Effects of Tartrazine on Some Sexual Maturation Parameters in Immature Female Wistar Rats.

Over the past century, the average age for onset of puberty has declined. Several additives present in our food are thought to contribute significantly to this early puberty which is recognized to also affect people's health in later life. On this basis, the impact of 40-days unique oral administration of the food dye tartrazine (7.5, 27, and 47 mg/kg BW doses) was evaluated on some sexual maturation parameters on immature female Wistar rats. Vaginal opening was evaluated during the treatment period. At the end of the treatments, animals were sacrificed (estrus phase) and the relative weight of reproductive organs, pituitary gonadotrophin and sexual steroids level, cholesterol level in ovaries and folliculogenesis were evaluated. Compared to the control group, animals receiving tartrazine (47 mg/kg BW) showed significantly high percentage of early vaginal opening from day 45 of age, and an increase in the number of totals, primaries, secondaries, and antral follicles; a significant increase in serum estrogen, LH and in uterine epithelial thickness. Our findings suggest that tartrazine considerably disturbs the normal courses of puberty. These results could validate at least in part the global observations on increasingly precocious puberty in girls feeding increasingly with industrially processed foods.

Effects of Hibiscus sabdariffa Calyxes Aqueous Extract on Antioxidant Status and Histopathology in Mammary Tumor-Induced in Rats.

Breast cancer is a major threat worldwide. Hibiscus sabdariffa is widely consumed as beverage in sub-Saharan Africa for its anticancer potential. The present study therefore aimed at scientifically verifying its anticancer effect in rats. For this, 48 Wistar rats (∼55 days) were treated either with tamoxifen at 3.3 mg/kg BW (standard) or with a decoction of H. sabdariffa (125, 250, and 500 mg/kg BW) or distilled water (vehicle). Breast cancer was induced by a single dose of 50 mg/kg of 7,12-dimethylbenz(a)anthracene (DMBA). At the end of the 21 weeks of treatment, the tumor incidence, tumor morphology, histopathology, as well as some biochemical parameters in the tumors were assessed. As a result, 86% of DMBA's rats developed mammary tumors. The H. sabdariffa extract (125 and 250 mg/kg) reduced tumor incidence by 63% and 75%, respectively; inhibited tumor burden by 84.86% and 38.78%, respectively, and decreased tumor volume by more than 72% compared to the DMBA group. It also protected rats against DMBA-induced diffuse breast neoplasia, and the optimal effect was recorded at 125 mg/kg. Furthermore, it significantly increases the SOD activity and decreases the MDA level. In summary, H. sabdariffa has antibreast tumor and antioxidant properties in rats, which could justify its common use to treat cancer.

Anti-breast cancer potential of Anonidium mannii (Oliv.) Engl. & Diels barks ethanolic extract: UPLC-ESI-QTOF-MS detection of anticancer alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: Breast cancer is a serious threat in low-income as well as developed countries. To face this, many herbal preparations are prescribed by traditional healers in Cameroon, among which is Anonidium mannii commonly called "wild soursop". AIM: This study was undertaken to assess the anti-tumor effect of A. mannii ethanolic extract on cancer cell growth and against DMBA-induced mammary tumors in rats. MATERIALS AND METHODS: The well characterized MTT bioassay was used to assess the cytotoxic potential of A. mannii ethanolic extract in liver (HepG2), prostate (DU145 & PC3) and breast (MCF-7) cancer cell lines. Considering the fact that breast cells were the most sensitive to the extract, a 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast tumor rat model was used to assess the possible anticancer effect of A. mannii extract. Indeed, rats were treated with either tamoxifen (3.3 mg/kg BW) or A. mannii extract (16.5, 50 and 150 mg/kg BW) or vehicle (2% ethanol) for 20 weeks. Tumor incidence, tumor mass and volume, oxidative stress status in tumor as well as tumor histoarchitecture were evaluated. RESULTS: A 24 h incubation of tested cells with the A. mannii extract significantly slowed cell growth in a concentration-dependent manner with an interesting effect in breast cells (IC50 ~61.5 µg/mL). As compared to the DMBA rats, those treated with A. mannii extract (50 and 150 mg/kg) showed reduced breast tumor incidence (28%), tumor burden (95.34% at 50 mg/kg and 99.14% at 150 mg/kg) and tumor volume (~92%). A. mannii extract counteracted the high proliferation of terminal mammary ducts induced by DMBA, mainly at 50 mg/kg. Furthermore, the extract decreased MDA and nitrite levels but increased SOD activity in the mammary gland. High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS) analysis detected potential anticancer and antioxidant alkaloids in A. manni extract, which are close to those found in Annona muricata. CONCLUSION: These results provide evidence on the in vitro and in vivo anticancer effects of A. mannii, and therefore support its use in traditional medicine system to fight against cancer.

