Botanical from the Fruits Mesocarp of Raphia vinifera Displays Antiproliferative Activity and Is Harmless as Evidenced by Toxicological Assessments

Raphia vinifera is widely used to treat several diseases including digestive disorders, dysentery, and genitourinary infections. In this study, the mineral contents, the cytotoxicity, and the toxicological effect of the crude CHCl3/MeOH extract (RVM) from the mesocarp of Raphia vinifera were evaluated. The mineral contents were evaluated using the method described by the Association of Official Analytical Chemists (AOAC). The cytotoxicity of both extract and chemical compounds from the plants was determined by a resazurin reduction assay (RRA). The toxicological studies were carried out using the experimental procedure of the Organization for Economic Cooperation and Development (OECD). After killing the rats, biochemical, histopathological, and hematological studies were performed. The result indicated that RVM is rich in zinc (6.52 mg/100 g of DM) and sodium (194.5 mg/100 g of DM). RVM had a cytotoxicity effect with IC50 values lower than 30 μg/mL in 18/18 cancer cell lines tested. These recorded IC50 values were between 12.35 µg/mL (toward CCRF-CEM leukemia cells) and 26.66 µg/mL (toward SKMel-505 BRAF wild-type melanoma cells). Raphvinin 4 displayed good cytotoxicity against MaMel-80aBRAF-V600E homozygous mutant with the IC50 of 10.42 μM. RVM was relatively nontoxic to rats, the median lethal dose (DL50) being above 5000 mg/kg body weight. However, during the oral administration period extending for 28 days, precautions should be taken due to the increase in urinary creatinine level and decrease in spleen weight in the male rats given the highest dose (1000 mg/kg) of extract. Conclusively, the extract of Raphia vinifera is weakly toxic in rats and could be further used in the development of anticancer phytomedicines.


Introduction
Despite the numerous means to fight against cancer, the number of deaths caused by this disease is increasing significantly in many countries [1]. In 2018, WHO (World Health Organization) recorded 9.6 million deaths and 18.1 million new cases because of cancer [2]. Due to morbidity and mortality that it generates, cancer represents a major health problem both nationally and globally. is pathology becomes increasingly difficult to diagnose and to treat when cancer cells develop resistance mechanisms against the usual chemotherapeutic agents [3]. Regarding the increasing resistance developed by these cells, research for alternative treatments should be performed. e varieties of secondary metabolites contained in medicinal plants are responsible for the pharmacological effects including cytotoxic activity [4].
Raphia vinifera (Arecaceae) is a plant from the genus Raphia; medicinal properties of different parts of the plants have been demonstrated. Raffia wine from Raphia vinifera is rich in lactic acid bacteria [5], which prevents the incidence of diarrhea and promotes the course of the immune response in rats; these probiotic isolates could strengthen the immune system in children [6]. Also, many medicinal plants used in Africa have shown interesting antiproliferative properties against the sensitive and multi-drug-resistant (MDR) cancer cells linked to their secondary metabolites [4,7]. e boiled solution of apical bud of Raphia vinifera is used to treat some diseases like genitourinary infections and gonorrhea in West Cameroon. e leaf is used to fight against poison and for various sexually transmitted diseases and witchcraft [8]. To solve liver problems, the young leaves of this plant are used, and the crushed fruits are poured into water to capture fish easily [9,10].
Palm has been proven to have minerals like calcium [11]. e Raphia vinifera fruit pulp and pericarps were found to contain a high concentration of saponins, alkaloids, and oxalate; a moderate concentration of tannin, flavonoid, and steroid; and a low concentration of phytate, phenol, and glycoside, which are responsible for its therapeutic activity [12]. is plant has provided steroidal saponins [13], which are beneficial in preventing tumors and treating many cancers with high efficiency associated with weak toxicological effect [13]. In addition, saponins are also cytotoxic and act by blocking the cell cycle and could significantly disrupt the mitochondrial membrane potential and selectively upregulate the protein levels of Bax, cytochrome C, and cleaved caspase 3/9 and downregulate the levels of Bcl-2 [14]. e pulp of Raphia vinifera contains oil that was extracted and characterized physically and chemically by Igwenyi et al. [15].
Many investigations have demonstrated the ability of medicinal plants in the prevention and treatment of many diseases [4,16]. However, little information is provided on the toxicological effect of plants on consumers. e research on toxicological effects of medicinal plants and their extract is crucial in the development of drugs and to rise human safety [17]. Many toxicological studies have been carried out using Raphia vinifera on fish [11], but studies hardly describe the biochemical toxicity of this plant on rats. is investigation was therefore carried out to evaluate antiproliferative potential of Raphia vinifera extract and its constituents, as well as the toxicity of the crude extract.

