medicinal plants

DOI 10.1007/s11094-015-1268-y Pharmaceutical Chemistry Journal, Vol. 49, No. 4, July, 2015 (Russian Original Vol. 49, No. 4, April, 2015)

MEDICINAL PLANTS REPRODUCTIVE TOXICITY OF CASSIA ABSUS SEEDS IN FEMALE RATS: POSSIBLE PROGESTERONIC PROPERTIES OF CHAKSINE AND b-SITOSTEROL Azadeh Hamedi,1,2,* Mohhamad Javad Khoshnoud,3 Nader Tanideh,4 Farzaneh Abbasi,5 Masood Fereidoonnezhad,6 and Davood Mehrabani7 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 49, No. 4, pp. 10 – 10, April, 2015.

Original article submitted August 11, 2014. This study was designed to evaluate the reproductive toxicity of Cassia absus seeds, which have been used in Persian folk medicine. Female rats in treatment groups (N = 5) received C. absus n-hexane fraction (CAF) at oral doses of 100, 200, and 500 mg/kg b.wt./day, once per day on days one to five post-coitum (pc). The control group received 0.2 mL of olive oil (vehicle). Fertility, maternal index, hormone level, and teratogenicity were evaluated. Phytoconstituents of the seeds were detected by HPTLC and GC-MS. The presence of phytoprogesterones was evaluated by docking software. The fertility index and number of fetuses in treated groups decreased significantly. Missed abortion was 50% after administration of 500 mg/kg/day of CAF. The maternal body weight, uterine weight, and levels of FSH, LH, and estradiol exhibited insignificance changes. The serum concentration of progesterone also changed significantly in a dose-dependent manner. No teratogenic effect was found. Oleic acid (35.89%) and linoleic acid (24.22%) were the major constituents of the seed oil. In addition, chaksine and b-sitosterol showed potential phytoprogesteronic properties in docking studies. The reproductive toxicity of C. absus seeds may be related to a hormonal imbalance, a decline in the fertility index, and an increase in the rate of missed abortions of fetuses. Keywords: anti-fertility; Cassia absus; female; hormone; progesterone; b-sitosterol.

treat conditions such as premenstrual syndrome (PMS), menopausal symptoms, hormonal imbalance, infertility, or as contraceptives [1, 2]. In many societies, for economic or cultural reasons, medicinal plants are the most – or even the only–available therapeutics. Thus, evaluating their efficacy is important for health organizations. Medicinal plants and herbal remedies are not currently subject to the same regulations as conventional drugs. There are some issues in determining their purity, safety and authenticity, which is mostly due to a lack of knowledge about their constituents [3]. On the other hand, many herbal drugs – which may be used for other therapeutic properties – can produce unwanted side-effects on a pregnant woman or her embryo, but the safety of most medicinal plants during pregnancy is still unknown [4]. Scientific evaluation of the potential effects of medicinal plants on the reproductive hormonal system or human fertility may prevent a notable number of unwanted infertility

INTRODUCTION Medicinal plants and herbal remedies have long been used by women in traditional and folk healing systems to 1 2 3 4 5 6 7 *

Medicinal Plants Processing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Department of Pharmacognosy, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Science, Shiraz, Iran. Department of Pharmacology, School of Medicine, Shiraz University of Medical Science, Shiraz, Iran. Student Research Committee, International Branch, Shiraz University of Medical Sciences, Shiraz, Iran. Student Research Committee, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran. Department of Pathology, Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. e-mail: [email protected].

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Reproductive Toxicity of Cassia absus Seeds in Female Rats

