Stéphane Zingue

Safety evaluation (acute and sub-acute studies) of the aqueous extract of the leaves of Myrianthus arboreus P. Beauv. (Cecropiaceae) in Wistar rats

ETHNOPHARMACOLOGICAL RELEVANCE Myrianthus arboreus P. Beauv (Cecropiaceae) is a medicinal plant distributed in forests and damp places of tropical Africa. Its leaves are widely used as food and/or for the treatment of various ailments including dysmenorrhoea, female infertility, tumors and diarrhea. However, to the best of our knowledge, no safety assessment of this plant has been reported yet. AIM OF STUDY The present study aimed at evaluating the safety of the aqueous extract of leaves of Myrianthus arboreus (MAA) in Wistar rats through an acute and sub-acute oral administration. MATERIAL AND METHODS In acute oral toxicity, the test was performed according to the Organization for Economic Cooperation and Development (OECD) guidelines Nr. 423 (acute toxicity class method, ATC) with slight modifications. Female Wistar rats were orally treated with the aqueous extract of M. arboreus at the doses of 2000 and 5000mg/kg. In sub-acute toxicity study, using the OECD guidelines Nr. 407, the extract was administered by gavage at the doses of 20, 110 and 200mg/kg/day for 28 consecutive days. RESULTS A single oral administration of 2000 or 5000mg/kg of the extract induced neither mortality nor exterior signs of toxicity indicating a LD50 >5000mg/kg. In sub-acute study, the extract decreased triglycerides, total cholesterol/high density lipoproteins ratio and atherogenic index of plasma in both sexes at all tested doses. Alanine transaminase decreased in both sexes at 200mg/kg and serum creatinine levels decreased at all tested doses in females. Moreover, significant increases in ovarian and uterine wet weights, red blood cell count, hematocrit, mean corpuscular hemoglobin and hemoglobin were observed at 200mg/kg in females. In males, this extract decreased white blood cell count, lymphocytes and relative weight of seminal vesicles and ventral prostate at 200mg/kg. CONCLUSION The aqueous extract of Myrianthus arboreus leaves was non-toxic in acute administration and exhibited a relatively low toxicity potential on accessory sex organs in both sexes, and leukocytes in males following the repeated 28-days oral administration of the dose 200mg/kg.

Estrogenic effects of Ficus umbellata Vahl. (Moraceae) extracts and their ability to alleviate some menopausal symptoms induced by ovariectomy in Wistar rats

ETHNOPHARMACOLOGICAL RELEVANCE Ficus umbellata Vahl. (Moraceae) is a medicinal plant used in Cameroon to treat amenorrhea as well as other physiological disorders related to menopause. AIM OF STUDY In order to justify scientifically its traditional use, the estrogen-like properties of the aqueous (AE) and methanol (MeOH) extracts of F. umbellata were investigated. MATERIAL AND METHODS In vitro, the ability of different extracts of F. umbellata to activate estrogen receptors α (ERα) and β (ERβ) in cell-based reporter gene assays using human embryonic kidney (HEK293T) cells transfected with ERs was tested. In vivo, a 3-day uterotrophic assay and the capacity of the extracts to alleviate hot flushes in ovariectomized adult rats were tested. Using a bioassay-guided fractionation the major compound of F. umbellata was isolated and tested in vitro on HEK293T-ERα and ERβ cells. RESULTS AE and MeOH extracts significantly altered ERα as well as ERβ activities. In vivo, both extracts significantly increase the uterine and vaginal epithelium thickness, and uterine total protein levels in a dose dependent manner. Interestingly, both extracts of F. umbellata at the dose of 100 mg/kg BW significantly decreased the total number, average duration as well as frequency of hot flushes in experimental rats compared to age-matched OVX controls. Finally, 7-methylumbelliferone, a coumarin was characterized as the major compound of F. umbellata; however this compound did not transactivate ERα as well ERβ in vitro. CONCLUSION These aforementioned results suggest that F. umbellata extracts as used by the traditional practitioner have estrogen-like effects and may alleviate some menopausal problems such as vaginal dryness and hot flushes.

Crateva adansonii DC, an African ethnomedicinal plant, exerts cytotoxicity in vitro and prevents experimental mammary tumorigenesis in vivo

