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Biomedicine & Aging Pathology 4 (2014) 43–47

Available online at

ScienceDirectwww.sciencedirect.com

riginal article

ontraceptive activity of-(4-hydroxy-3-methyl-hex-5-enyl)-chroman-2,7-diol via inhibitingvulation in Gonadotropin-primed immature rat model

shok Kumara, Sumeet Gullaiyab, Vishal Dubeya, Ashish Nagara,arun Singhc,∗, Shyam S. Agrawalb

Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), Pushp Vihar, Sector III, M.B. Road, New Delhi, IndiaDepartment of Pharmacology, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, IndiaDepartment of Biochemistry, Central Research Institute, Kasauli, Himachal Pradesh, India

a r t i c l e i n f o

rticle history:eceived 28 October 2013ccepted 12 December 2013vailable online 17 January 2014

a b s t r a c t

Gonadotropin-primed immature rats (GPIR) were used to study the anti-ovulatory effect of 4-(4-hydroxy-3-methyl-hex-5-enyl)-chroman-2,7-diol (chroman derivative) isolated from seeds of Ensete superbumon ovulation. Present results depicted dose-related anti-ovulatory effect of the chroman derivative at2 mg/kg bw and 4 mg/kg bw. Chroman derivative showed dose-dependent anti-estrogenic activity on

eywords:hroman derivativenti-ovulatorynti-estrogenicnsete superbum

MCF-7 cell line. Attenuation of ovulation was associated with a lowered ovarian estrogen and pro-gesterone levels along with reduced levels of some major cytokines, i.e. TNF�, VEGF, IL-6 and IL-1�,indicating the disrupted cytokinine signaling. Unruptured preovulatory follicles were found in all theovaries extracted from animals treated with chroman derivative. Our findings demonstrated that chro-man derivative has potential contraceptive activity through its anti-estrogenic activity which leads toinhibition in ovulation and attenuated ovarian cytokinine signaling.

. Introduction

Ovulation, the process by which oocytes are released fromhe graffian follicle in the ovary, is an inflammatory process andequires proteolytic degradation of the follicle wall, as well asemoval of the overlying ovarian tissues accompanied by induc-ion of prostaglandin synthesis [1]. For successful ovulation, follicleupture has to occur just at the site of the follicle wall facinghe ovarian surface, which permits release of the Cumulus-Oocyteomplex (COC) to the periovarian space, while preventing pro-eolytic damage of the perifollicular tissues at the basolateralollicle sides. Several mutant rodent models have this type ofnovulatory phenotype, including animal null for the progesteroneeceptor [2], cyclooxygenase-II [3], the prostaglandin E2 receptor4], which indicate that follicular rupture is a biologically regu-ated morphological phenomenon, that must occur at a precise and

ell-regulated cascade of events leading to the transformation of

n ovarian follicle into a progesterone producing corpus luteum.

Ensete superbum Cheesm is a stoloniferous stout herb of 3–6 mall, naturally occurring all around in India, mainly in Western Ghats

∗ Corresponding author.E-mail addresses: varunsinghbiochem@gmail.com,

arunsinghbiochem@yahoo.co.in (V. Singh).

210-5220/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved.ttp://dx.doi.org/10.1016/j.biomag.2013.12.001

© 2013 Elsevier Masson SAS. All rights reserved.

of India. The flesh of the fruits is given to diabetics under Ayurvedicpractice. The ground seeds are also used in ayurveda and certainfractions isolated from the seeds have shown to possess anti-variolaand anti-vaccinia properties [5]. Its leaf ash is used for treatmentof leucoderma. Leaves smeared with warm coconut oil are kept onhead of children for infantile cold along with leaf juice given orallydaily in the morning for three days [6]. Earlier study done in ourlab reported that chroman derivative 4-(4-hydroxy-3-methyl-hex-5-enyl)-chroman-2,7-diol possesses anti-implantation activity inrats [7] and their histopathological examination suggested thatit may have anti-ovulatory activity. Our present work is to eval-uate its anti-estrogenic activity and anti-ovulatory activity onGonadotropin-primed immature rat model and find out the possi-ble mechanism of action of its effect on various important cytokinesinvolved in the ovulation process for its contraceptive ability anduse.

