Antifertility efficacy of Drynaria quercifolia (L.) J. Smith on female Wister albino rats

Journal of Ethnopharmacology 153 (2014) 424–429

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Research Paper

Antifertility efficacy of Drynaria quercifolia (L.) J. Smith on female Wister albino rats Banani Das a, Amitabha Dey b, Anupam Das Talukdar a, Kh. Nongalleima b, Manabendra Dutta Choudhury a, Lokesh Deb b,n a

Ethnobotany and Medicinal Plant Research laboratory, Dept.Department of Life Science and Bioinformatics, Assam University, Silchar 788011, India Pharmacology Laboratory, Natural Product Chemistry and Pharmacology Programme, Institute of Bioresources and Sustainable Development (IBSD), Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur 795001, India

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art ic l e i nf o

a b s t r a c t

Article history: Received 15 September 2013 Received in revised form 25 December 2013 Accepted 20 February 2014 Available online 4 March 2014

Ethnopharmacological relevance: Plant based traditional medicines for birth control has been in practice in rural populations of North-East India, since time immemorial. Fresh rhizome of Drynaria quercifolia (L.) J. Smith is one of the plant parts used traditionally by different ethnic communities in Tripura, India for birth control. The present investigation is aimed to justify the scientific basis in traditional use of Drynaria quercifolia rhizome as anti-fertility agent. Materials and methods: Ex-vivo uterotonic activity was done on uterine tissue. Acute toxicity test of the plant extract was carried out in rats of both sexes. The abortifacient and anti-implantation activities of the extract were investigated, in-vivo and estimated the level of different hormones release. Results: The result revealed the effectiveness of methanol (87%) and aqueous (68%) extract of the plant on uterotonic activity. The extracts showed relatively non-toxic effect in acute toxicity study. Methanolic extract has shown higher efficacy for both abortifacient (nnpo 0.01) and anti-implantation performance (nnpo 0.01) and also effected hormone release level (nnpo 0.01). Conclusion: Methanolic extract of Drynaria quercifolia (L.) J. Smith rhizome has been proved to have significant anti-fertility activity. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Medicinal plants Antifertility agent Contraceptive Drynaria quercifolia (L.) J. Smith

1. Introduction Growing population is one of the major problems that the developing countries are facing at present in the present days. The current world population is 7.2 billion and India represents 17.3% of total world population. Worldwide explosion of population projected to increase by almost one billion people within the next twelve years, reaching 8.1 billion in 2025 and 9.6 billion in 2050. The population is expected to become 1.40 billion by 2025 in India (UN Press Release, 2012). Because of this, the resources in developing countries are falling short leading to economic crisis. As such it is the urgent need to control population growth, in order to ensure food, shelter and better health to those countries. Scientists have started tackling this serious problem by developing antifertility agents. With the advent time, hormonal (synthetic) oral contraceptives, specifically containing estrogen and progesterone; has created mounting popularity. But the risks associated with these contraceptives (Petiti et al., 1996; Lewis et al., 1997;

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Corresponding author. Tel: þ 91 3852051278x255; mobile: þ 91 9436890969. E-mail addresses: [email protected] (M.D. Choudhury), [email protected] (L. Deb). http://dx.doi.org/10.1016/j.jep.2014.02.047 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

Stampfer et al., 1998), which have triggered the need to develop new alternatives, with lesser side effects. Most of the plant based contraceptives are self-administrable and reversible (Bhattacharya and Saha, 2013) in action. Plants based drug showing antifertility activity are mainly due to their content of estrogenic compounds which act as suppressor of gonadotrophin secretion, with consequent inhibition of ovulation (Laurence and Bacharach, 1964; Bullock et al., 1995; Soejarto et al., 1978). Hence the fertility regulatory activity of the plants in females may be due to alternation of steroids levels. Drynaria quercifolia (L.) J. Smith belongs to the family Polypodiaceae of Pteridophyta which is a promising plant in this regard. The rhizome of the plant is reported to be used by different ethnic groups of India as a natural source of anti-fertility agent (Rajendran and Rajan, 1996; Das et al., 2012). It is also used for the treatment of various other ailments such as diarrhoea, typhoid, cholera, liver disease, fever, skin diseases and headache (Chopra et al., 1956; Nandkarni, 1976; Nejad and Deokule, 2009). The ‘Tripuri’ tribe of Tripura state uses both the leaves and rhizome of this plant to treat intestinal worms and abdominal pain (Das et al., 2009). Rhizome is also reported to be effective for rheumatic pain, lower the back and ligament injuries and used topically in traditional Chinese medicine to stimulate hair growth to treat baldness and having anti-inflammatory action (Asolkar et al., 1956; Nandkarni, 1976; Irudayaraj and Senthamarai, 2004). But

