Antihypertensive and antidiabetic activities of Erythrina senegalensis DC (Fabaceae) stem bark aqueous extract on diabetic hypertensive rats

Journal of Ethnopharmacology 246 (2020) 112200

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Antihypertensive and antidiabetic activities of Erythrina senegalensis DC (Fabaceae) stem bark aqueous extract on diabetic hypertensive rats

T

Danielle Claude Bilandaa,∗, Ronald à Goufani Bidinghaa, Paul Désiré Djomeni Dzeufieta, Yannick Bekono Foudaa, Rodrigue Fifen Ngapouta, Yannick Tcheutchouaa, Pascal Emmanuel Owonaa, Steven Collins Njonte Wouambab, Lucie Tanfack Tatchoua, Théophile Dimoa, Pierre Kamtchouinga a b

Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Science, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon Laboratory of Natural Substances Chemistry, Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, PO Box 812, Yaoundé, Cameroon

A R T I C LE I N FO

A B S T R A C T

Keywords: Hypertension Diabetes Antioxidant Erythrina senegalensis Rat

Ethnopharmacological relevance: Erythrina senegalensis is traditionally used in Cameroon for its relaxing and hypoglycemic properties in the treatment of cardiovascular diseases and diabetes. Aim of the study: High blood pressure and diabetes mellitus are frequently linked. These pathologies represent major risk factors for cardiovascular and renal diseases. The present study was designed to evaluate the antidiabetic and antihypertensive activity of the stem bark of Erythrina senegalensis aqueous extract in male hypertensive diabetic rats (HDR). Materials and methods: Hypertension and diabetes were induced by oral administration of sucrose (15%) and ethanol (40°) at doses of 1.5 g/kg and 5 g/kg respectively for 30 days, followed by an intravenous injection of streptozotocin (STZ; 40 mg/kg). A control group of 5 rats received distilled water (10 mL/kg) followed by intravenous injection of 0.9% NaCl (1 mL/100 g). HDR were divided into 4 groups of 5 rats each according to their blood glucose level and continued to receive ethanol in association with: distilled water (10 mL/kg); group I, metformin (200 mg/kg)+nifedipine (10 mg/kg); group II, plant extract (100 and 200 mg/kg) group IV and V, respectively for 28 days. At the end of the treatment, hemodynamic parameters were recorded by the direct method. Animals were sacrificed; blood and organs (aorta, heart, liver, and kidneys) were collected for biochemical and histological analysis. Phytochemistry and HPLC–DAD–HRESI-MS were used to determine the major compounds of the extract. Results: The administration of sucrose, alcohol, and STZ resulted in a significant increase in blood glucose, hemodynamic parameters, and body weight loss. A significant decrease in pancreatic islets size, nitrite, GSH, SOD and catalase activity was observed in HDR. There was also a significant increase in serum triglycerides, total cholesterol, creatinine, bilirubin, and transaminases activity in HDR. The aqueous extract of E. senegalensis, as well as the metformin + nifedipine combination, significantly improved all these parameters. HPLC coupled to both diode array and mass spectrometry detectors revealed the presence of 15 compounds and 11 of them were identified. Conclusion: These results suggest that the aqueous extract of E. senegalensis possess antihypertensive, hypoglycemic, hypolipidemic, cardiomodulator and antioxidant properties involved in the improvement of the metabolic disorders found in HDR. This may be due at least in part to the presence of Erysenegalensein (D, O, N, E), Warangalone, senegalensin and 6,8-diprenylgenistein identified in the extract.

1. Introduction

use the insulin it produces (WHO, 2018). WHO estimates that diabetes was the seventh leading cause of death in 2016; with 1.6 million deaths directly caused by diabetes (WHO, 2018). High blood pressure is most commonly associated with type 2 diabetes mellitus (T2DM) as part of

Diabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively

∗

Corresponding author. E-mail address: [email protected] (D.C. Bilanda).

https://doi.org/10.1016/j.jep.2019.112200 Received 21 March 2019; Received in revised form 23 August 2019; Accepted 27 August 2019 Available online 28 August 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.