2. Materials and methods

2.1. Isolation of standard chroman derivative

Seeds of Ensete superbum (Cheesm) were purchased fromthe local supplier. Seeds were dried in the shade at the tem-perature of 28 ± 2 ◦C and were authenticated at Department

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4 A. Kumar et al. / Biomedicine

f Botany, by botanist Dr. H.B. Singh, HOD, Dept. of Botanyt National Institute of Science Communication and Informa-ion Resources (NISCAIR) on 21st September, 2010 referenceo. NISCAIR/RHMDC/Consult/2010-11/1536/135, New Delhi, India.ernels of the dried seeds of Ensete superbum (2.5 kg) werextracted with 95% (v/v) ethyl, alcohol in Soxhlet apparatus, a redolored substance was obtained (crude extract, 1.92%) which wasurther dissolved in water and treated in basic lead, sodium sul-hate and calcium carbonate to obtain a yellowish white solid.

t was further confirmed through FTIR, NMR, GC-MS and C-NMRhat it was 4-(4-hydroxy-3-methyl-hex-5-enyl)-chroman-2,7-diolf molecular formula C16O4H22 as described earlier [8].

.2. Animals and drugs

Female albino wistar rats (25–26 days), procured from thenimal house of Delhi Institute of Pharmaceutical Sciences andesearch (DIPSAR), New Delhi, were used. Rats were housed inemperature controlled room at 25 ± 2 ◦C with a 12-hour light/darkycle and 57 ± 7% relative humidity under standard hygienic con-itions and had free access to fresh tap water and pelleted dietAmrut rat feed, Pranav agro Ltd., India). The study was approvedy the Institutional Animal Ethics Committee (IAEC/DIPSAR/2010-

/02) of DIPSAR, New Delhi, and experiments were carried out inccordance with the guidelines laid down by the Committee forhe Purpose of Control and Supervision of Experiments on AnimalsCPCSEA), New Delhi, India.

.3. Gonadotropin-priming and drug treatment

Wistar immature rats were injected s.c. with 10 IU of Equinehorionic Gonadotropin (eCG) at 17 00 h on 25 or 26 days of age,nd 48 h later were injected (s.c.) with 10 IU of hCG [9]. Chromanerivative was administered at dose level 2 mg/kg bw and 4 mg/kgw per day from eCG priming day till animals were sacrificed (6 perroup) at 09 00 h on the day following hCG injection.

.4. Tissue homogenate, RNA isolation and histopathology

Both the ovaries were recovered surgically and from each ratight ovary were fixed in 10% formalin [10]. Tissues embedded inaraffin blocks were used for histopathological examination. Leftvaries were divided into two equal parts. One part weighed andomogenized in PBS (0.1 g/mL) on ice bath by an ultra turrax. Theomogenate obtained was processed as follows: 500 �L were cen-rifuged at 2000 × g for 10 min at 4 ◦C and supernatant was storedt −40 ◦C until TNF�, IL-6, IL-1�, estradiol and progesterone deter-ination. The protein concentration was measured using Bradford

rotein assay. For expression of VEGF and COX-II total RNA wassolated from the second half of the ovaries with minor modifica-ion as per earlier described methods [11] using the trizol reagent.riefly, tissue was weighed and 500 �L trizol reagent/100 mg tis-ue weight added. Total RNA was separated from DNA and proteinsy adding 250 �L chloroform and precipitated with isopropanolovernight, 20 ◦C). The precipitate was washed twice in ethanol,ir-dried, and resuspended in 75% diethylpyrocarbonate (DEPC)-reated water and quantified (A260 nm). One microgram of totalNA was reverse-transcribed to cDNA using iScriptTM cDNA Syn-hesis Kit (Bio-RAD Laboratories Inc., CA, USA) in a final volume of0 �L, according to the manufacturer’s instruction.

.5. Real-time PCR

Primers express software (Version 2.0., Applied Biosystems)as employed for designing and synthesizing primers of quan-