ETHNOPHARMACOLOGICAL RELEVANCE Crateva adansonii DC is a plant traditionally used in Cameroon to treat constipation, asthma, snakebites, postmenopausal complaints and cancers. AIM The anticancer potential of the dichloromethane/methanol extract of C. adansonii stem barks was investigated using human breast cancer cell and 7,12 dimethylbenz(a)anththracene (DMBA)-induced mammary tumorigenesis model in rats. MATERIAL AND METHODS The cytotoxicity of C. adansonii extract was assessed in vitro towards breast carcinoma (MCF-7 and MDA-MB-231) and non-tumoral cell lines (NIH/3T3 and HUVEC) by Alamar Blue assay. Furthermore, in vivo studies were performed on female Wistar rats treated either with C. adansonii extract at a dose of 75 or 300mg/kg body weight or with tamoxifen (3.3mg/kg body weight), starting 1 week prior DMBA treatment and lasted 12 weeks. The investigation focused on tumour burden, tumour DNA fingerprint, morphological, histological, hematological, and biochemical parameters. RESULTS CC50 values for the in vitro assays were 289µg/mL against MCF-7 cells and >500µg/mL in others cells, leading to a selectivity index ≥1.73. C. adansonii extract significantly (p<0.001) revealed in vivo the reduction of the cumulative tumour yield (87.23%), total tumour burden (88.64%), average tumour weight (71.11%) and tumour volume (78.07%) at the dose of 75mg/kg as compared to DMBA control group. A weak effect was also observed at 300mg/kg. This extract showed a moderate hyperplasia at the dose of 75mg/kg while at 300mg/kg no significant change was noted as compared to DMBA group. It protected rats from the DNA alteration induced by DMBA and increased antioxydant enzymes activities in mammary gland tissue homogenates. In addition, Ultra-High Performance Liquid Chromatography/ESI-QTOF-Mass Spectrometry analysis of C. adansonii extract detected structure-related of many well-known anticancer agents such as flavane gallate, flavonol, phenylpropanoïds, sesquiterpene derivatives, gallotannins and lignans. The LD50 of C. adansonii was estimated to be greater than 5000mg/kg. CONCLUSIONS These aforementioned results suggest that the C. adansonii extract may possess antitumor constituents, which could combat breast cancer and prevent chemically-induced breast cancer in rats.

Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity

Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs.

In vivo and in vitro estrogenic activity of extracts from Erythrina poeppigiana (Fabaceae)

Abstract In developing countries, around 80% of the population still resorts on traditional medicine for their primary health care. Erythrina poeppigiana (Walp.) O.F. Cook, one of these medicinal plants, was found to be particularly rich in isoflavonoids which exhibited, individually, significant estrogenic activity in vitro. The possible combined effects of these bioactive isoflavones, as they are naturally found in the crude extracts of E. poeppigiana, prompted us to assess their in vivo estrogenicity. We first tested the ability of the extracts to transactivate estrogen receptor alpha (ERα) in vitro using U2OS human osteosarcoma cells. We next investigated their effects in vivo in an uterotrophic assay, using ovariectomized rats treated with the extracts at the doses of 50, 100, 200, and 400 mg/kg BW/d orally for 3 days. Finally, we assessed their ability to relieve hot flushes, using data loggers. At the end of treatments, animals were sacrificed, and organs (mammary glands, vagina, and uteri) were collected for histo-morphometric analyses. The methanol extract significantly and dose-dependently transactivated ERα at all tested doses. All extracts induced significant increases of vaginal and uterine epithelial heights. Only the dichloromethane extract could significantly relieve hot flushes as estradiol. These results indicate that E. poeppigiana extracts have estrogen-like effects in vivo, suggesting that its active principles act in synergy when they are taken in combination in the crude extract. These findings, therefore, support the traditional use of E. poeppigiana to alleviate some menopausal problems; our previous phytochemical investigations contribute to the standardization of this phytomedicine.

Ficus umbellata Vahl. (Moraceae) Stem Bark Extracts Exert Antitumor Activities In Vitro and In Vivo

A Ficus umbellata is used to treat cancer. The present work was therefore designed to assess antitumor potentials of F. umbellata extracts in nine different cell lines. Cell cycle, apoptosis, cell migration/invasion, levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspases activities as well as Bcl-2 and Bcl-xL protein content were assessed in MDA-MB-231 cells. The 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenesis in rats were also used to investigate antitumor potential of F. umbellata extracts. The F. umbellata methanol extract exhibited a CC50 of 180 μg/mL in MDA-MB-231 cells after 24 h. It induced apoptosis in MCF-7 and MDA-MB-231 cells, while it did not alter their cell cycle phases. Further, it induced a decrease in MMP, an increase in ROS levels and caspases activities as well as a downregulation in Bcl-2 and Bcl-xL protein contents in MDA-MB-231 cells. In vivo, F. umbellata aqueous (200 mg/kg) and methanol (50 mg/kg) extracts significantly (p < 0.001) reduced ovarian tumor incidence (10%), total tumor burden (58% and 46%, respectively), average tumor weight (57.8% and 45.6%, respectively) as compared to DMBA control group. These results suggest antitumor potential of F. umbellata constituents possibly due to apoptosis induction mediated through ROS-dependent mitochondrial pathway.

The methanol-soluble fraction of Millettia macrophylla (Fabaceae) stem bark endowed with estrogenic properties has adverse effects on the male reproductive system of Wistar rats

Abstract Background: The use of traditional medicinal plants to treat various diseases is common in Sub-Saharan African countries, including Cameroon. Millettia macrophylla, one of such plants, was previously found to exhibit estrogenic properties in female Wistar rats. However, its effects on the male reproductive system are unknown. Based on literature evidence that phytoestrogens impair male sexual behavior, we aimed at assessing the effect of the extracts of M. macrophylla stem bark on the male reproductive system of Wistar rats. Materials and methods: We evaluated the effects of the dichloromethane (DCM) and methanol (MeOH) soluble fractions of M. macrophylla stem bark on male rat sexual behavior, as well as androgen-dependent parameters for 60 days. Results: Data showed a significant decrease (p<0.05) in the wiring-touch frequency, mount frequency, intromission frequency, ejaculatory frequency, penile-licking frequency, and computed indices of sexual behavior, throughout the experimental period, as well as a significant increase (p<0.001) in mount, intromission, and ejaculatory latencies as well as post-ejaculatory interval. Moreover, we observed a significant decrease (p<0.05) in the androgen-dependent sexual parameters evaluated. The DCM extract did not induce significant effects on the assessed parameters. Conclusions: These results suggest that long-term exposure to the estrogenic MeOH fraction of M. macrophylla stem bark negatively alters sexual behavior and spermatogenesis.

