Assessment of Ferula hermonis Boiss fertility effects in immature female rats supported by quantification of ferutinin via HPLC and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferula hermonis is a small shrub renowned for its aphrodisiac abilities. Middle East herbalists have utilized Ferula hermonis seed and root as an aphrodisiac folk medicine to treat women's frigidity and male erectile and sexual dysfunction. AIM OF THE STUDY: Assessment of follicle-stimulating hormone-like (FSH), luteinizing hormone-like (LH), and estrogenic activities of the methanolic extract (ME) of the roots of Ferula hermonis on female reproductive function. MATERIALS AND METHODS: The methanolic extract was prepared from the root of F. hermonis and studied at dose level 6 mg/kg in immature female rats for FSH-like, LH-like, and estrogenic activities. These activities were determined by analyzing gross anatomical features, relative organ weight, and serum level of FSH, LH, progesterone and estrogen hormones, and histopathological characteristics. Quantification of the main phytoestrogenic component ferutinin carried out by HPLC. In addition, molecular docking for the binding affinity of ferutinin inside active sites of both estrogen receptor alpha (ERα) and FSH receptor (FSHR) was performed to predict the potential role of ferutinin in regulating the female reproductive process. RESULTS: Ferula hermonis (ME) showed potent FSH-like, LH-like activities and moderate estrogenic effect at the dose of 6 mg/kg. The content of ferutinin in F. hermonis was estimated to be 92 ± 1.33 mg/g of the methanolic extract. Molecular docking of ferutinin with ERα and FSHR displayed strong interaction with target proteins. CONCLUSIONS: Based on results, it can be concluded that Ferula hermonis can be considered as a suitable female fertility improving agent.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells.

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5-15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-ß-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

Ferutinin: A phytoestrogen from ferula and its anticancer, antioxidant, and toxicity properties.

This study was performed to evaluate the antioxidant, anticancer, and toxicity properties of ferutinin, a phytoestrogen derived from Ferula species. The human Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line and normal human fibroblast (HDF) were cultured and treated with different ferutinin concentrations. The cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell death-defining tests (a comparative real-time polymerase chain reaction [for Bax and Bcl-2 genes], flow cytometry, and acridine orange/propidium iodide cell staining). Moreover, 15 white male balb/c mice were divided into three groups of five (one untreated control group and two groups), which received different doses of ferutinin-supplemented water (500 and 1000 µg/kg mice weight) to check the mice liver and kidney pathomorphological alterations and to determine the antioxidant enzymes' expression profile (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase) in the mentioned tissues. Finally, the liver lipid peroxidation of mice was analyzed. The results of MTT and cell death-defining tests indicate the significant reduction in cell viability and induction of apoptotic death in MCF-7 cells (enhanced sub-G1 peaks, Bax overexpression, Bcl-2 downregulation, and increased apoptotic cells). The antioxidant enzymes (SOD and CAT) in the mice liver and kidney cells were found to be upregulated (p < .05) in response to the increasing doses of ferutinin. Besides, the lipid peroxidation of the liver tissue of mice was significantly reduced. According to the results, we suggest that ferutinin has the potential to be served as a selective anticancer compound for breast cancer treatment.

Ferula L. Plant Extracts and Dose-Dependent Activity of Natural Sesquiterpene Ferutinin: From Antioxidant Potential to Cytotoxic Effects.