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antifertility activity of rhizome of this plant has escaped from proper scientific attention, though it was available in practice. Therefore, the scientific validation is important. Further, the information regarding isolation of chemical compounds with contraceptive efficacy from this plant is also lacking, though 3,4-dihydroxybenzoic acid (Khan et al., 2007), Friedelin, epifriedelinol, β-amyrin, β-sitosterol, β-sitosterol 3-βD-glucopyranoside and naringin (Ramesh et al., 2001) have been reported from different parts of this plant. With this rationale at the backup, we considered to revalidate the traditional uses of Drynaria quercifolia (rhizome) as contraceptive, following reverse pharmacology method.

2. Materials and methods

425

intervals (Calixto et al., 1991). The contractile activity was then measured using an isometric force transducer (KYMOGRAPH, INCO E8REC DRUM). An isotonic contraction of the uterine muscle with of the standard drug oxytocin (0.01 ml) was conducted. Subsequently, the effect of the extracts EADQ, ACDQ, MEDQ and AEDQ (50 ml) with oxytocin (0.01 ml) was observed, where 10:1 was the extract concentration in medium and concentration–response curves (CRC) were constructed. Extracts were added to the organ bath and left for 5 min before exposure of the tissue to the oxytocin, maintaining contact time of 60 s. The responses of the extract with oxytocin were compared with that of oxytocin (reference standard drug) response. 2.5. Determination of acute toxicity (ALD50)

Drynaria quercifolia (L.) J. Smith rhizomes was collected from Dharmanagar, Tripura, North-East India in the month of July, 2011 and authenticated by curator of the Assam University Herbarium (AUS/2503). The rhizomatous part of the material was cleaned; shade dried and reduces into coarse powder form with the help of an electrical blender.

The acute toxicity for MEDQ and AEDQ was determined in albino rats, maintained under standard conditions. The animals (n¼3) in each group was fasted overnight prior to the experiment. The fixed dose (OCED Guideline no. 423 and Annexure 2b) method of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was adopted for toxicity studies. The extracts in suspension were administrated orally. The mortality and behavioral abnormality were observed after administering samples at a dose of 2000 mg/kg in all animals (Veeraraghavan, 2001).

2.2. Extraction and sample preparation

2.6. Determination of abortifacient activity

The powdered rhizome of Drynaria quercifolia (5 kg) was obtained and soaked in petroleum ether to remove wax and fat materials and then subjected to successive soxhlet extraction with ethyl acetate, acetone, methanol and water one after another as solvents in 1:4 (w/v) ratios which were standardize in laboratory condition before extraction. The respective extracts (Pet-ether extracts¼PEDQ, ethyl acetate extracts¼EADQ, acetone extract¼ ACDQ, methanol extracts¼MEDQ and aqueous extract¼AEDQ) were concentrated under reduced pressure in vacuum evaporator (Buchi Rotavapors R-210) and dried in vacuum desiccators. Following drying all products was stored in refrigerator (872 1C) and same extracts were used for present invivo and in vitro studies.