Cell Cultures and
Origins. 18 cancer cell lines and normal hepatocyte AML12 were used in the present study.

Experimental Animals.
For the toxicological studies, adult Wistar rats (8 to 9 weeks old) of the 2 sexes were selected. To ensure their growth, the animals engaged in the animal house received food daily and tap water. ey were maintained at standard laboratory conditions of regular 12 h light/12 h dark cycle. is work was carried out with respect to the well-being of rats like the Institutional Ethical Review Committee of the University of Dschang Cameroon recommended.

Determination of the Mineral Contents.
Mineral contents (Ca, P, Mg, Fe, Na, Zn, and K) were determined by the extract using the AOAC method [31]. Raphia vinifera fruit extract was introduced into a porcelain crucible and calcinated at 450°C for 2 hrs. e contents of potassium (K), magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe), zinc (Zn), and phosphorus (P) were determined colorimetrically by UV-visible spectrophotometer (Technel 752 P), according to AOAC procedure. Mineral contents of the sample were determined from calibration curves of standard minerals. All minerals were analyzed in duplicate.

Cytotoxicity
Assay. Different types of human cancer cell lines were used in this study. e resazurin reduction assay (RRA) as previously described [24,32] with similar experimental conditions to those reported earlier [26][27][28][29][30] was used to measure the cell cytotoxicity. Fluorescence was measured on an Infinite M2000 Pro ™ plate reader (Tecan, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. e viability was determined based on a comparison with untreated cells. e values representing the sample's concentrations required to inhibit 50% of cell proliferation (IC 50 ) were calculated from a calibration curve by linear regression using Microsoft Excel 2013 [33,34].

Acute Toxicity Study in Rats.
is test was realized under the OECD guidelines [35]. We followed the methods described by Nguenang [36]. ree adult female rats (8-9 weeks) were treated orally with one dose of extract (5000 mg/kg), after 12 hrs of fasting. ese rats were individually and frequently observed to check any signs of toxicity during the first day; observation was continued daily for a total of 14 days of the experiment. e body weight of animals on the 15 th day was measured. Subsequently, they were anesthetized through intraperitoneal injection with a solution containing diazepam and ketamine (0.2/0.1 ml per 100 grams of the animal), the vital organs such as lung, spleen, heart, kidneys, and liver were removed and weighed, and the macroscopic examinations were performed on those organs. e relative organ weight was determined.

Treatments.
is study was performed under the protocol of the OECD Guidelines [37]. We followed the methods described by Nguenang [36].
irty-two Wistar rats (16 males and 16 females) aged from 08 to 09 weeks were distributed in 4 groups of 4 rats per group. e groups treated received the doses of 250, 500, and 1000 mg/kg b.w. of extract, while the control group received only distilled water during 28 days of treatment. ese rats were individually and frequently observed to check any signs of toxicity. e body weights of all animals were measured after every four days during the experimental period. All animals were weighed on the 29 th day, and subsequently, they were anesthetized with solution containing diazepam and ketamine (0.2/0.1 ml per 100 grams of the animal). Blood samples were collected into EDTA and nonheparinized tubes for the measurement of hematological and biochemical parameters, respectively. e organs such as heart, lung, liver, kidneys, and spleen were weighed, and one part of each organ was conserved in the solution of formalin (10%) for histopathological examination. e relative organ weight was determined.

Histopathological Examination.
After killing the rats, kidneys and liver were removed and cleaned in saline solution. e parts of these organs were collected for histological studies. ese tissues were fixed in formalin (10%) during at least 24 h, dehydrated in a graded series of ethanol (80-100°), and enclosed (embedded) in paraffin. ereafter, 5-µm sections were prepared using a microtome and stained with hematoxylin-eosin before the microscopic examination. e microscopic features of the animal's (male and female) organs-treated groups were compared with the control group [36,38].

Statistical Analysis.
e data are expressed as mean-± standard deviation (SD). ese results have been submitted to the analysis of variance (ANOVA) at one factor according to the general linear model. Statistical analysis was done using version 21 of the IBM-SPSS statistical program, and statistical comparisons were made using the test of Waller Duncan for the subchronic toxicity at the 5% probability level.