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(Mbam and Inoubou Department), Central Region of Cameroon in June 2017. The plant was authenticated at the National Herbarium of Cameroon where voucher specimen N° 13752 SRF Cam has been deposited. The barks were then shade dried and crushed. The powder (100 g) was added to 3 L of distilled and allowed to macerate at room temperature for 24 h and then filtered with Wattman No. 3 filter paper. The filtrate obtained was evaporated in an oven (40–45 °C) to afford 8 g of crude extract, the yield of 8%. The stem bark aqueous extract was then analyzed for the presence of phytochemical compounds, using the method described by Odebiyi and Sofowora (1987). The compounds tested were alkaloids, flavonoids, saponins, tannins, triterpenes, steroids, polyphenols, anthraquinones, and anthocyanins.

the metabolic syndrome. The comorbidity of hypertension and T2D aggravates arterial injuries via endothelial dysfunctions responsible for the risks of kidney and cardiovascular diseases (Bruno et al., 2012). Data report a prevalence of about 422.7 million cases of cardiovascular diseases (CVD) and 17.92 million CVD patients died in the world in 2015 (Roth et al., 2017). The addition of hypertension which itself is a risk factor for cardiovascular disease (CVD) worsens the complications of diabetes. Many people with T2DM die from cardiovascular complications, mostly due to high blood pressure. The prevalence of hypertension among diabetic patients is from more than 55%–85.6%, depending on the area (Kaleab Tadesse et al., 2018; Nouh et al., 2017). In fact, the association of the two conditions is a risk factor of CVD often accompanied with dyslipidemia and impaired hepatic and renal function (Albai et al., 2017). The co-existence of the two pathologies multiplies the sources of the free radical formation. In addition, oxidative stress from the imbalance between pro and antioxidants is one of the factors linking diabetes and high blood pressure (Moon and Won, 2017). The treatment for this combined pathologies require early diagnosis, lifestyle changes, and heavy medications, sometimes difficult to follow. Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology, target identification and in the evaluation of novel therapeutic agents and treatments in vivo. Today there are many animal models of diabetes and hypertension, and each model has its advantages, disadvantages and its molecular mechanism target (Al-awar et al., 2016). Other studies focus on cellular or molecular targets such as cellular senescence or miRNAs (Childs et al., 2018; Zhou et al., 2018). More than 80% of the world's population uses medicinal plants for their primary health problems (OMS, 2002). Many plants have already proved their effectiveness against chronic diseases such as diabetes and/or hypertension (Delfan et al., 2014; Asuk et al., 2015). Most of them contain natural antioxidants that can prevent or minimize the complications by fighting against ROS formation and its damages. Erythrina senegalensis (E. senegalensis) used in the present study is a spiny shrub or tree with an open crown; the bole has a thick, corky, dark grey bark armed with stout prickles slightly recurved from a woody base (Von Maydell, 1990). The plant belongs to the family of Fabaceae and is found in west tropical Africa, from Senegal to northern Cameroon. Though the raw seeds are poisonous, E. senegalensis is used in traditional medicine in the treatment of many pathologies including arterial hypertension and diabetes (Focho et al., 2009; Togola et al., 2008; Von Maydell, 1990). The stem bark is febrifuge; the decoction is taken in the treatment of yellow fever, malaria, bronchitis, rachitis, liver and gallbladder problems, amenorrhea and women sterility. The bark and the roots are commonly used in the treatment of gastrointestinal disorders, leprosy, hemorrhoids, as an anti-abortive and as a general tonic (Focho et al., 2009; Togola et al., 2008). The wood is chewed as an aphrodisiac. Most of these effects result from its actions on smooth muscle and antimicrobial activity (Otimenyin and Uzochukuw, 2010; Doughari, 2010). In addition, some extracts of the E. senegalensis have revealed cardiovascular, hypoglycemic and antioxidant activities (Eka et al., 2011; Atsamo et al., 2013; Nembo et al., 2015). Moreover, many flavonoids isolated from the stem bark extract have shown to affect diacylglycerol acyltransferase (DGAT) activity, and are then good targets in the treatment of diabetes (Oh et al., 2009). However, the curative effects of the plant stem back on any model of hypertension and/or diabetes has yet not been studied. Therefore, the present study was designed to evaluate the activity of the aqueous extract of E. senegalensis stem bark aqueous extract on diabetic hypertensive rats and some of its complications.