Mature fertile female Wister rats (150–200 g) were selected for this study and were left overnight with fertile male Wister rat in the ratio of 3:1 (female:male). Every next morning vaginal smear was taken with normal saline and examined under microscope for detection of spermatozoa. Mating was confirmed by the presence of spermatozoa in the vaginal smear and considered as pregnancy day one (Oshima et al., 1987). Rats at day 1 of pregnancy were divided into four groups, consisting of six animals in each group. On the tenth day laparotomy was carried out under ketamine anesthesia and semisterile condition. The uteri were examined to determine the number of implantation sites. The number of corpora lutea in ovaries was also recorded. The abdomen was sutured and animals left in cages. The group I served as normal and received vehicle only (0.5 ml distilled water/day). Group II serves as standard (Ethinyl estradiol 0.1 mg/Kg) and test groups III and IV received suspension of 200 mg/kg of MEDQ and AEDQ respectively from day 10th to 18th of pregnancy. During the experiment animals were observed for vaginal bleeding. On 21st day, animals were again laparotomised under ketamine anesthesia and observed for number of litters and percentage of resorption (Dhanasekaran et al., 1993; De Freitas et al., 2005) and blood was drawn by cardiac puncture. The blood was allowed to coagulate for an hour. The separation of the serum from other cellular components of the blood was done by centrifuging the coagulated blood at 380g for 10 min. The sera were collected and stored in deep freezer ( 20 1C). After 1 week the sera were analyzed for Estradiol (E2) and Progesterone, Gonadotropic Releasing Hormone (GnRH), Lutenizing Hormone (LH), by using Enzyme-linked Immunosorbent Assay (ELISA) commercial kit, which read in 96 micro-plate reader (MULTISKAN SPECTRUM, Thermo Scientific) and Prostaglandin-F2α (PGF2α) and ProstaglandinE2 (PGE2) were analysed by Enzyme Immuno Assay (EIA) using EIA commercial kits. Uterus of all the animal groups were removed and rinsed in cold PBS and stored at  70 1C until used.

2.1. Plant materials

2.3. Animals Wister albino rats weighing 150–200 g of either sex were used in this study. They were procured from Regional Institute of Medical Sciences (RIMS), Imphal. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages and maintained at 27 1C72 1C and 12 h dark/light cycle. They were fed with soya bean choke, Gram and water ad libitum. The litter in the cages was cleaned daily to ensure hygienic condition and maximum comfort for animals. Ethical clearance for handling the animals was obtained from the Institutional Animals Ethical Committee (IAEC), IBSD, Imphal (approval No.–IBSD/IAEC/Trainee/Ph.cology/9) prior to the beginning of the study. 2.4. Ex-vivo assay for uterine contractile activity Female rat (300–500 g) during metaoestrus stage (induced by injecting 0.1 mg/kg S.C. and wait for 24 h) of estrous cycle was sacrificed by cervical dislocation, and the two horns of the uterus were dissected out and freed from surrounding tissues. One horn of uterus was mounted on De Jalon's Solution (DJS) with the following composition (Mmol) NaCl 154.0, KCl 5.6, CaCl2, 0.5, NaHCO3, 6.0 and glucose 2.8. This solution was constantly aerated with an aerator. The bath temperature was adjusted at 3272 1C and uterine segment was placed under optimum resting tension of 0.75 g and equilibrated for 45 min before the start of the experiment. During the equilibration period the tissue preparation was washed with DJS in every 10 min

2.7. Determination of anti-implantation activity Mature fertile female Wister rats (150–200 g) were selected for this study and were left overnight with fertile male Wister rat in the ratio of 3:1 (female:male). Every next morning vaginal smear was taken with normal saline and examined under microscope until detection of spermatozoa. Mating was confirmed by the

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presence of spermatozoa in the vaginal smear and considered as pregnancy day one. After mating all the female rats were grouped accordingly and each group contained six female rats. Group I (Normal) animals were treated orally with vehicle (0.5 ml of distilled water/day). Group II served as standard (Ethinyl estradiol 0.1 mg/kg/day, orally). Groups III and IV animals were administered orally with 200 mg/kg suspension of MEDQ and AEDQ respectively from day 1 to 7 of pregnancy (Dhanasekaran et al., 1993). On day 10th of pregnancy animals were sacrificed with cervical dislocation and uterine horns were inspected for number of implants (Khanna and Chaudhury, 1968). Blood was drawn and allowed to coagulate. The separation of the serum from other cellular components of the blood was done by centrifuging the coagulated blood at 380g for 10 min. The sera were collected and stored in deep freezer (  20 1C) prior to its enzymatic and hormonal estimation. The sera from each group were analyzed for the presence of Estradiol (E2), Progesterone, GnRH and LH by the methods of Enzyme linked Immunosorbent Assay (ELISA) using ELISA commercial kits. 2.8. Statistical analysis Data were expressed as mean7Standard Error Mean (SEM). Differences were considered significant at nnnpo0.001, or nnpo0.01 or n po0.05 when comparing test groups vs control group. For numerical results, one-way analysis of variance (ANOVA) with Dunnett's test was performed using GraphPad InStat Version 3 (GraphPad Software).

