Biologia https://doi.org/10.2478/s11756-020-00607-7 ORIGINAL ARTICLE Cytotoxic and cancer chemopreventive potentials of the Anthonotha macrophylla P. Beauv aqueous extract on 7,12-dimethylbenz[a] anthracene-induced breast cancer in rats Telesphore Nanbo Gueyo 1 & Stéphane Zingue 2,3 & Marie Alfrede Mvondo 4 & Edwin Mmutlane 3 & Derek Tantoh Ndinteh 3 & Constant Anatole Pieme 5 & Dieudonné Njamen 1,3 Received: 24 May 2020 / Accepted: 16 September 2020 # Institute of Molecular Biology, Slovak Academy of Sciences 2020 Abstract Breast cancer is one of the leading causes of cancer deaths in women worldwide. Many women rely on plants as alternatives to prevent/treat cancer. Anthonotha macrophylla (Ceasalpiniaceae) is one of this ethnomedicinal plant used to cure cancer in Cameroon. This study was therefore undertaken to bring scientific evidence to this claim. The in vitro cytotoxicity of A. macrophylla aqueous extract was evaluated using resazurin reduction assay in five tumors and two non-tumor cell lines. Moreover, the chemopreventive potential of A. macrophylla aqueous extract was evaluated on 7,12 dimethylbenz[a]anthracene (DMBA) induced breast cancer in rats. The research focused on the incidence, burden, volume and histological analysis of breast tumors. In vitro, A. macrophylla extract exhibited cytotoxic effect in all tested cell lines with a CC50 ~ 279 and 132 μg/mL in human (MCF-7 and MDA-MB-231) and rodent breast cancer cells, respectively after 24 h. In vivo, the untreated DMBA-rats presented 100% of tumor incidence, while no tumor was detected in normal rats. Interestingly, a seven-month oral administration of A. macrophylla extract at the doses of 75 and 150 mg/kg BW resulted in a significant decrease of tumor incidence (p < 0.01 and p < 0.05), burden (70.01% and 53.28%) and volume (p < 0.001 and p < 0.01) compared to DMBA rats. The presence of polyphenols in the aqueous extract of Anthonotha macrophylla, as well as its antiradical properties, may account for its antitumor effects. These results therefore support the traditional use of Anthonotha macrophylla against breast cancer. Keywords Anthonotha macrophylla . Breast cancer cell . Cytotoxicity . DMBA . Resazurin assay Abbreviation AM Anthonotha macrophylla AI Atherogenic index BW CC50 DMEM * Stéphane Zingue stephanezingue@gmail.com * Dieudonné Njamen dnjamen@gmail.com Telesphore Nanbo Gueyo nanbogueyotelesphore@yahoo.fr Marie Alfrede Mvondo mvondo.mariealfrede@yahoo.com Constant Anatole Pieme conanpieme@gmail.com 1 Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon 2 Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, P.O. Box 55, Maroua, Cameroon 3 Department of Chemical Sciences, Faculty of Science, University of Johannesburg, Doornfontein 2028, South Africa 4 Department of Animal Biology, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon 5 Department of Biochemistry and Physiological Sciences, Faculty of Medicine and Biomedical Sciences, University of Yaounde 1, P.O. Box 1364, Yaounde, Cameroon Edwin Mmutlane edwinm@uj.ac.za Derek Tantoh Ndinteh dndinteh@uj.ac.za Body weight Cytotoxic concentration for 50% of the cells Dulbecco’s Modified Eagle Medium Biologia DMBA DMSO ER FBS GSH Hb Ht HEPES MDA MCH MCHC MCV NHC NOR RBC RPMI SEM TAM 7,12 Dimethylbenz(a)anthracene Dimethylsulfoxide Estrogen receptor Fetal bovine serum Glutathione Hemoglobin Hematocrit 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid Malondialdehyde Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume National Herbarium of Cameroon Normal control Red blood cell Roswell Park Memorial Institute Standard Error of Mean Tamoxifen Background Breast cancer is the most frequent neoplasia and the second cause of cancer deaths in women worldwide (Siegel et al. 2018). In Cameroon, 2625 new cases of breast cancer are catalogued every year and this incidence is supposed to increase by 2030 (WHO 2014; Kemfang Ngowa et al. 2015). Chemical compounds found in the environment such as polycyclic aromatic hydrocarbons (PAHs) are recognized as cancer initiators (Gelboin 1980). These PAHs are metabolized and transformed into DNA that attack electrophiles in the body, producing