Safety assessment of the methanol extract of the stem bark of Amphimas pterocarpoides Harms: Acute and subchronic oral toxicity studies in Wistar rats

Amphimas pterocarpoides Harms (Leguminosae) is widely used traditionally in Central and West Africa for the treatment of various ailments. However, no data regarding its safety have been published until now. Thus, the present study aimed to investigate the potential toxicity of the methanol extract of the stem bark of Amphimas pterocarpoides (AP) in Wistar rats following the OECD guidelines. In acute oral toxicity, female rats received a single dose of 2000 mg/kg of AP and were observed for 14 days. In subchronic toxicity, doses of 150, 300, 600 mg/kg/day of AP were given per os to rats (males and females) for 28 days. No death and abnormal behaviors were observed in acute toxicity and the LD50 was estimated higher than 5000 mg/kg. In the subchronic study, AP induced no significant variation in body weight and relative weight of organs, whereas a delayed decrease of white blood cell count and granulocytes was observed. Inconsistent increase of the total cholesterol/high density lipoprotein was observed at 600 mg/kg in males. Such variation (not dose dependent) and without biological relevance indicate a wide margin of safety for the traditional use of AP.

Estrogenic properties of spices of the traditional Cameroonian dish “Nkui” in ovariectomized Wistar rats

Abstract Background: Besides the basic role to flavor and color foods, several health benefits have been attributed to spices. The traditional Cameroonian food “Nkui” is prepared using several spices (Afrostyrax lepidophyllus Mildbr., Capsicum frutescens Linn., Fagara leprieurii Guill. et Perr., Fagara tessmannii Engl., Mondia whitei Hook. F. Skell., Pentadiplandra brazzeana Baill., Solanum gilo Raddi., Tetrapleura tetraptera Taub. and Xylopia parviflora A. Rich. Benthane) that are believed to have a positive impact on the female reproductive physiology. Aiming to determine the potential effect of this food on the female reproductive tract, we evaluated the estrogenic properties of aqueous and ethanol extracts of Nkui using a 3-day uterotrophic assay in ovariectomized (OVX) rats. Methods: OVX female Wistar rats were randomly separated in several groups of five animals each and submitted to a 3-day uterotrophic assay (per os). At the end of treatment, animals were sacrificed and uterus, vagina and mammary gland collected and fixed in 10 % formalin for histological analysis. Results: These extracts increased the uterine wet weight, the uterine and vaginal epithelial heights, and the lumen and diameter of alveoli in the mammary glands. They also altered the estradiol-induced increase of uterine wet weight. The dichloromethane and methanol fractions of the ethanol extract exhibited estrogenic properties as well by increasing uterine and vaginal endpoints. Conclusions: These results suggest that the spices of “Nkui” contain estrogenic phytoconstituents and this traditional food may be considered as functional.

Erythrina lysistemon-derived flavonoids account only in part for the plant’s specific effects on rat uterus and vagina

Abstract Background: The stem bark ethyl acetate extract of Erythrina lysistemon was found to induce vaginal proliferation in ovariectomized rats orally treated. Alpinumisoflavone (AIF) and abyssinone V-4′-methyl-ether (AME), isolated as its major constituents, were reported to separately provoke uterine growth and/or vaginal proliferation. The present study aimed at evaluating the effects of the mixture of AIF and AME (51 mg/kg [AIF]+153 mg/kg [AME]) following their relative abundance in the extract, in order to compare these effects to those of E. lysistemon. Methods: The study was performed in ovariectomized rats treated intraperitoneally for 3 days. Estradiol valerate (E2 V) and AME were used for positive controls. Morphological and histological changes of animals’ uterus and vagina were used as the hallmark of estrogenicity. Results: E. lysistemon extract induced estrogen-like effects only on the uterus and significantly increased uterine wet weight (p<0.01) and uterine epithelial height (p<0.01). These results suggest a tissue-selective action of E. lysistemon extract depending on the route of administration. The mixture of AIF and AME induced E. lysistemon-like effects only at a dose of 1 mg/kg BW/d (0.25 mg/kg+0.75 mg/kg), although these effects were lower in magnitude (p<0.05) compared to those induced by E. lysistemon extract. Conclusions: Effects induced by the mixture of AIF and AME are analogous to those of E. lysistemon, but the low magnitude of these effects suggests that there are minor metabolites that interact with AIF and AME to provoke the specific effects of E. lysistemon.