3. Results 3.1. Ex-vivo assay for uterine contractile activity Initially all test extracts (EADQ, ACDQ, MEDQ, and AEDQ) were produced a tension in the uterine tissue and a synergistic effect Table 1 Ex-vivo uterine contractile activity. Sample Oxytocin Oxytocin Oxytocin Oxytocin Oxytocin

% Agonism (0.01 ml) (0.01 ml) þ EADQ (50 ml) (0.01 ml) þ ACDQ (50 ml) (0.01 ml) þ MEDQ (50 ml) (0.01 ml) þ AEDQ (50 ml)

 9 25 87 68

was observed on tension of the tissue upon addition of oxytocin in organ bath. The intrinsic activity of the extracts was obtained higher in combination with oxytocin. The percentage agonistic effect of EADQ, ACDQ, MEDQ, and AEDQ were calculated to be 9%, 25%, 87% and 68% respectively (Table 1).

3.2. Determination of acute toxicity study The test samples MEDQ and AEDQ employed in acute toxicity study did not show any sign of abnormality such as respiratory distress, salivation, weight loss, dull eyes, diarrhoea and any change in the appearance of fur and mortality at the dose of 2000 mg/kg body weight in experimental animals for a period of 48 h primarily and then after continuous 7 days observation. Therefore 2000 mg/kg body weight (BW) dose was considered as average lethal dose 50 (ALD50), cut off the dose under Globally Harmonised Classification System 5 (safe dose) for Chemical Substances and Mixtures described in OECD guideline 423 (Annexure 2b and 3b). However, in the present study 1/10th of ALD50 cut off dose i.e. 200 mg/kg body weight/days dose was selected as therapeutic dose for in-vivo experiments.

3.3. Abortficient activity Both the plant extracts at 200 mg/kg dose have shown good abortificient activity, where survival ratio was 20.12% and 76.47% for MEDQ and AEDQ treated groups respectively as against 100% in the control animals (Table 2). On the contrary, the feed and water intake was not altered in all the groups. The number of live fetus was decreased significantly (nnpo0.01) in the animals treated only with the MEDQ whereas, no live fetus was recorded in the animals treated with standard (Ethinyl estradiol 0.1 mg/kg body weight). There was major variation in implantation index among standard treated and extracts (AEDQ and MEDQ) treated groups compare to control group. The resorption indices were 99.52%, 47.09% and 97.65% in the standard, AEDQ and MEDQ treated animals, respectively; whereas it was 0 in the control animals. Differences were observed in the preimplantation loss and post-implantation loss in all the treated groups. Details of the results are illustrated in (Table 2). In other assay we observed a significant (nnpo0.01) difference in the level of PGF2α in the serum of all treated groups compared to control animals. Whereas, there was minimum differences in the level of PGE2 in the extracts treated groups as compared to control animals (Table 3).

Table 2 Effect of test samples (AEDQ and MEDQ) on abortifacient activity. Parameters

Normal (0.5 ml distilled Standard (Ethinyl estradiol water/day) 0.1 mg/kg/day)

AEDQ (200 mg/ kg bw/day)

MEDQ (200 mg/ kg bw/day)

Maternal initial weight (g) Maternal final weight (g) Number of live fetus Number of dead fetus Survival ratio (%) {(Live/Dead)  100} Number of rats that aborted Percentage of rats that aborted (%) Number of implantations Number of corpora lutea Implantation index (%) Resorption index (%) Pre-implantation loss (%) {(No. of corpora lutea No. of implantation)/No. of corpora lutea}  100 Post-implantation loss (%) {(No. of implantation  number of live fetus)/No. of implantation}  100

140.5 72.849 150.3 74.153 4.0 7 0.91 07 0 100 0 0 4.137 1.53 5.82 7 2.31 70.96 0 29.03

149.83 73.885 150.5 7 4.522 07 0nn 2.09 7 0.70 0 6 100 2.17 3.21 4.09 7 1.54 51.34 99.52 48.65