PAH-DNA adducts that are found in human breast tumors (Leung et al. 2009). Although modern medicine has established treatments for breast cancer, however, there are adverse effects to these therapies. In fact, paclitaxel (an antimicrotubular agent), induces myelosuppression that affects the immune system; doxorubicin (a cytotoxic antibiotic) leads to a serious irreversible cardiotoxicity; while tamoxifen (an antiestrogen) increases the risk of endometrial cancer (Wu 2012; Lazzeroni and DeCensi 2013). The development of more efficient and safe alternatives for the treatment of breast cancer is therefore needed for better management of this malignant tumor. Anthonotha macrophylla (Ceasalpiniaceae), is a medicinal plant widely distributed in Tropical Africa including Cameroon and traditionally recommended as a cure for various diseases including cancer, venereal diseases and intestinal worms (Ugoeze et al. 2014). The leaves are used to treat diarrhea, dysentery and skin infections (Burkill 1985). A gas chromatography coupled with mass spectrometry analyses revealed the presence of phytosterols in A. macrophylla. Phytosterols, especially campesterol was reported to have anticancer properties (Awad et al. 2000; Li et al. 2001). In addition, this extract also showed in vitro and in vivo antiestrogenic effects on ovariectomized Wistar rats (Gueyo et al. 2019). However, this plant has not yet been investigated for its probable anticancer properties. The present study therefore aimed at investigating the in vitro cytotoxicity of the aqueous extract of A. macrophylla in a panel of cancerous and non-cancerous cell lines, and its protective effects against 7,12 dimethylbenz[a]anthracene (DMBA)-induced breast cancer in female Wistar rats. Breast tumors obtained after exposing prepubescent female rats (~55 days) to the environmental carcinogen DMBA is similar to that of women in their molecular and genetic characteristics, biochemical properties, hormonal reactivity and histology (Russo and Russo 1996). Methods Chemicals and reagents Fetal bovine serum and antibiotics, 2-[4-(2-hydroxyethyl) piperazin-1- yl] ethane sulfonic acid (HEPES), DMBA and Tamoxifen citrate were bought from GIBCO (Grand Island, NY, USA), Ludwig Biotecnologia Ltda (Alvorada, RS, Brasil), Sigma-Aldrich (Stanford, Germany) and Mylan SAS (Saint-Priest, France) respectively. Trypan blue (0,4%), Alamar blue and cell culture mediums were bought from Sigma-Aldrich (St. Louis, MO, USA). Plant material and extraction The stem barks of Anthonotha macrophylla were harvested in August 2015 in Yato, Cameroon Littoral region and the plant was authenticated at the Cameroon National Herbarium (CNH) where a voucher specimen has been deposited under the number 37148 CNH. Four kilograms of powder from dried stem barks of A. macrophylla were boiled in 13 L of potable water for 25 min and then filtered with Wattman paper N04. The filtrate was freeze-dried and a total mass of 80 g (2%) of the crude aqueous extract was obtained. In vitro antioxidant assay Antioxidant activity by DPPH radical scavenging assay Free radical scavenging activity of A. macrophylla was measured using DPPH radical scavenging assay (Katalinić et al. 2004). For the assay, 500 μL of A. macrophylla extract at different concentrations (100–300 μg/mL) were introduced into test tubes and 500 μL of the freshly prepared solution of 400 μmol/L DPPH in methanol were then added. The mixture was incubated at 37 °C for 30 min in the dark and the absorbance was measured at 517 nm using a UV-1605 Biologia Shimadzu spectrophotometer and Ferulic acid was used as the positive control. The DPPH radical scavenging effect was calculated using the following formula: percentage of inhibition ð%Þ ¼ f½ðcontrol absorbance−sample absorbanceÞ=control absorbanceŠ  100g ABTS radical scavenging assay for antioxidant activity of the extract ABTS free radicals scavenging activity was evaluated according to Re et al. (Re et al. 1999). In brief, 1 mL of ABTS reagent was added to 100 μL of A. macrophylla extract at different concentrations (100–300 μg/mL). The blend was stirred and kept in the dark for 30 min. The absorbance was measured