Sperm immobilization activity of Allium sativum L. and other plant extracts

Asian J Androl 2003 Jun; 5: 131-135             

Keywords: Allium sativum; plant extracts; sperm; immobilization; farm animals; ram; human; spermicidal agents; thermostability

Abstract

Aim: To identify possible spermicidal agents through screening a number of edible medicinal plants with antimicrobial activity. Methods: Initial screening was made on the basis of ram cauda epididymal sperm immobilization immediately after addition of extracts. The most potent extract was selected and was evaluated on both ram and human spermatozoa. To unravel its mode of action several sperm functional tests were carried out, namely viability of cells, hypo-osmotic swelling test for membrane integrity and assays of membrane-bound enzyme 5?nucleotidase and acrosomal marker enzyme acrosin. Results: The crude aqueous extract of the bulb of Allium sativum L. showed the most promising results by instant immobilization of the ram epididymal sperm at 0.25 g/mL and human ejaculated sperm at 0.5 g/mL. Sperm immobilizing effects were irreversible and the factor of the extract responsible for immobilization was thermostable up to 90 . On boiling at 100 for 10 minutes, this activity was markedly reduced. Moreover, this extract was able to cause aggregation of ram sperms into small clusters after 30 minutes of incubation at 37 . However this property was not found in human spermatozoa. More than 50 % reduction in sperm viability and hypo-osmotic swelling occurred in treated sperm as compared with the controls, indicating the possibility of plasma membrane disintegration which was further supported by the significant reduction in the activity of membrane bound 5?nucleotidase and acrosomal acrosin. Conclusion: The crude aqueous extract of A. sativum bulb possesses spermicidal activity in vitro.

1 Introduction

There are many Indian medicinal plants, which were reported to possess antifertility property; they acted either by preventing implantation or by suppressing spermatogenesis [1, 2]. The most potent spermicidal agent presently available in the market was a formulation of nonoxynol-9, however the product had been observed to cause inflammation and genital ulceration and thereby increased the risk of HIV-1 infection on repeated use [3, 4]. On the other hand, gossypol, a seed extract of Chinese cotton plant Gossypium herbaceum, was studied extensively but the programme was discontinued due to its side effects, mainly hypokalaemia [5]. Considering these problems, the present investigation had been carried out on screening a number of edible medicinal plants, namely Allium sativum (family Lilliaceae), Zingiber officinale (Zingiberaceae), Curcuma longa (Zingi-beraceae), Curcuma amada (Zingiberaceae), Allium cepa (Lilliaceae) and so forth at an aim to identify active extracts for the future development of herbal spermicidal agents.

2 Materials and methods

Ram testes were obtained from slaughterhouse in the local market and human ejaculates, from the Scientific Clinical Research Laboratory Pvt. Ltd. (Kolkata, India). Plant materials were collected from the local market. a-N-benzoyl-L-arginine ethyl ester (BAEE), HCl and adenosine 5-monophosphate sodium salt were purchased from Sigma (St.Louis, USA) and other chemicals, from E-merck (Germany).

For ram sample, the cauda portion of epididymes was isolated and minced in 0.9 % saline solution (pH 7.5) and filtered through a piece of cheese cloth to get sperm suspension. For human sample, ejaculates (n=10) from normal subjects after 72 h~96 h of sexual abstinence were subjected to routine semen analysis following liquefaction at 37 . Sperm count above 100 million/mL and viability above 60 % with normal morpho-logy, rapid and progressive motility was employed for the tests.

The fresh plant materials, Allium sativum bulb, Zingiber officinale stem, Curcuma longa stem, Curcuma amada stem and Allium cepa bulb, were homogenized separately with the help of a mortar in physiological saline (pH 7.4). Homogenates were centrifuged at 10,000 g at 4 for 30 minutes. The pellet was discarded and the supernatant was preserved at 4 for experimental purposes.

Crude extracts of the plants and either ram epididymal sperm suspension (100 million/mL~200 million/mL) or human ejaculate (100 million/mL~150 million/mL) were mixed thoroughly in 1:1 ratio according to a modified method of Waller [6]. A drop of the mixture was placed immediately on a slide and at least five fields were microscopically observed under high power (400) for assessment of sperm motility. The mixture was then incubated at 37 for 30 minutes and the above process was repeated.

The effective concentration that causes 50 % immobilization of highly motile cells (EC50) [7] was determined by different dilutions of garlic extract using physiological saline as the dilution medium. Sperm suspension and garlic extract were mixed in 1:1 ratio. The concentrations ranged from 0.25 g/mL to 0.8 mg/mL for ram epididymal sperm and 0.5 g/mL to 0.11 g/mL for human ejaculated sperm (Figure 2).

Different concentrations of Allium sativum (ranging from 0.25 g/mL to 0.8 mg/mL) were treated with ram sperm suspension in 1:1 ratio and kept at 37 C for 1 h. Then from the bottom of the microcentrifuge tube, one drop of the sedimented sperm was placed on a slide and the percent aggregation was examined microscopically under 400 magnification. Considering that the non-aggregated spermatozoa will remain in the supernatant, the latter was collected and the turbidity determined spectrophotometrically [8] at 545 nm. This experiment was not performed with human ejaculates, as human sperm did not show any sign of aggregation after incubation with the plant extract.