147.83 7 2.587 159.337 2.906 1.3 7 0.73 1.7 7 0.67 76.47 4 66.66 3.617 2.99 5.87 7 3.23 61.49 47.09 38.50

147.5 71.996 147.337 2.940 0.677 0.33nn 3.337 0.95nn 20.12 5 83.33 3.417 1.31 5.88 7 0.97 57.99 97.65 42.00

3.14

100

63.98

80.35

All data were expressed as Mean7Standard Error Mean (SEM). Differences were considered significant at normal (n¼6).

nnn

po0.001,

nn

po0.01, npo0.05, when compared test groups vs

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427

3.4. Determination of anti-implantation activity

3.6. Determination of serum biochemical assay

MEDQ and standard treated animals were found to reduce the total number of implantation sites compared to control animals. Whereas, AEDQ treated animals have not shown any such differences. Percentage decrease in survivability of fetus in standard, AEDQ and MEDQ treated animals were calculated as 0%, 52% and 70% respectively when compared with 100% normal control animals (Table 4). In the other hand, weight of different body organs did not show any major differences among all treated groups compared to the control (Table 5).

Serum levels of SGOT, SGPT, Total Bilirubin (BIT), Cholesterol (CHO), Triglyceride (TRI), ALP, Urea and Uric acid were estimated for anti-implantation activity. Where only MEDQ has shown significant increase in SGPT and uric acid levels, while AEDQ did not show any significant changes in the levels of these biochemicals (Table 7).

3.5. Determination of hormonal assay Serum levels of estradiol, progesterone, LH and GnRH were estimated for both abortifacient and anti-implantation activity. Whereas, AEDQ shows significant decrease in GnRH level in antiimplantation animal model, while MEDQ shows significant increase in level of progesterone in both the activities and significant (nnpo0.01) decrease in the level of serum GnRH in anti-implantation activity (Table 6).

Table 3 Effect of test samples (AEDQ and MEDQ) on PGE2, PGF2α level. Group

Control (1 ml/kg saline) Standard (0.1 mg/kg) DRY-AQ (200 mg/kg) DRY-ME (200 mg/kg)

ABORT serum PGE2 (ng/ml)

PGF2α (ng/ml)

5.1847 1.95 20.64nn 71.09 6.790 7 5.701 14.98 71.26

13.08 7 3.604 46.48nn 7 2.56 61.185nn 7 2.947 176.93nn 7 2.26

All data were expressed as Mean 7 Standard Error Mean (SEM). Differences were considered significant at nnnp o0.001, nnp o 0.01, np o 0.05, when compared test groups vs normal (n ¼6).

4. Discussion Evidences from different sources revealed that herbal drugs are useful agents for antifertility purposes in rodents and humans (Noumi and Tchakonang, 2001; Mukherjee and Mitra, 2009; Kumar and Mishra, 2011). Literature scrutinization (Yadav et al., 2006; Mukherjee and Mitra, 2009; Kumar and Mishra, 2011) revealed the fact that there is no report available to the best of our knowledge which confirms the scientific evaluation of the rhizome part of the Drynaria quercifolia as antifertility agent (Rajendran and Rajan, 1996; Das et al., 2012). The use of Drynaria quercifolia in contraception could be of obvious interest in human fertility control. The scientific parameters evaluated in this study provided a guide way to judge the potentiality of the plant or its part and its mechanism of action as antifertility agent. The experimental results suggest the absence of discomfort and physiological abnormality in animals treated with extract at high dose (2000 mg/kg BW). In ex-vivo uterine contractility assay, all the extracts have shown contraction in isolated rat uterus preparation. Among all the test extracts, only AEDQ and MEDQ showed the highest contractility activity. Because of this finding we decided to proceed further for the in-vivo experiments with AEDQ and MEDQ only. The results of anti-implantation experiments revealed that oral administration of test samples (AEDQ and MEDQ) at a dose of 200 mg/kg showed significant decrease in number of implantation sites compared to that of control animals. Thus, it is seen the crude extracts have inhibited the implantation process. This is possibly due to antizygotic, antiblastocytic as well as antiestrogenic property of the extracts. This result showed similarity with the reports that, some