at 734 nm using UV-VIS 1605 Shimadzu spectrophotometer. Ferulic acid was used as the positive control. The ABTS radical scavenging effect was calculated as aforementioned in “Antioxidant activity by DPPH radical scavenging assay” section. In vitro cytotoxicity assessment mL. The fluorescent intensity was determined by a Perkin Elmer LS55spectrofluorimeter (Becton Dickinson, San Jose, CA) with excitation at 530 nm and emission at 590 nm. The cytotoxic concentration which kill 50% of cells (CC50) was determined by nonlinear regression analysis of the logarithm of concentration in function of the normalized response (percentage of cell viability) using the software GraphPad Prism 6.0. The assay was done in triplicate. In vivo experiment Experimental animals Healthy female Wistar rats aged 30–37 days (49–55 g) were supplied by the production facility of the Animal Physiology Laboratory, University of Yaounde I (Cameroon). Rats were housed in plastic cages and maintained at room temperature in the Laboratory of Animal Physiology, University of Yaounde I (Cameroon). The animals had access to a soy-free diet established by the Laboratory of Animal Physiology and water ad libitum. The composition of animal diet was: corn (36.7%), bone flour (14.5%), wheat (36.6%), fish flour (4.8%), crushed palm kernel (7.3%), sodium chloride (0.3%) and vitamin complex (Olivitazol® - 0.01%). Cell lines and culture Ethics and consent to participate The in vitro anticancer activity was evaluated on five tumor cell lines to evaluate the difference in the sensitivity depending on the tissue of origin where cancer is developed: MCF-7 [human estrogen receptor (ER)-positive breast adenocarcinoma cells], MDA-MB-231 (human ER-negative breast adenocarcinoma cells), SK-MEL-28 (human melanoma cells), 4 T1 (mouse mammary tumor cells), SF-295 (human glioblastoma cells) and two non-tumors: HUVEC (human umbilical vein endothelium cells) and MCR-5 (human fetal lung fibroblast cells) procured from the Rio de Janeiro Cell Bank, Brazil. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI1640) medium completed with 10% of FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 10 mM HEPES in humidified atmosphere of 37 °C in a 5% CO2. The number of viable cells was assessed by the trypan blue method and the count was made in a Neubauer chamber. Cell viability assay This assay was done following the method depicted by O’Brien et al. (O’Brien et al. 2000) which assesses the mitochondrial production as a measurement of cell viability. To do this, a density of 1 × 104 cells/well in 100 μL of culture medium was seeded in a 96-well plate and allowed to adhere overnight. After 24 h, cells were exposed to different substances at concentrations ranging between 50 and 500 μg/ Housing of animals and all experiments were approved by the Cameroon Institutional National Ethic Committee (Ref n°.Fw-IRb00001954), which adopted all procedures recommended by the European Union on the protection of animals used for scientific purposes (CEE Council 86/609). Determination of doses The environmental carcinogen DMBA was used in this study to induce mammary tumors in prepubescent rats at a single oral dose of 70 mg/kg as previously reported by Dias et al. (2000) and Mohamed and Alshaimaa (2016).The doses of A. macrophylla extract were acquired based on the preparation method of the extract and the posology recommended by the traditional healer. For the pharmacological dose obtained (150 mg/kg) in rats, a middle dose of 75 mg/kg and a lower dose of 37.5 mg/kg were obtained by dividing the pharmacological dose by a factor of 2 twice. Tamoxifen was administered at the dose of 3.3 mg/kg (Maltoni et al. 1997) and served as the positive control. Induction of breast tumors in rats After 15 days of acclimatization, sixty animals were randomized and assigned to six treatment groups (n = 10 animals per group) as follows: Group I, normal control (NOR); Group II, Biologia negative control (DMBA); Group III, positive control (Tamox+DMBA); Group IV to VI, animals treated with A. macrophylla extract at the doses of 37.5, 75 and 150 mg/kg (AM 37.5 + DMBA, AM 75 + DMBA, AM 150 + DMBA). Breast cancer