does-dependent manner and 100% of the sperms became immotile at a concentration of 100 mg/ml but other parameters were intact. After washing the sperms, motility was returned in 30.8 ± 3.2% of the sperms, besides coiled tails in 38.6 ± 5.5% of the treated cells, in comparison to 12.5 ± 2.0% of the control group (p = 0.001). The part of the extract, responsible for immobilization of the sperms was stable upon boiling. Conclusions: As the cells were alive and immotile, probably some ionic currents are blocked by a thermostable component of the plant which can be promising as a new male channel blocker contraceptive. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ruta graveolens L.; Iranian traditional medicine; Male contraception; Sperm motility; Channel blocker

1. Introduction Male contraception is a worldwide interesting matter of concept and investigation. Even the World Health Organization (WHO) formed coalitions with governments and international agencies to support research and activities in this filed (Waites, 2003). A lot of efforts have been made to find a safe and effective method with the least side effects but beyond condom and vasectomy there is no other choices for male contraception (Nieschlag and Henke, 2005). Many hormonal, immunological and chemical substances are being investigated but none of them is completely desirable and without side effects (Jensen, 2002). Looking through history, ancient people were also interested in

∗

Corresponding author. Tel.: +98 21 22432024; fax: +98 21 22432021. E-mail address: [email protected] (M.R. Sadeghi).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.09.004

contraception and had their own way of controlling their family size. Some of these methods are still popular among folks (Adsersen et al., 2006). Medicinal plants were one of those methods which nowadays are one of the matters of interest among researches in different countries. Effects of some plants like Gossypium herbaceum (Hoffer et al., 1987), Azadirachta indica (Garg et al., 1994; Khillare and Shrivastav, 2003), Tripterygium wilfordii (Bai and Shi, 2002), Echiveria gibbiflora (Delgado et al., 1999; Reyes et al., 2002), Allium sativum (Chakrabarti et al., 2003), Carica papaya (Lohiya et al., 2000), Alstonia macrophylla (Chattopadhyaya et al., 2005), Ricinus communis (Sandhyakumary et al., 2003), Achyranthes aspera and Stephania hernandifolia (Paul et al., 2006) on human or animal sperm had been studied which all had been chosen by folks or according to history they had been of indigenous usage. Ruta graveolens L. (RGL), is a member of Rutaceae family. It is a hardy, evergreen shrub of up to one meter tall, with a

Z.N. Harat et al. / Journal of Ethnopharmacology 115 (2008) 36–41

characteristic grayish green color and a sharp unpleasant odor. The leaves are small, oblong, deeply divided, pinnate, glandular dotted. The stems are much ramified. Its flowers are small, yellow and in clusters during spring and summer. They have 4 petals, except for the central flower, which has 5 petals. The fruits are round, brown, small and lobulated. The taste is slightly stinging but is masked by the strong bitter odor (Zargari, 1990). This plant have different established effects like antimicrobial, cytotoxic (Ivanova et al., 2005), fungicide (Oliva et al., 2003; Meepagala et al., 2005), herbicide (Hale et al., 2004), anti-inflammatory (Raghav et al., 2006) and hypotensive properties (Chiu and Fung, 1997). It is one of the most ancient and effective contraceptive plants (Maurya et al., 2004). Its potent female antifertility and abortive effects have been reported from countries like Brazil (de Freitas et al., 2005), India (Gandhi et al., 1991), Peru (Gutierrez-Pajares et al., 2003) and Mexico (Conway and Slocumb, 1979), both according to their traditional usage and in animal studies. In Iranian traditional and folk medicine, Ruta graveolens L. (the common rue which is called Sodab in Iran and grows in northern part of Iran, especially in Gilan) was one of the plants used for contraception. Contraceptive usage of this plant was both in females and males. Females used it in two forms: immersing a cotton wool in its fresh leaves extract and inserting it into vagina before intercourse or drink its tea after coitus. In males, they have applied its fresh leave extract on to their penis during intercourse or they have drunk its tea before intercourse. It also was known as a multipotential effective herbal plant with different usages for different diseases. According to the most famous ancient Iranian medical book, “The canon of medicine” which is written by Avicenna, it can be used as an antiinflammatory, sedative, antifungal, analgesic, antispasmodic, antihelmintic and abortive agent. Usually aerial parts including leaves, stem, flowers, and in some cases seed of this plant were used in Iranian traditional medicine (Avicenna, 1997). In addition to its potent abortive effects, it has been mentioned by the folk and traditional medicine to have a male contraceptive effect. Recently, Khouri and El-Akawi (2005) have reported its antiandrogenic effects in male albino rats and its decreasing effects on sexual and aggressive behaviors in the animal. Also, they have reported a decrease in sperm motility and density, but there are no other reports about the effects of this plant as a male contraceptive in the literature. Only in 1995, a report on the sperm immobilization effects of Ruta chalepensis L. extract, another member of Rutaceae family, was published (Gijon et al., 1995). Therefore, for the first in this time, we decided to experience the in vitro effects of Ruta graveolens L. on human sperm and find out its mode of action on some sperm parameters as a probable new male contraceptive. 2. Materials and methods 2.1. Specimens Human ejaculates, obtained from male partners of those referring to Avesina Infertility Clinic for IVF and had been proved to be normal according to WHO criteria (1999). The part of semen

