Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 4, 2011, pp. 419-423

Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 419-423

Research Article

Fertility, developmental toxicity and teratogenicity in albino rats treated with methanol sub-fraction of Carica papaya seeds

S Shrivastava, AS Ansari, NK Lohiya

Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur, India

Correspondence Address: N K Lohiya Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur India lohiyank@hotmail.com

Date of Submission: 03-Aug-2010
Date of Decision: 16-Mar-2011
Date of Acceptance: 25-Apr-2011

Code Number: ph11112

DOI: 10.4103/0253-7613.83113

Abstract

Keywords: Carica papaya seeds, methanol sub-fraction, gravid uterus, implantation, teratogenicity

Introduction

The seed extracts of Carica papaya have been proven to possess male antifertility properties. ^[1] Treatment with the chloroform extract of the seeds of Carica papaya showed a total inhibition of sperm motility in rats ^[2] and langur monkeys, ^[3] while in rabbits it exhibited uniform azoospermia. ^[4] Purified benzene chromatographic fraction of the chloroform extract inhibited cauda epididymal sperm motility, reduced sperm density and altered sperm morphology in rats and rabbits. ^[5],[6] The methanol sub-fraction of the benzene chromatographic fraction of the chloroform extract of Carica papaya seeds (MSF) exhibited sperm motility-inhibiting property in rats and langur monkeys ^[1],[7] and azoospermia in rabbits leading to contraception without adverse systemic effects. ^[1] Toxicological investigations carried out so far with MSF following a single high dose or daily administration up to 10 times of contraceptive dose (CD) for a period of 28 and 90 days in rats indicated no major changes in biochemical markers of reproductive organs, hematological and clinical parameters. ^[8] Moreover, long-term daily administration up to 10 times the CD for two years in rats indicated no associated health hazards. ^[9] The MSF is safe for long-term treatment and the mechanism of contraception is shown by its effect on spermatid differentiation in the testis, possibly mediated by the Sertoli cell factors. ^[10] The present study was undertaken to investigate the developmental toxicity and teratogenicity of the methanol sub-fraction of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in albino rats following the guidelines of reproduction/developmental toxicity. ^[11]

Materials and Methods

Animals

Adult Wistar albino rats, three to four months old, weighing between 150 to 180 g were used and maintained in the animal house, with 12:12 h light and dark schedule. Rats were housed in polypropylene cages (size 43 x 27 x 15 cm), fed with rat pellet diet (M/s Ashirwad Industries Limited, Chandigarh, India) and had free access to safe drinking water. The Guidelines for Care and Use of Animals in Scientific Research of the Indian National Science Academy, New Delhi ^[12] were strictly followed. The experimental protocol was approved by the Institutional Ethical Committee.

Test Material

The seeds of Carica papaya L (family: Caricaceae, Voucher No. RUBL 16590) of pure honey dew variety were obtained commercially, shade-dried and powdered. The powdered material was refluxed in chloroform for 12 x 3 h at 58-60°C. The resultant chloroform extract was subjected to silica gel column chromatography (60-120 mesh) eluted with benzene. The benzene chromatographic fraction of the chloroform extract was concentrated under reduced pressure and sub-fractionated with methanol, and termed as methanol sub-fraction.

Characterization of the Carica papaya seed products through ¹ H-NMR, IR, Mass Spectra (E1/C1/FAB) and Gas Chromatography revealed several sub-components and appeared to be a mixture of compounds. Spectral data showed evidences of long-chain fatty alcohols, long-chain ester, glycerides of unsaturated fatty acids and long-chain unsaturated acids. Based on the available evidences, the most probable fatty acids could be saturated myristic acid with molecular formula (CH ₃ [CH ₂ ] ₁₂ COOH), palmitic acid with molecular formula (CH ₃ [CH ₂ ] ₁₄ COOH) and stearic acid with molecular formula (CH ₃ [CH ₂ ] ₁₆ COOH). Further purification through analytical High Pressure Liquid Chromatography (HPLC) using the solvent system acetonitrile: tetrahydrofuran: methanol: water in 45:5:35:15 ratio resulted into non-homogenous mixture of several compounds. ^[13]

The methanol sub-fraction (MSF) of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya, that possesses the desired antifertility activity in all animal trials, was used as an Investigational New Drug (IND), for preclinical reproductive toxicity testing.

