Contraception 83 (2011) 167–175

Original research article

Potent spermicidal effect of oleanolic acid 3-beta-D-glucuronide, an activeprinciple isolated from the plant Sesbania sesban Merrill☆

Nilanjana Das, Poornima Chandran, Smritinath Chakraborty⁎

Reproductive Biology Research Department, Cell-Biology and Physiology Division, Indian Institute of Chemical Biology (A Unit of CSIR),Kolkata-700 032, India

Received 10 November 2009; revised 12 May 2010; accepted 12 May 2010

Abstract

Background: The spermicidal activity of oleanolic acid 3-β-D-glucuronide (OAG), an active principle isolated from root extracts ofSesbania sesban, was evaluated.Study Design: Under the Sander–Cramer test criteria, the sperm-immobilizing activity of OAG was studied using highly motile rat sperm.Sperm mortality and membrane integrity were assessed by supravital staining, hypo-osmotic swelling (HOS), transmission electronmicroscopy (TEM) and sperm membrane lipid peroxidation (LPO). In vitro microbicidal potential and hemolytic index of OAG wereexamined in Lactobacillus culture and rat red blood corpuscles (RBCs), respectively. Post-intravaginal OAG application, the in vivocontraceptive efficacy was evaluated in rats. Ames test determined the carcinogenic potential of OAG.Results: The minimum effective concentration (MEC) of OAG was 50 mcg/mL. More than 97% of the OAG-treated sperm lost their HOSresponsiveness in a dose-dependent manner. TEM and LPO revealed that OAG affected the sperm membrane integrity. OAG declinedfertility to zero, was nonmutagenic and was not harmful to lactobacillus.Conclusion: OAG has significant spermicidal activity that may be explored further.© 2011 Elsevier Inc. All rights reserved.

Keywords: Oleanolic acid 3-beta-D-glucuronide; Saponin; Spermicides; Vaginal contraceptive

1. Introduction

The explosive growth of the global population coupledwith the relentless spread of sexually transmitted diseases(STDs), especially human immunodeficiency virus (HIV),has adversely affected the socioeconomic equilibrium. Thepredicament is more profound in the developing andunderdeveloped nations wherein lack of education,consumer unwillingness and disparity in widespreaddissemination of health services have prevented theembracement of safe contraceptive options [1–6]. There-fore, our goal was to explore active spermicidal principle(s) that could be developed as potential safe vaginalcontraceptive formulation(s).

☆ This research was financially supported by the CSIR, New Delhi,India.

⁎ Corresponding author.E-mail address: smritinath.chakraborty@yahoo.com (S. Chakraborty).

0010-7824/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.contraception.2010.05.009

Plants have served as natural sources of medicinal agentsfor thousands of years since the dawn of civilization. Themedicinal plants, especially those used in traditionalmedicine, are presently being revisited as sources for novelbioactive agents. Quite a good number of plants are reportedto possess spermicidal activity [7,8]. The scientific quest forunique active component(s) from traditional medicinecomplements the success of modern medicine. The presentcompound oleanolic acid 3-beta-D-glucuronide (OAG) wasisolated from Sesbania sesban Merrill, a shrub belonging tothe Leguminosae family. It is a soft-wooded fast-growingplant cultivated throughout India up to an altitude of 1200 m[9] and can also be found in the wild. Different parts of theplant are used in various tribal medicines. Seeds areemmenagogue, stimulant, astringent and are used also forthe treatment of diarrhea. The root is anti-helminthic andcarminative. The Kandha tribe in the Kandhamal district ofOrissa, India, uses the root extracts as contraceptive [10].The seed and flower extracts have been reported to havepotent antifertility activity in rodents [11]. Phytochemical

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examination revealed that different parts of the plant containderivatives of oleanolic acid. Oleanolic acid was reported toinhibit sperm motility [12]. In seeds, oleanolic acid,stigmastane-5.24(28)-diene-3β-O-β-D-galactopyranosideand galactomannan are present [13,14].