itative PCR. The VEGF primers were designed to amplify a

g Pathology 4 (2014) 43–47

region common to all VEGF isoforms, so the sense VEGF primerwas 5’-CAGCTATTGCCGTCCAATTGA-3’ (114-124) and the anti-sense VEGF primer was 5’-CCAGGGCTTCATCATTGCA-3’ (244-226)where a 131-bp (NCBI accession No. AF215726), COX-2 primerwas 5’-CCCAATTTGTTGAATCATTT-3’ (958-977) and antisense 5’-TCTCATCTCTCTGCTCTGGT-3’ (1057-1076) (NCBI accession No.AF207824) a product of 119 bp PCR product was expected. Thesense �actin primer was 5’-AGGGAAATCGTGCGTGACAT-3’, andthe antisense �actin primer was 5’-AACCGCTCATTGCCGATAGT-3’(NCBI accession No. 55574), giving rise to an expected PCR prod-uct of 149 bp. Real-time quantitative PCR was performed in theiCycler Thermal Cycler (Bio-RAD Laboratories Inc., Hercules, CA,USA) using SYBR green I detection. The following reaction compo-nents were prepared to the indicated end-concentrations: 0.6 �Mof each primer, 1X IQ SYBR Green BioRad Supermix (Bio-RAD Lab-oratories Inc.), 150 ng of cDNA and nuclease free double distilledwater were added to a final volume of 25 �L. All samples wereperformed in duplicate for all genes. The real-time PCR protocolwas followed according to the manufacturer’s instructions with aheated lid (105 ◦C), an initial denaturation step at 95 ◦C for 10 min,followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. Thespecificity of the amplified PCR products was verified by analy-sis of the melting curve. The relative mRNA level was determinedas the PCR cycle number that crosses an arbitrarily placed signalthreshold (Ct). The Ct value correlates inversely with the amountof target mRNA in the sample. The relative changes in VEGF, Cox-IIwith respect to �actin expressions were examined using the ��Ctmethod �Ct = Cttarget − Ct �actin. As PCR amplification is an expo-nential process, a �Ct difference denotes a shift in regulation by afactor of two (2-��Ct). To validate a real-time PCR, standard curveswith r2 > 0.95 and slope values between 3.0 and 3.4 were required.Real-time efficiencies were acquired by amplification of a standard-ised dilution series and corresponding slopes and PCR efficiencieswere calculated using iCycler iQ Real-Time PCR Detection System(Bio-RAD Laboratories Inc.).

2.6. Histological analysis and follicle rupture and ovulation

The right ovaries were serially sectioned (5.0 �m) and stainedwith hematoxylin and eosin. All sections were examined underthe microscope. The total number of cumulus-oocyte complexes(COCs) per ovary were counted as described [12] and number ofCOCs trapped inside the corpus luteum, released to the ovarianinterstitium, retained in the bursal cavity or found in the oviducts,were recorded.

2.7. Cell culture and anti-estrogenic activity

Estrogen receptor-positive human breast cancer MCF-7 cellsline is the model used to evaluate anti-estrogenic effect of a NewChemical Entities (NCEs). MCF cell line was obtained from NationalCentre For Cell Science (NCCS), Pune, and routinely maintained aspreviously described [13] in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with 1 nM Estradiol (E2), 2 mg/mL insulin,1 mM sodium pyruvate, 1 mM nonessential amino acids, 4 mMglutamine, and 10% fetal bovine serum. Exponentially growing cul-tures were maintained in a humidified atmosphere of 5% CO2 at37 ◦C. Experiments were performed in 96-well plates containingphenol red-free DMEM supplemented with 5% CS-FBS. MCF-7 cellswere seeded at a plating density of 2 × 103 cells/well in 200 �L ofmedium and then cultured for 24 h to allow their adhesion to theplate. Two days later, the medium was replaced, and two sets of

experiment were performed. Set-I control group cells were exposedto estradiol 1.0 nM and chrome derivative group’s cells wereexposed with estradiol 1.0 nM at 0.01–100 �g/mL doses of chro-man derivative for 4 days. Set-II control group cells were exposed

A. Kumar et al. / Biomedicine & Aging Pathology 4 (2014) 43–47 45

Fig. 1. Serial sections of the ovary of a rat primed with eCG and treated with 4 mgoec

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Fig. 2. Dose-response effects of the chroman derivative on the growth of MCF-7cells in estrogen medium. Cell viability was determined by the MTT assay. Resultsare expressed as percentages of proliferation relative to the untreated control(mean ± SD, n = 3). *Significantly different from control, P < 0.01.