The employment studies of natural extracts in the prevention and treatment of several diseases highlighted the role of different species of genus Ferula L., belonging to the Apiaceae family, dicotyledonous plants present in many temperate zones of our planet. Ferula communis L. is the main source of sesquiterpene ferutinin, a bioactive compound studied both in vitro and in vivo, because of different effects, such as phytoestrogenic, antioxidant, anti-inflammatory, but also antiproliferative and cytotoxic activity, performed in a dose-dependent and cell-dependent way. The present review will focus on the molecular mechanisms involved in the different activities of Ferutinin, starting from its antioxidant potential at low doses until its ionophoric property and the subsequent mitochondrial dysfunction induced through administration of high doses, which represent the key point of its anticancer action. Furthermore, we will summarize the data acquired from some experimental studies on different cell types and on several diseases. The results obtained showed an important antioxidant and phytoestrogenic regulation with lack of typical side effects related to estrogenic therapy. The preferential cell death induction for tumor cell lines suggests that ferutinin may have anti-neoplastic properties, and may be used as an antiproliferative and cytotoxic agent in an estrogen dependent and independent manner. Nevertheless, more data are needed to clearly understand the effect of ferutinin in animals before using it as a phytoestrogen or anticancer drug.

Ferula hermonis: A Review of Current Use and Pharmacological Studies of its Sesquiterpene Ester Ferutinin.

Ferula hermonis Boiss, is an endemic plant of Lebanon, locally known as "shilsh Elzallouh". It has been extensively used in the traditional medicine as an aphrodisiac and for the treatment of sexual impotence. Crude extracts and isolated compounds of ferula hermonis contain phytoestrogenic substances having a wide spectrum of in vitro and in vivo pharmacological properties including anti-osteoporosis, anti-inflammatory, anti-microbial and anti-fungal, anti-cancer and as sexual activity enhancer. The aim of this mini-review is to highlight the traditional and novel applications of this plant's extracts and its major sesquiterpene ester, ferutinin. The phytochemical constituents and the pharmacological uses of ferula hermonis crude extract and ferutinin specifically will be discussed.

Comparison the Effect of Ferutinin and 17ß-Estradiol on Bone Mineralization of Developing Zebrafish (Danio rerio) Larvae.

There is an urgent need to develop novel drugs for osteoporosis which occurs due to estrogen deficiency. Phytoestrogens derived from medicinal plants would be the best alternative to chemical drugs with harmful side effects. The main purpose of the present study was to investigate the effect of ferutinin compared to 17ß-estradiol (E2) on bone mineralization of zebrafish larvae. Regarding the lack of publications, the histology analysis was performed after exposure to E2 to find effective treatment on bone mineralization of developing zebrafish larvae. Then, the larvae were exposed to four concentrations of ferutinin at three time points to assess the mortality, the expression of some related genes and histology of the ceratohyal and hyomandibular of treated larvae. The RT-PCR result of the treatment groups demonstrated the similar expression pattern in the larvae which were exposed to 1.25 µg/mL of ferutinin and 2 µM of E2 at 2 dpf, which confirmed the result of histology analysis. In addition, RT-qPCR of high concentration of ferutinin and E2 demonstrated that bmp2a/b and esr1 were downregulated and upregulated when the larvae were exposed to 5 µg/mL of ferutinin and 10 µM of E2, respectively.

Phytochemistry and Pharmacology of Ferula hermonis Boiss. - A Review.

Ferula hermonis, a well-known species of the genus Ferula found in Lebanon and Syria, has a brilliant history in traditional medicine as it has been used for the treatment of erectile dysfunction in men and menopausal disturbances in women. Thanks to modern pharmacological and clinical investigations, F. hermonis is a valuable medicinal and condimental plant that may be used for the treatment of impotence and diabetes, the prevention of osteoporosis, and possesses anti-microbial and anti-inflammatory properties. Phytochemical investigations have shown that this plant contains daucane aryl esters such as ferutinin, which has exhibited various biological activities including hypoglycemic and estrogenic activities. Ferutinin is one of the strongest natural phytoestrogen which has agonistic activity on estrogen receptors, particularly α receptor. It seems that ferutinin and its derivatives play an important role in F. hermonis biological activities, mainly the beneficial effects of this plant on impotence, diabetes and osteoporosis. The present review discusses the available data on the active constituents and biological activities of F. hermonis and their possible underlying mechanisms of action.

Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: effect of oral ferutinin treatment in rats.