2.2. Qualitative determination of compunds contents of E senegalensis using HPLC–DAD–HRESI-MS 2.2.1. Sample preparation Aqueous preparations extracts were separately dissolved in HPLC grade methanol in a concentration of 5 mg/mL then filtrated through a syringe-filter-membrane. Aliquots of 5 μL were injected into the LC–DAD/MS Dionex Ultimate 3000 HPLC (Germany), used to perform the analyses. 2.2.2. HPLC-MS conditions High resolution mass spectra were obtained with an OTOF Spectrometer (Bruker, Germany) equipped with a HRESI source and a UV–vis absorbance detector. The spectrometer was operated in positive mode (mass range: 100–1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 2 ppm deviation using Na Formate as calibrant. Mass spectra were simultaneously acquired using electrospray ionization in the positive ionization mode. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200 °C. Nitrogen was used as sheath gas (10 L/min). The spectrometer was attached to an Ultimate 3000 (Thermo Fisher, USA) HPLC system consisting of LCpump, UV traces were measured at 215, 218, 254, 280 and 330 nm and UV spectra―Diode Array Detector― (DAD) were recorded between 190 and 600 nm, auto sampler (injection volume 5 μL) and column oven (35.0 °C). The separations were performed using a Synergi MAX-RP 100 A (50 × 2 mm, 2.5μ particle size) with a H2O (+0.1% HCOOH) (A)/acetonitrile (+0.1% HCOOH) (B) gradient (flow rate 500 μL/min). Samples were analyzed using a gradient program as follows: 95% A isocratic for 1.5 min, linear gradient to 100% B over 6 min, after 100% B isocratic for 2 min, the system returned to its initial condition (90% A) within 1 min, and was equilibrated for 1 min. 2.2.3. Identification of peaks Identification of all constituents was performed by HPLC–DAD–MS analysis and by comparing the UV, MS spectra and MS/MS fragmentation of the peaks in the samples with those of data reported the literature of scifinder data base. 2.3. Drugs and chemicals All the drugs and biochemicals used in this experiment were purchased from Sigma Chemical Company (St. Louis, MO, USA). The chemicals were of analytical grade. 2.4. Animal material Ten to twelve weeks old male albinos Wistar rats, weighing between 200 and 220 g were used. The animals were raised at the animal house of the Laboratory of Animal Physiology of the University of Yaounde I (5 animals per cage). They were kept at room temperature under a natural light cycle with adequate ventilation and free access to tap water and standard animal diet. Prior authorization for the use of

2. Materials and methods 2.1. Plant extraction and phytochemistry The stem barks of E. senegalensis were harvested at Bafia-goufan 2

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whereas liver and kidney were homogenized in Tris-HCl 50 mM buffer solution. The homogenates were centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant collected was stored at -20 °C for further biochemical assays. The total protein was determined by the method of Gornall et al. (1949) and superoxide dismutase (SOD) activity was assessed according to Misra and Fridovish (1972). Reduced glutathione (GSH) and catalase were assayed following the method described by Ellman (1959) and Sinha (1972) respectively. Nitrites content was estimated according to Slack (1987) while malondialdehyde (MDA) was determined by the method described by Wilbur et al. (1949). For microscopic evaluation, the other part of investigated organs was fixed in 10% formalin for 7 days and paraffin embedded according to routine laboratory procedure. Paraffin sections of 4 μm were prepared and stained with hematoxylin and eosin (H&E) for histological examination. A digital camera and minisee software was used for images capture. The histomorphometry of pancreatic islets was performed using Image J software 1.49v.

laboratory animals in this study was obtained from the Cameroon National Ethics Committee (Reg. Nº. FWA-IRD 0001954). 2.5. Hypertension and diabetes induction and treatment Hypertensive rats (HTR) were obtained from normal rats (NR) as already described with some modifications (Dzeufiet et al., 2014). Sucrose (15%) at the dose of 1.5 g/kg followed by 40° ethanol (5 g/kg) were given orally daily for 30 days. Another group of 5 rats was receiving distilled water (10 mL/kg). After 30 days, the animals received an intravenous injection of streptozotocin (STZ) at the dose of 40 mg/kg for the first group and NaCl 0.9% for the second group. Serum glucose was determined 72 h later using an ACCU-CHEK ® Active blood glucose meter and strips. Animals with blood glucose ≥200 mg/dL were considered diabetic and maintained for one week in order for diabetes to stabilize. The diabetic hypertensive rats (20) continued to receive ethanol and were divided into 4 groups of 5 aminals each, treated for 28 more days as follows: one group receiving distilled water (10 mL/kg) a diabetic hypertensive control (HD), another group receiving metformin (200 mg/kg)+nifedipine (10 mg/kg) positive control (MN) group; two test groups (ES 100 and ES 200) receiving aqueous extract of E. senegalensis (100 and 200 mg/kg respectively). Another group of five healthy normotensive animals received distilled water (10 mL/kg), thus constituting the normal control group (NC). During the experimental period, body weight and glycemia were assessed twice a week. At the end of the treatments, blood pressure and heart rate of all rats were recorded according to the method previously described (Bilanda et al., 2010). Briefly, each rat was anesthetized using an intraperitoneal injection of urethane (1.5 g/kg). The trachea was exposed and cannulated to facilitate spontaneous breathing. The arterial blood pressure and heart rate were measured from right carotid artery via an arterial cannula connected to a pressure transducer coupled with a hemodynamic recorder Biopac Student Lab. (MP35) and computer. Thirty minutes of equilibration period was observed before each measurement.