cases, abortions or fetal abnormalities and can also lead scientists to find new active molecules for use as contraceptives or fertility therapeutics [5]. The plant of Cassia absus L, Sesse & Moc. (Synonym: Senna insularis (Britton & Rose) H. S. Irwin & Ba and Chamaecrista absus (L.) H. S. Irwin & Barneby), with the common name Pig’s Senna, belongs to family Caesalpiniaceae. The seeds of this plant – which is known as chaksu in Ayurveda and cheshmizeh and tashmizaj in Persian traditional medicine – have been used as bitter tonics, attenuants, diuretics and astringents for the bowels. Preparations of C. absus seeds have been used to treat eye ailments and infections, diarrhea, hemorrhages, leprotic and syphilitic ulcers, leukoderma and some skin ailments [6]. Some studies have investigated the phytochemical constituents of the seeds, roots and leaves of C. absus [7], and metabolites including galactomannan [8], chaksine, isochaksine and 9-ketooctadec-cis-15-enoic acid [9] have been isolated from the seeds of this plant [10]. Also, antimicrobial [11, 12], cardiovascular [13], anti-inflammatory and antihistaminic activity [14] of C. absus seeds or their preparations have been evaluated scientifically, but we could not find any reports concerning their anti-fertility effects. This study was designed to investigate the effects of C. absus seeds extract on female fertility, its safety when used during pregnancy, and the phytochemical constituents of the seeds, as well as evaluating the phytoprogesterone and phytoestrogenic properties of the known constituents of seeds by molecular docking. EXPERIMENTAL CHEMICAL PART Chemicals All solvents and reagents were purchased from Merck (Germany) or Sigma Aldrich (United States). Hormonal ELISA kits were supplied from Monobind Inc. (United States). Plant Material C. absus seeds were purchased from a local herbal market at Shiraz, Iran. The seeds were authenticated by a taxonomist, Miss Sedigheh Khademyan, and its voucher specimen was held in the Department of Pharmacognosy, School of Pharmacy, Shiraz University of Medical Sciences with the code 159 PM. The seed powder (500 g) was extracted with petroleum ether through application of Soxhlet apparatus for 6 h. The residuum was dried, macerated with dichloromethane, and then with ethanol (48 h each). n-Hexane, dichloromethane, and ethanol fractions were concentrated with a rotary evaporator and dried in a speed vacuum apparatus at 40°C. The dried extracts were weighed and stored at –20°C.

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Estimation of Total Phenolic Compounds Hexane (50 mL) was added to 8 mg of dried n-hexane fraction and this mixture was diluted with 60% methanol (100 mL ´ 3). The methanol phase was dried, dissolved in water, and then extracted with petroleum ether. The separated aqueous phase was saturated with NaCl and extracted by ethyl acetate (´4). The collected ethyl acetate fraction was dried with anhydrous sodium sulphate fallowed by rotary operator. Total phenol content was determined by the Folin–Ciocalteu method (Folin, et al., 1927). UV absorbance of the sample was read at 765 nm. Serial dilutions of standard gallic acid were used to draw a calibration curve. All experiments were performed in triplicate. High-Performance Thin-Layer Chromatography (HPTLC) Solutions of 5 mg/mL of different fractions were prepared and 15 mL of each fraction was applied with HPTLC system (CAMAG) to a silica gel plate 60F254 (10 ´ 20 cm) from Merck. The plates were run in three mobile phases of different polarity including: nonpolar (toluene : acetone, 80 : 20), semi-polar (toluene : chloroform : acetone, 40 : 25 : 35) and polar (n-butanol : glacial acetic acid : water, 50 : 10 : 40) [15]. Chromatographic spots were visualized first using UV254, UV365 nm and then using different spray reagents (including phosphomolybdic acid, Dragendorff, 5% potassium hydroxide, NP (ethanolamine diphenylborate)/PEG, Liebermanne–Burchard, FeCl3, vanillin–sulphuric acid, and anisaldehyde–sulphuric acid reagents). All chemicals, solvents, and reagents of analytical grade were purchased from Merck (Darmstadt, Germany) or Sigma Aldrich (St. Louis, Mo., United States). Determination of the Fatty Acid Profile of C. absus Seeds For GC-MS analysis of fatty acids extracted in petroleum ether, methyl ester derivatives were prepared according to the AOAC (AOAC, 1990). Palmitic acid was added as an internal standard. GC-MS analysis was carried out using a Hewlett Packard 6890 system. The gas chromatograph was equipped with an HP-5MS phenyl methyl siloxane capillary column (25 m, 0.25 mm i.d.). The mass spectrometer operated in the EI mode at 70 eV. The interface and injector temperature was 250°C. The masses were detected in the m/z range of 30 – 600. The oven temperature was held at 160°C for 2 min, increased from 160 to 230°C at a rate of 8°C/min, and held at 230°C for 20 min. Helium was used as the carrier gas at a flow rate of 1 mL/min and a split ratio of 1:10 [16]. Fatty acid profile was determined based on standard fatty acid spectra and Wiley 275 database.