was induced by a single dose of DMBA (70 mg/kg) dissolved in olive oil and orally given to all experimental animals except normal control. Treatment continued in the same way from the day of cancer induction until the 196th day. After 28 weeks of experimentation, all survivor rats were euthanized after a 12 h overnight nonhydric fasting under diazepam and ketamin anesthesia (respectively 10 mg/kg and 50 mg/kg BW; i.p.). Blood was collected with and without anticoagulant tubes for hematological analysis and centrifuged at 3000×g for 15 min for biochemical analysis. Moreover, all tumors were removed, counted and weighed. A 1 mm precision sliding caliper (IGAGING®) was served to measure the size of tumors. Tumorous incidence was noticed, and tumor volume was calculated using the following formula: length × weight × height ×π/6 (FaustinoRocha et al. 2013). Uterus, vagina, mammary glands (estrogen target organs), femur, brain, liver, lungs (metastatic organs of breast cancer), spleen and kidneys (other toxicity organs) were removed and weighed. At last, all these organs were fixed in 10% formalin solution for histological analysis. Histological analysis The histological analysis of different organs was done using the techniques described by Cannet (Cannet 2006). An axioskop 40 microscope connected to a computer where the image was transferred using MRGrab1.0 and axio vision 3.1 soltware (Zeiss, Hallbergmoos, Germany) was used to determine histomorphological changes. Histologic classification of tumors of rat mammary gland from Russo and Russo (Russo and Russo 2000) were used in this study. Hematological and biochemical analyses Hematological parameters were assessed using a Mindray BC-2800 Auto Hematology Analyzer. These parameters include white blood cell (WBC), lymphocytes, monocytes, granulocytes, red blood cell (RBC) count, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelets. The determination of Malondialdehyde (MDA) and glutathione reduced (GSH) levels as well as FRAP activity were performed according to the methods of Wilbur et al. (Wilbur et al. 1949), Ellman (Ellman 1959) and Benzie and Strain (Benzie and Strain 1996) respectively. Statistical analysis Results were expressed as mean ± standard deviation (SD) for each experimental group and each in vitro experiment which were performed in triplicate and repeated three independent times. Statistical significance was evaluated by one-way analysis of variance followed by Dunnett’s post hoc test using the Graphpad Prism software version 5.03. the p value <0.05 was considered as significant. Results Scavenging free radical activities The concentrations of A. macrophylla extract which resulted in 50% of radical scavenging (EC50) were 198.7 μg/mL (DPPH) and 198.4 μg/mL (ABTS) (Table 1). Cytotoxicity of A. macrophylla extract Cytotoxic activity of A. macrophylla extract on cancer cell lines (MCF- 7, MDA-MB-231, 4 T1, SK-MEL-28, and SF295) and non-tumors cell lines (HUVEC and MRC-5) is presented on Table 2. A. macrophylla extract induced a cytotoxic effect on all cancer cell lines with a less pronounced effect in MCF- 7 (CC50 = 300 μg/mL) and MDA-MB-231(CC50 = 279 μg/ mL) cancer cells, while the most important effect (CC50 = 132 μg/mL) was observed in mouse 4 T1 cells. Moreover, A. macrophylla exhibited a CC50 of 147 and 155 μg/mL in SF-295 and SK-MEL-28, respectively. Cancer chemopreventive effects of aqueous extract of a. macrophylla Effects on mammary tumors Data related to the chemopreventive activity of A. macrophylla aqueous extract on mammary tumor incidence, total tumor Table 1 extract Scavenging free radical activities of A. macrophylla aqueous EC50 (μg/mL) Ferulic acid (control) A. macrophylla DPPH ABTS 3.44 ± 0.46 198.71 ± 8.92 3.68 ± 0.81 198.42 ± 11.3 EC50 = concentration of Anthonotha macrophylla extract which results in 50% of scavenging. The results are expressed as mean ± SD of at least 3 independent experiments Biologia Table 2 CC50 values of aqueous extract of Anthonotha macrophylla in tumoral and non tumoral cell lines A. macrophylla CC50 (μg/mL) MCF-7 MDA-MB-231 300 ± 31.2 279 ± 16.9 Selectivity index (SI) HUVEC/MCF-7 0.52 HUVEC 156 ± 18.9 