2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism 5.03. All data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey post hoc test and expressed as a mean ± standard error of the mean (SEM). The difference was considered significant at p < 0.05.

3. Results 3.1. Phytochemistry of the aqueous extract of the stem bark of E. senegalensis The results of the phytochemical screening of the aqueous extract stem bark of E. senegalensis revealed that the extract contains alkaloids, flavonoids, saponins, tannins, triterpenes, steroids, polyphenols, and anthraquinones. Glucosides and anthocyanins were not present.

2.6. Biochemical and histological analysis After hemodynamic parameters recording, rats were sacrificed and the blood was collected and centrifuged at 3000 rpm for 15 min. The serum obtained was stored at -20 °C for the assessment of some biochemical markers. Serum samples were assayed for glucose, triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol, albumin, creatinine, transaminases (ALT and AST) activity using the commercial diagnostic kits (Inmesco, Germany). The atherogenic index (AI) was calculated by the following formula of Wakayashi and Kobaba (2002): AI = ([total cholesterol] – [HDLcholesterol)])/[HDL-cholesterol]. After blood collection, organs (aorta, heart, liver, and kidney) of each animal were dissected out and part of each was homogenized to make 20% homogenates. Aorta and heart were mixed to Mc Even solution,

3.2. Qualitative determination of compunds contents of E senegalensis using HPLC–DAD–HRESI-MS E. senegalensis crude extract sample was analyzed by HPLC coupled to both diode array and mass spectrometry detectors. The latter was used with an electrospray ionization source in positive ion mode. A representative base peak chromatogram and all ions MS is shown in Fig. 1 and Table 1 indicating that the used HPLC conditions allowed a good separation of a large percentage of compounds. The compounds were recognizable from their characteristic UV spectra, which were identified based on the HPLC–DAD–HRESI-MS data and subsequent confirmation by comparison with literature data.

Fig. 1. Chromatographic profiles of E. senegalensis aqueous extract. 3

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Table 1 Main signals exhibited in the HPLC-DAD-MS spectra of compounds detected in E. senegalensis and proposed attribution. N°

Tr (min)

[M+H]+

Molecular Formular

Name of Compund

Not Identified warangalone 6,8-diprenylgenistein

Exp.

Calcd. 325.2733 405.1697

C20H36O3 C25H24O5

407.1853

C25H26O5

1 2 3

5.2 5.1 5

325.2735 405.1697 407.1853

4 5

4.9 4.8

407.1852 423.1795

6 7 8

4.8 4.6 4.7

423.1797 423.1801 337.1069

9

4.4

439.1745

erythrisenegalone 8- prenylluteone 423.1802

C25H27O6

337.1071

C20H17O5

Fig. 3. Effects of E. senegalensis on blood glucose during 28 days of treatment.

Auriculatin

Each bar represents the mean ± SEM; n = 5; a P < 0.05, b P < 0.01, c P < 0.001: significantly different from normal rats 2 P < 0.01, 3 P < 0.001: significantly different from hypertensive diabetic rats; DBP diastolic blood pressure (mm Hg); SBP systolic blood pressure (mm Hg); MABP mean arterial blood pressure (mm Hg); NC: normal control rats, HD: hypertensive diabetic rats receiving ethanol, MN: hypertensive diabetic rats receiving metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving the extract from E. senegalensis at the respective doses of 100 and 200 mg/kg.

