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Spermicidal action of a protein isolated from ethanolic root extracts of Achyranthes aspera: an in vitro study.

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Keywords: Achyranthes aspera (Ap)

Nonoxynol 9

Sperm immobilization

Hypo osmotic swelling

Acrosome

In vitro

ABSTRACT

A previous study conducted in our department, showed that 50% ethanolic extract of the roots of Achyranthes aspera possess spermatotoxic effects. Preliminary studies also revealed that the active principle may be a protein. In this study a 58 kDa Achyranthes protein (Ap) was isolated from Achyranthes aspera using standard protocols and their effects on the rat sperm was studied in vitro in comparison with nonoxynol-9 (N-9). The sperm immobilization studies showed that about 150[micro]g of Ap was able to immobilize sperms completely within seconds at a lower concentration than N-9 (250[micro]g). The sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in the Ap-treated and N-9 treated groups in comparison to the control. In the Ap and N-9 treated groups the number of acrosome reacted cells were found to be high and it also caused agglutination of the sperms indicating the loss of intactness of the plasma membrane which was further supported by the significant reduction in the activity of membrane bound 5' nucleotidase and acrosin enzyme. Hence this study showed that the protein isolated from the roots of Achyranthes aspera possess spermicidal activity in vitro and can act as a spermicide similar to that of nonoxynold 9. Ap also possessed spermicidal activity against human sperms in vitro.

[c] 2011 Elsevier GmbH. All rights reserved.

Introduction

Achyranthes aspera belongs to the family of Amaranthaceae. The whole plant is used as estrogenic, antibacterial, anti inflammatory and diuretic. There are many reports on the antifertility action of Achyranthes aspera. The ethanolic and chloroform extracts of the roots of Achyranthes aspera possess anti implantational and estrogenic properties (Vasudev and Sharma 2007). Studies using the ethanolic leaf extracts also showed anti androgenic and aborti-ficant property (Workinesh et al. 2007). A composite extract of 50% ethanolic leaf extract of Achyranthes aspera and Stephania her-nandifolia possess contraceptive and spermicidal activity in male rats (Paul et al. 2006). There are hardly any reports on the active component from Achyranthes aspera which possess antifertility effects.

Earlier studies conducted in our department showed that 50% ethanolic extract of the root of Achyranthes aspera possess reversible spermatotoxic effects (Sandhyakumari et al. 2002). Preliminary studies also showed that a protein present in the ethanolic extract was responsible for the spermatotoxic effects. Hence the objective of this study was to isolate the protein from these extracts and to study its effects on sperm in vitro in comparison with nonoxynol-9, a known spermicide.

Nonoxynol-9 is the most common spermicide in the world market. Among the different nonoxynol derivatives designated as N1-N15, p-nonyl phenoxy polyethoxy ethanol (N-9) is reported to be the most potent spermicide (Shah et al. 2008)

Materials and methods

Preparation of the extract and isolation of the active protein

Roots of Achyranthes aspera (Voucher No. KUBH 5788) were collected from Thiruvananthapuram, India, in 2009, authenticated by Dr. Valsala Devi and kept in the herbarium of the Department of Botany, University of Kerala, Thiruvananthapuram, India. About 10 g of the shade-dried root was r efluxed in 100 ml of 50% ethanol at 65 C for 90min, then filtered and evaporated to dryness at 65 C. The yield of the extract was 5g/100g dry root. About 1 g of the extract was dissolved in 25 ml saline, stirred, centrifuged and the supernatant was dialyzed against double-distilled water. Dialyzed sample was evaporated to dryness at 50 C. The sample was then dissolved in potassium phosphate buffer (pH 7.0) and diethyl amino ethyl cellulose (DEAE) ion exchange chromatography (Plummer 1988) was carried out. Gradient elution was carried out using 0.1 M potassium phosphate buffer (pH 7.0) containing KCl (50-200 mM) and each fraction was tested for sperm immobilization activity. The 200 mM fraction which showed maximum sperm immobilization activity was pooled, dialyzed against double-distilled water to remove salt and concentrated. It was further subjected to gel filtration (Sephadex G100) using phosphate-buffered saline. The fractions showing sperm immobilizing activity were pooled, concentrated, dialyzed against double-distilled water and subjected to 20% native polyacrylamide gel electrophoresis (native PAGE) (Reisfield et al. 1962). A protein of molecular weight 58 kDa (Fig. 1) showed sperm immobilization activity. This protein has been named Ap. This active protein was electro-eluted, dialyzed against distilled water, concentrated and used for the studies. The molecular weight was quantified by Bio-Rad Gel Doc (California, USA) using Quantity One Imaging Software. The N-terminal ten aminoacids of the 58 kDa protein was also sequenced in Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram.

