|Year : 2011 | Volume
| Issue : 4 | Page : 419-423
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
|Date of Submission||03-Aug-2010|
|Date of Decision||16-Mar-2011|
|Date of Acceptance||25-Apr-2011|
|Date of Web Publication||22-Jul-2011|
N K Lohiya
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur
Objective: To evaluate the status of fertility, developmental stages during gestation and teratological changes, if any, following oral administration of methanol sub-fraction (MSF) of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats.
Materials and Methods: The MSF was administered at the doses of 50 mg contraceptive dose (CD), 100 mg (2x CD), 250 mg (5x CD) and 500 mg (10x CD)/kg body wt/day along with vehicle-treated control using 10 male and 20 female Wistar rats in each group. Necropsies were performed one day before the expected parturition. Status of gravid/non-gravid uterus, the number of corpora lutea in the ovary, implantation status, fetal wellbeing, fetal resorption, fetal body weight, external, visceral and skeletal malformations were recorded.
Results: Pregnancies were recorded in vehicle-treated control animals and in the animals treated with 50 mg/kg body wt/day. The animals treated with 2x CD, 5x CD and 10x CD did not get pregnant. The fetuses and the status of the ovary, uterus and implantation, fetal body weight, soft tissues and skeletal structures were recorded normal. Data were comparable to those of control.
Conclusion: The results suggest that the test substance had no developmental toxicity and teratogenicity which could affect pregnancy, implantation and gestation.
Keywords: Carica papaya seeds, methanol sub-fraction, gravid uterus, implantation, teratogenicity
|How to cite this article:|
Shrivastava S, Ansari A S, Lohiya N K. Fertility, developmental toxicity and teratogenicity in albino rats treated with methanol sub-fraction of Carica papaya seeds. Indian J Pharmacol 2011;43:419-23
|How to cite this URL:|
Shrivastava S, Ansari A S, Lohiya N K. Fertility, developmental toxicity and teratogenicity in albino rats treated with methanol sub-fraction of Carica papaya seeds. Indian J Pharmacol [serial online] 2011 [cited 2013 May 23];43:419-23. Available from: http://www.ijp-online.com/text.asp?2011/43/4/419/83113
| » Introduction|| |
The seed extracts of Carica papaya have been proven to possess male antifertility properties.  Treatment with the chloroform extract of the seeds of Carica papaya showed a total inhibition of sperm motility in rats  and langur monkeys,  while in rabbits it exhibited uniform azoospermia.  Purified benzene chromatographic fraction of the chloroform extract inhibited cauda epididymal sperm motility, reduced sperm density and altered sperm morphology in rats and rabbits. , 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 , and azoospermia in rabbits leading to contraception without adverse systemic effects.  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.  Moreover, long-term daily administration up to 10 times the CD for two years in rats indicated no associated health hazards.  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.  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. 
| » Materials and Methods|| |
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  were strictly followed. The experimental protocol was approved by the Institutional Ethical Committee.
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. 
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.
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.
The body weight was noted weekly during the entire study period.
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  and uterine weight, number of corpora lutea, number of implantations, early resorptions/embryonic deaths and late resorptions/fetal deaths were observed.
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:
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.
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.
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. 
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.  The skeletal malformations, viz., skull, vertebral column, sternebrae, forelimbs and hind limbs, fore and hind paw, etc., if any, were observed using magnifying glass.
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).
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.
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].
|Table 1: Pregnancy, implantation and offspring records of female rats mated with male rats treated with different doses of MSF Carica papaya seeds|
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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].
|Figure 1: Gravid uteri of vehicle (Group I) and MSF (Group IIb)-treated female rats at 50 mg /kg body wt./day; p.o. Uterine horns of both groups appeared alike|
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Developmental Toxicity and Teratogenicity
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 examination of all fetuses revealed no abnormality in Groups IIa and IIb when compared with vehicle-treated control rats.
There were no noticeable visceral malformations observed in the offspring of vehicle-treated control as well as in Group IIa and IIb rats.
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].