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which was going to be discarded, used for the experiments, and an institutional consent form was signed by each of the participants. Semen samples (n = 14) which were taken 72–96 h of sexual abstinence, were subjected to routine semen analysis following liquefaction at 37 ◦ C. Amounts of semen containing 106 cells were chosen for the experiments. 2.2. Test materials Dried RGL herb was purchased from Zard-Band herbal medicine factory. The plant was cultured in botanic farm of Zardband herbal drug company. The farm is in Zard-band village in north of Tehran with these geometrical characteristics: Latitude 35◦ 47 north, longitude 51◦ 37 east, altitude 1548 m from sea. In last 10 years, maximum temperature was 39 ◦ C, minimum temperature was −6.8 ◦ C and average humidity was 489.2 mm in this area. The plant was harvested before the seeds were completed (in June 2005) and dried in dark room with free ventilation. We bought the dried herb in August 2005 and kept it in dark and cold place in a sealed box for farther experiments. The plant was approved in the herbarium of Pharmacognosy Department of Pharmacy Faculty of Shahid Beheshti University of Medical Sciences by Mr. Mohammad Kamalinejad (Botanist) with herbarium number 660. Hams F-10 and bovine serum albumin were purchased from USB, staining dyes, and salts were obtained from Sigma. All materials and equipment were warmed to 37 ◦ C while using them. 2.3. Preparation of the plant extract Fifty grams of dried crushed herb was immersed in 500 ml of distilled water, put on stirrer for 48 h in room temperature and in a dark place, and then filtered through a paper filter. The filtrate was lyophilized by an Eyela lyophilizator (Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The brownish yellow powder was kept in a dark clean jar in room temperature for farther experiments. About 12 g of the lyophilized powder was obtained. 2.4. Immobilization assay Different amounts of the lyophilized powder (6.25, 12.5, 25, 50,100 and 200 mg) were dissolved in 1 ml of phosphate buffer saline (PBS). After filtration through a 0.2-␮m filter (Schleircher & Schuell FP 30/0.02 CA-S), the filtrate was added to a semen sample in a 1:1 volumic ratio (semen volume was in an amount to give 106 cells). In the control group, PBS was added to the same volume of semen. For the assessment of sperm motility, 10 ␮l of the mixture was immediately placed on a prewarmed slide and at least 10 fields were viewed and 200 sperms were counted by high power microscope (400× magnifications). Motility was assessed by using the WHO classification system (1999). The total motile sperms (a + b + c) were summed up. The lowest dose causing 100% immobilization in aliquots was called the minimum effective dose (MED).

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2.5. Extract stability evaluation To find out the effects of heat on denaturing the structure of effective RGL components, crude extract of plant were boiled for 2 h, then lyophilized and eventually an immobilization assay was performed with a MED. 2.6. Sperm revival test After incubating MED treated and the control spermatozoa for 2 h in RT, both were washed twice in a 10-fold volume PBS and incubated at 37 ◦ C for 30 min to observe the reversal of sperm motility and morphological changes. 2.7. Assessment of sperm viability and membrane integrity Sperm viability tests were done by using one step EosinNigrosin (EN) staining method (Bjorndahl et al., 2003), for MED treated samples and that of the control group. Briefly 25 ␮l of dye and the same amount of the test sample were mixed for 30 s. Then a smear was obtained by a 15 ␮l droplet of the mixture. After air drying the smear was viewed microscopically, under ×1000 magnification. The percentages of alive (white) and dead (red) cells were calculated by at least counting 200 cells. As the sperms with intact membrane do no undergo staining, this staining shows the integrity of cell membrane too (Juhasz et al., 2000).