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EXPERIMENTAL BIOLOGICAL PART Adult female Sprague-Dawley rats (weighing between 150 – 220 g) were housed in standard cages, five per cage, in a controlled-temperature room (23 ± 2°C) at a humidity of 45 – 50% with a 12/12 h light and dark cycle. Animals had free access to standard chow food and tap water ad libitum. All the experimental procedures were performed according to the ethical guidelines for animal studies established by the Ethical Committee of Shiraz University of Medical Sciences. All doses of C. absus n-hexane fraction (CAF) were prepped freshly on each day of the experiment. The animals in group I (as the control group) received 0.2 mL/rat of vehicle (olive oil). Animals in groups II, III and IV received CAF at doses of 100, 200 or 500 mg/kg b.wt./day, suspended in olive oil, respectively. The animals received the treatment orally, in a fixed volume, once per day from day one to five post-coitum (pc). All animals were weighed at the beginning and end of the experiment [17]. Reproductive Toxicity In order to evaluate the post-coital reproductive toxicity of C. absus seeds, normal-cycling proestrous or oestrous female rats were caged overnight with male rats of proven fertility with the ratio 2 : 1. Insemination was confirmed the next morning by detecting the presence of the vaginal plug and spermatozoa in the vaginal smear. The day of mating was considered as day 0 of pregnancy. The mated female rats were isolated, weighed and divided into four groups of five animals each. To detect the effects of C. absus seeds on uterus and implantation, all the treated and control female rats were sacrificed on day 15 pc and, after washing the uteri with normal saline, their weights were recorded. For hematological studies, blood samples were collected directly from the cardiac puncture of the rats. Fertility index (FI) was calculated using the formula: FI = (Total number of females pregnant/Total number of females mated) ´ 100. By incision into the uterus, both of the uterine horns were examined for the number of implantation sites, live or dead/resorbed fetuses and then all fetuses were transferred into a petri dish containing PBS to wash the fetuses and remove the presence of any blood. The embryos were evaluated for any macroscopic abnormalities. Embryos with bright reddish aspects and clear margins were considered to be normal and those with a dull blue colour, no clear margin, smaller in size and with some surrounding exudate were considered to be resorbing. One half of the foetuses from each animal were fixed in 10% neutral phosphate buffered formalin. Serial sections were prepared from the head, thorax and abdomen and tissue samples were stained with haematoxylin and eosin dyes. All of the tissue samples were histologically

Azadeh Hamedi et al.

evaluated to detect abnormality and teratogenic effects in bone, cartilage, digestive tract, kidney, liver, spleen and spinal cord tissues. MOLECULAR DOCKING Molecular docking was performed for known constituents of C. absus seeds. Docking procedure was as follows: the crystal structure of the progesterone receptor ligandbinding domain bound to levonorgestrel (3D90.pdb), and the crystal structure of the estrogen receptor-alpha ligand binding domain bound to estradiol (1A52.pdb) were obtained from the RCSB protein data bank [5]. Initially there was a pre-treatment process for both the ligands and the enzymes. All the pre-processing steps for receptor and ligand files were done by AutoDock Tools 1.5.6 (ADT). For ligand preparation, the 3D structures of compounds were generated with ChemBioOffice 2012 and optimized with different minimization methods such as the quantum-based semi-empirical method (AM1) or the commonly used molecular mechanics method (MM+) using HyperChem 8. For receptor preparation, repair of missing atoms followed by the addition of hydrogen and Gasteiger charge computation was performed and crystallographic waters were removed. Molecular docking was performed using the AutoDock 4.2 program with a genetic algorithm method and the receptors were kept rigid. Having completed the docking process, the protein–ligand complex was analysed to investigate types of interactions. One hundred docking poses saved for each compound were ranked according to their dock score function. AutoDock gives total binding energies of the compounds as well as steric and electrostatic lowest binding energy (LBE) for individual atoms as an output [18, 19]. This information was used and the results were summarized for each ligand. PoseViewWeb [6] was used for visual representation of the ligand-protein complex. Statistical Analysis Numerical results of this study were expressed as means ± standard deviation (SD) and subjected to statistical analysis using SPSS16 software. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons was used to analyse the data. The level of significance was considered at p < 0.05. RESULTS AND DISCUSSION Phytochemical Constituents of C. absus Seeds Application of HPTLC in the testing of medicinal plants and herbal formulations and their fingerprinting for identification of their phytoconstituents, authentication of medicinal plants, and detection of their adulterations are very popular


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Fig. 2. Effects of C. absus n-hexane fraction (CAF) on the fetus size (A) and fetus containing uterus weight (B).

