Archives of Medical Research 39 (2008) 631e638
ORIGINAL ARTICLE
Spermicidal Activity of Bivittoside D from Bohadschia vitiensis Vijai Lakshmi,a Ajet Saxena,a Sunil Kumar Mishra,a Ram Raghubir,b Mahendra N. Srivastava,c Rajeev K. Jain,d Jagdamba P. Maikhuri,d and Gopal Guptad a
Medicinal and Process Chemistry, bPharmacology, cBotany, dEndocrinology Division, Central Drug Research Institute, Lucknow, India Received for publication April 21, 2008; accepted June 19, 2008 (ARCMED-D-08-00175).
Background. A recent revelation about increased susceptibility to HIV by use of nonoxynol-9 (N-9) has called for identification of novel molecules with potent spermattenuating activity and lower side-effect profile, as suitable alternatives. The present study was designed to investigate spermicidal activity in Bohadschia vitiensis whole-body extracts followed by isolation and characterization of bioactive molecule. Methods. Bohadschia vitiensis (Semper) was collected from the Southern Andaman coast of India. Freshly collected marine animals were extracted with methanol. A portion of the crude extract was fractionated into four fractions by macerating with hexane, chloroform, and n-butanol successively. All fractions were evaluated for spermicidal activity. Because maximum activity was localized in the n-butanol soluble fraction, it was chromatographed over a silica gel column, and elution with chloroform-methanol-water (35:10:2, v/v) yielded the major compound bivittoside D (400 mg). Bivittoside D [molecular weight (MW) 1426] is a lanostane triterpenoid with six monosaccharide units. The structure of the compound was established on the basis of physicochemical data, acid hydrolysis of saponin, identification of sugar units and aglycon, melting point, and by comparison with data reported in the literature. Results. The aqueous methanol extract of the Bohadschia vitiensis caused 100% mortality of human sperm at 0.01% concentration in vitro, whereas N-9 (reference control) exhibited an equivalent activity at 0.05%. On further fractionation, activity was localized in n-butanol soluble fraction from which the major compound purified was a lanostane triterpenoid called bivittoside D. Bivittoside D was found to be a more potent spermicide (|2.3 times) than N-9 and killed 100% human sperm at the concentration of 350 mM in |20 sec in vitro. Supravital staining and hypoosmotic swelling test revealed sperm membrane permeabilization by bivittoside D as the major mode of spermicidal action. However, bivittoside D was much safer than N-9 towards normal vaginal flora (Lactobacillus) in vitro, although it affected the viability of HeLa cells like other surfactants. Conclusion. Bivittoside D from B. vitiensis can adequately replace N-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly. Ó 2008 IMSS. Published by Elsevier Inc. Key Words: Bohadschia vitiensis, Holothurideae, Bivittoside D, Spermicidal, Saponins.
Introduction With a view to explore the possibilities of finding new molecules with proven therapeutic efficacy for human use,
Address reprint requests to: Dr. Vijai Lakshmi, Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226 001, India; E-mail:
[email protected]
a program is operational at the Central Drug Research Institute (Lucknow, India) for screening of extracts of marine organisms for a wide range of biological activities. The program consists of collection, identification and extraction of marine flora and fauna along the Indian coasts for biological screening. Bohadschia vitiensis was selected for detailed chemical and biological investigations with a view to isolate bioactive compounds (if any).