Crude extract was heated in water bath at different temperatures (30 , 50 , 70 , 90 and 100 ) and aliquot was taken, cooled and centrifuged. The supernatant and the pellet were kept separately. The supernatant was tested for its effect on the sperm motility and the pellet was similarly tested after resuspended in physiological saline. The crude extract was lyophilized and the residue resuspended in physiological saline. It was then centrifuged to discard undissolved matter, if any. The clear supernatant was again tested microscopically as described earlier and the loss of activity was re-corded.

After completion of the experiment, the spermatozoa were washed twice in physiological saline and incubated once again in the same medium free of plant extract at 37 for 30 minutes to observe the reversal of sperm motility.

Sperm viability and hypo-osmotic swelling (HOS) tests were done according to WHO [9], Eliasson & Treichl [10] and Jeyendran et al. [11] for assessing plasma membrane functional integrity. Ram sperm suspension (100 million/mL~200 million/mL) and human ejaculated sperm (100 million/mL~150 million/mL) were mixed separately with Allium sativum extract at the minimum effective concentration i.e., 0.25 g/mL and 0.5 g/mL, respectively at a ratio of 1:1 and incubated for 30 min at 37 . Similarly, sperm samples in saline served as the controls. For viability assessment one drop each of 1 % aqueous solution of eosin Y and of 10 % aqueous solution of nigrosin was placed in a microcentrifuge tube. A drop of well mixed sperm sample was added to it and mixed thoroughly. The mixture was dropped onto a glass slide and observed under 400 magnification. For HOS 0.1 mL of aliquot was taken from each of the treated and control sample, mixed thoroughly with 1mL of HOS medium (1.47 % fructose and 2.7 % sodium citrate at 1:1 ratio), incubated for 30 minutes at 37 and the curling tails were examined under phase contrast microscope using 100 magnification.

The activity of 5-nucleotidase was determined by measuring the rate of release of inorganic phosphate from adenosine 5-monophosphate according to Heppel and Hilmoe [12] with a minor modification in substrate concentration as standardized in our laboratory. After incubating the sperm suspension with the plant extract, the sperm pellet was collected by centrifugation at 3,000 g at 37 , washed twice in 0.9 % saline and then suspended in 0.1 mol/L Tris-HCl buffer (pH 8.5) with each reaction system containing (100~200) million sperma-tozoa. An aliquot of 0.1mL suspension of sperm was added to 0.9 mL of buffered substrate containing 3 mmol/L adenosine 5-monophosphate and 50 mmol/L MgCl2 dissolved in 0.1 mol/L Tris-HCl buffer. The tubes were incubated at 37 for 30 minutes and 0.5 mL 20 % TCA (0 ~4 ) was added to the mixture to stop the reaction. The mixture was then centrifuged at 10,000 g at 4 . The pellet was discarded and the supernatant was kept for phosphate estimation [13]. The activity of 5-nucleotidase was expressed in terms of g of phosphate released.(hour.10⁸ cells)^-1.

The most widely studied acrosomal enzyme is the acrosin that has been shown to be associated with acrosomes of all mammalian spermatozoa and the highest substrate specificity was obtained with BAEE. The pellets were extracted with 3 mmol/L HCl at pH 3 and the enzyme activity was measured according to Bhatta-charyya, et al. [14, 15] following the hydrolysis of 0.5 mmol/L BAEE dissolved in 0.05 mol/L Tris HCl buffer containing 0.05 mol/L CaCl₂ at pH 8. The activity of acrosin was expressed in terms of mIU.(min.10⁸ cells)^-1. One mIU activity means the amount of enzyme, which caused the hydrolysis of one nanomole of BAEE in one minute at 25 .

Data were expressed in meanSEM. Students t-test was employed for statistical comparison.

The crude extracts of all the five plants at 1 g/mL concentration level were able to immobilize the ram spermatozoa instantly. A. sativum crude extract was the most effective and a concentration of 0.5 g/mL was able to immobilize human spermatozoa instantly at 1:1 ratio.

Figure 1 showed the relative efficacy of the extracts at the minimum effective concentration of 0.25 g/mL and Figure 2, the EC₅₀ values of A. sativum extract with ram (0.01 g/mL) and human (0.16 g/mL) spermatozoa. The data on nonspecific aggregation of ram spermatozoa were not presented but it suggested that the higher the concentration of A. sativum extract, the greater the percent of sperm aggregation and the less the optical density of isolated sperm floating in the supernatant.

Figure 1: In vitro effect of aqueous extract of five medicinal plant parts on motility of goat cauda epedidymal spermatozoa at a concentration of 0.25 g/mL.

Figure 2: EC₅₀ of A. sativum extract with ram epididymal sperm (triangles) and human ejaculated sperm (circles).