Fig. 3. Dose-response effects of the chroman derivative on the growth of MCF-7cells in estrogen-depleted medium, with 1.0 nM E2 as positive control cell viabilitywas determined by the MTT assay. Results are expressed as percentages of prolif-

TN

Vw

f chroman derivative, stained with hematoxylin and eosin. The ovarian vein ismbolized with folicular fluid and two COCs. A COC released to the ovarian hilusan be also observed.

o vehicle, E2 group cells were exposed estradiol 1.0 nM and chromeerivative groups cells were exposed at 0.01–100 �g/mL doses ofhroman derivative in DMSO (Dimethyl sulfoxide, Sigma, USA)0.1%) for 4 days. MTT stock solution (20 �L, 5 mg/mL, Sigma) wasdded to each well, and the plates were further incubated for 4 ht 37 ◦C. The supernatant medium was aspirated and precipitatedormazan salt crystals were dissolved with DMSO and then read at90 nm with an Elisa Micro-plate Reader (Bio-Rad). All measure-ents were performed in triplicate. Results were expressed as the

ercentages proliferation with respect to vehicle-treated cells.

.8. Statistical analysis

Statistical analysis was performed by ANOVA followed by thetudent-Newman-Keuls method for multiple comparisons amongeans. Significance was considered at the P < 0.01 level.

. Result

.1. Effects of chroman derivative on ovarian histopathology

Absolute numbers of COCs, that match the numbers of corporautea per ovary, are presented in Table 1. The total number of COCser ovary is not significantly different in treated rats compared toontrol. Immature rats primed with eCG at dose 2 mg/kg bw and

mg/kg bw showed significant ovulatory defects. COC trapped inontrol group were 40% of the luteinized follicles in rats primedith eCG with a significant dose-dependent reduction (P < 0.001)

0% and 3% in treated rats at dose levels 2 mg and 4 mg respec-ively. In treated groups many luteinized follicles containing theOC showed rupture of the theca layers and release of follicularuid to the ovarian interstitium. Furthermore, 5–15% of the COCs

ere released to the ovarian interstitium. Also the treated animals

howed marked degradation of the ovarian stroma with invasion ofhe blood and lymphatic vessels and formation of emboli containinghe COC and follicular fluid as shown in Fig. 1.

able 1umber of COCs per ovary in rats primed with eCG.

Treatment Total COCs in COCs located in

Ovarian section Corpus luteum

Control group 36.17 ± 0.83 13.17 ± 0.70

2 mg/kg p.o. body weight 34.83 ± 0.54 22.83 ± 0.60a

4 mg/kg p.o. body weight 34.5 ± 0.78 29.17 ± 0.93a,b

alues are the mean ± SEM for n = 6. ANOVA and Student-Newman-Keuls multiple rangeight of chroman derivative.

eration relative to the untreated control (mean ± SD, n = 3). *Significantly differentfrom control, P < 0.01.

3.2. Effects of chroman derivative on the growth of MCF-7 humanbreast cancer cells

In set-I, chroman derivatives show dose-dependent sig-nificant reduction in cell proliferation activity at dose lev-els 0.10–100 �g/mL (P < 0.01) compared to control and dose-dependent significant anti-estrogenic activity compared to estra-diol 1.0 nM/mL (P < 0.01) at dose level 0.001–100 �g as shownin Fig. 2. In set-II, chroman derivatives show dose-dependentsignificant reduction in cell proliferation activity at dose levels0.10–100 �g/mL (P < 0.01) compared to control after comple-

menting with estradiol (Fig. 3). Proliferation of MCF cell lineindication of estrogenic activity of drug and any reduction in cellproliferation signified anti-estrogenic activity.

Interstitium Ovarian bursa Oviduct

1.5 ± 0.22 0.0 ± 0.0 21.5 ± 0.623.5 ± 0.42a+ 1.67 ± 0.21a 6.83 ± 0.65a

2.67 ± 0.33a++ 1.5 ± 0.22a 1.17 ± 0.40a,b

es test. aP < 0.001, a+P < 0.01, a++P < 0.05, vs control, bP < 0.001 vs 2 mg/kg p.o. body

46 A. Kumar et al. / Biomedicine & Aging Pathology 4 (2014) 43–47

Fig. 4. Effects of chroman derivative on ovarian IL-�, IL-6, TNF-�, progesterone andestradiol levels. Values are the mean ± SEM for n = 6. ANOVA and Student-Newman-Keuls multiple ranges test. *Significantly different from control, P < 0.01.