senegalensin Derrone or Alpinumisoflavone erysenegalensein D erysenegalensein N

439.1751 10 11 12 13 14 15

4.3 4.2 3.8 2.9 2.8 1.2

439.1741 439.1747 439.1736 565.1542 595.1648 405.0984

C25H27O7

erysenegalensein O Erysenegalensein E

565.1552 595.1657 405.0969

C26H29O14 C27H31O15 C23H17O7

Not Identified Not Identified Not Identified

3.4. Effects of E. senegalensis on blood glucose The effects of the aqueous extract of E. senegalensis on the serum glucose level are shown in Fig. 3. During the 28 days of treatment (Fig. 3), the administration of the extract at different doses as well as the combination of metformin and nifedipine resulted in a significant decrease (P < 0.001) in the glycaemia of treated rats compared to HD rats. There was 11.45% and 10.69% decrease on day 7 respectively in ES 100 and ES 200; 13.45%, 17.38% and 26.87% decrease on day 14, 33.52%, 36.88% and 43.14% on day 21 and 50.82%, 55.60% and 50.62% decrease on day 28, respectively in the MN, ES 100 and ES 200. At the end of experimental period (Fig. 3), the results showed a significant (p < 0.001) increase (169.03%) in serum glucose levels in HD group as compared with normal controls (NC). The treatment with plant extract (100 mg/kg; ES 100 and 200 mg/kg; ES 200) or the association metformin (200 mg/kg) + nifedipine (10 mg/kg) MN significantly decreased (p < 0.001) the serum glucose (50.82%, 55.60% and 50.64% respectively) as compared with HD group. The reduction with plant extract even dropped serum glucose level to values close to NC. Each bar represents the mean ± SEM; n = 5; c P < 0.001: significantly different from normal rats, 1 P < 0.05, 2 P < 0.01, 3 P < 0.001: significantly different from hypertensive diabetic rats; NC: normal control rats, HD: hypertensive diabetic rats receiving

3.3. Effects of E. senegalensis on blood pressure and heart rate Fig. 2 shows the effect of the aqueous extract of E. senegalensis on blood pressure and heart rate. Daily oral administration of sucrose (15%) and ethanol (5 g/kg) for 30 days followed by intravenous injection of STZ (40 mg/kg) resulted in a significant increase (p < 0.001) in hemodynamic parameters. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP) and heart rate (HR) were significantly increased in hypertensive diabetic rats (HDR) as compared to normal control (NC). That increase was of 44.35%, 82.50%, 65.16% and 7.55% respectively. The administration of the plant extract at the dose of 100 mg/kg significantly decreased the SBP (p < 0.01), the DBP, the MABP and the HR (p < 0.001) respectively by 20.44%, 31.95%, 15.04% and 8.62%. The extract (200 mg/kg) and the metformin + nifedipine combination (200 and 10 mg/kg) significantly (p < 0.001) decreased the SBP (27.49% and 26.13%), the DBP (25.82% and 29.62%), the MABP (24.54% and 28.21%), and the HR (11.15% and 18.52%) respectively as compared to HDR. The plant extract (100 and 200 mg/kg), as well as the reference treatment (metformin + nifedipine), reduced arterial blood pressure to the values close to NC.

Fig. 2. Effects of E. senegalensis on blood pressure (A) and heart rate (B). 4

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Fig. 4. Effects of the aqueous extract of E. senegalensis on lipid profile.

Fig. 5. Effects of the aqueous extract of E. senegalensis on some parameters of the oxidative stress.

3.5. Effect of E. senegalensis on lipid profile

ethanol, MN: hypertensive diabetic rats receiving metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving the extract from E. senegalensis at the respective doses of 100 and 200 mg/kg. The data of the day 28 represent the values obtained with colorimetric assay.

The effect of the aqueous extract of E. senegalensis on the lipid profile is summarized in Fig. 4. Animals in the HD group showed a significant increase (p < 0.001) in total cholesterol (CT), LDL5

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that increase (p < 0.01) was of 23.07% and of 45.67% in aorta and liver respectively, and (p < 0.001) of 54.99% and of 573.38% in heart and kidneys respectively as compared with HDR. Animals in the HD group showed a significant decrease in superoxide dismutase (SOD) activity of 22.68% (p < 0.001) in aorta, of 28.90% (p < 0.01) in heart, of 40.15% (p < 0.001) in liver and of 31.43% (p < 0.05) in kidneys as compared to the NC group. The treatment with the plant extract (100 mg/kg) resulted in a significant increase in SOD activity of 35.48% (p < 0.001) in the aorta. At the dose of 200 mg/kg, the extract favored a significant increase in SOD activity of 45.04% (p < 0.001) in aorta and of 48.11% (p < 0.05) in kidneys. The combination of metformin + nifedipine significantly increased SOD activity by 23.46% (p < 0.01) in aorta and by 108.61% (p < 0.001) in kidneys as compared to HD group. The extract (100 and 200 mg/kg) increased SOD activity in the aorta, liver and kidneys to values close to NC (Fig. 5D). The treatment with the plant extract (100 mg/kg) significantly increased the nitrites by 817.44% and by 93.32% (p < 0.001) in the aorta and heart respectively, and by 67.29% (p < 0.01) in the liver as compared to HDR. At the dose of 200 mg/kg, the plant extract favored a significant (p < 0.001) increase in nitrites of 510.28%, 121.00% and of 86.41% respectively in aorta, liver, kidneys, and of 43.18% (p < 0.05) in the heart as compared to HDR. The aqueous extract, as well as the combination of metformin + nifedipine, increased the nitrite concentration to values very close to NC group (Fig. 5 E1 and E2). Each bar represents the mean ± SEM; n = 5; 2P < 0.05, b P < 0.01, c P < 0.001: significant difference compared to normal rats; 1 P < 0.05, 2 P < 0.01, 3 P < 0.001: significant difference from hypertensive diabetic rats; NC: normal control rats; HD: hypertensive diabetic rats receiving ethanol, MN: hypertensive diabetic rats receiving metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving the extract of E. senegalensis at the respective doses of 100 and 200 mg/kg.