Mineral Content in Extract.
e mineral composition of RVM is presented in Table 1. e data obtained from the mineral levels showed that RVM contains more zinc (6.52 mg/100 g of DM) and sodium (194.5 mg/100 g of DM) and less potassium (575.4 mg/100 g of DM), calcium

Cytotoxicity of Extract.
e RRA was used to evaluate the effects of RVM, compounds 1-9, and doxorubicin on the proliferation of 18 cancer cell lines and normal AML12 hepatocytes (Tables 2 and 3; Figure 1). e degree of resistance (D. R.) was calculated as the ratio of the IC 50 value of the resistant cell line divided by that of the corresponding parental sensitive cell line. e D. R. lower than 0.9 was defined as hypersensitivity or collateral sensitivity; D. R. around 1 was interpreted as normal sensitivity, while D. R. greater than 1.2 was signified as cross-resistance. e botanical RVM and doxorubicin revealed antiproliferative effects against the 18 cancer cell lines (Tables 2 and 3 Table 2). In addition to the fact that RVM had recordable   (1), raphvinin (2), raphvinin 2 (3), raphvinin 3 (4), diosgenin (5), trillin (6), deltonin (7), (8), and sitosterol (9)

Acute Toxicity of RVM.
During this experiment, no animals died among the female rats receiving 5000 mg/kg of RVM. e signs of toxicities were not detected based on the behavior of rats during the observation period (14 days). erefore, lethal dose (LD 50 ) of this extract was estimated greater than 5000 mg/kg in female rats. Tables 4 and 5 represent the body weights (g) and relative organ weights in the female rats during acute toxicity, respectively.

Food Consumption.
e food consumption changes in both female and male rats treated with different doses (250, 500, and 1000 mg/kg b.w.) of extract are presented in Figures 2(a) and 2(b). During the treatment period, both sexes of rats showed reduction in the food intake compared with the control group. However, the reduction of food consumption was significant from the 16 th day of treatment in male rats treated at highest with respect to controls.

Body Weight.
e body weight gain changes in both female and male rats treated with different doses (250, 500, and 1000 mg/kg b.w.) of extract are represented in Figures 3(a) and 3(b). At all doses during the treatment period, the female and male rats showed a decrease in their   body weight compared with the control group. is body weight of treated groups reduced inversely proportional to doses administered with respect to the control group. However, the decrease of body weight in male rats treated at highest dose (1000 mg/ kg) with respect to the control group was significant from the 16 th day of treatment. Table 6 represents the effect of RVM on organ weights (g) of both female and male rats during subchronic toxicity. e results show that no significant difference (p < 0.05) was remarked in organ weights of treated rats with respect to those of the controls. Nevertheless, the spleen weight of male rat was significantly decreased at the dose 1000 mg/kg compared with the control group.

Biochemical Parameters
(1) Effect of Extract on ALT, AST, ALP Activity, and Total Serum Protein Levels. e effect of different doses of extract on the activity of transaminases (ALT and AST), total serum protein levels, and alkaline phosphatase levels is shown in Table 7. After repeated administration doses of extract, the results showed that, in female and male rats, the activity of serum total proteins and alkaline phosphatase was significantly reduced at doses 250 and  500 mg/kg compared with the control group. In female rats, the activity of ALT and AST was significantly decreased (p < 0.05) at all doses compared with controls. No significant difference was observed in the activity of transaminases at doses 500 and 1000 mg/kg in the male rats. However, in male rats, the significant reduction of these parameters was observed at dose 250 mg/kg compared with the control group.