0188-4409/08 $esee front matter. Copyright Ó 2008 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.06.007
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Lakshmi et al./ Archives of Medical Research 39 (2008) 631e638
B. vitiensis (Semper) belongs to the family Holothurideae. Sea cucumbers are utilized as therapeutic agents as well as for food in the Malaysia peninsula. The body of B. vitiensis is cylindrical in shape with an average length of 30 32 cm, thickness of |7 mm and an average weight of |1.2 kg. The upper side is slightly wrinkled and brown to brown/black in color. The underside is grainy and brown. It is distributed in the South Pacific and the Indian Ocean at depths of 0e20 m in the coastal lagoons and inner reef flats, often burrowed in the sandy muddy bottoms. Several species of Bohadschia such as B. argus, B. bivittata, B. marmorata, B. graeffii, B. tenuissima, and B. vitiensis have been chemically investigated. Sea cucumbers possess a peculiar specialized defense system that is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments called Cuvierian tubules. The adhesiveness combined with high tensile strength allows Cuvierian tubules to entangle and immobilize potential predators. The Cuvierian tubule of the organism is particularly rich in holothurid saponins. A literature search revealed that few compounds have been reported from B. vitiensis. Two saponins, bivittoside C and bivittoside D (1) and four triterpenoid sapogenins (2) have been isolated from this species. The high rates of unintended pregnancies and STDs (including HIV) call for the development of novel strategies to help individuals avoid these risks (3). Contraceptive microbicides are intended to interact with sperm and pathogens in the vagina to irreversibly inactivate them. Surface active molecules, acid buffers, receptor blockers, antimicrobial substances, enzyme inhibitors, etc. are currently under development for dual protection. Although functionally different, the basic plasma membrane structure of sperm and of lymphocytes and monocytes (from which HIV takes its envelope) is highly susceptible to the disruptive action of surface-active molecules (3). Nonoxynol-9 (N-9), the active ingredient used in the majority of over-the-counter local contraceptives, has been evaluated as a dually active contraceptive microbicide. However, in spite of potent microbicidal activity in vitro, N-9 not only failed to protect against HIV but also increased the incidence of this infection in users (4,5). This anomaly was largely attributed to the strong detergent nature of N-9 that caused pro-inflammatory reaction in the vagina resulting in recruitment of HIV host (immune) cells to the site of viral entry and increased susceptibility to HIV. On the other hand, saponins are weaker surfactants that partially permeabilize the cell membrane but are not classified as detergents (6), although some are equipotent to N-9 in killing human sperm (7,8). Lipid rafts are cholesterol-rich cell membrane microdomains that play an important role in functional survival of both sperm and HIV (3). Saponins are known to form complexes with cholesterol and are therefore potentially capable of inactivating both sperm and HIV. Some spermicidal phytosaponins have shown promising dual activity (9). It
was thus decided to evaluate the spermicidal activity of the saponin fraction of B. vitiensis in comparison to N-9 and conduct some preliminary safety studies.
Materials and Methods Collection of Marine Animals B. vitiensis (Semper) was collected from the Southern Andaman coast of India at 2e3 m depths in the month of March. A specimen sample has been preserved in the herbarium of the Botany Division of the Institute as specimen number 326. Extraction, Fractionation, and Isolation of Pure Compounds The freshly collected marine animals (2.0 kg) were thoroughly washed with distilled water to remove extraneous materials and then filled in steel containers and soaked in methanol. This was then transported to the laboratory of Central Drug Research Institute, Lucknow, India. The methanol extract was drained off and the animals were chopped into small pieces and filled in a glass percolator soaked in 80% aqueous methanol and left for 24 h at room temperature. The process of extraction was repeated four times and the combined percolates were concentrated under reduced pressure !50 C to a green viscous mass. It was further dried under high vacuum to remove last traces of solvent (weight 60.0 g). A portion of the crude extract (25.0 g) was fractionated into four fractions by macerating with hexane, chloroform, and n-butanol successively. The hexane soluble (0.25 g), chloroform soluble (1.25 g), n-butanol soluble (2.25 g), and n-butanol insoluble (20.0 g) fractions were evaluated for spermicidal activity. Because maximum activity was localized in the n-butanol soluble fraction, it was chromatographed over a silica gel column, and elution with chloroform-methanol-water (35:10:2, v/v) yielded the major compound bivittoside D (400 mg). Bivittoside D (MW 1426) is a lanostane triterpenoid with six monosaccharide units. The structure of the compound was established on the basis of physicochemical data, acid hydrolysis of saponin, identification of sugar units and aglycon, melting point, and by comparison with those given in the literature (10). The structure of bivittoside D is given in Figure 1. Biological Evaluations Human Semen Samples. Fresh human semen samples obtained by masturbation into a sterile vial from healthy, young, fertile donors were liquefied for 45 min at 37 C and used for in vitro spermicidal and mechanism of action assays. The semen characteristics, viz., volume, pH, viscosity and morphology, were determined according to World Health Organization guidelines (11). Sperm count and
Spermicidal Activity of Bivittoside D
HO
O
O
633
O
O
OH CH 2OH CH 2OH O OCH 3 OH
H OH
CH 2OH O OCH 3 OH
O OH
H
O
CH 3 H OH
O OH
CH 2OH O
O
H OH
O OH
H
O
O
H OH
OH
Figure 1. Bivittoside D (MW 1426).