Flavonols and anthocyanidins were reported to have beenderived from flowers [15,16]. Gluconide derivatives ofoleanolic acid, 3-O-[α-L-rhamnopyranosyl-(1,3)-β-D-glu-curonopyranosyl]-oleanolic acid, extracted from leavesshowed molluscidal activity [15,16]. Lack of specific anddetailed information pertaining to the fertility-regulatingproperty and chemical constituents of the root extract formedthe basic premise of the present study. Fractionation of thealcoholic part revealed two glycosides as stigmasterol 3-O-β-D-glucoside and OAG. OAG is a derivative of oleanolicacid. OAG is reported to have different biological activitiesincluding anti-neoplastic effect and spermicidal action[17,18]. All of the above information instigated us toinvestigate the spermicidal activity of OAG. Moreover,determining the contraceptive efficacy of OAG, compatibil-ity with vaginal flora, irritability index and probablemutagenic potential compounded further mechanistic details.

2. Material and methods

2.1. Plant material

The roots of S. sesban were collected from the Midnaporedistrict of West Bengal and authenticated by Dr. DebjaniBasu [Assistant Director, Botanical Survey of India, JC Bose(National) Botanical Garden, Howrah, West Bengal, India].Roots were washed thoroughly, dried and then pulverized inan industrial blender.

2.2. Preparation of extracts

Two kilograms of the powdered roots was percolated withrectified spirit (90% ethanol). The extract was concentratedunder reduced pressure to obtain 32 g of brown solid. Thesolid material was then suspended in distilled water (200 mL)and extracted successively with ethyl acetate (3×100 mL) andn-butanol saturated with water (3×100 mL). The organiclayers were dried by passing through anhydrous Na2So4, andthe solvents were evaporated under reduced pressure using arotary evaporator (Eyela Aspirator Model A-3s, TokyoRikakikai Co., Ltd., Japan) to yield 12 g of ethyl acetateand 17 g of n-butanol extracts, respectively.

2.3. Purification of the butanolic extract

The brown gummy mass obtained from the n-butanolicportion was subjected to column chromatography over silicagel (100-200 mesh). A 20-in.×2-in.-radius column was usedfor extraction, and 25 mL of solvent was collected for eachfraction. The fraction obtained from 95:5 CHCl3/MeOHyielded (520 mg) OAG as an amorphous solid (MP 256–258°C) [19,20]. Oleanolic acid 3-O-β-D-glucuronide IR

Vmax (KBr) in per centimeter: 3426 (–OH), 2942.1701 (C=Oof acid), 1618.1389.1036. Negative ESI-MS, m/z (%): 631.4([M-H], 100), 455.2 ([M-H] −176.22 for loss of glucronicacid moiety). 13C NMR (C5D5N,75MHz) δ in parts permillion: 38.7 (C-1), 26.5 (C-2), 89.2 (C-3), 40.0 (C-4), 55.8(C-5), 18.6 (C-6), 33.1 (C-7), 39.6 (C-8), 48.1 (C-9), 37.1(C-10), 23.9 (C-11), 122.5 (C-12), 144.7 (C-13), 42.2 (C-14), 28.1 (C-15), 23.8 (C-16), 46.8 (C-17), 42.1 (C-18), 46.5(C-19), 31.1 (C-20), 34.2 (C-21), 33.2 (C-22), 28.1 (C-23),17.1 (C-24), 15.4 (C-25), 17.4 (C-26), 26.3 (C-27), 180.2 (C-28), 33.2 (C-29), 23.8 (C-30), 107.1 (C-1), 75.4 (C-2), 78.0(C-3), 73.4 (C-4), 77.7 (C-5), 173.3 (C-6). The authenticityof the compound (Fig. 1) was compared with the valuesreported in the literature.

2.4. Chemicals

Chemicals and solvents were of analytical grade and wereprocured from Merck India Ltd. LIVE/DEAD spermviability kit (cat. L-7011) was from Invitrogen (MolecularProbes Inc., Eugene, OR, USA) and the other chemicalswere from Sigma (St. Louis, MO, USA). The media forbacterial culture were procured from M/s Hi-Media Labo-ratories Pvt. Limited, Mumbai, India. For the mutagenicitytest, the Muta-chromoplate™ basic kit version 3.1 wasprocured from Environmental Biodetection Product Inc.(EBPI), Ontario, Canada.

2.5. Animals

Adult Sprague-Dawley rats (180–220 g) were acquiredfrom the animal house of the Indian Institute of ChemicalBiology. The animals were maintained at laboratoryconditions (12:12, dark/light cycle) and fed with standardpellet diet and water was supplied ad libitum. All experi-ments were performed in accordance with the guidelinesrecommended by the institute's animal ethics committee.