[FIGURE 1 OMITTED]

Semen preparation

Semen was collected from cauda epididymis of healthy male rats and was diluted with normal saline and kept at 37 C Sperm count above 100 millions/ml and viability above 60% with normal morphology, rapid and progressive motility were used for in vitro analysis (WHO manual 1992). Semen samples from healthy fertile men with the above properties were also used for in vitro analysis studies.

Test samples

150[micro]g/m1 of Ap and 250[micro]g/ml of nonoxynol-9 were used as the test samples for in vitro analysis studies. Sperm suspension in saline served as the control. The dose of N-9 was taken according to the reference of Xu et al. (2005).

Experimental procedure

Sperm immobilization assay

Sperm immobilization assay was done by treating the diluted semen sample with isolated protein Ap at different concentrations ranging from 50 to 250[micro]g/ml. The sample was added to the diluted semen (1:1) and the time taken for immobilization was recorded using a phase contrast microscope.

Sperm revival test

The sperms were first immobilized separately with the isolated protein (150[micro]g/m1 of Ap) in vitro, then the sperms were washed twice with physiological saline and incubated in the same medium free of protein sample at 37[degrees]C for 30 min to observe the revival of sperm motility, if any. Immotile sperms showing vibratory movement to progressive motility after incubation were considered revived.

Assessment of plasma membrane integrity

Sperm viability and hypo osmotic swelling (HOS) tests were done according to WHO manual (1992) for assessing plasma membrane functional integrity. Sperm suspension (100 million/ml) was mixed with the samples separately (150[micro]g/mlofAp and 250[micro]g/ml of N-9) for in vitro analysis. Similarly sperm suspension in saline served as the control.

Assay of acrosin

Double staining method using Bismarck brown and Rose Bengal was done for the assessment of acrosome (Zenevald et al. 1991). Acrosin, the most widely used acrosomal enzyme associated with the acrosome of all mammalian spermatozoa was assessed by the method of Bhattacharya and Zeneveld (1978), Bhattacharya et al. (1979).

Agglutination reaction

An aliquot of sperm suspension was mixed with 150[micro]g/mlofAp and 250[micro]g/ml of N-9 at room temperature and incubated at 37[degrees]C for 15 min. A drop of the mixture was placed on the microscopic slide and was examined microscopically (Namjuntra et al. 1985). Sperm suspension in saline served as the control.

Assay of 5' nucleotidase

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 (1964).

Toxicity evaluation of Ap in vitro

Blood was collected from rats in heparinised tubes and was diluted three times with isotonic saline and centrifuged at 1000 rpm for 5 min.The pelleted RBC's were washed five times with saline and diluted to about 2 x [10.sup.5] cells/ml. 10[micro]1 of Ap was added to 50[micro]1of the RBC preparation and observed under the microscope for hemolysis if any.

Effect of temperature on the spermicidal action of Ap

The isolated protein Ap was heated in a water bath for 20 min at different temperatures (30[degrees]C, 40[degrees]C, 55[degrees]C, 70[degrees]C 85[degrees]C, and 100[degrees]C) and their action on spermicidal action in vitro was assayed.

Statistical analysis

The results were analyzed using a statistical programme SPSS/PC+, Version 5.0 (SPSS Inc., Chicago, IL, USA). A one way ANOVA was employed for comparison among the groups. Duncan's post hoc multiple comparison tests of significant differences among groups were determined, p < 0.05 was considered to be significant.

Results

Isolation of active protein (Ap)

The results of native polyacrylamide gel electrophoresis (native PAGE) analysis revealed the presence of a 58kDa protein (Fig. 1). This protein was electro-eluted and the in vitro sperm immobilization was checked. The results showed that the active protein caused the complete immobilization of sperms within one minute. This native protein was named as Ap. The N terminal 10 aminocids of Ap (58 kDa) is Glycine, Asparagine, Arginine, Serine, Glycine, Serine, Alanine, Aspartic acid, Aspartic acid. Alanine.

Sperm immobilization

The purified 58 kDa protein from Achyranthes aspera caused the immediate immobilisation of sperms as compared to that of control in a dose dependent manner. (Fig. 2). 25[micro]g/ml of the protein took 2 min for the complete immobilization of sperms. As the concentration of the protein increased there was a considerable decrease in the time taken for in vitro immobilization of sperms. "150[micro]g/ml of Ap was able to immobilize the sperms completely within 30 s. The minimum effective concentration of nonoxynol which caused complete immobilization of sperm was 250[micro]g/ml at 30 s (Xu et al. 2005).