motility analysis were also performed in a CASA system (HTM-IVOS; Hamilton Thorn Research, Beverly, MA) using a small drop of liquefied semen placed on a ‘‘Makler’’ counting chamber (Sefy´ Medica, Haifa, Israel) prewarmed to 37 C. Semen samples with normal sperm count (O60 million/mL), motility (O75% motile), sperm morphology (O70% normal), pH (7.4e8.0), viscosity, and volume were used. Ethical approval for this study was obtained from the Institute’s ethics committee. Minimum Effective Concentration (MEC) Determination The test compounds were dissolved in physiological saline to make a 1.0% (10 mg/mL) solution and diluted serially up to 0.01%. A spermicidal test was performed with each dilution starting from 1.0% until the MEC was arrived at, following the modified method of Sander and Cramer (12). Briefly, 0.05 mL of human semen was added to 0.25 mL of spermicidal compound solution and vortexed for 10 sec. A drop was immediately placed on a microscope slide, covered with a cover glass and examined under a phase-contrast microscope. The result was scored positive if 100% spermatozoa became completely immotile within 20 sec. The weakest dilution that completely immobilized all the spermatozoa in 20 sec was recorded as MEC in gram% (w/v). MEC was determined in three individual semen samples from different donors. Eosin Staining & HOST (Mechanism of Action) Supravital staining with eosin and the hypoosmotic swelling test (HOST) were used to assess the effect of spermi-
cides on sperm/membrane permeability (7). Then, 0.2 mL liquefied semen was treated with 1.0 mL of spermicide solution at the MEC (1.0 mL of buffer in control tubes) and incubated for 1 min at 37 C. Spermatozoa were pelleted (500 g for 5 min) and 0.1% eosin solution was added to the pellet, mixed and incubated for 15 min at 37 C. The numbers of stained and unstained spermatozoa were counted under a light microscope. The HOST of Jeyendran et al. (13) was used to determine the effect on the physiological integrity of the sperm membrane. Human spermatozoa treated with spermicide solution (as in the supravital staining experiment) were pelleted, treated with hypoosmotic solution and mixed gently. The suspension was incubated for 30 min at 37 C. A wet mount was prepared for each compound solution and observed under a phasecontrast microscope, and spermatozoa with and without tail curling were counted and recorded.
Safety Towards Normal Vaginal Flora (Lactobacillus) Safety of the compound towards Lactobacillus was determined by following the method of Ojha et al. (14). Briefly, Rogosa SL agar (7.5%; containing 0.132% acetic acid) plates were prepared with (experimental) or without (control) the addition of spermicidal agent at a final concentration of 0.05% and 0.1% (1 MEC & 2 MEC, respectively). N-9 and Sapindus saponins were used as reference controls. Plates were inoculated with L. acidophilus (|70 spores/10 cm2) and incubated at 37 C in 5% CO2 and 95% air for 72 h. Control plates were inoculated simultaneously and incubated similarly. Number and size of colonies were recorded
Lakshmi et al./ Archives of Medical Research 39 (2008) 631e638
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at the end of the experiment. The average colony size (% of control) was multiplied by the colony number and divided by 100 to arrive at the data presented. The average colony size of control was taken as 100%. N-9 and Sapindus saponins were used as reference controls.
Cytotoxicity Towards Human Cervical (HeLa) Cell Line The MTT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide) based colorimetric assay for evaluation of cytotoxicity of bivittoside D against human cervical (HeLa) cell line (NCCS, Pune, India) was adopted. Cells seeded at a density of 2.5 105 per well in 96-well plate were incubated in culture medium (DMEM) for 24 h at 37 C in 5% CO2 /95% air atmosphere. After 24-h, culture medium was replaced with fresh medium containing dilution of spermicidal compound (5 MECe1 MEC). Positive control wells contained an equal concentration of N-9 or Sapindus saponins, whereas control wells contained saline (0.85% NaCl). After a 3-h incubation, 10 mL of MTT solution (5 mg/mL) was added to each well. Formazan crystals formed inside the viable cells were solubilized in DMSO and the OD was read at 540 nm in a microplate reader (Microquant; Bio-Tek, Winooski, VT).
Sperm Motility Parameters Effect of bivittoside D on human sperm motility parameters [%Mot 5 percent motile; rapid 5 percent sperm with velocityOVAP; VAP 5 average path velocity (m/s); ALH 5 amplitude of lateral head displacement (m); BCF 5 beat cross frequency (Hz)] was assessed at ED50 concentration in vitro using Computer Assisted Sperm Analyzer (CASA) system, as detailed earlier (7).
Table 1. Spermicidal activity of bivittoside D against human spermatozoa in vitro
Compound
MW
Solubility
MEC (%, w/v)
MEC (mM)
B. vitiensis methanolic extract Bivittoside D Nonoxynol-9 Sapindus saponins
1426 616
Aqueous Aqueous Aqueous Aqueous
0.01 0.05 0.05 0.05
350 811
MW, molecular weight; MEC, mimimum effective concentration.