2.6. Isolation of rat caudal sperm

Males of proven fertility were used for sperm collectionfollowing sexual abstinence. The animals were necropsiedfollowing anesthesia with ketamine (100 mg/kg bodyweight). The caudal portion of the epididymis wasdissociated and minced in 2.5 mL of Briggers, Whittenand Whittengham's medium (BWW medium) [94 mMNaCl, 4.7 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2mM MgSO4·7H2O, 25 mM NaHCO3, 0.5 mM sodiumpyruvate, 19 mM sodium lactate, 5 mM glucose, 0.4%BSA, 0.1% antibiotic (penicillin/streptomycin) solution, pH7.2] [21]. The collected sperm exudates were thensuspended in BWW medium. The sperm were incubatedin the medium for 30 min under oil cover in a CO2

chamber. Sperm count was done using a Makler chamber.Highly motile sperm suspensions with a sperm count of20–25×106/mL were used.

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2.7. Assessment of spermicidal activity

The spermicidal activity of OAG was assayed accordingto the modified version of the Sander–Cramer method[22–24]. Varying concentrations of OAG solution wereprepared in physiological saline (0, 10, 30, 40, 50, 60 mcg/mL) and 100 μL of each was mixed with an aliquot of 20 μLof sperm suspension (25×106/mL). The specimens wereexamined under a phase contrast microscope (×100) after20 s of treatment and counted for motile sperm. The spermthat lost complete motility within 20 s were subsequentlytested for motility revival. About 250 μL of Baker's buffer(glucose 3%, Na2HPO4·2H2O 0.31%, NaCl 0.2%, KH2PO4

0.01%) was used to show total immobilization of the spermby incubating at 37°C for 60 min and observed if there wasagain any kind of motility. The minimum concentration ofOAG that caused 100% immobilization of sperm within 20 swith no revival of motility after subsequent 60 min ofincubation in Baker's buffer at 37°C was considered to bethe minimum effective concentration (MEC).

Fig. 2. Concentration-dependent inhibition of sperm motility by OAG.Sperm suspensions of fertile Sprague-Dawley rats were exposed to varyingconcentrations of OAG, and motility was observed under a phase contrastmicroscope. Each point represents themean±SEMof six sets of observations.

2.8. Hypo-osmotic swelling test

Control and OAG-treated sperm suspensions (at MEC,i.e., 50 mcg/mL) were exposed to HOS solution (75 mMfructose, 20 mM sodium citrate) for at least 30 min at 37°Cto detect changes in sperm membrane integrity [24–28].The number of spermatozoa showing characteristic tailcurling or swelling was counted under a phase contrastmicroscope (×400).

Fig. 1. Structure of the compound, oleanolic acid 3-beta-D-glucuronide.

2.9. Sperm viability test by fluorescent staining

Sperm viability was assessed by using a sperm viabilitykit according to the manufacturer's instructions where livespermatozoa fluoresced green (Sybr14dye) and dead sper-matozoa fluoresced red [propidium iodide (PI)]. OAG-treated (50 mcg/mL) and untreated sperm samples weresubjected to sperm viability assessment [29,30]. Sperm countwas taken from 200 spermatozoa under a phase contrastmicroscope (×200).

2.10. Lipid peroxidation of sperm membrane

Lipid peroxidation (LPO) was measured to determine theextent of oxidative damage induced by OAG on the spermmembrane as indicated by malondialdehyde (MDA) adductformation. MDA quantification was done by reaction withthiobarbituric acid and measurement of the pink chromo-phore was performed. MDA quantification was donespectrophotometrically [31].

2.11. Ultrastructural study

Aliquots of OAG-exposed and unexposed sperm suspen-sions were fixed with 2% glutaraldehyde in phosphate bufferat 48°C for 4 h. After three successive washings in buffer,post-fixation was done using 1% osmium tetraoxide forobservation under a transmission electron microscope(TEM) [24].

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2.12. Assessment of irritation potential throughhemolytic studies

The irritation potential of the compound was estimatedusing an RBC hemolytic assay. The RBC from rat blood wasisolated by repeated washing (three times) with isotonicphosphate buffered saline (PBS; pH 7.4). Rat RBCs withvarying concentrations (1, 5, 15, 30, 45, 60, 75 mcg/mL) ofOAG, 0.1% of Triton X-100 and PBS were used as treated,positive control and negative control, respectively. The testsamples were prepared in PBS and incubated at 37°C for 30min with occasional mild shaking to achieve completehemolysis. The reaction was quenched in ice and the sampleswere centrifuged at 1500 rpm for 2 min, and supernatantswere used for spectrophotometer reading at 576 nm. Thepercentage hemolysis was then calculated by the followingformula [32]:

kH = 100k Abs − Abscontrolð Þ= Abs100 − Abscontrolð Þ

where Abs is the absorbance of the sample, Abscontrol is theabsorbance of the control sample (negative control) andAbs100 is the absorbance of the sample where 100%hemolysis occurred. Hemolytic index (50% hemolysis) wasdetermined by comparing control and positive control.