The leaf aqueous extract of Myrianthus arboreus P. Beauv. (Cecropiaceae) improved letrozole-induced polycystic ovarian syndrome associated conditions and infertility in female Wistar rats.

BACKGROUND: Myrianthus arboreus P. Beauv. (Cecropiaceae) is a medicinal plant used to treat female infertility. The aqueous extract of M. arboreus leaves was found to improve the fertility of healthy female Wistar rats. In the present study, we proposed evaluating the effects of such an extract on an animal model of infertility caused by polycystic ovary syndrome (PCOS), in order to bring scientific evidence to the curative action of this plant against female infertility. METHODS: Following a 21-day administration (gavage) of letrozole (1 mg/kg), animals with PCOS, indicated by overweight and an estrous cycle blocked in the diestrus phase, were co-treated with letrozole (1 mg/kg) and the aqueous extract of M. arboreus leaves at doses of 20, 110 and 200 mg/kg. The positive control received clomiphene citrate (1 mg/kg) and metformin (200 mg/kg). The negative control received distilled water. Each group of animals was made up of 10 female rats. Vaginal smear was examined 7 days before and during co-treatments. Co-treatments were orally administered for 30 consecutive days and 5 animals per group were sacrificed thereafter for biochemical and histological analyses. The 5 remaining animals in each group were crossbred with males of proven fertility for 5 consecutive days. The daily examination of vaginal smears allowed evaluating fertility index. Following parturition, gestation rate was calculated. RESULTS: The aqueous extract of M. arboreus leaves reversed letrozole effects by decreasing body weight, abdominal fat accumulation, and serum levels of LH and testosterone (p < 0.001). Ovarian dynamic was improved and the number of tertiary, Graafian follicles (p < 0.001) and corpus luteum increased while that of cystic (p < 0.001) and atretic follicles (p < 0.01) decreased. These effects were associated with increased serum levels of estradiol, decreased ovarian oxidative stress, the resumption of the estrous cycle, the hypertrophy of uterine epithelial cells and increased fertility index and gestation rate. CONCLUSIONS: These results justify at least in part, the traditional use of M. arboreus against female infertility and suggest that this plant could be a promising alternative treatment to improve symptoms associated with different PCOS phenotypes.

Soy Intake Since the Prepubertal Age May Contribute to the Pathogenesis of Endometriosis in Adulthood.

High prevalence of endometriosis was reported in Asian women as a result of their traditionally high intake of soy foods during infancy. Soy is widely used in infant feeding after weaning from breast milk or cow milk. This study thus aimed to determine to what extent soy intake before puberty may contribute to the development of endometriosis. For this purpose, immature (6-week old) female rats were fed with various soy formulas (0%, 10%, 20%, 30%, 40%, 50%, and 60%). Normal control animals were fed with a soy-free diet. At 13 weeks of age, animals (except the normal control) underwent a transplantation surgery to establish endometriosis. Estradiol valerate and oxytocin were used to induce pelvic pain. Endometrial implant levels of glutathione (GSH) and malondialdehyde (MDA) allowed estimating tissue oxidative status. Physiological ovarian function was assessed by histological analysis of ovaries. Results showed that soy-fed animals grew faster than animals receiving a soy-free diet (P < .001). In animals supplemented with more than 10% of soy, the intensity of pelvic pain increased (P < .001) as well as the volume of ectopic foci. In addition, tissue levels of MDA and GSH increased (P < .001). The ovarian function was altered and the number of luteinized unruptured follicles increased. In conclusion, although animals supplemented with soy at the prepubertal stage displayed a good growth performance, regular soy consumption may promote the development and progress of endometriosis in adulthood, especially when soy content in food is more than 10%.