Table 4 Effect of test samples (AEDQ and MEDQ) on anti-implantation activity. Group

Normal Standard AEDQ (200 mg/kg/day) MEDQ (200 mg/kg/day)

Body weight difference on 8th day

17.6774.09 5.0 71.69nn 8.3371.52n 5.83 71.70nn

Weight of uterus (g)

1.107 0.13 0.8147 0.098 0.737 0.05n 0.46 7 0.07nn

Total no. of implants

4.337 0.5 2.6 7 0.42 4.2 7 0.54 2.83 7 0.54

Survivability of Implants on day 8 Live

Dead

4.3370.49 0 70nn 2.0 70.45nn 0.83 70.40nn

0 70 2.17 70.48n 2.17 70.31n 2.0 70.52n

% decrease in survivability

100 0 47.62 29.32

All data were expressed as Mean 7Standard Error Mean (SEM). Differences were considered significant at nnnpo 0.001, nnp o0.001, npo 0.05, when compared test groups vs normal (n¼ 6).

Table 5 Effect of test samples (AEDQ and MEDQ) on different organ weights of the female rats in anti-implantation activity. Sample

Normal Standard AEDQ (200 mg/kg/day) MEDQ (200 mg/kg/day)

Mean organ weight (g)7 SEM Vagina

Uterus

Ovary

Kidney

Liver

Adrenals

0.082 7 0.006 0.0707 0.004 0.0767 0.005 0.068 7 0.007

1.56 7 0.04 0.45 7 0.04 0.58 7 0.02 0.497 0.02

0.08 70.002 0.092 70.003 0.112 70.005 0.085 70.001

1.447 0.3 1.34 7 0.03 1.7 7 0.08 1.577 0.08

57 0.1 4.9 7 0.2 5.054 7 0.2 5.247 0.12

0.0637 0.006 0.05 7 0.003 0.0727 0.002 0.0647 0.004

All data were expressed as Mean 7 Standard Error Mean (SEM).

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Table 6 Effect of test samples (AEDQ and MEDQ) on different female hormones level anti-implantation activity. Group

Control (1 ml/kg saline) Standard (0.1 mg/kg) AEDQ (200 mg/kg) MEDQ (200 mg/kg)

Estradiol (ng/ml)

Progesterone (ng/ml)

LH (ng/ml)

GnRH (ng/ml)

Implt

Abort

Implt

Abort

Implt

Abort

Implt

Abort

4.5317 1.367 13.63 7 8.97 12.4667 3.778 12.187 3.80

9.525 7 6.63 17.62 7 2.58 16.6127 1.181 16.60 7 8.522

2.96 7 6.6 19.357 2.34 4.5047 3.96 48.96nn 7 5.567

2.722 7 2.47 22.02nn 7 1.76 4.82 7 1.00 24.59nn 7 1.86

14.226 7 4.76 83.81nn 7 2.98 14.4857 1.53 18.34 7 4.22

18.8017 3.27 89.70nn 7 2.35 16.117 7.56 18.45 7 3.35

63.63 7 1.72 4.85nn 7 2.61 1.544nn 7 5.69 4.026nn 7 8.07

15.690 77.292 3.63 71.2 5.97 76.43 6.482 72.78

All data were expressed as Mean 7 Standard Error Mean (SEM). Differences were considered significant at normal (n¼ 6).

nnn

po 0.001,

nn

p o 0.01, np o0.05, when compared test groups vs

Table 7 Effect of test samples (AEDQ and MEDQ) on different biochemical parameters of the female rats. Group

SGOT

SGPT

BIT

CHO

TRI

ALP

Urea

Uric acid

Control (1 ml/kg saline) Standard (0.1 mg/kg) AEDQ (200 mg/kg) MEDQ (200 mg/kg)

333.5 7 1.19 358.47 4.00 362. 87 1.80 358.27 1.24

113.73 76.9 81.73 75.3n 170.85 72.5 175.80 76.2nn

1.03 7 0.05 0.7 7 0.07 1.137 0.0 1.017 0.07

37.737 2.5 45.337 2.3 37.707 2.3 45.137 4.6

228.80 7 1.6 157.187 4.6n 280.337 1.5 294.80 7 0.9

98.55 7 1.4 166.037 8.6nn 131.88 7 1.11 128.78 7 5.82

38.8 72.4 46.5 75.0 47.5 74.4 53.3 71.7

14.8 71.1 15.4 71.2 13.94 71. 4 90.53 74.2n

All data were expressed as Mean 7 Standard Error Mean (SEM). Differences were considered significant at normal (n¼ 6).