Fig. 1. The effects of varying amounts of the aqueous extracts of Ruta graveolens L. on human sperm immobilization.

stain in the neck area of sperms was assumed as functional mitochondria. 2.10. Statistical analysis The gathered data were expressed as mean ± S.E. percentiles. Student’s t-test was employed for statistical comparisons, p < 0.05 was considered significant. 3. Results

2.8. Evaluation of DNA status

3.1. Sperm immobilization assays

In order to find out the action site of RGL aqueous extract on sperm cells, the effects of the minimum effective dose on DNA status and mitochondrial function were assessed. MED treated semen samples and the control group were stained by acridin orange dye as modified method of Tejada et al. (1984). Briefly, a smear was obtained from a 15 ␮l samples and then fixed in a 1:1 ethanol–acetone solution for 30 min at 4 ◦ C. After drying at RT, the slides were placed in a jar containing acridin orange dye for 5 min in RT. After washing slides with distilled water, they were immediately viewed under 1000× magnification using florescent microscopy in 470 nm florescent beam and 200 cells were counted. The red to yellow stained cells showed a denatured chromatin with single-stranded DNA and the green ones showed an intact chromatin with double-stranded DNA.

Different amounts of lyophilized RGL aqueous extracts were tested as described and immediate immobilizing effects of the material were seen in a dose-dependent manner (Fig. 1). All of the sperms become immotile in a 100 mg/ml of the extract and higher concentrations. This concentration was chosen as the minimum effective dose (MED) in other experiments. Effects of amounts lower than 25 mg/ml were not much different from that of the control group. The total motile sperm counts were significantly decreased in both 25 and 50 mg/ml extract-treated groups (Table 1).

2.9. Evaluation of mitochondrial function MED treated Semen samples and the control group were stained by Rhodamine 123 (Rh123) dye as the method used by Evanson (Evenson et al., 1982). Briefly, aliquots of 106 cells were incubated in PBS, containing 10 mg/ml Rh123 for 10 min in RT in a dark place. Then the samples were washed by PBS and immediately after, 10 ␮l of the specimen was put on a slide and viewed under 1000× magnification by florescent microscopy, in 470 nm florescent beam, and 200 cells were counted. Presence of the green florescent

3.2. Extract stability Hundred mg/ml of the boiled lyophilized extract of plant could immediately immobilize all sperm cells in a sample containing 106 cells in a 1:1 ratio. This indicates that the active components of RGL aqueous extract are thermostable and no chemical or physical changes occur in them. 3.3. Sperm revival test After removing the extract from the media, it was surprisingly seen that motility returned in 30.8 ± 3.2% of the sperms. It was noticed that sperm tails had a rapid whip-like movement. MEDtreated washed cells, showed morphological changes in the forms of tail coiling and bending as seen in Fig. 2 (40.8 ± 3.7% versus 14.1 ± 1.7% in the control group, p < 0.0001).

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Table 1 Total motile sperm count percentile in control and aqueous extracts of Ruta graveolens L. treated group Groups 200 mg/ml

100 mg/ml

50 mg/ml

25 mg/ml

12.5 mg/ml

6.25 mg/ml

Control

Total motile sperm count% (mean ± S.E.)

0

0

53.2 ± 4.7

65.5 ± 2.4

72.4 ± 3.8

70.6 ± 6.2

74.1 ± 3.3

p

0.000

0.000

0.001

0.048

0.73

0.63

–

t-Test was employed for comparison of treated groups with control.

Fig. 2. Morphological changes in the form of tail coiling (black arrows) and bending (dashed arrows) in sperm cells treated with Ruta graveolens L. aqueous extract (a) compared with the control group only receiving PBS solution (b).

3.4. Sperm viability and membrane integrity EN staining showed that although treated cells were totally immotile but there were no significant differences between viability of the two groups (Table 2). The results also showed that membrane integrity was intact after treating with RGL aqueous extract. 3.5. DNA status and mitochondrial function Acridin orange and Rhodamine123 staining did not show any differences between MED-treated and the control group chromatin denaturation and mitochondrial function (Table 2). 4. Discussion More than 50 plants had been known to be anti-fertility agents in men with anti-spermatogenetic or sperm immobilizing effects (Unny et al., 2003). Immobilization effect of these plants were due to sperm death, so motility revival test had negative results (Lohiya et al., 2000; Reyes et al., 2002; Chakrabarti et al., 2003; Khillare and Shrivastav, 2003).