Fig. 5. �-Actin mRNA levels in the ovaries of each sample were used as thehousekeeping gene to normalize VEGF expression between them. The averageVEGF/�-actin ratio in each group was used to compare mRNA VEGF levels among thegroups. In order to normalize VEGF expression levels, the VEGF/�-actin ratio in thecoK

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Fig. 6. �-Actin mRNA levels in the ovaries of each sample were used as thehousekeeping gene to normalize COX-II expression between them. The averageCOX-II/�-actin ratio in each group was used to compare mRNA VEGF levels amongthe groups. In order to normalize COX-II expression levels, the COX-II/�-actin ratio in

ontrol group was assigned the value 1 and used to normalize VEGF expression in thether groups. Values are the mean ± SEM for n = 6. ANOVA and Student-Newman-euls multiple ranges test. *Significantly different from control, P < 0.01.

.3. Effects of chroman derivative on ovarian hormones andytokines levels

Significant difference at dose of 2 mg/kg and 4 mg/kg in ovar-an estradiol and progesterone levels between treated and controlnimals was observed with marked reduction in estradiol levelsn 57.25% and 48.53% and progesterone levels 70.10% and 55.52%.ytokines levels in ovarian tissues were also significantly reduced

or IL-1� and IL-6 levels. IL-1� levels were reduced to 70.75% and9.83% and IL-6 to 75.66% and 57.34% compared to control rats. TNF-

levels also showed the similar trends with reduction of 69.34%nd 44.05% of control as shown in Fig. 4.

.4. Expression of COX-II and VEGF levels in ovarian tissue

Expression of VEGF and COX-II significantly reduced comparedo control group at dose level 2 mg and 4 mg (P < 0.01) as shown inigs. 5 and 6.

. Discussion

In gonadotropin-primed immature rat model, eCG stimulates

he growth of large number of follicles, that reach preovulatoryize in about 48 h and are then induced to ovulate by hCG [14]. Inhe present study, we evaluated the ovulatory process in immatureats due to an additional advantage of this model is the absence

the control group was assigned the value 1 and used to normalize COX-II expressionin the other groups. Values are the mean ± SEM for n = 6. ANOVA and Student-Newman-Keuls multiple ranges test. *Significantly different from control, P < 0.01.

of regressing corpora lutea of previous cycles. Our result are pro-viding support to our previous studies suggesting anti-ovulatoryactivity of chroman derivative [7], the proportion of COCs foundin the oviducts were significantly reduced in treated rats 19.62%and 3.38% at dose levels of 2 mg and 4 mg compared to con-trol rats 59.44%. Antiestrogens exerted a similar inhibitory effectagainst ovulation when administered at the early stage of theestrus cycle [15]. The present findings indicate chroman deriva-tive significantly inhibits the ovarian estradiol levels and growth ofER-dependent MCF-7 breast cancer cells supplemented by 1.0 nMestradiol. Results demonstrated that the growth-inhibiting effect ofthe chroman derivative was mediated by ER receptors and fully abetwith our previous suggestive finding. Ovarian progesterone, IL-6,IL-1� and TNF-� level were also significantly reduced in compar-ison to control group. The sites of ovulation-related progesteronesynthesis are the theca interna and the granulosa layer [16] and theovulatory effects of the gonadotropin surge are mediated at leastin part by induction of the progesterone receptors (PR). Rodentslacking PR fail to ovulate further supporting the present finding.TNF�, IL-1� and IL-6 are primarily pro-inflammatory cytokines,with their most salient and relevant properties being their abil-ity to initiate expression of COX-2, phospholipase A2, eicosanoids,iNOS, NF-�B-inducing kinase [17]. IL-1� and IL-6 gene expres-sion in the ovary and transcripts of these genes increase in thegranulosa and surrounding theca interna within 4 h after stimu-lation of the ovulation in rats. Reduction in TNF�, IL-1� and IL-6in treated rats compared to control suggestive of interruption inpro-inflammatory pathway of ovulation. Moreover, inflammatorycytokines TNF-� and IL-1� can induce VEGF expression during thetransformation of an ovulatory follicle into a corpus luteum, VEGFtranscripts are expressed primarily in ovarian granulosa cells, the-cal and stromal cells [18] support our finding of a reduction in VEGFexpression in ovaries of treated rats. Chroman derivative-treatedrats show reduction in ovarian COX-II expression. In our researchfinding, it shows that chroman derivative isolated from seeds ofEnsete superbum has potent anti-ovulatory activity which was dueto its anti-estrogenic property and ability to suppress TNF�, IL-1�,IL-6 and pro-inflammatory cytokines which lead to reduction inVEGF and COX-II expression in the ovaries and failure in ovulation.