AIMS: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. MAIN METHODS: In 12 week old male rats (n=10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold+ferutinin at a dose of 2mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 µm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. KEY FINDINGS: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistry performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ERα and GPR30) in all groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. SIGNIFICANCE: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen-AFSCs constructs.

Investigating anticancer properties of the sesquiterpene ferutinin on colon carcinoma cells, in vitro and in vivo.

AIMS: In this research, ferutinin was evaluated for its possible cytotoxic and apoptotic inducing effects in vitro and in vivo. MAIN METHODS: To determine IC50 values of ferutinin, CT26, HT29 and NIH/3T3 cells were treated with different concentrations of ferutinin. In addition to morphological changes in cells, the DNA damage was studied using DAPI staining, comet assay and PI staining. Ferutinin was also tested for its in vivo activity. KEY FINDINGS: Analyses of cell survival by MTT assay showed that the IC50 values of ferutinin on CT26 and HT29 cells were 26 and 29 µg/ml, respectively, while after treating nontumoural mouse cells even with 50 µg/ml ferutinin, 70% of cells was still surviving. The results of DAPI staining and comet assay revealed that ferutinin significantly induced DNA damage in treated cells. Induction of sub-G1 peak after PI staining was also indicative of apoptotic effects of ferutinin in cancerous cells. In vivo studies showed a significant regression in tumour size in mice treated with ferutinin as compared to control groups. Its antitumour effects were very similar to the cisplatin treated group. Histological studies demonstrated that apoptosis rate in tumour cells was increased in comparison to tumour cells in control mice without ferutinin treatment. Interestingly, haematoxylin and eosin staining showed no damage in the spleen and liver of ferutinin treated mice. SIGNIFICANCE: As ferutinin showed less toxic effects in nontumoural cells, and induced its effects via apoptosis induction, it could be considered as an effective anticancer agent for future preclinical experiments.

Ferutinin, an apoptosis inducing terpenoid from Ferula ovina.

A current hurdle in cancer management is the intrinsic or acquired resistance of cancer cells to chemical agents that restricts the efficacy of therapeutic strategies. Accordingly, there is an increasing desire to discover new natural compounds with selective toxicity to combat malignancies. In present study, the cytotoxic and apoptosis- inducing activities of ferutinin, a terpenoid derivative from Ferula ovina, were investigated on human breast (MCF7) and bladder (TCC) cancer cells as well as normal fibroblasts (HFF3).The toxicity and DNA damage inducing effects of ferutinin were studied by MTT and comet assays, DAPI and PI staining and DNA laddering. The IC50 values of ferutinin were identified and compared with routine prescribed drugs, doxorubicin and vincristine, by MTT test. Alkaline comet assay and DAPI staining revealed DNA damage due to ferutinin, which was significantly (p<0.001) higher in MCF7 and TCC than HFF3 cells. Apoptosis induction was evidenced by PI staining and DNA laddering. Our results suggest that ferutinin could be considered as an effective anticancer agent for future in vivo and clinical experiments.

Antifungal compounds from the rhizome and roots of Ferula hermonis.

The antifungal activity of hexane, dichloromethane, methanol and aqueous extracts from the rhizome and root of Ferula hermonis was assayed in vitro by the agar disk diffusion method against a panel of human opportunistic and pathogenic fungi. Among them, the hexane and dichloromethane extracts showed the highest activity particularly against the dermatophytes Microsporum gypseum and Tricophyton mentagrophytes as well as the yeast Candida lactis-condensi. Activity-guided fractionation of both extracts using an agar overlay bioautographic method led to the isolation of two antifungal compounds which were identified as the daucane aryl esters jaeschkeanadiol p-hydroxybenzoate (ferutinin) and jaeschkeanadiol benzoate (teferidin). Determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values of both compounds evidenced a stronger antifungal activity for ferutinin than for teferidin. Particularly, T. mentagrophytes was the most sensitive strain with MIC and MFC values ranging from 8 to 256 µg/mL.