(2) Effect of RVM on Level of Urea, Creatinine, and Urinary
Protein. e effects of RVM on the level of serum creatinine, serum urea, and urinary protein are represented in Table 8. Serum and urinary urea level and urinary creatinine level showed a significant reduction in female rats compared with their control group. Urinary urea and serum creatinine levels were significantly reduced in male rats treated at all doses with respect to control groups. However, urinary   Evidence-Based Complementary and Alternative Medicine creatinine level was significantly decreased in rats treated at doses 250 and 500 mg/kg but significantly increased in male rats treated at dose 1000 mg/kg with respect to the control group.
(3) Effect of RVM on Serum Lipid Profile. e effect of administration of extract on lipid profile in both female and male rats is represented in Table 9. e HDL cholesterol levels reduced in males treated at three doses compared with the control group. An increment in triglyceride levels (TG) was observed in male rats treated at three doses of extract compared with controls. As compared to the control groups, other parameters measured did not show significant differences. Table 10 presents the effect of RVM on hematological parameters of the rats treated with RVM. A significant decrease was observed in the level of monocytes, hemoglobin, and hematocrit in the female rats treated with extract at the highest dose compared with controls, while lymphocyte levels significantly reduced in the same group of rats with respect to controls. A significant increase was remarked in the level of platelets in both sexes   Evidence-Based Complementary and Alternative Medicine treated with extract at dose 1000 mg/kg compared with control groups. In male rats, granulocytes were significantly higher in treated animals who received highest dose of extract; the lymphocyte level indicated significant reduction in same group of rats compared with the control group. e significant differences did not show in the rest of hematological parameters measured compared with the control group.

Histopathological Examination.
Histopathological examinations were performed on the liver and kidneys to verify whether these organs or tissues had been damaged. No remarkable pathological change was shown on all organs after the microscopic observation compared with the control group. e effect of RVM on liver and kidneys histology in female and male rats during subchronic toxicity study is presented in (Figures 4(a) and 4(b)) and (Figures 5(a) and 5(b)).