Myrianthus arboreus P. Beauv (Cecropiaceae) Extracts Accelerates Sexual Maturation, and Increases Fertility Index and Gestational Rate in Female Wistar Rats.

Background: Despite the wide use of leaves of Myrianthus arboreus (Cecropiaceae) in several African countries including Cameroon as food and against amenorrhea and female infertility, it has never been tested for this purpose. Methods: Using immature female Wistar rats, the impact of M. arboreus on the sexual maturation parameters (vaginal opening, ovarian relative weight and follicle maturation, gonadotropins and ovarian hormones serum levels) and fertility index has been evaluated through a 30-day oral administration of aqueous and methanol extracts of leaves at the doses of 20, 110 and 200 g/kg/day. Results: Aqueous extract increased the ovarian relative weight (p < 0.001), progesterone (p < 0.001) and gonadotropins (p < 0.001) serum levels, and induced the maturation of ovarian follicles. The methanol extract additionally induced an early vaginal opening (p < 0.001), uterine growth (p < 0.01) and increased estradiol (p < 0.001) serum levels. The fertility index generally increased following treatments, while the gestation rate remained almost unaffected except at the highest tested dose of M. arboreus extracts where lowest values were observed. Conclusion: Globally, M. arboreus induced an early puberty onset and an increased fertility rate validating at least in part its traditional use for female infertility.

Natural Terpenoids Against Female Breast Cancer: A 5-year Recent Research.

BACKGROUND: The approval of Taxol® in 1993 marked the great entrance of terpenoids in the anti-cancer area and this drug is still highly important in the treatment of refractory ovarian, breast and other cancers. Over decades, other prominent natural terpenoids have become indispensable for the modern pharmacotherapy of breast cancer. However, given the rapid evolution of drug resistance, effective treatments for advanced breast cancers requiring cytotoxic chemotherapy represent a major unmet clinical need. Therefore, innovative agents effective in long-term chemotherapy are urgently needed. OBJECTIVE: This review examines recent advances/research about natural terpenoids, and their mechanisms against female breast cancer over the period covering January 1st, 2012 to December 31st, 2016. RESULTS: Carcinogenesis constitutes a multistep process wherein each stage is characterized by distinct phenotypic changes. Numerous chemicals recorded in this review have been shown to significantly inhibit proliferation, migration, apoptosis resistance, tumor angiogenesis or metastasis in different breast cancer cells/tumours in vitro and in vivo. Targeting simultaneously several or all these aspects/steps of cancer progression could be an advantage. In line with this, phytochemicals such as thymoquinone (8), costunolide (46), tanshinone IIA (132), triptolide (136), cucurbitacin B (179), celastrol (226) and lycopene (238) had caught our attention. CONCLUSION: These compounds appear to be promising to overcome breast cancer treatment failure. However, despite the interesting activities, additional preclinical investigations are needed in further breast cancer cell/tumor models in vitro and in vivo.

Emmenagogue properties of Milicia excelsa (Welw.) C.C. Berg (Moraceae) based, at least in part, on its ability to correlate the activity of the hypothalamic-pituitary axis to that of the ovaries.