Experimental Design

Adult male rats were divided into following five groups of 10 animals each:

Group A : The animals served as the vehicle-treated control

Group B: The animals were treated orally with 50 mg contraceptive dose (CD) MSF/kg body wt./day

Group C: The animals were treated orally with 100 mg (2 x CD) MSF/kg body wt./day

Group D: The animals were treated orally with 250 mg (5 x CD) MSF/kg body wt./day

Group E: The animals were treated orally with 500 mg (10 x CD) MSF/kg body wt./day

Adult female rats were divided into following groups of 20 animals each:

Group I: The animals served as the vehicle-treated control and mated with males of Group A

Group IIa: The female rats served as untreated control and mated with five males of Group B

Group IIb: The animals were treated orally with 50 mg contraceptive dose (CD) MSF/kg body wt./day and mated with the remaining five males of Group B

Group IIIa: The female rats served as untreated control and mated with five males of Group C

Group IIIb: The animals were treated orally with 100 mg (2 x CD) MSF/kg body wt./day and mated with the remaining five males of Group C

Group IVa: The female rats served as untreated control and mated with five males of Group D

Group IVb: The animals were treated orally with 250 mg (5 x CD) MSF/kg body wt./day and mated with the remaining five males of Group D

Group Va: The female rats served as untreated control and mated with five males of Group E

Group Vb: The animals were treated orally with 500 mg (10 x CD) MSF/kg body wt./day and mated with the remaining five males of Group E

The male rats were dosed for a minimum period of 28 days that included a minimum of 14 days prior to mating, during the mating period and approximately 14 days post-mating. The female rats were treated 14 days prior to mating with the objective of covering at least two complete estrous cycles, during the mating period and from P0 (i.e., the day on which spermatozoa were seen in vagina) to 20 days of pregnancy (P20). The following observations were carried out:

Visible Toxicological Symptoms

Mortality and morbidity, changes in fur, skin, eyes and mucous membrane, tremors, convulsions, salivation, diarrhea, lethargy, animal behavior, feeding pattern, changes in the level of motor activity, gait and posture, reactivity to handling or sensory stimuli, grip strength and bizarre behavior such as self-mutilation, walking backward, etc., were recorded daily in all groups. Libido was recorded weekly in male animals.

Food and Water Intake

Individual consumption of food and water was measured daily. Rats were fed individually with 150 g of rat pellets and 200 mL of drinking water. On the next day the remaining food and water were measured and the consumed food and water was recorded.

Body Weight

The body weight was noted weekly during the entire study period.

Terminal Sacrification

All the pregnant rats on Day 20 of gestation were sacrificed with an overdose of diethyl ether after completion of the experiment schedule. Uterine horns and ovaries were removed, freed from fat and adherent tissues. Non-gravid uteri were subjected to ammonium sulphide staining for confirmation of non-pregnant status ^[14] and uterine weight, number of corpora lutea, number of implantations, early resorptions/embryonic deaths and late resorptions/fetal deaths were observed.

Fetal Observations

The fetuses were removed by opening the uterus and were placed in a sequential manner in 0.9% saline solution. All dead and live fetuses were counted and the following observations were made:

Sex

The sex of the fetus was identified by observing the anogenital distance. The numbers of male and female fetuses were counted and sex ratio was noted.

Fetal Body Weight and Length

Individual fetuses were weighed to the nearest milligram on an electronic balance and fetal length (crown to rump) was measured.

External Examination

The fetuses were sacrificed using diethyl ether vapor. All the fetuses were examined for external malformations in an orderly manner starting from head, face, nostrils, eyes, external ears (pinna), trunk to tail and limbs.

Visceral Examination

Half the number of male and female fetuses from each group were fixed overnight in 70% ethanol and examined by the modified Wilson′s technique/fetal necropsy. ^[15]

Skeletal Examination

The remaining (half) number of the fetuses from each of the control and treated groups were skinned, fixed in 70% ethanol, macerated in 1% KOH and stained with alcian blue and Alizarin red S according to the method of Taylor. ^[16] The skeletal malformations, viz., skull, vertebral column, sternebrae, forelimbs and hind limbs, fore and hind paw, etc., if any, were observed using magnifying glass.

Statistical Analysis

Values were represented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was employed for statistical comparison. The difference between means was analyzed by the Holm-Sidak multiple comparison test to detect the inter-group difference by using the statistical software SPSS Version 10.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered significant.

Results

Visible Toxicological Symptoms

No pre-terminal deaths were recorded. Daily observation of morphological characteristics, viz., skin, fur, eyes and nose showed normal characteristics. No neurological symptoms like tremors, convulsions, autonomic activity, viz., salivation, lethargic and bizarre behavior such as self-mutilation, walking backward, etc. were observed. Diarrhea was observed, temporarily in one male rat each in Groups A and D and in one female rat in Group IIb during the initial treatment period. Motor activity, gait and posture, reactivity to handling or sensory stimuli and grip strength were recorded normal. All rats were active throughout the treatment period.

Food and Water Intake

Food and water intake of vehicle-treated control and MSF-treated rats fluctuated throughout the investigation in all males and non-pregnant female rats of Groups III, IV and V. However, in pregnant female rats, in Groups I, IIa and IIb food and water intake were significantly increased (P < 0.001).

Body Weight

A gradual significant increase (P < 0.001) in the body weight gain was observed in pregnant females of Groups I, IIa and IIb as compared to pre-mating weights. Daily oral administration of MSF in male and female rats (Groups III-V) showed the initial and final body weights comparable to those of vehicle-treated control rats.