cholesterol (LDL-C), triglyceride (TG) and atherogenic index (IA). That increase was of 79.13%, 182.15%, and 44.78% and of 166.67% respectively as compared to NC group. A significant decrease of 32.91% (p ˂ 0.001) in HDL-cholesterol (HDL-C) was also observed. Treatment with aqueous extract of E. senegalensis (100 mg/kg) resulted in a significant decrease of 52.59%, 29.34%, 72.72% and of 64.69% in the TC, TG, LDL-C and atherogenic index (p ˂ 0.01) respectively, as compared to HDR. An increase of 34.39% (p ˂ 0.001) was observed in HDL-C. The treatment with the plant extract (200 mg/kg) resulted in a significant decrease (p ˂ 0.001) of 25.14%, 45.48%, 21.25% and of 54.06% in TC, LDL-C, TG, and AI respectively as compared to HDR. It was also observed an increase of 58.33% (p ˂ 0.001) in HDL-C of HDR. Administration of metformin + nifedipine decreased significantly (p ˂ 0.001) by 38.48%, 52.14% and 50% respectively, the CT, LDL-C and atherogenic; 27.60% (p ˂ 0.01) triglyceride levels and a significant increase (p ˂ 0.001) of 51.39% HDL-C as compared to HDR. The extract, as well as metformin + nifedipine combination, reduced lipid metabolism values close to those of NC. Each bar represents the mean ± SEM; n = 5; a P < 0.05, b P < 0.01, c P < 0.001: significant differences from normal rats 2 P < 0.01, 3 P < 0.001: significantly different from hypertensive diabetic rats; HD: hypertensive diabetic rats receiving ethanol, MN: hypertensive diabetic rats receiving ethanol, metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving ethanol and the extract of E. senegalensis at the respective doses of 100 and 200 mg/kg; chol: cholesterol. 3.6. Effects of E. senegalensis on oxidative status The effects of the aqueous extract of stem bark of E. senegalensis on some oxidative stress parameters in the aorta, heart, liver, and kidney are shown in Fig. 5. It is observed a significant decrease (p ˂ 0.05) in reduced glutathione (GSH) of 25.73% and 47.95% respectively in the aorta and heart (p ˂ 0.001) of 46.44% and 64.20% respectively in the liver and kidneys of HDR as compared to NC (Fig. 5A). The treatment with plant extract (100 mg/kg) resulted in a significant increase of 48.78% (p ˂ 0.01), 107.28% (p ˂ 0.05) and of 76.48% (p ˂ 0.01) respectively in the aorta, heart, and liver; and of 122.02% (p ˂ 0.01) in the kidneys at the dose of 200 mg/kg as compared with HDR. The treatment with the combination of metformin + nifedipine resulted in a significant increase of 111.83% and 97.89% (p < 0.05) in the heart and kidneys respectively as compared to the HDR. The extract (100 and 200 mg/kg) succeeded to bring GSH level close to the values NC. Fig. 5B revealed a significant (p < 0.001) increase in malondialdehyde (MDA) concentration in the HD group as compared to the NC group. This increase was of 135.56% in the aorta, and of 35.06%, 38.33%, and 89.33% respectively in the heart, liver, and kidneys. Administration of the extract (100 mg/kg) resulted in a significant decrease (p < 0.001) in MDA of 47.90%, 36.17% and 32.42% respectively in the aorta, liver, and kidneys as compared with HDR. At the dose of 200 mg/kg the plant extract reduced significantly MDA by 79.93% (p < 0.001) in the aorta, by 27.29%, 26.99%, and by 18.85% (p < 0.01) respectively in the heart, liver and kidneys. The combination of metformin and nifedipine significantly decreased (p < 0.001) the MDA level by 78.34% in the aorta, (p < 0.05) by 18.38%, 20.40% and by 39.59% in the heart, liver, and kidneys respectively as compared with HDR. The extract, as well as the metformin + nifedipine combination, brought the MDA concentration to the values close to NC. From Fig. 5C, the animals in the HD group showed a significant (p < 0.05) decrease of 20.25% and of 38.3% in catalase activity in aorta and liver respectively; a decrease (p < 0.001) of 35.06% and of 89.33% in heart and kidneys respectively as compared to NC group. The treatment with plant extract (100 mg/kg) significantly (p < 0.001) increased catalase activity by 28.64% and by 615.97% in the aorta and kidneys respectively as compared to HDR. At the dose of 200 mg/kg