In this study, RGL aqueous extracts’ immobilizing effects on human sperm cells were reported for the first time. The effect was dose dependent and in a concentration of 100 mg/ml of the lyophilized form, in a 1:1 volumic ratio, could immediately immobilize 106 sperm cells in a semen sample. Boiled aqueous extracts did the same too. This effect was without any impairment in cell viability, mitochondrial function and chromatin structure. After 2 h, sperm cells were still alive and some extent of motility returned after washing them. So it seems that immobilization of sperm cells is neither to cell death nor ATP depletion or chromatin damage. RGL is a medicinal plant which its roots and aerial parts contain more than 120 compounds of different classes of natural products such as acridone alkaloids, coumarines, essential oils, flavonoids, and furoquinolines (Kostova et al., 1999; Kuzovkinaa et al., 2004). This plant is the main source of furanocoumarins such as psoralen, xanthotoxin (8methoxypsoralen; 8-MOP) and bergapten (5-methoxypsoralen; 5-MOP) (Milesi et al., 2001). Neurophysiologic studies had shown that RGL has a potassium channel blocking effect in myelinated nerves (Bethge et al., 1991; Bohuslavizki et al., 1992). In further studies, it

Table 2 Sperm viability, chromatin status and mitochondrial function in treated and control group Sperm parameters

Percentage of viable sperm (mean ± S.E.) Percentage of denatured chromatin (mean ± S.E.) Percentage of functional mitochondria (mean ± S.E.)

Groups 100 mg/ml

Control

60.4 ± 4.2 28.7 ± 2,2 57.5 ± 2.9

71.2 ± 5.1 29.0 ± 2.8 54.2 ± 2.3

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was revealed that benzofurans, acridinons, psoralens and other coumarines present in this plant could block potassium currents in a time-dependent manner, producing the so-called K+ transients in intact myelinated nerve fibers. Among them, 5-MOP is a largely selective blocker of predominantly the axolemmal potassium channels (Bohuslavizki et al., 1994). Although it had been shown that 5-MOP and 8-MOP can block voltage K channels in rats T cell and lymphocytes (Strauss et al., 2000). Psoralen can also block opening of Kv1.5 channels cloned from human heart (Darszon et al., 1999; Eun et al., 2005). On the other hand, the most of physiological functions in human sperm cell such as motility, capacitation, acrosomal reaction, hyperactivation and volume regulation are mediated by ionic flux through the sperm membrane and also it had been established that sperm membrane has different kind of Ca2+ , K+ and Cl− channels (Darszon et al., 1999). Nowadays these channels are a target for non-hormonal male contraception (Barfield et al., 2005; Yeung et al., 2006). K+ channels are involved both in sperm motility and volume regulation (Darszon et al., 1999; Barfield et al., 2005) and potassium blocking agents could impair both of them (Yeung and Cooper, 2001). Volume regulation is one of the important physiological properties of each cell, especially in sperm cell; it has a key rule in fertilizing ability of spermatozoa (Yeung et al., 2006). When volume regulation is impaired in sperm cell, it swells to a certain extent (Yeung et al., 2002) and change its shape in order to avoid excessive stretching of the plasma membrane by flagellar coiling or angulation, even in an isoosmotic medium (Yeung et al., 2003; Yeung et al., 2006). In the present experiment, the sperm cells were immediately immotile and after washing, they were significantly underwent changes in volume as demonstrated by tail coiling. This means that volume regulation has also been impaired in them. Considering RGL’s potassium channel blocking effect and the results of the present study that exhibits both macerated and boiled aqueous extract of RGL could immobilize human sperm cell and impair their volume regulation without killing them, it can be concluded that may be coumarines which are exist in this plant, block sperm potassium channels and induce such effects. As the coumarines have a melting point more than 145 ◦ C (2001), so they remain intact after boiling the extract. In that case, RGL aqueous extract could be a natural channel blocker compound and be used as a sperm channel blocker. To establish this hypothesis, we have designed some more trials to perform in our laboratory and we have got some promising unpublished results. Besides, RGL is a potent antimicrobial and antifungal and anti-inflammatory agent (Ojala et al., 2000; Meepagala et al., 2005; Raghav et al., 2006). If this plant is used in vaginal contraception, it also may prevent some sexually transmitted disease. Considering RGL as a potent abortive agent (Conway and Slocumb, 1979; Gandhi et al., 1991; Gutierrez-Pajares et al., 2003; Unny et al., 2003; Maurya et al., 2004; de Freitas et al., 2005) and our preliminary data, it seems that Ruta graveolens L. can act as a novel contraception both in men and women. Its exact mechanism of action is not understood yet and more experiment shall be done to reveal its real effect as a contraceptive plant.

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