Fig. 1. HPTLC fingerprints of n-hexane (H) and ethanol (E) fractions of C. absus seeds treated with Liebermanne–Burchard reagent in polar mobile phase under visible light (A) and non-polar mobile phase UV365 lamp (B).

in pharmacognosy research—especially as HPTLC fingerprinting is economical and rapid [20]. Thin-layer chromatography of C. absus seed fractions revealed the presence of alkaloids, steroids, saponins, and flavonoids in different fractions of the seeds. Figure 1 shows the HPTLC fingerprints of n-hexane and ethanol fractions. b-Sitosterol and its glycoside form were the major steroids of the n-hexane and ethanol fractions of the seeds compared with standard steroids. Total phenolic content was determined as 9.13 ± 0.03 mg/g of the seeds. We could not find any published report on phytochemical constituents of C. absus seed fractions to compare the results, but alkaloids chaksine and isochaksine from the seeds were previously reported [21]. In addition, chrysophanol, aloe-emodin (from roots), quercetin and rutin (from leaves) have been reported as phytochemicals of this plant [22]. As seen in Table 1, 35.89 ± 0.63 (W/W %) of the fixed oil was 9-octadecenoic acid (oleic acid) as the major fatty acid followed by 24.22 ± 0.92 (W/W %) for 9,12-octadecadienoic acid. About 22% of the oil consisted of methylated fatty acids. Methylated fatty acid (9-keto-octadec-15-enoic acid with a concentration of 52%) in C. absus seed oil was previously reported by Hosamani [9] but the methylated fatty acids in this study were 15-methyl heptadecanoic acid (22%) and 12-methyl tridecanoic acid (2.07%). He also reported the presence of linoleic acid, eicosanoic acid, and oleic acid, which is inconsistent with the results of our study.

Maternal Observation The entire female rat population in the control group became pregnant while in all groups receiving CAF at different doses, the pregnancy fertility index decreased to 60%. Also, the total number of fetuses and the fetus/rat ratio in groups receiving CAF was decreased, but this decline was only significant for groups receiving 200 and 500 mg/kg b.wt./day of the fraction (Table 2). In this study we did not evaluate the effects of CAF on the implantation of blastocysts but instead a significant decrease in the fertility index and fetus/rat ratio in groups showed that CAF possesses anti-fertility and abortifacient properties. The significant increase in the number of missed-aborted fetuses might be due to the direct reproductive toxicity effect of the CAF on the foetuses, which showed its toxicity in a dose-dependent manner.

TABLE 1. Constituents found in C. absus seed oil (n-hexane fraction) Number

Name

Percentage ± SD

1

Nonadecane

0.45 ± 0.14

2

Dodecanoic acid

0.44 ± 0.09

3

12-Methyl tridecanoic acid

2.07 ± 0.11

4

Octadecane

1.77 ± 0.17

5

Hexadecanoic acid

6

9,12-Octadecadienoic acid

24.22 ± 0.92

7

9-Octadecenoic acid

35.89 ± 0.63

8

15-Methyl heptadecanoic acid

22.00 ± 0.58

9

Eicosanoic acid

3.44 ± 0.72

10

Unknown

7.94 ± 0.37

1.78 ± 0.08

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as well as their size, it can be concluded that the insignificant changes in uterine weight might be due to the observed oedema of the uterus in treated female rats. Results of Reproductive Hormonal Assay

Fig. 3. Effects of C. absus n-hexane fraction (CAF) on maternal weight and hormonal levels: estradiol (A) and foetus progesterone (B), FSH and LH (C), and maternal weight before and after pregnancy (D).

Administration of CAF at different doses from day one to 15 pc did not result in any significant changes in maternal body weight. One of the female rats, which was receiving 500 mg/kg b.wt./day of CAF, died on the third day of the experiment. We could not find any reports on the anti-fertility properties of C. absus, but previous studies on other species of Cassia genus, C. fistula [17, 23] and C. occidentalis [24], showed that, despite the presence of anti-fertility characteristics in the plants, maternal body weight did not change significantly, in agreement with the results of our study. In all treated groups, the fetus size decreased significantly (Fig. 2A). The relative uterine weight was decreased in the female rats of all treated groups when compared with controls, but this decline in uterine weight was not significant (Fig. 2B). Considering the decrease in number of fetuses