MCR-5 140 ± 12.3 HUVEC/MBA-MB-231 0.56 MCR 5/MCF-7 0.46 4 T1 132 ± 30.25 SK-MEL-28 155 ± 12.6 SF-295 147 ± 11.4 MCR-5 /MDA-MB 231 0.50 CC50 = Concentration of A. macrophylla aqueous extract which results in 50% of cell viability. The results are expressed as mean ± SD of at least 3 independent experiments. SI = CC50 of Anthonotha macrophylla on non-tumoral cell lines (MCR-5 and HUVEC) divided by CC50 determined for cancer cells (MCF-7, MDA-MB-231 and 4 T1) burden, average tumor weight and tumor volume after 28 weeks of treatment are presented in Table 3. No tumors were observed in normal animals while animals in the DMBA group presented 100% of mammary tumors with a tumor volume of 1.25 cm3 at the end of experiment. In the tamoxifen group, animals showed a significant (p < 0.001) reduction in tumor incidence (25%), tumor volume (0.25 cm3) and an average tumor weight of 72.33% as compared to DMBA group. A. macrophylla extract (75 and 150 mg/kg) significantly (p < 0.01 and p < 0.05 respectively) reduced the tumor incidence (30% and 50%), the inhibition related to average tumor weight (70% and 53.28%) and tumor volume (0.26 cm3 and 0.59 cm3) as compared to DMBA group. Effects on oxidative stress status in mammary glands Oxidative stress status in mammary glands homogenates was evaluated. An increase in MDA levels and a non-significant decrease of GSH level and FRAP activity were observed in mammary glands of DMBA rats as compared to normal animals (Fig. 1). A. macrophylla extract significantly (p < 0.01) decreased MDA levels in mammary glands at the doses of 37.5 and 150 mg/kg BW. Furthermore, A. macrophylla extract Table 3 induced a significant increase (p < 0.05) in GSH level at the doses of 37.5 and 75 mg/kg and FRAP activity at the dose of 150 mg/kg as compared to DMBA group. Effect on the Histomorphology of mammary glands After 7 months of treatment, in situ carcinoma of mammary glands of the DMBA group was observed. This was characterized by a hyperplasia of the lobular units of mammary and the dilated ducts with a diminution of the conjunctive tissue (Fig. 2). No sign of hyperplasia was observed in the mammary glands of animals treated with tamoxifen. Rats treated with A. macrophylla extract showed hyperplasia at the dose of 37.5 and 150 mg/kg. However, animals that received A. macrophylla extract at the doses of 75 presented quasinormal histoarchitecture. Effects of a. macrophylla on relative organ weights Only tamoxifen and A. macrophylla extract at dose of 75 mg/kg (at day 56 and 84) significantly (p < 0.01) lowered body weight as compared to the normal control group (data not shown). Breast cancer chemopreventive activity of A. macrophylla extract after 28 weeks of treatment Items N° of rats with tumors/total rats Tumor incidence (%) Average tumor weight (mg/kg) % Inhibition related to tumor weight Total tumor burden (mg) % Inhibition related to tumor burden Tumor volume (cm3) NOR 0/10 0 – – 0 – – DMBA 10/10 100### 318.3 ± 136.4 – 3183.2 – 1.25 ± 0.11 TAMOX + DMBA 2/10 20*** 88.1 ± 31.5** 72.3 880.5 72.34 0.25 ± 0.07*** A. macrophylla (mg/kg) + DMBA 37.5 75 150 6/10 60 298.7 ± 100.2 6.2 2987.2 6.16 0.86 ± 0.04 3/10 30** 98.2 ± 49.5.1** 70.0 981.6 70.01 0.26 ± 0.09*** 5/10 50* 148.7 ± 62.2* 53.3 1487 53.28 0.59 ± 0.18** NOR = Normal control rats receiving vehicle (distilled water); DMBA = Rats serving as a negative control, receiving vehicle; TAMOX + DMBA = rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); A. macrophylla + DMBA = Rats receiving the aqueous extract of A. macrophylla at doses of 37.5, 75 and 150 mg/kg BW. Data are represented as mean ± SD (n = 10). * p < 0.05, ** p < 0.01 and ***p < 0.001 as compared to negative control. ###p < 0.001 as compared to normal control Biologia the spleen wet weight which significantly (p< 0.001) decreased as compared to normal rats. Treatment of rats with tamoxifen significantly decreased uterine wet weight (p< 0.001) and increased brain (p< 0.001), lungs (p< 0.05) and spleen (p< 0.05) wet weight as compared to DMBA control group. Concerning treatment with A. macrophylla extract, it induced a significant (p < 0.001) decrease in the uterine wet weight at