[FIGURE 2 OMITTED]

Sperm revival test

None of the spermatozoa, once immobilized, recovered their motility following the removal of the protein and re-suspending in physiological saline followed by incubation at 37[degrees]C for 30 min. Similar results were observed in the case of nonoxynol 9 also.

Sperm viability and membrane integrity

The viable sperms appear to be unstained while the non-viable sperms are stained in pink on eosin-nigrosin staining. There was a significant increase in the number of non-viable sperm in the treated groups in comparison to the control group. Thus there was a significant decrease in sperm viability in the Ap and N-9 treated groups when compared to the control group (Fig. 3 and Table 1). N-9 group showed maximum number of non-viable sperms. When subjected to hypo-osmotic test morphological changes were reduced in treated groups indicating the impairment of the functional integrity of plasma membrane. In our experiment the control showed a higher percentage of tail curling, whereas in Ap and N-9 treated groups tail curling was significantly reduced (Fig. 4 and Table 1).
Table 1

Effects of Ap and N-9 on rat sperms in vitro.

Experiments Control Ap N-9

Viability (%) 78 [+ or -] 17.55 [+ or -] 12.30 [+ or -]
 7.48 1.68 (a) 1.12 (a)

HOS (%) 76.0 =b 7.25 36.08 [+ or -] 27.3 [+ or -]
 3.47 (a) 2.62 (a)

5"-Nucleotidase [[micro]g 6.42 [+ or -] 2.99 [+ or -] 2.97 [+ or -]
Pi released [(h 1.24 0.98 (a) 0.93 (a)
[10.sup.8]
cells)].sup.-1]

Acrosin[mIU[(h 239.85 [+ or -] 154.05 [+ or -] 146.25 [+ or -]
[10.sup.8] 23.01 14.78 (a) 14.03 (a)
cells).sub.-1]]

Acrosome reaction (%) 30.75 [+ or -] 60.12 [+ or -] 62.40 [+ or -]
 2.81 6.08 (a) 5.99 (a)

Agglutination reaction 12.25 [+ or -] 66.33 [+ or -] 71.27 [+ or -]
(%) 1.12 6 3(a) 6.85 (a)

Values are expressed as mean[+ or -]SD of 6 experiments. (a) p<0.05
between control and treated groups.


[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Assessment of agglutination and acrosome reaction

The number of acrosome reacted cells were found to be very high in both the treated groups as compared to control (Table 1 and Fig. 5). The protein of the plant extract agglutinated and immobilized sperms significantly at higher percentage in comparison to the control. Head to head agglutination was prominent in both groups (Fig. 6).

[FIGURE 5 OMITTED]

Assays of 5' nucleotidase and acrosin

The release of inorganic phosphate was maximum in the control and was significantly reduced in Ap and N-9 treated groups. Similar results were obtained with acrosin.

Toxicity evaluation ofAp in vitro

All the RBC's were found to be intact even after 30 min indicating that this protein is without any haemolytic activity.

Effect of temperature on the spermicidal action of Ap

The result showed that Ap was thermostable upto 70 C (Table 2). The results also showed that spermicidal activity of Ap was lost at temperatures above 70[degrees]C.
Table 2

Effect of temperature on the spermicidal action of Ap.

Time intervals 0' 5' 10' 15' 20' 25' 30'
(min)
 Percentage of moiility at
 different time intervals (%)

Control 100% 90% 80% 60% 40% 20% 5%
Ap incubated at

30[degrees]C 50% 30% 10% 5% 0% 0% 0%
40[degrees]C 50% 30% 20% 10% 5% 0% 0%
55[degrees]C 50% 30% 20% 10% 5% 0% 0%
70[degrees]C 50% 35% 20% 10% 5% 0% 0%
85[degrees]C 80% 60% 40% 30% 20% 10% 5%
100[degrees]C 90% 75% 60% 40% 20% 10% 5%

Values are expressed as mean [+ or -] SD of 6 animals


In vitro effect of Ap on human sperms in vitro

Ap took 40 s for the complete immobilization of sperms in comparison to the control group (Table 3) where as nonoxynol took only 20 s for the complete immobilization of sperms.
Table 3

Effects of Ap and N-9 on human sperms in vitro.

Groups Time taken for the complete immobilization of sperms

Control semen 30[+ or -]2.8min

Ap treated 41 [+ or -]X74(a)s
semen

N-9 treated 20 [+ or -] 1.4(a) s
semen

Values are expressed as mean [+ or -] SD of 6 experiments.
(a) p<0.05 between control and treated groups.