2.13. Evaluation of effect on microflora

The effect of OAG against nonpathogenic beneficialvaginal microflora was tested in an in vitro culture of Lac-tobacillus acidophilus. The commercially available lactoba-cillus spores were suspended in deMan, Rogosa and Sharpe(MRS) broth. Overnight log phase cultures were then seriallydiluted in sterile saline (10−1–10−6). Bacterial dilutions ofL. acidophilus (10−4–10−6) with countable number of colonieswere plated on control MRS agar plates, while agar platessupplemented with varying concentrations of OAG rangingfrom 20 to 80 mcg/mL comprised the treated plates [4].

2.14. Contraceptive efficacy

The contraceptive efficacy of the compound was assayedin rats via intrauterine administration of OAG and subse-quent evaluation of mating outcomes. Sexually mature cyclicadult Sprague-Dawley rats (n=16) were subjected to lightether anesthesia on the day of proestrous phase of their cycle,then one very small mid-ventral abdominal incision wasmade through which the uterine horns were gently pulled outcarefully. In one horn, 50 μL of OAG solution (300 mcg/mLof sterile physiological saline) was introduced through atuberculin syringe fitted with a 24-G needle that penetratedthrough the cervical end (treated horn). The contralateral

Fig. 3. HOS responsiveness of rat sperm as tail coiling: (A) Untreatedcontrol. (B) OAG-treated rat sperm (50 mcg/mL) examined under a phasecontrast microscope (40×). (C) Over 94% of untreated control sperm wereHOS-positive, whereas OAG-treated sperm showed no significant HOSresponse; ⁎pb.001.

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uterine horn received 50 μL of sterile physiological saline(control horn). The incision was closed by suture andMitchel's clip. The animals were maintained in individualcages and distributed into two groups with eight animals pergroup. In the evening, they were exposed to males of provenfertility in 1:1 ratio. The presence of sperm the next morningin vaginal lavage confirmed mating. Mated rats of one groupwere sacrificed on Day 10 of gestation. The uterine hornswere examined and the number of implantation sites/fetuswas counted. Animals of the other group were allowed todeliver pups. After parturition, the litter size was measuredand pups were examined for any abnormalities.

2.15. Mutagenicity test (Ames test)

Mutagenicity of OAG was assayed by using thecommercial Muta-chromoplate™ kit (EBPI) which follows

Fig. 4. Sperm viability assessment by double fluorescent staining method.(A) Control live sperm appear green; Sybr14 dye entered only into thehead portion of the spermatozoa. (B) OAG-treated dead sperm appear red byPI staining.

Fig. 5. Effects of OAG at MEC (50 mcg/mL) on sperm membrane lipid weremeasured by MDA concentration. The treated sperm showed significantlyhigher MAD value. Each bar represents the mean±SEM of six sets ofobservations; ⁎pb.001.

Ames's principle and uses Salmonella typhimurium(TA100), carrying mutation(s) in the operon coding forhistidine biosynthesis with reversion property [33–35]. Theprotocol as described in the product insert was followed toassess the mutagenicity of the test compound. Overnightnutrient broth culture of the bacteria S. typhimurium wasincubated at 37°C. The samples to be tested were dissolvedin distilled water and filter sterilized by passing it via a 0.22-μm filter. To 2.5 mL of the reaction mixture, 17.5 mL of theOAG sample was added to obtain the desired drugconcentration (10, 50, 100 mcg/mL). Then 5 μL of S.typhimurium was mixed. Finally, 200 μL of the mixture wasdispensed into each well microtritration plate. Plates werecovered with a lid and sealed in sterile air-tight plastic bagand then incubated at 37°C for 5 days.

The sample was tested at least five times in independentexperiments. The plates were scored visually: all yellow,partially yellow or turbid wells were scored as positives andall purple wells were scored negative. The result of eachtreatment plate was evaluated against the backgroundmutation, and the number of wells was scored as positive

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or negative in treated and background plates. For eachtreatment plate, the statistically significant level wasdetermined using the table provided with the kit.