all tested doses, while the lung weight increased (p< 0.001) at the dose of 150 mg/kg. Effects on toxicological parameters No statistical significance was observed between the different groups in all measured hematological parameters (Table 5). Exception made for the A. macrophylla extract at the dose of 75 mg/kg (p < 0.05), which significantly increased hematocrit and hemoglobin as compared to DMBA group. No metastasis and damages were noted in the histoarchitectures of the liver, lungs, kidneys, bone and brain (Fig. 3). Discussion Fig. 1 Effects of A. macrophylla aqueous extract on GSH (a) and MDA (b) levels as well as FRAP (c) activity in mammary glands. NOR = Normal control rats receiving vehicle (distilled water); DMBA = Rats serving as a negative control, receiving vehicle; TAMOX + DMBA = rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); A. macrophylla + DMBA = Rats receiving the aqueous extract of A. macrophylla at doses of 37.5, 75, and 150 mg/kg BW. Data are represented as mean ± SD (n = 10). * p < 0.05, ** p < 0.01 as compared to negative control Table 4 presents the relative weights of various organs following 28 weeks of treatment with A. macrophylla. No significant difference was observed in the weight of various organs in animals that received DMBA, exception made with New anticancer molecules are continuously being targeted and developed from medicinal plants, which in general are less toxic and have presented effective results on cancer (Burkill 1985). As a contribution to this search, we focused on Anthonotha macrophylla, a medicinal plant used in Cameroon to cure cancer. The aqueous extract of A. macrophylla exhibited cytotoxic effect on 5 cancer cell lines with a more pronounced effect on mouse mammary tumor cells (4 T1). The fact that A. macrophylla extract induced quite similar cytotoxic effect on both ER+ (MCF-7) and ER(MDA-MB-231) cells suggests that it killed the cells by an estrogen receptor independent pathway. Furthermore, A. macrophylla extract was not selective to cancer cells (with a selectivity index <1), which is a drawback in the research for an alternative therapy for breast cancer. However, the LD50 > 2000 mg/kg obtained with A. macrophylla extract (Gueyo et al. 2019) encourages in-depth phytochemical study to isolate its bioactive components, which could have better selectivity for cancerous cells (> 3). Available phytochemical reports on A. macrophylla extract shows that it contains flavonoids, particularly flavonols, which might account for its cytotoxicity; given that quercetin the most abundant flavonol in plants has been described to be cytotoxic in vitro (Murakami et al. 2008; Gibellini et al. 2011). DMBA, an organic environmental pollutant was used in this study to induce cancer in rats. It is highly lipophilic and requires metabolic activation for its carcinogenicity. Several tissues, including the mammary glands are capable of activating DMBA. In the breast, DMBA is converted to epoxides; Biologia Fig. 2 Effects of A. macrophylla aqueous extract on the microarchitectures of mammary glands. a Normal control rats receiving vehicle (distilled water); b Rats serving as a negative control, receiving vehicle; c rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); d, e and f Rats receiving the aqueous extract of A. macrophylla at doses of 37.5, 75, and 150 mg/kg BW, respectively. Ac = Acinus; AT = Adipose tissue; FT = Fibrous tissue; L = Lobule; AD = Atypical duct; HLU = Hyperplastic lobular unit active metabolites with the capacity to damage DNA molecules and this is the main event in carcinogenicity initiation (Clarke 1997; Balogh et al. 2003). In this study, DMBA was administrated at a single dose of 70 mg/kg BW by gavage to Wistar rats to induce mammary tumors. Due to the active proliferation of the terminal ducts in breast tissue of rats in this age range (45 to 60 days), they become very susceptible to carcinogens and tumor development (Ariazi et al. 2005). After 28 weeks of treatment, it was observed that DMBA induceed 100% tumors in the experimental animals that only received DMBA whereas animals in the normal group did not exhibit tumors. This result is in