3.7. Effects of E. senegalensis on liver and kidney function The effects of the aqueous extract of E. senegalensis on some parameters of liver and kidney function are presented in Fig. 6. The results showed a significant increase (p < 0.001), of transaminase (ALT and AST) activity, albumin, bilirubin, and creatinine serum concentrations in diabetic hypertensive rats (HDR) as compared to normal controls (NC) rats. That increase was 143.68%, 257.85%, 43.04%, 119.43% and 158.97% respectively while a significant (p < 0.01) decrease of 19.61% was observed in serum proteins. The treatment with the plant extract (100 and 200 mg/kg) or with the combination of metformin (200 mg/kg) + nifedipine (10 mg/kg); induced a significant (p < 0.001) decrease in ALT activity of 49.44%, 60.77% and of 58.01% respectively; in the ASAT activity of 61.13%, 71.61% and of 35.92% (p < 0.05) respectively; bilirubin of 67.51%, 63.34% and of 62.31% respectively; in creatinine of 59.41%, 57.43% and of 33.66% respectively as compared to HDR. The serum albumin decreased by 28.35% and by 20.70% respectively with plant extract (100 mg/kg) or metformin + nifedipine combination (MN). The serum total protein significantly (p < 0.05) increase by 15.18% in ES 200 group as compared with HDR. The treatment with the plant extract (200 mg/kg) reduced transaminase activity, creatinine, bilirubin and protein levels to the values close to that of NC rats. Each bar represents the mean ± SEM; n = 5; a P < 0.05, b P < 0.01, c P < 0.001: significantly different from normal rats; 1 P < 0.05, 3 P < 0.001: significantly different from hypertensive diabetic rats; NC: normal control rats; HD: hypertensive diabetic rats receiving ethanol, MN: hypertensive diabetic rats receiving metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving the extract of E. senegalensis at the respective doses of 100 and 200 mg/kg. 6

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Fig. 6. Effects of the aqueous extract of stem bark E. senegalensis on some parameters of liver and kidney functions.

3.8. Effects of E. senegalensis on the pancreas

4. Discussion

Fig. 7 show the effects of the extract on pancreatic islet area (A) and architecture (B). Animals in the HD group had a significant decrease in pancreatic islet size and area (p < 0.001) of 41.02% compared with those in the NC group. The extract (100 and 200 mg/kg) as well as the treatment combining metformin + nifedipine significantly increased (p < 0.001) the pancreatic islet area and size of the order of 77.92%, 56.53% and 38.99% respectively as compared to HDR. Each bar represents the mean ± SEM; n = 5; a P < 0.05, c P < 0.001: significant difference from normal rats; 3 P < 0.001: significant difference from hypertensive diabetic rats; Is = Islet; Enp = Endocrine pancreas; Exp = Exocrine pancreas; NC: normal control rats; HD: hypertensive diabetic rats receiving ethanol, MN: hypertensive diabetic rats receiving metformin and nifedipine at the respective doses of 200 and 10 mg/kg, ES 100 and ES 200: hypertensive diabetic rats receiving the extract of E. senegalensis at the respective doses of 100 and 200 mg/kg.