|Figure 2: Evaluation of skeletal malformation in fetuses by Alizarin – Red S & Alcian blue double staining of vehicle (Group I) and MSF (Group IIb)-treated female rats at 50 mg /kg b.wt./day; p.o. Note normal skeletal morphology in both groups|
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| » 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.,  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.  Raji and associates  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  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  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.  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.,  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|| |
|1.||Lohiya NK, Manivannan B, Mishra PK, Pathak N. Prospects of developing a plant based male contraceptive pill. Current status in fertility regulation: Indigenous and modern approaches. Lucknow: Central Drug Research Institute; 2001. p. 99-119. |
|2.||Lohiya NK, Goyal RB. Antifertility investigations on the crude chloroform extract of Carica papaya Linn. seeds in male albino rats. Indian J Exp Biol 1992;30:1051-5. |
|3.||Lohiya NK, Manivannan B, Mishra PK, Pathak N, Sriram S, Bhande SS, et al. Chloroform extract of Carica papaya induces long-term reversible azoospermia in langur monkey. Asian J Androl 2002;4:17-26. |
|4.||Lohiya NK, Pathak N, Mishra PK, Manivannan B. Reversible contraception with chloroform extract of Carica papaya Linn. seeds in male rabbits. Reprod Toxicol 1999a;13:59-66. |
|5.||Pathak N, Mishra PK, Manivannan B, Lohiya NK. Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats. Phytomedicine 2000;7:325-33. |
|6.||Lohiya NK, Mishra PK, Pathak N, Manivannan B, Jain SC. Reversible azoospermia by oral administration of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rabbits. Adv Contracept 1999b;15:141-61. |
|7.||Lohiya NK, Manivannan B, Goyal S, Ansari AS. Sperm motility inhibitory effect of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in langur monkey, Presbytis entellus entellus. Asian J Androl 2008;10:298-306. |
|8.||Lohiya NK, Manivannan B, Garg S. Toxicological investigations on the methanol sub-fraction of the seeds of Carica papaya as a male contraceptive in albino rats. Reprod Toxicol 2006;22:461-8. |
|9.||Goyal S, Manivannan B, Ansari AS, Lohiya NK. Safety evaluation of long term treatment of methanol sub-fraction of seeds of Carica papaya as a male contraceptive with particular emphasis on carcinogenicity in albino rats. Int J Pharmacol 2009;5:114-25. |
|10.||Manivannan B, Mittal R, Goyal S, Ansari AS, Lohiya NK. Sperm characteristics and ultrastructure of testes of rats after long-term treatment with the methanol subfraction of Carica papaya seeds. Asian J Androl 2009;11:583-99. |
|11.||Office of Prevention, Pesticides and Toxic Substances 870.3550. Health effects test guidelines. Reproduction/developmental toxicity screening test guidelines for developmental toxicity risk assessment. United States Environmental Protection Agency 712-C-00-367. 2000. |
|12.||Indian National Science Academy. Guidelines for care and use of animals for scientific research. New Delhi: INSA; 2000. |
|13.||Bhande S. Contraceptive Evaluation and Toxicological Screening of the Isolated Compounds of the Seeds of Carica papaya in Langur Monkey - A Preclinical Study. PhD thesis. Jaipur: University of Rajasthan; 2004. |
|14.||Kopf R, Lorenz D, Salewski E. The effect of thalidomide on the fertility of rats in reproduction experiments over 2 generations. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 1964;247:121-35. |
|15.||Monie LW, Kho KG, Morgan J. Dissection procedures for rat fetuses permitting alizarin red staining of skeleton and histological study of viscera. Supplement to teratology workshop manual. California, Berkeley; 1965. |
|16.||Taylor P. Practical Teratology. New York: Academic Press; 1986. |
|17.||Al-Hamood MH, Elbeteiha A, Alkofahi A, Bataineh H. Reproductive toxicity potentials of Salvia fruticosa (Labiatae) in rats. J Ethnopharmacol 1998;61:67-74. |
|18.||Anuar NS, Zahari SS, Taib IA, Rahman MT. Effect of green and ripe Carica papaya epicarp extracts on wound healing and during pregnancy. Food Chem Toxicol 2008;46:2384-9. |
|19.||Raji Y, Morakinyo AO, Akinsomisoye OS, Oloyo AK, Esegbue-Peters PR, Kunle-Alabi OT. Evaluation of the abortifacient properties of chloroform extract of Carica papaya L. seed in female albino rats. Int J Phamacol 2005;2:20-3. |
|20.||Oderinde O, Noronha C, Oremosu A, Kusemiju T, Okanlawon OA. Abortifacient properties of aqueous extract of Carica papaya Linn. seeds on female Sprague Dawley rats. Niger Postgrad Med J 2002;9:95-8. |
|21.||Schmidt H. Effect of papain on different phases of prenatal ontogenesis in rats. Reprod Toxicol 1995;9:49-55. |
|22.||Moallem SA, Niapour M. Study of embryotoxicity of Perovskia abrotanoides, an adulterant in folk-medicine, during organogenesis in mice. J Ethnopharmacol 2008;117:108-14. |
|23.||Gebrie E, Makonnen E, Debella A, Zerihun L. Phytochemical screening and pharmacological evaluations for the antifertility effect of the methanolic root extract of Rumex steudelii. J Ethnopharmacol 2005;96:139-43. |
[Figure 1], [Figure 2]