Discussion
Several mineral elements and metabolic products of plant cells are capable to influence the metabolism. ese minerals are very important as they have several biological functions, and their deficiency generally leads to nutritional disorders [39]. In this study, zinc (Zn) and sodium (Na) were detected. Zinc plays a vital role in human growth and development.
High zinc content was observed in RVM (6.52 mg/100 g DM). e recommended daily dose is between 0.3 and 1 mg/ kg in adults [40]. is result is not the same as that of Doungue [41] who obtained 0.88 mg/100 g of DM in Raphia (Raphia hookeri). is variation of values might be due to the difference in Raphia species used.
Medicinal plants are good cytotoxic agents if their IC 50 value is below 20 µg/mL; phytochemicals are significantly cytotoxic if their IC 50 < 10 µM and moderately cytotoxic if 10 µM < IC 50 < 50 µM [42]. Also, according to Suffness and Pezzuto, if the IC 50 values of plant extracts are lower than or around 30 µg/mL, they deserve to be purified in order to find active components [43]. Hence, plant extract with IC 50 values lower than 20 µg/mL and 30 µg/mL as obtained in this assay against 15/18 and 18/18 cancer cell lines, respectively. Compound 4 showed IC 50 equal to 10.42 against MaMel-80aBRAF-V600E homozygous mutant; compounds 4 and 1 showed 10 µM < IC 50 < 50 µM against 8/9 and 3/9 cell lines, respectively. Regarding criterion of anticancer activities, the plant extracts (RVM), compound 4 (Raphvinin), could therefore be considered as potential cytotoxic drug towards sensitive and resistant phenotypes. ose activities are due to different chemical compounds present in the plant extract. is result is in agreement with those of some authors [18,26], which have shown that the Raphia vinifera compounds have cytotoxicity activity against cancer cell lines. Previous research showed that saponins from Raphia vinifera (Progenin III) induced necroptosis, autophagy, and apoptosis in leukemia cells [26]. Zhao   previously exhibited cytotoxic effects by blocking the S phase of interphase [14]. e undesirable effects observed in animals after substance intake predict the toxic effects in humans after its administration [44]. e dosage of markers that ensures the correct functioning of the organism in rats can provide information on the toxicological effect of a substance. For the acute toxicity, the single dose (5000 mg/kg b.w.) of extract administered in rats did not cause animal deaths. erefore, LD50 was estimated to be greater than 5000 mg/ kg since no acute toxicity was detected. e extract has low toxicity when their LD 50 is between 2000 mg/kg and 5000 mg/kg after oral administration [35]. Some authors had obtained LD 50 higher than 4000 mg/kg b.w. after administration of the root extract of Raphia spp. (Raphia hookeri) in same experience [45]. e high safety margin presented by this genus proof its safety for consumers [45].
For the subchronic toxicity study, the rats received 3 doses (250, 500, and 1000 mg/kg) of extract. e results showed that the amount of food intake had a direct effect on animal growth. e significant reductions of food consumption and animal growth were observed in male rats treated at highest dose (1000 mg/kg) from the 16 th day of treatment with respect to the control group. e weight loss of animals during this work can be explained by the presence of tannins and saponins (antinutritional substances) in this plant extract. ese substances that have the ability to reduce absorption of nutrients in the body [46] would be responsible for the reduction of food consumption, and thus, the reduction in body weight of rats treated at the highest dose of extract. is is agreed with that of Felix and Mello [47] who have reported that tannins showed inhibitory activities on digestive enzymes and decrease the protein quality of foods. e levels of transaminases and ALP are generally used as biomarkers associated with liver damage [48,49]. e decrease in serum transaminase, total proteins, and alkaline phosphatase levels at all doses observed in the female rats with respect to control groups could reflect the hepatoprotective activity of secondary metabolite contents in RVM. is result is in agreement with those obtained by Kamga and Russell [9,12] who have shown that the young leaves of Raphia vinifera are used against liver problems. Also, many types of research showed that many flavonoids and saponins present in root, leaf, and epicarp of Raphia ssp (R. hookeri) have hepatoprotective, antioxidative, anti-inflammatory, and anticancer activities [50][51][52].
is hepatoprotective activity is proven by histopathological analysis of the liver that revealed no damage in both sexes in rats treated at all doses. e significant increase of triglyceride levels (TG) was remarked in male rats treated at all doses of extract with respect to their control groups. ese could be due to the fact that the extract contains oil, which is rich in triglyceride. is is in accordance with the idea of Igwenyi et al. [15] who have extracted oil from the dried pulp of Raphia vinifera. Noubangue et al. [39] have extracted oil from the dried pulp of Raphia spp using the maceration method. Kidney is an organ, which excrete waste product of metabolism outside organism. However, prolonged exposure of the kidney to toxic substances may be altered the renal tubules [53]. e significant reduction of urea and creatinine levels in rats treated with lower doses (250 mg/kg and 500 mg/kg) of extract compared with the control group would be due to the fact that the extract contains secondary metabolism responsible for nephroprotective effect. Some studies have shown that the phenolic compound contents in the extract have nephroprotective activities [52]. However, the apical bud is taken to treat gonorrhea and other genitourinary infections [8]. ese results are reinforced by the kidney sections of rats, which present no alteration. e possible kidney malfunction is suspected when the serum levels of creatinine and urea are abnormally high [54]. e increase in urinary creatinine levels in rat treated at dose 1000 mg/kg is due to antinutrient (oxalate) contained in the extract. In fact, the oxalic acid is nocive to the kidney and heart [55] and the symptoms of mild oxalate poisoning include kidney diseases [56].
Hematological components are useful for assessing food's toxicity [57]. e significant increase of lymphocyte levels in female rats treated at highest dose (1000 mg/kg) of extract with respect to their control group was observed since extract contains lactic acid responsible for immunoprotection effect. e investigations performed by some authors showed that raffia wine of Raphia vinifera contains lactic acid bacteria [5] that stimulate the immune system of rats [6]. ese results agree with those of some authors [58] who have shown that lymphocyte and monocyte levels increase at dose 1000 mg/kg in rat treated with ethanol extracts. e significant increase in blood platelets in both sexes treated at highest dose would indicate thrombocytosis. Moreover, the investigation carried out showed that extracts of Raphia vinifera fruit showed an increase in platelet indices counts [58]. e significant reduction of hemoglobin and hematocrit level in female rats treated at highest dose of extract could be because these rats had anemia. Several types of research demonstrate that, when the hemoglobin level is decrease, the patient has anemia [59]. However, investigations carried out by Ogidi et al. [58] showed that methanol and ethanol extracts of RVM showed an increase in red blood cell with respect to control groups. ese contradictory results could be explained by the difference in the qualitative or quantitative composition of those two extracts of Raphia.

Conclusion
e aim of this investigation was to evaluate the antiproliferative potential of Raphia vinifera extract and its constituents on cancer cells, as well as the toxicity of the crude extract. It also showed that this extract is relatively nontoxic. However, caution should be taken when consuming the extract of the fruit mesocarp of Raphia vinifera during 28 days of treatment at highest dose, as it may induce some liver and kidney injuries. In general, Raphia vinifera is a safe medicinal plant that deserves further investigation to afford an anticancer phytomedicine.

Data Availability
All data obtained or generated during this work are incorporated in this published article.