Discussion

There is a growing interest in the search for spermicides of natural origin which can be used to coat condoms and can also be developed as gels. There are many reports on the sperm immobilization action of active compounds isolated from many plant extracts. There are also reports that the peptides from natural origin also have sperm immobilization activity. Nisin, a naturally occurring antimicrobial cationic peptide produced from Lactococcus lactis (Reddy et al. 2004), subtilosin, a cyclopeptide from Bacillus subtilis (Sutyak et al. 2008) and magainins, a class of peptides from the skin of African clawed frog, Xenopus laevis (Edelstein et al. 1991) caused complete immobilization of human spermatozoa. The mechanism of action of many of the sperm immobilization compounds of plant origin seems to be by surface action, disrupting the plasma membrane of the spermatozoa (Bhattacharya et al. 1979).

In this study we have isolated a protein of Ap 58kDa from the 50% ethanolic extracts of the roots of Achyranthes aspera. The isolated protein was found to have significant spermicidal action against human and rat sperm. Hence in this study our aim was to compare the action of our protein Ap with a known spermicide available in the market, N-9. Since all the small ions have been removed by dialysis and the protein has been electro eluted, the observed spermicidal activity is due to the protein only. The sperm immobilizing effect was dose dependent. There was a progressive increase in sperm immobilization rate with the increased concentration of protein sample. The sperm revival test showed that the effect was spermicidal and not spermiostatic as there was no revival of motility after incubation with the physiological saline medium free of proteins. Similar results were observed in the case of N-9 also. But the minimum effective concentration at which the Ap caused complete immobilization of sperm was much lower than that of N-9.

[FIGURE 6 OMITTED]

In our study damage to membrane integrity was evaluated by the reduction in sperm viability and tail curling in the Ap and nonoxynol treated groups. A functional plasma membrane is important for maintaining homeostasis, sperm motility, capacita-tion, acrosome reaction and other events related to fertilization (Jayendran et al. 1984). HOS test measures the ability of sperm membrane to transport fluid selectively, thereby providing an indication of functional and physical integrity. On incubation with the Ap and N-9 treated samples this intactness was lost. Similar results were also observed in the study of the composite extract of the leaf of Stephania hernandifolia and the root of Achyranthes aspera (Paul et al. 2006).

The number of acrosome reacted cells were increased in Ap and N-9 treated groups. The acrosome of mammalian spermatozoa contains various hydrolytic enzymes which are required for the process of fertilization and is directly involved in sperm-egg fusion. Treatment with the protein caused the breakage of outer acrosomal membrane. Ap and N-9 contains certain sperm agglutination factors which agglutinates rat spermatozoa. In both cases head to head agglutination was prominent. This is in agreement with the studies on the purified fraction from the aqueous crude extract of Echeveria gibbiflora in guinea-pig spermatozoa (Delgado etal. 1999).

There are hardly any reports on the sperm immobilization activity of the proteins except for few peptides. Nisin, a 34 amino acid cationic peptide produced by Loctococcus lactis caused sperm immobilization of human spermatozoa at a dose of 300-400[micro]g (Reddy et al. 2004). The spermicide nonoxynol also caused complete immobilization of sperms at a dose of 250[micro]g/ml (Xu et al. 2005). The dose at which Ap possess complete sperm immobilization action is much lower (150[micro]g/ml) than N-9. Toxicity studies on the hemolysis of RBC showed that the protein is non-toxic in nature and protein is also thermostable upto 70[degrees]C.

One of the most promising actions of Ap is its quick, complete, irreversible immobilization of sperms at very low concentrations. The mechanism of action seems to be by disrupting the plasma membrane integrity as they caused drastic inhibition on sperm membrane specific enzymes like acrosin and 5' nuleotidase. Hence in short the protein isolated from 50% ethanolic extract of the root of Achyranthes aspera (Ap) possess spermicidal activity in vitro similar to nonoxynol-9, a potent spermicide.

Conclusion

The protein isolated from the alcoholic extract of the root of Achyranthes aspera (Ap) possess spermicidal activity in vitro and, the mechanism of action seems to be by acting on the membrane integrity.

Acknowledgement

The financial support from Kerala State Council for Science Technology and Environment (KSCSTE) is gratefully acknowledged.

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doi:10.1016/j.phymed.2011.01.007

M.M. Anuja, R.S. Nithya, S.S. Swathy, C Rajamanickam, M. Indira *

* Corresponding author. Tel.: +9I 471 2308078; fax: +91 471 2308078, E-mail address: indiramadambath@gmail.com (M. Indira).

Department of Biochemistry. University of Kerala, Kariavartom, Thiruvananthapurarn, Kerala 695 581, India
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Author:Anuja, M.M.; Nithya, R.S.; Swathy, S.S.; Rajamanickam, C.; Indira, M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jun 15, 2011
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