Pregnancy Record

No pregnancy was observed in all female rats in Groups III-V. However, in Groups IIa and IIb, the pregnancy record, viz., weights of gravid uterus, ovary and placenta and the fetal sex ratio was comparable to those of vehicle-treated control [Table - 1].

Implantation Status

Post-implantation loss was minimum and not different in Groups IIa and IIb when compared to vehicle-treated control [Table - 1]. The gravid uterine horns with healthy implantations were observed in pregnant females of both Groups I and IIb [Figure - 1].

Developmental Toxicity and Teratogenicity

Offspring Record

The litter size, body weight and body length of litters in vehicle-treated control, respectively, were 6.80 ± 0.69, 4.49 ± 0.32 g and 4.41 ± 0.24 cm. No significant difference was noticed in the litter size, body weight and body length of litters of Groups IIa and IIb when compared with vehicle-treated control [Table - 1].

External Malformations

External examination of all fetuses revealed no abnormality in Groups IIa and IIb when compared with vehicle-treated control rats.

Visceral Malformations

There were no noticeable visceral malformations observed in the offspring of vehicle-treated control as well as in Group IIa and IIb rats.

Skeletal Malformations

No noticeable skeletal and teratogenic malformations in the skull, vertebral column, sternebrae, forelimbs, hind limbs, fore and hind paws were observed in the fetuses of vehicle-treated control as well as in Group IIa and in IIb rats [Figure - 2].

Discussion

Herbal drugs have been used since ancient times as medicines for the treatment of a wide range of diseases. One of the major challenges in drug development is the accurate assessment of human drug toxicity. In the history of male contraceptive research, three plant products, viz. gossypol, Tripterygium wilfordii and Ecballium elaterium, claimed to have better contraceptive efficacy in clinical trials. However, they failed to reach the level of commercial launch due to the side-effects.

In the present study, the pregnancy record in animals treated with 50 mg MSF/kg body wt. was comparable to that of vehicle-treated control. This is in agreement with Al-Hamood et al., ^[17] who observed that the ingestion of aqueous extract (800 mg/kg) or ethanolic extract (400 mg/kg) of Salvia fruticosa for 30 consecutive days by female rats had no effect on the occurrence of pregnancy. However, the higher dose of the drug caused sterility within 15 days of the pre-mating study period. Therefore, observation on teratogenic effects was not feasible.

Several studies on plants have been reported for reproduction and developmental toxicity. Oral administration of aqueous extract of ripe papaya epicarp (RPE), at the dose regimen of 1 mg/g body weight/day, to pregnant mice from day 10 and onwards after conception resulted into embryonic resorption. Whereas, the average body weight of live pups delivered by mice following the treatment with aqueous extract of green papaya epicarp (GPE) was significantly lower. ^[18] Raji and associates ^[19] reported that oral administration of chloroform extract (25 and 50 mg/kg body wt./day for two weeks pre-coital) of Carica papaya seeds exhibited antiimplantational and abortifacient properties in female rats. Oderinde and associates ^[20] reported that the aqueous extract of Carica papaya seeds at the dose regimen of 100 mg/kg body wt. once a day on Days 1-10 post-coitum orally in Sprague-Dawley rats, significantly decreased fetal weight. At 800 mg/kg body wt. dose regimen there were complete resorptions of about 30% of fetuses. In the present study, there were no significant differences in implantation status and offspring record in untreated control and MSF treated (50 mg/kg body wt./day) rats. The findings are in agreement with Schmidt ^[21] who observed that the oral administration of papain up to a dose of 800 mg/kg from Days 0-6 of gestation did not adversely affect prenatal development and did not cause signs of maternal toxicity.

Teratogenic effects of a number of medicinal plants have been reported. The ethanolic and aqueous extracts of Perovskia abrotanoides, respectively, at the dose 0.125 and 0.25 g/kg during organogenesis in mice caused minimal maternal toxicity, while resorption, stillborn, fetal malformations and numerous skeletal abnormalities were detected particularly in the ethanolic extract groups. ^[22] In our experiment no external, visceral and skeletal malformations were recorded in untreated control and MSF (50 mg/kg body wt./day) treated groups when compared with vehicle treated control group. Our findings are in agreement with Gebrie et al., ^[23] who reported that the methanolic extract of the Rumax steudelii root at the dose 2.2 g/kg body wt. administered intragastrically for 10 days from Day 1 to Day 10 has antifertility effect but did not show teratogenic effect in rats. In conclusion, MSF at the contraceptive dose regimen exerted no developmental toxicity and teratogenicity which could affect pregnancy, implantation and gestation.

Acknowledgments

The investigation was supported by the Indian Council of Medical Research, New Delhi. The authors are grateful to the Head/Coordinator, Centre for Advanced Studies (CAS), Department of Zoology, University of Rajasthan, Jaipur for providing infrastructural facilities.

References

Copyright 2011 - Indian Journal of Pharmacology

The following images related to this document are available:

Photo images