Discovery of small molecules that inhibit melanogenesis via regulation of tyrosinase expression.

5,6,7,8-Tetrahydro-4H-cyclohepta[d]isoxazole derivatives were synthesized and evaluated as a novel class of inhibitors for α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in a mouse melanoma B16F10 cell line. Compound 8e (IC(50)=0.67 µM), 8h (IC(50)=1.01 µM) and 9b (IC(50)=0.99 µM) exhibited a potent inhibitory activity approximately 85- to 126-fold greater than kojic acid, a well-known potent inhibitor. A biochemical study indicates that the activity of this series should be displayed via down-regulation of the expression of tyrosinase.

Antimicrobial antioxidant daucane sesquiterpenes from Ferula hermonis Boiss.

Seventeen daucane sesquiterpenoid esters, including a new one (4), were isolated from the root of Ferula hermonis Boiss. The structures of the isolated compounds were elucidated on the basis of spectroscopic evidence and correlated with known compounds. The relative stereochemistry of the new compound was determined using 2D NOESY and the most stable and the lowest energy conformation was determined using molecular modelling. The antimicrobial activity was evaluated by determination of MIC using the broth microdilution method against six bacterial strains and one fungal strain (Pseudomonas aeruginosa PAO1, Escherichia coli, Bacillus subtilis ATCC6633, Mycobacterium bovis BCG Pasteur, Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC6538 and Candida albicans SC5314). There was a significant indication that compounds 15, 16, 17 demonstrated potent activity against Gram +ve (S. aureus, B. subtilis), as well as Mycobacterium strains M. bovis BCG and M. tuberculosis H37Rv. None of the isolated compounds exhibited a significant antifungal activity. In the antioxidant study using the DPPH assay method, the highest radical scavenging activity was observed for compounds 15, 16, 17.

Structural and histomorphometric evaluations of ferutinin effects on the uterus of ovariectomized rats during osteoporosis treatment.

AIMS: The effects of chronic administration of Ferutinin (phytoestrogen found in the plants of genus Ferula), compared with those elicited by estradiol benzoate, were evaluated, following ovariectomy, on the uterus of ovariectomized rats as regard weight, size, structure and histomorphometry. MAIN METHODS: The experimental study included 40 female Sprague-Dawley rats, assigned to two different protocols, i.e. preventive and recovering. In the preventive protocol, ferutinin (2mg/kg/day) was orally administered for 30days, starting from the day after ovariectomy; in the recovering protocol, ferutinin was administered, at the same dosage, for 30days starting from the 60th day after ovariectomy, when osteoporosis was clearly established. Its effects were compared with those of estradiol benzoate (1.5µg per rat twice a week, subcutaneously injected) vs. vehicle-treated ovariectomized controls and vehicle-treated sham-operated controls. Uteri were removed, weighed and analysed under both the structural and histomorphometrical points of view. KEY FINDINGS: Our data show that ferutinin acts, similarly to estradiol benzoate, on the uterus stimulating endometrial and myometrial hypertrophy; this notwithstanding, the phytoestrogen ferutinin, in contrast to estrogen treatment, appears to increase apoptosis in uterine luminal and glandular epithelia. SIGNIFICANCE: Ferutinin, used in osteoporosis treatment primarily for bone mass recovering, seems in line with an eventual protective function against uterine carcinoma, unlike estrogens so far employed in hormone replacement therapy (HRT).

The antidepressant-like effect of Hedyosmum brasiliense and its sesquiterpene lactone, podoandin in mice: evidence for the involvement of adrenergic, dopaminergic and serotonergic systems.