accord with the study of Zingue et al. (2016) who also reported 100% tumors with DMBA in female Wistar rats. The significant reduction in tumor volume and average tumor weight observed with A. macrophylla extract at the doses of 75 and 150 mg/kg BW suggests protective effects of this extract on the mammary tumorigenesis. These effects could be explained by the ability of compounds of A. macrophylla extract to kill cancer cells, as observed in vitro in this study. These results suggest that the acute effect of A. macrophylla involves inhibition of cell proliferation. Flavonols detected in A. macrophylla extract, might have counteracted the estrogen activity in vitro and in vivo via ER-binding to inhibit tumor growth. This is in line with the lately reported antiestrogenic effects of A. macrophylla aqueous extract (Gueyo et al. 2019). Histopathological Biologia Table 4 Effects of A. macrophylla aqueous extract on relative weights of organs after 28 weeks of treatment Organs (mg/kg) Uterus Ovary Liver Lungs Spleen Adrenals Kidneys Femur Brain NOR DMBA 2510.8 ± 392.7 515.7 ± 72.3 32,180.8 ± 2415.8 7519.7 ± 711.4 4494.1 ± 925.5 287.1 ± 69.1 5762.8 ± 217.14 2889.8 ± 296.78 7996.2 ± 456.3 3119.4 ± 562.5 553.0 ± 84.1 33,445.9 ± 2448.38 7438.2 ± 710.2 2432.2 ± 435.4### 304.5 ± 52.2 5336.3 ± 417.8 2569.7 ± 215.2 8137.3 ± 271.5 TAMOX + DMBA 860.7 ± 151.6*** 271.3 ± 34.5*** 32,885.5 ± 1403.2 8683.4 ± 694.1* 3589.4 ± 1150.2* 278.1 ± 27.7 5831.2 ± 317.9 2776.2 ± 354.4 9335.1 ± 350.7*** A. macrophylla (mg/kg) + DMBA 37.5 75 150 1995.6 ± 474.9** 508.1 ± 126.7 32,666.7 ± 2733.3 7829.4 ± 718.7 2796.9 ± 363.4 312.3 ± 45.5 5482.3 ± 504.7 2574.5 ± 171.6 8016.7 ± 606.9 1918.3 ± 580.1*** 633.1 ± 86.7 31,340.7 ± 1222.1 7072.6 ± 412.5 3430.7 ± 492.14 315.7 ± 53.5 5644.4 ± 396.4 2710.9 ± 200.6 8158.2 ± 324.1 1886.6 ± 292.2*** 560.67 ± 96.2 32,650.9 ± 2642.6 9722.9 ± 577.9*** 3308.5 ± 203.7 308.5 ± 68.9 6236.3 ± 279.6 2459.4 ± 186.2 7877.8 ± 295.5 NOR = Normal control rats receiving vehicle (distilled water); DMBA = Rats serving as a negative control, receiving vehicle; TAMOX + DMBA = rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); A. macrophylla + DMBA = Rats receiving the aqueous extract of A. macrophylla at doses of 37.5, 75 and 150 mg/kg BW. Data are represented as mean ± SD (n = 10). * p < 0.05, ** p < 0.01 and ***p < 0.001 as compared to negative control. ###p < 0.001 as compared to normal control examination of the mammary gland sections revealed in-situ carcinoma in DMBA control animals as compared to the normal group that exhibited quite-normal histological sections. This result is in accordance with several studies (Russo et al. 1982; Silihe et al. 2017). Tamoxifen and A. macrophylla extract at the doses of 75 and 150 mg/kg protected the mammary gland against DMBA-induced histological alterations. Based on the fact that the extract of A. Macrophylla was administered for a long time (7 months), its impact on body weight and relative organ weight was assessed. These are indicators of toxic effects when testing substances (Michael et al. 2007), and the fact that no changes were seen at this level, suggest that the aqueous extract of A. Macrophylla is not toxic. Table 5 The antioxidant activity of A. macrophylla extract could be due to the presence of the analogues of quercetin, given that its chemopreventive properties have been linked to its antioxidant properties (Murakami et al. 2008; Gibellini et al. 2011). Antioxidant-based drug formulations are used for the prevention and treatment of complex diseases including cancer (Khalaf et al. 2008). It is worth noting that DMBA metabolites are toxic and cause oxidative stress, leading to cell structural damage and possibly cell necrosis (Patri and Padmini 2009). In this study, MDA level increased following DMBA exposure in mammary glands, while, GSH level and FRAP activity decreased. A. macrophylla extract significantly decreased MDA levels in mammary glands at the doses of 37.5 and 150 mg/kg and induced a significant Effects of A. macrophylla aqueous extract on hematological parameters after 28 weeks of treatment Items Control DMBA TAMOX + DMBA A. macrophylla (mg/kg) + DMBA 