Administration of 500 mg/kg of CAF produced an increase in serum FSH (4.25 ± 0.57), LH (2.15 ± 0.34) and estradiol (41.35 ± 5.39) compared to the control group (3.58 ± 0.58, 1.96 ± 0.35 and 39.32 ± 6.45). In the group receiving 100 mg/kg of the fraction, the serum level of FSH (2.76 ± 0.49), LH (1.84 ± 0.23) and estradiol (35.88 ± 5.65) decreased, however none of these changes were statistically significant. As seen in Fig. 3, progesterone serum level was changed by administration of CAF in a dose-dependent manner compared to the control group (20.56 ± 2.59). Administration of 500 mg/kg of CAF significantly increased the serum concentration of progesterone (27.00 ± 3.12) while 100 mg/kg of CAF resulted in a significant decrease in the progesterone serum level of the female rats (15.50 ± 2.21). Considering the significant decrease in fertility index, it can be suggested that, although the changes in FSH, LH and estradiol are not statistically significant, the imbalance in these hormonal levels in conjunction with significant changes in progesterone serum level made a significant clinical presentation. The pregnancy interceptory effect of CAF might be due to the inhibition of circulating estrogen–progesterone balance which may result in a non-receptive stage in the uterus by changing the reproductive biochemical milieu, especially the uterine environment which is directly involved in implantation and thus results in significant anti-fertility effects. Such effects were previously reported for C. fistula by Yadav, et al. [25], although they suggested a mild estrogenic property for this plant. There is another possibility that the constituents of CAF have the potential to directly bond to the estrogenic or progesterone receptors and induce activities such as oral estrogenic and progesteronic contraceptives: in other words, the plant may have phytoestrogen or phytoprogesterone constituents.

TABLE 3. Docking validation results TABLE 2. Effects of C. absus n-hexane fraction (CAF) on the rate of pregnancy and fetus health Control

CAF CAF CAF (100 mg/kg) (200 mg/kg) (500mg/kg)

Fertility index

100

60

60

60

ratio of fetus/rat in group

8.6

6.2

5.4

5.2

Missed-aborted fetuses

3

2

1

11

PDB code

Number of runs Number of evaluations

PDB ID: 3D90 PDB ID: 1A52

100

100

2500000

2500000

Number of conformations in optimum cluster (out of 100)

100

100

RMSD (A°) from reference structure

0.47

0.76

Estimated binding energy (kcal/mol)

-11.54

-9.90


Histopathologic Evaluation No teratogenic effects were found histologically in any of the embryonic tissues isolated from pregnant mice on day 15 of pregnancy. Considering the high number of missedaborted fetuses (Table 2), and significant decrease in the fetus size (Fig. 2A), the results of histopathologic evaluation are interesting and show that the observed toxicity of CAF on fetuses’ health is not due to teratogenicity or induction of organ malformation and may be due to induction of functional or biochemical toxicity, which may not be detectable in histopathologic studies. Results of Docking In order to find possible active constituents with phytoprogesteronic or phytoestrogenic properties, the binding affinity of detected phytochemicals of the CAF (chaksine and b-sitosterol) to the progesterone and oestrogen receptors was evaluated with docking studies. Ligands were submitted to 100 independent genetic algorithm (GA) runs for this search. For the Lamarckian GA method, a 150 population size, a maximum number of 2,500,000 energy evaluations and 27,000 maximum generations were used. A grid of 50, 60, and 50 points to x, y, and z, respectively, for estrogen receptors and a grid of 40, 40, and 40 points in x, y, and z, for progesterone receptors with a grid spacing of 0.375A° was built centred on the catalytic site of the receptors. The docking validation step was performed by re-docking of the co-crystallized conformation of cognate ligand (levonorgestrel as a known progestin contraceptive bound to progesterone receptor and estradiol bound to estrogen receptor) into the 3D structure of its receptor. Thus, validation of the method for forecast of the known binding poses would be maintained. If the RMSD is less than 2 A°, it is generally considered a successful docking prediction [2]. The validation results for our targets are shown in Table 3. Cluster analysis was performed on the docked results using a RMS tolerance of 2 A°. Docking results are shown in Tables 4 and 5. In each case, the best docking result was considered to be the conformation with the lowest binding energy. Hydrogen bindings between docked potent agents and

TABLE 4. Docking results of ligand 1 – 4 docked into progesterone receptor (PDB ID: 3D90)