The supernatant of A. sativum showed 100 % immobilization (both with ram and human sperm) on heating up to 90 , but its sperm immobilization activity was reduced to 60 % (ram epididymal sperm) or 70 % (human sperm) after boiling for 10 minutes at 100 . The pellet obtained from each fraction, when resuspended in physiological saline, did not show immobilization activity. Crude extract at room temperature (23 ~ 26 ) was able to aggregate ram spermatozoa, but on heating (50 ~100 ) none of the aliquots showed this property. After lyophilization of the crude extract, the sperm immobilization property remained unaltered, but the ram sperm aggregation property was lost.

None of the spermatozoa, once immobilized, recovered their motility following removal of plant extracts and 30 minutes incubation with physiological saline.

3.4 Serm membrane integrity

The significant decrease in sperm viability on treatment with the extract indicated the spermicidal property of A. sativum crude preparation. Ram as well as human spermatozoa showed typical morphological changes when subjected to hypo-osmotic shock. These changes were clearly visible by phase contrast microscopy. In our experiment, the controls showed the maximum amount of tail curling, while in A. sativum extract treated sperma-tozoa, tail curling was significantly reduced (P<0.01), indicating the impairment of functional integrity of the plasma membrane. From Table 1, it could be seen that the release of inorganic phosphate was the maximum in the controls and was significantly reduced in the treated group, indicating inactivation and/or maximum expulsion of the enzyme 5-nucleotidase following A. sativum extract treatment and thereby hampering the breakdown of the substrate by the enzyme which is known to be plasma membrane associated. A similar result was obtained with acrosin, indicating the vesiculation or perforation of membrane system.

Table 1. Effect of A. sativum extract in vitro (n=10). All values are statistically significant (P<0.01) in comparison to controls. Sp.Sus. = Sperm suspension; P.Ext. = Plant Extract.

4 Discussion

Farnsworth and Waller [16] have screened a large number of plants for spermicidal property and reported that the majority of plant-derived spermicides were triterpene saponins of several structural types, flavonoids and phenol compounds. The saponins of Cyclomen persicum, Primula vulgaris and Gypsophyla paniculata have been reported to cause almost instant immobilization of human spermatozoa within 20 seconds [17]. Carica papaya seed extract has also been shown to possess sperm immobilizing effect in human spermatozoa in vitro [18]. The purified fraction from the aqueous crude extract of Echeveria gibbiflora had sperm immobilizing activity as well as strong agglutinating property in guinea-pig spermatozoa [19].

The present paper here reported for the first time the sperm immobilization activity of the aqueous homogenate of five edible plant parts having antimicrobial activities [20]. A. sativum extract was shown to be the most active. It was indicated previously that allitridum, an active principle of A. sativum, showed spermicidal effect on rat and hamster spermatozoa [21]. The present study pointed out that at a concentration of 0.5 g/mL, A. sativum crude aqueous extract was able to immobilize human spermatozoa instantly. The extract of A. sativum contained certain sperm agglutination factor as it agglutinated the ram epididymal spermatozoa, however, the human sperm in the presence of seminal fluid were not agglutinated.

Most of plant spermicidal compounds act on the sperm surface, disrupting the plasma membrane [16]. The currently used active principle of vaginal spermicide, nonoxynol-9, acted in a similar manner. It produced disruption of lipids within the sperm membrane, par-ticularly, on the acrosome and mid-piece causing rapid loss of sperm motility [22, 23]. Inhibition of sperm specific enzymes, as acrosin and hyaluronidase, which play important roles in the fertilization process, by plant derivatives has also been reported [16].

In the present study the damage to the membrane architecture was evidenced by the significant reduction in sperm viability and tail curling and a marked decrease in the 5-nucleotidase and acrosin activities in the treated group. A property of the cell membrane was its ability to permit the transport of molecules selectively. This is not only essential for the maintenance of sperm motility, but also for the induction of the acrosome reaction and possibly other key events related to fertilization [11]. When exposed to hypo-osmotic conditions, water will enter the spermatozoon in an attempt to reach osmotic equilibrium, and as a result the sperm volume increased and plasma membrane bulges. This ability of spermatozoa implied intact membrane function. On incubation of motile sperm with A. sativum extract this general property of the plasma membrane was lost, and moreover, the plasma membrane marker enzyme 5?nucleotidase was also getting released, possibly due to disstabilization of plasma membrane. The loss of acrosin from the acrosomal structure also indicated the damage of at least the outer acrosomal membrane with plant extract treatment. What we have attempted to show here, however, was that aqueous extract of very commonly used edible bulbs of A. sativum possess a potent sperm immobilizing/ spermicidal factor which is thermostable up to 90 . The mode of action appeared to involve the disruption of membrane architecture leading to the release of membrane associated key molecules, thereby causing impairment of functional competence of the cells.

The work has been done under partial financial support of Indian Council of Medical Research, New Delhi and University of Calcutta. Authors are thankful to Dr. S. K. Datta (Scientific Clinical Research Laboratory Pvt.Ltd. Kolkata.) for supplying human samples.

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Correspondence to: Professor A. K. Bhattacharyya, Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Calcutta 700019, India.
Tel: +91-33-2466 1264, Fax: +91-33-2476 4419
E-mail: ashokbha@cal2.vsnl.net.in
Received 2003-03-12    Accepted 2003-05-09

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