plant extracts can cause endometrial alterations resulting in nonreceptive endometrium and thus cause implantation failure (Soejarto et al., 1978; Bullock et al., 1995; Simon et al., 2003). It was reported that administration of high levels of exogenous estrogenic substances to rats causes implantation failure (Ements, 1970; Simon et al., 2003). In rodents and humans estrogen plays a crucial role in implantation because it participates in estrogen– progesterone balance and therefore it affects the uterine receptivity to the embryo (Farnsworth et al., 1975). Administration of compounds with estrogenic activity at low concentrations during early pregnancy results in rapid ova passage through the oviduct along with expulsion from oviduct. Furthermore, transportation of degenerated fertilized ova during early transportation into the uterus decreases the number of implants resulting in decreased fertility (Farnsworth et al., 1975). In our study a sharp rise in serum estrogenic activity in extracts treated animals was observed, indicating the estrogenic property extract of the rhizome of Drynaria quercifolia. Administration of the extracts at a dose of 200 mg/kg body weight caused significant change in the number of live fetuses, increase in the percentage of the post-implantation embryonic loss and decrease in fetal survival percent indicating the abortifacient activity of the extracts. The published report on abortifacient activity of Senna alata leaves (Yakubu et al., 2010) also supports this result. Various cytotoxic agents interrupt pregnancy possibly by interference with mitotic division of the foetus, misbalance with serum biochemical and hormone levels both before and after the implantation process which ultimately leads to pre- and post-implantation embryonic loss (Yakubu et al., 2010). In our study maternal homeostasis balance was checked periodically by evaluating maternal body weight changes, vaginal bleeding, food and water intake. The implantation index, resorption index and pre-implantation loss are important parameters for evaluating the number of blastocytes implanted in the uterus and the underdeveloped. Therefore, the increase in the resorption index by the extracts is a sign of failure in the embryo development. Such incidence of fetal resorption indicates the termination of pregnancy after implantation. The post-implantation loss due to administration of the extracts indicated the abortifacient or fetal resorptive properties of the Drynaria quercifolia rhizome extracts. This result approves the folklore use of the plant as a postcoital abortion promoter. The measurement of the concentration of several female reproductive hormones such as progesterone, GnRH and LH in the body

nnn

po 0.001,

nn

p o 0.01, np o0.05, when compared test groups vs

fluid plays a significant role in determining ovulation and characterizing luteal phase defects. Quantitative determination of the hormone concentration in serum was used as a diagnostic tool for detecting ectopic or failure of pregnancy (Yakubu and Bukoye, 2009). In this study, specific hormones were assayed based on their roles in maintaining pregnancy, because, failure of pregnancy could be associated with the levels of different hormones in body fluids. The fall in the concentration of GnRH is an indication of impairment in the reproductive cycle of the animals. LH is required for continuous development and normal functioning of the corpora lutea. The level of serum LH is linked with the physiological process of luteolysis former parturition. Pregnancy failure in the present study after the extract treatment is possibly due to disturbed luteal phase. Moreover, progesterone plays a vital role in preserving the condition and is an important factor in the implantation process during pregnancy (Waterhoff et al., 1994; Bullock et al., 1995). Therefore, increase in the serum levels of progesterone may contribute to abortion and anti-implantation activity of the Drynaria quercifolia. Similarly, the observed level of serum progesterone describes the reason of destruction in the endometrial function which avoids the secretion of special protein needed to promote an implanted fertilized egg in the extract-treated animals. Furthermore, the increased level of PGE2, PGF2α plays a key role on abortion by inducing separation of fetal membrane from uterine wall and uterine contraction (Chandra et al., 1991; Romagnoli et al., 1993; Atad et al., 1997). In our study the levels of PGE2 and PGF2α were also increased in AEDQ and MEDQ extract treated animal groups suggesting their abortifacient activity. The differences in the hormone levels in extracts treated groups compared to control animals as observed in this study possibly facilitate the onset of labor. Our findings in this study were supported by the reported findings of Al-Dissi et al. (2001). Result in ex-vivo uterotonic response by AEDQ and MEDQ was characterized by an increase in the scale and occurrence of uterine contractions when given with oxytocin depicted the abortifacient effect of the extracts. This might be another mechanism of antifertility action of Drynaria quercifolia.