5. Conclusions

The present study confirmed that chroman derivative (4-(4-hydroxy-3-methyl-hex-5-enyl)-chroman-2,7-diol) isolated fromseeds of Ensete superbum has anti-estrogenic activity to produce

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A. Kumar et al. / Biomedicine

nti-ovulatory effect in ovulation induced gonadotropin-primedmmature rats via disrupting major cytokinine signaling in ovu-ation process which can be utilised for the development of a newontraceptive method.

isclosure of interest

The authors declare that they have no conflicts of interest con-erning this article.

cknowledgment

We are thankful to Department of Training and Technical Edu-ation, Government of NCT Delhi for the funding assistance.

eferences

[1] Epsey LL. Current status of the hypothesis that mammalian ovulation is com-parable to an inflammatory reaction. Biol Reprod 1994;49:233–8.

[2] Jaeyeon K, et al. Control of ovulation in mice by progesterone receptor-regulated gene networks. Mol Hum Reprod 2009;15(12):821–8.

[3] Lim H, et al. Multiple female reproductive failures in cyclooxygenase 2-deficientmice. Cell 1997;91:197–208.

[4] Davis BJ, et al. An ovulation in cyclooxygenase-2 deficient mice is restored by

prostaglandin E2 and interleukin-1b. Endocrinology 1999;142:3187–97.

[5] Cheesman EE. Classification of the bananas. The genus Ensete Horan. News Bull1947;2:97–106.

[6] Kosalge SB, Fursule RA. Investigation of ethnomedicinal claims of some plantsused by tribals of Satpuda Hills in India. J Ethnopharmacol 2009;121:456–61.

[

g Pathology 4 (2014) 43–47 47

[7] Agrawal SS, Monica K. Isolation, characterisation & anti-fertility activity of theactive moiety from the seeds of Ensete Superbum Cheesm (Banakadali). J NatRemed 2009;9:12–20.

[8] Kachroo M, et al. Characterization of a Chroman derivative isolated from theseeds of Ensete superbum Cheesm. Pharmacog Mag 2008;4:114–7.

[9] Kurusu S, et al. Inhibition of ovulation by a lipoxygenase inhibitor involvesreduced cyclooxygenase-2 expression and prostaglandin E2 production ingonadotropin-primed immature rats. Reproduction 2009;137:59–66.

10] Singh V, et al. Antifertility activity of methanolic bark extracts of Aeglemarmelos in male wistar rats. DARU J Pharm Sci Biomed Central 2012;20:94,http://dx.doi.org/10.1186/2008-2231-20-94.

11] Singh V, et al. Modulation of disease related immune events by demethoxycur-cumin against autoimmune arthritis in rats. Biomed Aging Pathol 2013;3:7–13.

12] Gaytan M, et al. Immature rats show ovulatory defects similar to those in adultrats lacking prostaglandin and progesterone actions. Reprod Biol Endocrinol2004;2:63.

13] Maneesha P, et al. Antiproliferative effects of Datura innoxia extract in cervicalHeLa cell line. J Pharm Res 2011;4(4):1124–6.

14] Mori T, et al. Inhibition by indomethacin of ovulation induced by humanchorionic gonadotrophin in immature rats primed with pregnant mare serumgonadotrophin. J Endocrinol 1980;84:333–41.

15] Nishino T, et al. Antiovulatory effect of a single injection of pure antiestro-gen ZK 191703 at early stage of rat estrus cycle. J Steroid Biochem Mol Biol2009;114(4):152–60.

16] Richards JS, et al. Differentiation of rat ovarian thecal cells: evidence for func-tional luteinization. Endocrinology 1986;118:1660–8.

17] Rummel C, et al. Circulating interleukin-6 induces fever through a STAT3-linked activation of COX-2 in the brain. Am J Physiol Regul Integr Comp Physiol

18] Levitas E, et al. Periovulatory and interleukin-1�-dependent up-regulation ofintraovarian vascular endothelial growth factor (VEGF) in the rat: potentialrole for VEGF in the promotion of periovulatory angiogenesis and vascularpermeability. J Soc Gynecol Investig 2000;7:51–60.