ETHNOPHARMACOLOGICAL RELEVANCE: Milicia excelsa (Welw.) C.C. Berg (Moraceae) is a medicinal plant recommended over tropical Africa as a cure for several ailments including amenorrhea. This is the hallmark of an ovarian lesion or a dysfunction of the hypothalamic-pituitary-ovarian axis which may lead to infertility, osteoporosis or endometrial cancer. However, regardless these traditional claims, no scientific report dealing with emmenagogue properties has been reported yet. AIM OF THE STUDY: To bring scientific evidence to the curative action of the plant, we proposed evaluating the effects of a root aqueous extract of Milicia excelsa on female Wistar rat sexual maturation. MATERIAL AND METHODS: The study was performed in immature (45 days old) female Wistar rats orally treated with the root aqueous extract of Milicia excelsa at doses of 14, 77 and 140mg/kg BW/day for 7 and 15 consecutive days. Genistein (10mg/kg BW) served as the reference substance. Negative control animals, treated with the vehicle, were followed up for 7, 15, 30 and 45 days and rats were aged 52, 60, 75 and 90 days at these respective days. This allowed setting the nubile age of experimental animals and to determine the impact of treatments with genistein or the aqueous extract of Milicia excelsa on the age of rat sexual maturation. Since female rats do not have menstruation and that the normal menstrual cycle occurs because of changing levels of hormones made and secreted by the ovaries in response to hormonal signals from the pituitary gland, the effects of treatments were evaluated on the pituitary production of gonadotropins, the ovarian production of estradiol and progesterone, and uterine and vaginal growths. RESULTS: The sexual maturation of untreated rats was set at 90 days old. This sexual maturation was indicated by the simultaneous elevation of gonadotropins (FSH and LH (p<0.01)) and ovarian hormones (estradiol (p<0.001) and progesterone (p<0.05)) in animals aged 90 days. Uterine and vaginal growths (p < 0.001) observed in these animals appear as the result of elevated level of estradiol. The root aqueous extract of Milicia excelsa displayed genistein-like effects and increased FSH and estradiol serum levels following both treatment periods (7 and 15 days). Estradiol serum concentration significantly increased following a 7-day treatment at the dose of 14mg/kg BW (p<0.001). This resulted in an increase in the uterine wet weight, uterine and vaginal epithelial heights (p<0.05). These results suggest that the root aqueous extract of Milicia excelsa reduced rats' sexual maturation from 90 to 52 days. Moreover, animals' body weight was not affected following treatment with Milicia excelsa. CONCLUSION: The root aqueous extract of Milicia excelsa may solve the problem of amenorrhea by synchronizing the activity of the hypothalamic-pituitary axis to the ovarian production of estradiol and progesterone. The unaltered body weight following treatments justifies at least in part, the traditional use of Milicia excelsa for primary and secondary amenorrhea.

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.

Estrogenic and cytotoxic potentials of compounds isolated from Millettia macrophylla Benth (Fabaceae): towards a better understanding of its underlying mechanisms.

BACKGROUND: Millettia macrophylla was previously reported to have estrogenic effects and to prevent postmenopausal osteoporosis in Wistar rats. So, the study deals with the identification of its secondary metabolites and the evaluation of their estrogenicity and cytotoxicity toward tumoural cells. Thus, 13 known compounds were obtained from successive chromatographic columns and identified by NMR data compared to those previously reported. METHODS: In vitro estrogenicity of the isolates and the phenolic fraction (PF) of M. macrophylla were performed by E-screen and reporter gene assays, while their cytotoxicity was evaluated by Alamar Blue (resazurin) assay. A 3-days uterotrophic assay and the ability of PF to alleviate hot flushes in ovariectomized adult rats were tested in vivo. RESULTS: Seven of the 13 secondary metabolites turned to be estrogenic. Only two exhibited cytotoxic effects on MCF-7 and MDA-MB-231 with CC50 values of 110 µM and 160 µM, respectively. PF induced a significant (p < 0.01) MCF-7 cells proliferation and transactivated both ERα and ERß in the reported gene assay at 10-2 µg/mL. In vivo, PF acted more efficiently than the methanol crude extract, resulting to a significant (p < 0.01) increase in the uterine wet weight, uterine protein level, uterine and vaginal epithelial height at the dose of 10 mg/kg BW. In addition, PF reduced the average duration and frequency of hot flushes induced in rat. CONCLUSION: These aforementioned results indicate that PF is a good candidate for the preparation of an improved traditional medicine able to alleviate some menopausal complaints such as vaginal dryness and hot flushes. Estrogenic and cytotoxic potentials of compounds isolated from Millettia macrophylla Benth. (Fabaceae): towards a better understanding of its underlying mechanism.

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.

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.

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 ß (ERß) 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 ERß cells. RESULTS: AE and MeOH extracts significantly altered ERα as well as ERß 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 ERß 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.