5. Conclusion This study has demonstrated that the Methanolic extract of Drynaria quercifolia rhizome possesses significant abortifacient and

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anti-implantation activity which may be attributed to the phytoconstituents of the plant. The mechanism of abortifacient and antiimplantation activities of Drynaria quercifolia rhizome extract could possibly be through changes in implantation site, and altered hormone levels. The increase in uterine muscle contraction might also be another possible mechanism of abortion. This study thus justifies the traditional claim of the Drynaria quercifolia rhizomes as a fertility antidote. However, other efforts are on to establishing the potential of this plant as a useful source of fertility regulating agent.

Acknowledgement I would like to acknowledge Assam University, Silchar for providing financial assistance in the form of UGC non net fellowship. I would like to thank Dr. N.C. Talukdar, Director and Scientist – F, Institute of Bioresources and Sustainable Development (IBSD), Department of Biotechnology, Government of India, Imphal, Manipur and the technical staffs of Pharmacology Laboratory, IBSD, Imphal, Manipur for providing all possible facilities to successfully complete this research work. Authors are also thankful to Mr. Supriya Das, Assistant Professor, HCPG College, Varanasi and Sushmita Nath, Research Scholar, Department of Life Science and Bioinformatics, Assam University, Silchar for helping in manuscript correction. Authors are thankful to Bioinformatics Centre and Biotech Hub of Assam University, Silchar. References Al-Dissi, N.M., Salhab, A., Al-Hajj, H.A., 2001. Effect of Inula viscosa leaf extracts on abortion and implantation in rats. J. Ethnopharmacol. 77, 117–121. Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1956. Second Supplement to Glossary of Indian Medicinal Plants With Active Principles. Part-I(A-K). National Institute of Science Communication and Information Resources. CSIR, New Delhi. Atad, J., Hallak, M., Fruchter, O., Borenstein, J., Abramovici, H., 1997. Secondtrimester membrane rupture. Abortion induced with prostaglandin E2 after oxytocin failure. J. Reprod. Med. 42 (9), 581–584. Bhattacharya, P., Saha, A., 2013. Evaluation of reversible contraceptive potential of Cordia dichotoma leaves extract. Rev. Bras. Farmacogn. 23 (2), 242–250. Bullock, J., Boyle, J., Wang, M.B., 1995. In: Volker, J. (Ed.), Physiology, 3rd ed. Lippin Cott Williams and Wilkins Publishers, pp. 497–519. Calixto, J.B., Yunes, R.A., Rae, G.A., 1991. Effect of crude extract of Leonotis nepetaefolia (Labiatae) on Rat and Guinea pig smooth muscle and rat cardiac muscle. J. Pharm. pharmacol. 43, 529–534. Chandra, K., Gupta, I., Dhawan, V., Ganguly, N.K., 1991. Prostaglandin-F2 alpha levels in normal saline-induced mid-trimester abortions. Contraception 44 (1), 99–106. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian medicinal plants, Council of Scientific and Industrial Research India (CSIR). Publication and Information Directorate, New Delhi. Das, B., Choudhury, M.D., Talukdar, A.D., Choudhury, S., Nath, D., Chetia, P., 2012. Traditionally used Ethnomedicinal formulations for birth control by ethnic people of Tripura. Abstract No. SNPSJU0197. 12th International Congress of Ethnopharmacology. Jadavpur University, Kolkata, India, February 17–19. Das, H.B., Majumdar, K., Datta, B.K., Ray, D., 2009. Ethnobotanical uses of some plants by Tripuri and Reang tribes of Tripura. Nat. Prod. Rad. 8 (2), 172–180. De Freitas, T.G., Augusto, P.M., Montanari, T., 2005. Effect of Ruta graveolens L. on pregnant mice. Contraception 71, 74–77.

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