We have recently shown that the ethanol extract of the leaves of Hedyosmum brasiliense exhibits an antidepressant-like effect in the tail suspension and forced swimming tests in mice. The present study investigates the mechanisms involved in the antidepressant-like effect of H. brasiliense extract, together with the antidepressant potential of podoandin, an isolated sesquiterpenoid. H. brasiliense (50mg/kg, i.p.) and podoandin (10mg/kg, i.p.) decreased the immobility time in the forced swimming test, without any accompanying changes in ambulation in the open-field test. The anti-immobility effect of the H. brasiliense extract was prevented by pre-treating the mice with ondansetron, NAN 190, pindolol, prazosin, yohimbine, haloperidol, SCH23390, and sulpiride. On the other hand, pre-treating the mice with: p-chlorophenylalanine (4 consecutive days), ketanserin, naloxone, naltrindole, bicuculline, phaclofen, or l-arginine did not block the antidepressant-like effect of H. brasiliense. In addition, pre-treatment of the animals with methylene blue, NG-nitro-l-arginine or 7-nitroindazole, at subeffective doses, did not cause a synergistic effect with H. brasiliense extract at an effective dose in the forced swimming test. The anti-immobility effect of podoandin was also prevented by pre-treating the mice with NAN-190, ondansetron, prazosin, yohimbine, sulpiride and haloperidol. The results indicate that the antidepressant-like effect of H. brasiliense (and podoandin) is dependent on the serotonergic, noradrenergic and dopaminergic systems, but not on the GABAergic, opioid and oxidonitrergic systems.

Antiinflammatory sesquiterpenes from the root oil of Ferula hermonis.

Ferula hermonis Boiss. (Apiaceae), commonly known as 'Shilsh-el-zallouh', 'Hashishat-al-kattira' or 'The Lebanese viagra', is a small shrub that grows abundantly on the Hermon Mountain between Syria and Lebanon. The seeds and roots of this plant have long been used in the Middle East as an aphrodisiac, and for the treatment of frigidity and impotence for both men and women. The antiinflammatory properties of three major daucane esters, ferutinin (1) teferin (2) and teferidin (3), isolated from the root oil of Ferula hermonis, were assessed by the carrageenan-induced oedema model in rats. The antiinflammatory effect of both 1 and 2 was observed with a dose of 100 mg/kg, while compound 3 did not show any antiinflammatory activity; conversely it produced a significant proinflammatory effect 2 and 3 h after carrageenan injection.

Antimycobacterial activity of ferutinin alone and in combination with antitubercular drugs against a rapidly growing surrogate of Mycobacterium tuberculosis.

The aim of this study was to investigate the antimycobacterial activity of the major daucane constituent, ferutinin (jaeschkeandiol p-hydroxybenzoate, 1), four of its natural analogues, its hydrolysis products, as well as methyl p-hydroxybenzoate (methylparaben) against Mycobacterium smegmatis, a rapidly growing surrogate of Mycobacterium tuberculosis. The agar dilution assay was utilised for an antimycobacterial evaluation of single compounds. A modified agar dilution assay, the checkerboard method, was utilised for evaluating the potentiating effect of 1 on different antitubercular drugs, namely isoniazid, ethionamide, rifampin and streptomycin. In the agar dilution assay, 1 exhibited higher potency (minimum inhibitory concentration [MIC] 10 µg mL⁻¹) than streptomycin and rifampin (MIC 20 µg mL⁻¹ for each). Of the natural analogues, 8,9-epoxyjaeschkeandiol p-hydroxybenzoate and 8,9-epoxyjaeschkeandiol benzoate exhibited marginal activity (MIC ≥ 40 and 80 µg mL⁻¹, respectively). The checkerboard method showed that the combination of 1 with each antitubercular drug led to mutual enhancement of the antimycobacterial activity with isoniazid and ethionamide, while no such effect was observed with rifampin or streptomycin. Based on this study and earlier studies with Staphylococcus aureus, the major constituent 1 may be responsible for the major part of the antimicrobial activity of the root of Ferula hermonis.

Influence of ferutinin on bone metabolism in ovariectomized rats. II: role in recovering osteoporosis.