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the progesterone and estrogen receptors was analysed using AutoDock tools program (ADT, Version 1.5.6) and PoseViewWeb. As is shown in Table 4, the compounds chaksine and b-sitosterol have better binding energies than the co-crystallized ligand (levonorgestrel), and so their interactions with their receptors were investigated by PoseViewWeb. Possible key hydrogen bonds between compound b-sitosterol and the progesterone receptor active site were analysed. Having compared the results, we find that b-sitosterol may show better binding to progesterone receptor sites than levonorgestrel. The summarized results of ligand 1 – 4 docked onto estrogen receptor in Table 5, shows that only the compound b-sitosterol has a better binding energy than the co-crystallized ligand (estradiol), but the analysis of its binding interactions with receptor by PoseViewWeb shows no significant interaction. Meanwhile estradiol has a hydrogen-bond-donor interaction through its hydroxyl group hydrogen with the amino acids Gly521A and 524A, and a hydrogen-bond-donor interaction through its phenolic hydroxyl group hydrogen with the amino acid Glu353A. Despite lower calculated binding energy, such interactions are not evident in b-sitosterol. Baker, et al., [26] as well as Gutendorf and Westendorf [27] through their in vivo or in vitro experiments have reported that binding of b-sitosterol to estrogen receptors is non-existent or weak, which is in agreement with the molecular docking results in our study. According to the docking study, it can be concluded that only chaksine and b-sitosterol may have progesteronic properties but a notable estrogenic activity cannot be expected for these components. b-Sitosterol was previously reported as a weakly estrogenic phytosterol [28 – 30], but our work is the first report suggesting progesteronic properties for chaksine or b-sitosterol by molecular docking. We could not find any published report on the anti-fertility effects of chaksine, but there are several controversial reports on effects of b-sitosterol on male and female reproductive systems [31]. Some reports stated b-sitosterol can induce reproductive disturbances in mammals [32], fish and birds [33] which may be due to the endocrine-disruptive effects of b-sitosterol [34]. On the contrary, some reports have suggested the enhancing

TABLE 5. Docking results of ligand 1 – 4 docked into estrogen receptor (PDB ID: 1A52) Ligand name


1

Estradiol

100

-9.90

2

Chaksine

70

-8.00

3

b-Sitosterol

75

-13.09

Ligand name


LBE (kcal/mol)

Ligand Number

1

Levonorgestrel

100

-11.54

2

Chaksine

100

-13.21

3

b-Sitosterol

91

-13.40

Ligand Number

LBE (kcal/mol)

274 effects of b-sitosterol on reproduction possibly due to the modification of b-sitosterol into sex steroids [35]. b-sitosterol was reported to have a uterotonic effect and improve uterine contractions due to its nonestrogenic effects by inhibiting K+ channels and sarcoplasmic reticulum calcium ATPase [36]. These controversial results might be due to different animal models and b-sitosterol doses applied in experiments. On the other hand, most of the previous in vitro and in vivo studies considered b-sitosterol as a phytoestrogen rather than as a phytoprogesterone or a phyto-progestin. ACKNOWLEDGMENTS This study was part of the Pharm. D. thesis project of Farzaneh Abbasi and was financed by the International Branch of Shiraz University of Medical Sciences (grant # 18). CONFLICT OF INTERESTS The authors have no competing interests or conflicts of interest to declare. REFERENCES 1. M. L. Hardy, J. Am. Pharm. Assoc., 40, 234 – 242 (1999). 2. B. J. Tesch, Am. J. Obstet. Gynecol., 188, S44 – S55 (2003). 3. H. Nordeng and G. C. Havnen, Pharmacoepidem Dr. D., 13, 371 – 380 (2004). 4. E. Ernst, Brit. J. Obstet. Gynecol., 109, 227 – 235 (2002). 5. J. Adams, D. Sibbritt, and C. W. Lui, Birth, 38, 200 – 206 (2011). 6. M. Aghili, Tehran: Tehran University of Medical Sciences, 260 (2009). 7. H. Pandya, Y. Kachwala, L. Sawant, et al., Pharnacog. J., 2, 419 – 426 (2010). 8. V. Kapoor and S. Mukherjee, Phytochemistry, 10, 655 – 659 (1971). 9. K. M. Hosamani, Ind. Eng. Chem. Res., 33, 1058 – 1061 (1994). 10. K. Gupta and I. Chopra, Ind. J. Med. Res. (1953). 11. M. Wath, A. Khadatkar, and S. Deshmukh, Bioinfolet, 6, 626 – 630 (2009).


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