2.16. Statistical analysis

The data were analyzed using Microsoft Excel (Microsoft,Inc., Redmond, WA, USA) and expressed as mean±SEM, andpb.001 was considered as the statistically significant level.

ig. 7. Dose-dependent effect of OAG in rat RBCs. Isolated RBCs werecubated with varying drug dilutions, Triton X-100 and PBS. The extent ofemolysis was determined spectrophotometrically.

3. Results

3.1. Spermicidal activity

Fig. 2 depicts the effect of varying concentrations of OAGon sperm motility. There was a dose-dependent increase insperm immobilization with an increase in concentration ofOAG. The MEC that induced 100% immobilization of thesperm was 50 mcg/mL.

Fig. 6. Transmission electron microscopic illustration of (A) normalspermatozoa showing intact membrane and (B) OAG-treated spermatozoawith damaged membrane, respectively.

Finh

3.2. Sperm membrane integrity

More than 97% of the control sperm were HOSresponsive, whereas OAG-treated sperm was non-HOSresponsive as depicted via tail curling (Fig. 3A–C). Lossof HOS responsiveness indicated compromised spermmembrane integrity post OAG treatment, anticipating overallloss of sperm-membrane physiology.

3.3. Sperm viability test by fluorescent staining

The fluorescent green (Sybr14dye) and fluoresced red PIdye differently stain live and dead sperm, respectively. AtMEC (50 mcg/mL), treated sperm were all dead, whichfluoresced red, and control sperm fluoresced green (Fig. 4).

Fig. 8. Photograph of a uterus showing the presence or absence ofimplantation sites in the control (C) and OAG-treated (T) uterine horn of aSprague–Dawley rat.

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3.4. Sperm membrane lipid peroxidation assay

LPO assay revealed lower MDA levels (shown inFig. 5) in untreated sperm suspensions compared to highMDA levels in OAG-treated sperm suspensions. Thethreefold MDA adduct increase signified substantial spermmembrane disintegration.

3.5. Ultrastructural study

Imaging of the control sperm sample under TEMexhibited intact plasma membrane without any appreciablemorphological injury (Fig. 6A), while OAG-treated spermshowed severe damages via membrane pore formation anddistortion of intact membrane morphology (Fig. 6B).

3.6. Irritation potential as measured by hemolytic activity

The hemolytic activity of OAG as depicted in Fig. 7 wasdetermined by quantifying the hemoglobin released from theRBC following exposure to varying concentrations of OAG.At 24 mcg/mL of OAG, 50% hemolysis occurred. RBCtreated with 0.1% Triton X-100 served as positive control.

3.7. Effect on Lactobacillus acidophilus in vitro

OAG has no significant inhibitory activity on thegrowth of Lactobacillus acidophilus. It was observed thatat MEC (50 mcg/mL) and in higher (5× MEC)concentration, about ∼10% and ∼40% inhibition of growthoccurred, respectively.

3.8. Contraceptive efficacy in rats

All the females mated successfully and no deviation ofmating efficiency was observed. Not a single implantationsite was observed in the OAG-treated horns on Day 10 ofgestation. On the contrary, the number (mean±SEM) ofimplantation sites in the control horn was 7.0±0.95. Onparturition, 6±0.56 normal pups were delivered by the othergroups of rats. This observation (Fig. 8) suggests that even inthe uterine milieu, OAG could effectively block spermpotential to reach and/or fertilize oocyte, while fertilizationand implantation were unhindered in the control horn. Theresults suggest that OAG decreased fertility to zero.

3.9. Mutagenicity study (Ames test)

Observations from the Ames test revealed a greaternumber of revertant wells in all positive control plates ascompared to spontaneous revertants. On the contrary, thenumber of revertant wells of OAG-treated groups did notdiffer significantly with respect to negative control.

4. Discussion

The present study focuses on the assessment of the rootextract of S. sesban for its prospective sperm immobilizingor spermicidal activity. Activity-guided fractionation study

led to identification of the compound OAG as an activeprinciple. Sperm immobilization, contraceptive efficacy andother related parameters of OAG as spermicidal agent wereverified in a series of in vitro and in vivo experiments.Results demonstrated that the compound exerted a dose-dependent sperm immobilizing property. Irreversible loss ofsperm motility was observed at a concentration of 50 mcg/mL, which is quite low as compared to non-oxynol-9 (N-9)whose MEC is 350 mcg/mL.