The aim of the present investigation, which represents an extension of a previous study, was to investigate the effect of ferutinin in recovering severe osteoporosis due to estrogen deficiency after rat ovariectomy and to compare phytoestrogen effects with those of estrogens commonly used in hormone replacement therapy (HRT) by women with postmenopausal osteoporosis. The animal model used was the Sprague-Dawley ovariectomized rat. Ferutinin was orally administered (2 mg kg(-1) per day) for 30 or 60 days starting from 2 months after ovariectomy (i.e. when osteoporosis was clearly evident) and its effects were compared with those of estradiol benzoate (1.5 microg per rat twice a week, subcutaneously injected) vs. vehicle-treated ovariectomized (OVX) and sham-operated (SHAM) rats. Histomorphometric analyses were performed on trabecular bone of lumbar vertebrae (4th and 5th) and distal femoral epiphysis, as well as on cortical bone of femoral diaphysis. Bone histomorphometric analyses showed that ferutinin seems to display the same effects on bone mass recorded with estradiol benzoate, thus suggesting that it could enhance the recovery of bone loss due to severe estrogen deficiency in OVX rats. On this basis, the authors propose listing ferutinin among the substances representing a potential alternative for the treatment of postmenopausal osteoporosis, which occurs as a result of estrogen deficiency.

Calcium mobilization by the plant estrogen ferutinin does not induce blood platelet aggregation.

Platelet activation is closely associated with an increase in intracellular Ca(2+) concentration. Various compounds including Ca(2+) ionophores are able to trigger platelet aggregation by increasing intracellular Ca(2+) concentration in platelets. In the present study, we monitored the effect of the phytoestrogen ferutinin, which acts as a Ca(2+) ionophore in human blood platelets; its ionophore-like properties include upregulation of [Ca(2+)](in), activation of fibrinogen receptors and increased fibrinogen binding. Using spectrofluorometry and triple-color flow cytometry, we demonstrate that ferutinin increases [Ca(2+)](in) in both isolated platelets and platelets in whole blood from humans. This effect was almost completely blocked by the Ca(2+) chelator EGTA and was not sensitive to either Gd(3+) or econazole, which inhibit VOC and SOC channels, respectively. Nor was the effect sensitive to thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) ATPases. Ferutinin stimulated the expression of the active form of the GPIIb-IIIa complex and whole blood platelet aggregation only weakly and had no statistically significant effect on the binding of fibrinogen. These results demonstrate apparently inconsistent effects of ferutinin, which raises intraplatelet Ca(2+) concentration but fails to have an effect on spontaneous blood platelet aggregation. This pattern of responses may be caused by the combination of ferutinin's Ca(2+) ionophoric and estrogenic properties.

The phytoestrogen ferutinin improves sexual behavior in ovariectomized rats.

The present study was designed to examine the effect of ferutinin chronic administration on sexual behavior of ovariectomized non-estrogen-primed rats. Starting from 3 weeks after ovariectomy, female rats were orally treated with ferutinin at the doses of 0.2 and 0.5 mg/kg, daily for 4 weeks. Ferutinin's effect was compared with that of estradiol benzoate, subcutaneously injected at the dose of 1.5 microg/rat twice a week. Animals were tested for sexual motivation, receptivity and proceptivity after 1, 2 and 3 weeks of treatment and for paced mating behavior after 4 weeks of treatment. Before each experimental test, they received progesterone injection (500 microg/rat). Both dosages of ferutinin significantly increased the receptive behavior in a time-dependent manner, as well as estradiol benzoate did. Also proceptive behaviors increased in ferutinin-treated animals in comparison with control ones. During the partner preference test ferutinin was able to induce a significant preference for a sexually active male over a sexually receptive female. Moreover, ferutinin restored a normal paced mating behavior, which had been suppressed by ovariectomy. These results show that ferutinin exerts an estrogenic activity in ovariectomized non-estrogen-primed female rats.