37.5 75 150 WBC (×103 μL−1) Lymphocytes (%) 5.41 ± 0.68 78.18 ± 5.95 6.62 ± 1.13 71.00 ± 1.74 6.37 ± 0.98 66.77 ± 2.38 7.48 ± 1.01 68.70 ± 2.25 7.74 ± 0.81 74.18 ± 1.91 8.42 ± 1.21 67.04 ± 1.76 Monocytes (%) Granulocytes (%) RBC(×103 μL−1) Hematocrit (%) MCV (fL) Platelets (×103 μL−1) MCH (pg) Hemoglobin (g/dL) MCHC (g/dL) 5.88 ± 1.07 24.03 ± 0.92 5.68 ± 0.40 32.48 ± 2.73 57.82 ± 2.17 307.75 ± 13.57 18.66 ± 0.90 10.75 ± 0.99 32.42 ± 1.15 7.66 ± 0.74 21.34 ± 2.29 5.76 ± 0.27 32.46 ± 2.10 56.20 ± 1.02 320.60 ± 33.83 18.14 ± 0.66 10.54 ± 0.83 32.32 ± 0.74 6.55 ± 0.55 26.67 ± 2.32 6.35 ± 0.15 36.85 ± 0.74 58.10 ± 1.14 349.40 ± 40.13 18.82 ± 0.46 11.97 ± 0.32 32.45 ± 0.31 7.44 ± 0.72 23.86 ± 1.70 6.28 ± 0.61 39.26 ± 1.87 58.54 ± 0.66 402.60 ± 57.25 18.32 ± 0.87 12.73 ± 0.57 31.38 ± 1.28 7.40 ± 0.70 18.42 ± 1.62 7.01 ± 0.24 41.5 ± 1.29* 59.30 ± 0.22 421.20 ± 31.13 19.20 ± 0.31 13.48 ± 0.46* 32.44 ± 0.53 8.10 ± 0.68 24.86 ± 2.20 7.05 ± 0.31 40.18 ± 1.62 57.12 ± 0.78 383 ± 17.78 18.40 ± 0.28 13 ± 0.54 32.44 ± 0.53 NOR = Normal control rats receiving vehicle (2% ethanol); DMBA = Rats serving as a negative control, receiving vehicle; TAMOX + DMBA = rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); A. macrophylla + DMBA = Rats receiving the aqueous extract of A. macrophylla at doses of 75, 150 and 300 mg/kg BW. Data are represented as mean ± SEM (n = 10). * p < 0.05 as compared to negative control Biologia Fig. 3 Effects of A. macrophylla aqueous extract on microphotographs (H&E, × 400) of liver, lungs, kidneys, brain and bone after 28 weeks of treatment. a Normal control rats receiving vehicle (distilled water); b Rats serving as a negative control, receiving vehicle; c rats serving as a positive control, receiving tamoxifen (3.3 mg/kg BW); d, e and f Rats receiving the aqueous extract of A. macrophylla at doses of 37.5, 75, and 150 mg/kg BW mg/kg BW, respectively. Vp = portal vein; H = hepatocyte; S = sinusoids; A = alveoli; Ba = aveolar bag; G = glomerulus; Dt = distal tube; Ne = neuron; Co = cortex; TB = Trabecular bone; MB = marrow bone Biologia increase in GSH (75 mg/kg) and FRAP (150 mg/kg). Since flavonoids are well known natural antioxidant compounds, the effects of this extract could be due to the ability of its flavonoids to reduce oxidative stress and free-radical formation. The in vitro study showed that the concentrations of A. macrophylla extract which resulted in 50% of radical scavenging (EC50) were 198.7 μg/mL (DPPH) and 198.4 μg/mL (ABTS). Conclusion In summary, the aqueous extract of A. macrophylla induced cytotoxicity in a panel of cell lines with a CC50 ~ 132 μg/mL in rodent breast tumor 4 T1 cells. Moreover, A. macrophylla extract inhibits the DMBA-induced mammary glands hyperplasia in female rats at the dose of 75 and 150 mg/kg. These antitumor effects might be attributed partly to its flavonoids and to its ability to exert both antioxidant and antiestrogenic activities. Taken altogether, these results support traditional use of the aqueous extract of Anthonotha macrophylla to fight against breast cancer. Acknowledgments The authors would like to kindly thank Professor Tânia Beatriz Creczynski-Pasa (Federal University of Santa Catarina) for her collaboration. Prof. Dr. Stephane Zingue was CNPq -TWAS fellow (Grant no. 190741/2015-5). The authors would also like to thank Alexander von Humboldt Foundation for the material support offered to Dieudonné Njamen. Author contributions TNG, ZS, MMA and DN designed the experiments. ZS, CAP and TNG performed the in vitro experiment. TNG, ZS and MMA performed the in vivo experiment. ZS, DTN, EM and MMA analyzed the data. TNG and ZS wrote the first draft and EM, DTN MMA and ND reviewed the manuscript with editing. All authors read and approved the final version of the manuscript. Data availability The data and materials used in this study are available upon request from the authors. Please contact Prof. Dr. Stephane Zingue (stephanezingue@gmail.com). Compliance with ethical standards Conflict of interest The authors declare that there are no conflicts of interest regarding the publication of this paper. 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