Membrane permeability, indispensable for effectivesperm motility during transport and fertilization, is directlydependent on plasma membrane integrity. HOS test is anindicator of the structural and functional integrity of spermmembrane [27,28]. In living state, cell plasma membrane issemi-permeable, and in hypo-osmotic condition, intact cellmembrane facilitates free entrance of solvent into the cell toattain osmotic equilibrium rendering the cell to be turgid orswell. Since the plasma membrane around the sperm tailfiber is more loosely bound as compared to the rest of thesperm, sperm tail is particularly susceptible to hypo-osmoticfluctuation and responds by coiling or curling. Thischaracteristic feature was exhibited in N97% of the spermin the control set, while the OAG-treated sperm demonstrat-ed (100% HOS-negative) overall distraction/loss of sperm-membrane permeability. Thus, it could be attributed to thefact that the morphological/structural integrity of the spermmembrane was severely compromised by OAG. Thisobservation was validated by viability assay of the spermsamples using a membrane permeant nucleic acid stainSybr14 (stains live cells, i.e., control sperm) and PI (stainsdead cells, i.e., treated sperm) [30,31]. Nearly 100% of thesperm died following OAG treatment. TEM also illustratedthat OAG treatment affected sperm membrane integrity. Theobservation was supported biochemically by increased lipidperoxidation. Malondialdehyde is a product of LPO thatappears to be produced in relatively constant proportion fromthe breakdown of polyunsaturated fatty acids [31]. MDAresults from the decomposition of lipid endoperoxidesduring LPO and/or during the assay. Triterpenoid saponinshave previously been reported to induce permeability change[36]. OAG being a saponin exerts a similar mode of action.All the observations clearly demonstrated that OAGcompromised sperm membrane integrity which leads us todeduce that there was an ultimate loss of sperm functionality.There are reports that many spermicidal compounds exert asimilar type of effects [14,24,35]. Therefore, it could beinferred that OAG has potent sperm immobilizing activity.

The prospective contraceptive effect of OAG asevaluated by intrauterine application in rats is significantlyencouraging as fertility was reduced to zero. Rat is not aclassical model for evaluation of in vivo contraceptiveefficacy because during fertilization semen is deposited inthe cervix rather than in the vagina [24]. The presence ofthe bicornuate uterus in rat was exploited wherein oneuterine horn served as control and the other as experimen-tal, while both horns were exposed to identical systemic

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milieu. A point noteworthy of being mentioned is thepaucity of pure compound which deters the testing of OAGin other higher animals following intravaginal applicationuntil now. But this obstacle may be overcome by devisingnovel cost-effective strategies that yield optimal yields ofthe compound in question.

OAG was found to be non-mutagenic when assessedby the Ames test. Another remarkable aspect is that thebasic scaffold of OAG (Fig. 1) is oleanolic acid, which isreported to have diverse biological activities includingantiviral action against HIV, antifertility and anticancer[21]. Over the last 10 years, more than 700 articles havebeen published on oleanolic acid, reflecting a tremendousinterest in the compound, despite its limited solubility.But the compound OAG is unique in that it has one sugarmoiety in its structure. When compared to other activecomponents present in extracts from Azadirachta indica(neem) leaf (MEC ∼2.91 mg/million sperm) [8], acacia-side-B (Ac-B)-enriched fraction from the seeds of Acaciaauriculiformis (MEC ∼120 mcg/mL) [35], compositeextract of Achyranthes aspera and Stephania hernandifo-lia (MEC ∼0.08 g/mL) [25], and Mi-saponin A (MSA)(MEC ∼320 mcg/mL) from seed extracts of Madhucalatifolia [37], the MEC of OAG is very low. It could bespeculated that this rendered the compound morebioavailable and effective. Therefore, from the foregoingexperimental observations, it could be concluded thatOAG has very strong antifertility activity and otherproperties that qualify the agent to serve as an activeingredient of vaginal contraceptive. However, furthertesting in a primate model is obligatory. More investiga-tions on pharmacokinetic and pharmacodynamics areneeded to elucidate its mode of action.

Acknowledgments

We thank Mr. S.N. Dey for his help in the electronmicroscopic studies. We are also grateful to the Council ofScientific and Industrial Research (CSIR) and the UniversityGrants Commission, New Delhi, for providing fellowship toN. Das and P. Chandran (UGC/SRF), respectively.

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