Results Spermicidal Potential Bivittoside D, N-9, and Sapindus saponins exhibited a minimum effective spermicidal concentration (MEC or ED100) of 0.05% that irreversibly immobilized 100% human sperm in |20 sec in vitro. However, because bivittoside D has a MW of 1426, its molar concentration was |2.3 times lower than that of N-9 (MW 616). Hence, this natural compound was more than twice as potent as N-9 in killing human sperm. Because Sapindus saponins were a mixture of compounds, its molar concentration could not be calculated. Table 1 shows the spermicidal MEC of bivittoside D, N-9 and Sapindus saponins that killed 100% human sperm in Sander Cramer Assay in all the semen samples (minimum 3) from different donors. Safety Towards Lactobacillus Acidophilus In Vitro Bivittoside D was found to be significantly safer than N-9 towards normal vaginal flora (Lactobacillus). The compound also had a slight edge over Sapindus saponins in its safety towards the normal vaginal flora (Figures 2 and 3).
120
Lactobacillus Colonies
100
80
a
a 60
b
40
c
20
c 0 Cont
N-9(0.05%)
N-9(0.1%)
Sapindus (0.05%)
Sapindus (0.1%)
B-vit(0.05%) B-vit(0.1%)
Treatments Figure 2. Safety of spermicidal compounds towards normal vaginal flora (Lactobacillus acidophilus) in vitro (a 5 p !0.05; b 5 p !0.01; c 5 p !0.001).
Spermicidal Activity of Bivittoside D
635
Figure 3. In vitro safety of bivittoside D towards Lactobacillus acidophilus in comparison to nonoxynol-9 [A 5 control; B 5 bivittoside D (0.1%); C 5 bivittoside D (0.05%); D 5 nonoxynol-9 (0.05%)].
HOST & Eosin Staining
Effect on Motility Parameters
The hypoosmotic swelling test and supravital staining with eosin (Figure 4) reveals that the spermicidal compound bivittoside D exerts a surfactant type of effect on sperm plasma membrane, which is very similar to that exerted by Sapindus saponins and N-9. Bivittoside D disrupted the structural integrity of sperm membrane (as revealed by supravital staining with eosin) in |90% cells as compared to only 15% disrupted cells in untreated control. Similarly, the physiological integrity (as revealed by HOST) was damaged in O80% sperm by bivittoside D treatment as compared to only 23% in vehicle control. The physiological damage of sperm membrane was highest in the case of N-9.
Bivittoside D at ED50 concentration significantly reduced percentage of rapidly motile sperm (Rapid) and their average path velocity (VAP). Amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were also affected marginally. All motility parameters were similarly affected by Sapindus saponins and bivittoside D at ED50 concentration (Figure 6).
Cytotoxicity Towards HeLa Cells In Vitro Cytotoxicity of bivittoside D towards human cervical (HeLa) cell line in vitro is shown in Figure 5. The compound was marginally more cytotoxic at lower doses (1 & 2 MEC) and less cytotoxic at higher doses (3, 4 & 5 MEC) than N-9 and Sapindus saponins. Overall cytotoxicity was in the same range as that of N-9 and Sapindus saponins (Figure 4).
Discussion The vaginal mucosa of healthy women of childbearing age is populated by commensal bacteria typically dominated by Lactobacillus species, which plays a protective role in preventing urogenital infections (15). Depletion of vaginal lactobacilli is associated with establishment of opportunistic infections and increased risk of acquiring HIV and herpes simplex virus type 2 in women (16,17). The principal Lactobacillus species isolated from the vaginal mucosa of healthy women include L. acidophilus and was therefore
120
HOST Dye-Exclusion
100
c
Percent
80
60
40 c
20
0 Control
N-9
Sapindus
B-vit
Treatments
Figure 4. Percent human sperm with hypoosmotic swelling and eosin staining after treatment with the spermicidal agents (c 5 p !0.001 vs. control).
