Study of Foeniculum vulgare Effect on Folliculogenesis in Female Mice

Infertility is one of the main problems seen in young couples. Due to cost and social problems, some couples seek traditional methods to treat their infertility before meeting with specialists. Since folk medicine plays an important role in individual and community primary health care, the use of herbal products are increasing in many countries (1). At present, great efforts are underway to studies medicinal plants antioxidant and antimicrobial extract both in industry and for use in scientific research (2). It seems medicinal plant properties are related to many phytochemical components, including carotenoids, flavanoids, curcumins and terpenoids (3).

Foeniculum vulgare Mill (FVE; Fam. Umbellifarae), commonly known as fennel, is a small genus of annual, biennial or perennial herbs located in central Europe and the Mediterranean region. It is widely used as a culinary spice and grown in different tropical regions of the world for its aromatic fruits (4). The FVE fruit has a long history of use as both a food and medicine. Traditionally, it is said to act as a carminative (assists with flatulence control) and increase breast milk production. It has been reported to enhance libido, promote menstrual flow, soothe indigestion and cough (5).

FVE has a clear protective effect against ethanolinduced gastric lesions, which is related to decreases in lipid peroxidation and antioxidant activity (6). Additionally, FVE can be used for pediatric colic and some respiratory disorders due to its anti-spasmodic effects (7, 8). It is widely acknowledged that all parts of FVE, including the seed have considerable medicinal value (9). For centuries, FVE fruits have been used as traditional herbal medicines in many Asian countries (10). It is also used as a spice in tropical Asia (11). FVE is used in Indonesia to cure albuminuria, abdominalgia, insomnia, and menstrual disorder, and the seed’s oil is utilized for the treatment of carminative, colds, and asthma (12).

FVE is an indigenous herb in Iran, and the plant extract has been used as an antiseptic, palliative and anti-inflammatory. In a study, FVE essence and mefenamic acid were compared for the treatment of primary dysmenorrheal and it was reported that FVE could be used as a safe and effective herbal drug for primary dysmenorrhea (13).

FVE fruit also possesses emnenagague, galactagogue, carminative, diuretic, and lactation stimulant properties (14). It contains 1% - 3% of a volatile oil, which is composed of 50% - 85% of anethole and about 20% of d-fenchone (15, 16). Other compounds present in the fruit are d-apinene, d-a-phellandrene, dipentene, methyl chavicol , fenelon, anisaldehyde and anisic acid (17).

FVE has been known to be able to regulate menstruation, alleviate the symptoms of female climacteric syndrome, and increase libido (18). One study has shown that FVE seed increases the weight of the genital organs. Oral administration of FVE increases total protein concentration in seminal vesicles and prostate glands in male rats, and increases the weight of mammary glands, oviducts, endometrium, and myometrium in female rats (19). FVE essential oil affects uterine contractions and significantly reduces the intensity of oxytocin and Prostaglandin E2-induced contractions (14). In Iranian folk medicine, it has been claimed that FVE improves sexual function and infertility in women, however there is no documented study to clarify this effect.

This study researches the quantitative aspects of folliculogenesis in female albino mice after administration of FVE fruit extract.

The total number of follicles were 26.5 ± 5.24 in group 1(100 mg/kg FVE), 27.2 ± 4.1 in group 2 (200 mg/kg FVE), 10.1 ± 2.53 in group 3 (ethanol) and 17.2 ± 3.9 in group 4(saline). There were significant increases in the experimental groups compared to the control groups (p<0.05, Fig 1A). There were no significant differences between the FVE concentrations. Similarly a significant increase was observed in the number of graffian, antral and multilaminar follicles (p<0.05) in the FVE groups, but there was no significant difference between the 100 and 200 mg/kg doses of FVE (Fig 1B, C, D).

There was no significant difference in the number of unilaminar primary follicles between all experimental and control groups. Histological sections of control and experimental groups showed increased numbers of growing follicles in the FVE groups (Fig 2A-D). GC-MS analysis showed existence of diosgenin, an estrogenic compound in the FVE extract.

To our knowledge, there is no scientific report on the effect of FVE on the ovary. In the present study, the alcoholic extract of FVE fruit has induced a significant increase in the numbers of graffian, antral and multilaminar primary follicles and improved folliculogenesis in mice ovaries. This may explain traditional fennel consumption to improve female infertility. Follicular growth is regulated by endocrine hormones [follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin] and local (paracrine and autocrine) factors (27). The significant increase in the number of follicles in the presence of FVE may have been due to the estrogenic effects of this plant.

FVE constituent's dianethole and photoane hole resemble stilbene and diethylstilbestrol, and anethole is structurally similar to cathecolamines, which may influence secretion of prolactin (28). Steroids and prolactin are involved in ovarian follicullogenesis. In this study it has been determined that one of the active constitutes of FVE extract is diosgenin a steroid sapogenin which is the starting material for the synthesis of a number of hormonal products such as DHEA. The estrogenic effects of diosgenin have also been demonstrated (29). In addition, diosgenin has been used to treat osteoporosis in the ovariectomized adult rat model (30).

As described in many studies, FVE can affect some reproductive factors. FVE appears to induce estrus in rats (18). Malini et al. have shown that FVE seed extract had estrogenic activity on the female and male genital organs and increased the weight of genital organs. In female rats, oral administration of FVE for ten days increased the weight of the mammary glands, oviduct, endometrium, myometrium, cervix and vagina (19). The researchers did not study the histological changes of the ovaries and genital organs. Here, we have shown the effect of FVE on the histology of mouse ovaries over a shorter period of time (five days), which emphasized the potent folliculogenesis effect of FVE. FVE also showed radical scavenging and antioxidant properties (31) which can affect general cell growth.

Both FVE doses showed nearly the same folliculogenesis effect, but 100 mg/kg had more numbers of antral follicles (Fig 1C) and 200 mg/kg FVE had more numbers of multilaminar follicles (Fig 1D). We did not utilize a stereological study to calculate all follicle numbers in each ovary. Our method was an improvement of a previous technique reported by other researchers (25, 32). The method consists of a reliable, repeatable technique for studying the effect of herbal extracts on animal ovaries.

The present study elucidated the fact that FVE has a folliculogenesis effect in female mice consistent with its use in folk medicine as a fertility enhancing agent. Further studies are suggested for understanding the exact mechanism(s) underlying these actions and probable changes in hormonal levels.