Lakshmi et al./ Archives of Medical Research 39 (2008) 631e638
636 0.7
0MEC MEC 2MEC 3MEC 4MEC 5MEC
Absorbance at 540nm
0.6
b
0.5
0.4
c
c
c
0.3
c c
c
0.2
c
0.1
c
c
c
c
c
c
c
0 N-9
Sapindus
B-vit
Treatments Figure 5. Cytotoxicity of spermicides towards human cervical (HeLa) cell line at multiple MEC doses in vitro (0MEC 5 vehicle control; b 5 p !0.01; c 5 p !0.001).
chosen for evaluation of the safety of spermicidal compounds in the present study. Concentration of bivittoside D (0.05%) that killed 100% human sperm in vitro in 20 sec was found to be almost completely harmless to L. acidophilus for up to 48 h. Previously, we reported the safety of some spermicidal phytosaponins (Sapindus saponins) to Lactobacillus species (14). However, in the present study bivittoside D was found to be marginally safer than Sapindus saponins. Within the cervicovaginal environment, lactobacilli act as natural barriers to infection by human immunodeficiency virus type 1 (HIV-1) (18), which may be compromised by the vaginal application of topical compounds that have a less than ideal safety profile towards this
microorganism. The ideal topical contraceptive would not only be effective at preventing pregnancy (by effectively and efficiently killing sperm) but would also be safe for cervicovaginal application by preserving the inherent defense of the genital tract provided by the natural vaginal microflora (Lactobacillus). The extreme safety of bivittoside D towards the normal vaginal flora (Lactobacillus) even at multiple MEC doses implies that this compound is most likely to be vaginally ecofriendly and therefore safer as a topical contraceptive agent than N-9, which was found to be extremely toxic towards Lactobacillus at MEC. As already reported, the nonspecific surfactant action of N-9 kills both sperm and Lactobacillus with almost equal efficiency
90 Control Bivittoside-D Saponins
80 70
Value
60 50 40 30 20 10 0
% Mot
Rapid
VAP Motility Parameters
ALH
BCF
Figure 6. Effect of Bohdaschia vitiensis pure compound bivittoside D on motility parameters of human sperm in vitro at ED50 concentration [%Mot 5 percent motile; rapid 5 percent sperm with motilityOVAP; VAP 5 average path velocity (m/s); ALH 5 amplitude of lateral head displacement (m); BCF 5 beat cross frequency (Hz)].
Spermicidal Activity of Bivittoside D
(8,14). Hence, bivittoside D appears to be a promising molecule for safer vaginal contraception than that by N-9. One of the most promising actions of bivittoside D was its quick (within |20 sec), complete (100%) and irreversible immobilization of human sperm in vitro at micromolar concentration (350 mM) that was much lower than that of N-9 (811 mM) for a similar action. Hence, the effective molar concentration of bivittoside D in contraceptive preparations can be more than two times lower than that of N-9 for providing an equivalent pregnancy protection. Although bivittoside D at MEC was found to be marginally more cytotoxic in vitro than N-9 towards cervical cancer (HeLa) cell line, but at multiple MEC doses (which is normally used in contraceptive creams), it was found to be less toxic. The mechanism of action of bivittoside D appears to be chiefly through its surface active action on human sperm cells as seen in supravital staining and HOST. On the other hand, bivittoside D did not cause 100% permeabilization of cell membranes (as seen in the case of N-9 and Sapindus saponins), indicating a gentler action than other spermicidal surfactants. However, this gentle action was adequate to functionally attenuate 100% human sperm irreversibly in |20 sec for contraception. CASA analysis indicates that at ED50 concentration (concentration immobilizing |50% sperm in vitro) |50% sperm became immotile and the remaining 50% sperm showed major changes in motility pattern, which was characterized by significantly reduced sperm velocity and visibly reduced lateral head displacement and beat frequency. However, these changes were very similar to those seen in case of treatment with Sapindus saponins. Hence, spermicidal action of bivittoside D is similar to that of phytosaponins that have been used clinically as spermicides (9). Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other STD infections. Thus, it becomes a serious liability for vaginal products (19). Whereas N-9 has been shown to induce pro-inflammatory response in vaginal and cervical epithelial cells (20), saponins from a number of natural sources have been shown to have antiinflammatory activity (21e23). Hence, in the current scenario of unintended pregnancies and STD/HIV menace, saponins appear to be the most promising spermicide for local contraception as they are capable of killing sperm very efficiently and rapidly by their ‘gentle’ surfactant action while remaining vaginally ecofriendly (by preserving the vaginal microflora) and also being potentially capable of controlling vaginal inflammation caused by identified or unidentified factors. Acknowledgments This study was supported by a grant from the Department of Ocean Development and the Ministry of Health and Family Welfare, Government of India, New Delhi. Mr. N.P. Mishra and Mr. H.R. Mishra are acknowledged for their excellent technical assistance (CDRI communication number: 7426).
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