The purpose of this study was to evaluate the antihypertensive and antidiabetic activity of the aqueous extract of the stem bark of Erythrina senegalensis, on a diabetic hypertensive rat model. The results show that administration of sucrose 15% (1.5 g/kg) and ethanol 40° (5 g/kg/day) for four weeks resulted in a significant increase in arterial blood pressure (ABP) and heart rate (HR). The installation of this arterial hypertension (HTA) might involve complex and variety of mechanisms (Hussain et al., 2014). Combined with chronic consumption of sucrose, alcohol may be able to cause endothelial dysfunction resulting in a decrease in the level of NO responsible for HTA (Hussain et al., 2014). This was confirmed by the results on aorta nitrite that was significantly low in HDR as compared to NC group. Insulin involved in the use of sugar by the cells is produced by the pancreatic islets. Therefore hyperglycemia observed in hypertensive diabetic (HD) control rats might be due to the decrease in pancreatic islet area and size, leading to a decrease in insulin secretion. In fact streptozotocin (STZ) as shown by

Fig. 7. Effects of E. senegalensis on the size of pancreatic istels (A) and pancreas histology (B). 7

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E. senegalensis aqueous extract has proved to have beneficial effects on lipid profile. In fact, 6,8-diprenylgenistein inhibits fat absorption by the inhibition of pancreatic lipase, and reduces lipogenesis via AMPK activation and follow by fatty acid oxidation and inhibition of cholesterol synthesis, which might contribute the improvement of metabolic parameters (Yang et al., 2015). The antihypertensive and/or antidiabetic effect on several plant extract has already been linked to their lipid profile improvement (Bilanda et al., 2010, 2017; Dzeufiet et al., 2014, 2018). Therefore, the improvement of lipid profile could be one of the ways used by E.senegalensis aqueous extract to treat hypertension and diabetes as well as preventing its complications. Chronic diseases such as diabetes and hypertension are strongly associated to oxidative stress which is involved in their complications (Moon and Won, 2017), resulting in the alterations of the functions of some organs (Bilanda et al., 2017, 2018). HDR in this study was having a higher concentration of Malondialdehyde (MDA), one of lipid peroxidation products. Its increase indicates a state of oxidative stress (Moon and Won, 2017). In addition, glutathione (GSH) and nitrite concentration, as well as superoxide dismutase (SOD) and catalase (CAT) activities, were reduced in those rats. These results are in accordance with many authors who linked the oxidative stress to the depletion of those parameters in animals (Asuk et al., 2015; Moon and Won, 2017; Bilanda et al., 2017; Bilanda et al., 2018). The same is also observed in humans (Okoduwa et al., 2013). The improvement of those parameters after treatment with our plant extract suggests that E. senegalensis stem bark aqueous extract has antioxidative activity. In fact, the plant extract has succeeded to reduce the amount of MDA. Indeed, the protection of cell membranes from peroxidation by the extract may have led to a decrease in MDA. Our conclusions are consistent with many authors working with different plant extracts; (Dzeufiet et al., 2014; Asuk et al., 2015; Bilanda et al., 2018). Our plant extract also increased the GSH, a direct free radicals scavenger. GSH plays a multifactorial role in the antioxidant defense mechanism. Indeed, during diabetes, the relative depletion of NADPH due to the activation of aldose reductase alters the regeneration of GSH which leads to the depletion of its rate (Hussain et al., 2014). Its decrease could also reflect its destruction or an increase in the production of free radicals. Therefore the increase in GSH after treatment with E. senegalensis aqueous extract suggest that the plant may have some compounds like flavonoids that are free radical scavengers (Atsamo et al., 2013; Bilanda et al., 2017, 2018). E. senegalensis may also be able to prevent the overproduction of free radicals and then sparing the GSH. The maintenance of SOD and CAT activity by the plant extract confirms its antioxidant activity. These results are in agreement with those of several authors who have demonstrated that the antioxidant activity of plant extracts result in the maintenance of the activity of these enzymes (Asuk et al., 2015; Bilanda et al., 2017, 2018). All these observations confirm the antioxidant activity of the bioactive compounds of E. senegalensis aqueous extracts. Thus, preventing the oxidative stress, the link between diabetes and hypertension could be a novel treatment strategy in clinical practice (Moon and Won, 2017). The oxidative stress and impairment of lipid profile associated with hypertension and/or diabetes damage organs both in humans (Albai et al., 2017) and aminal models (Asuk et al., 2015; Bilanda et al., 2017, 2018). This can in part explains the decrease in pancreatic islets size. In fact, β-cells are more vulnerable to oxidative stress than other cells because their antioxidant capacity is weaker (Moon and Won, 2017). Those damages on liver and kidney were shown as expected, by increased ALT and ASAT activity, total bilirubin, albumin, and creatinine serum levels. Increased activity of ALT and ASAT in the blood assumes that cells containing these enzymes have been harmed. This lesion of cell membranes could have occurred during lipid peroxidation. This would still explain the high level of MDA observed in HDR. Indeed, lipid peroxidation damages cell membrane and lead to the leakage of these enzymes. Administration of the extract decreased the activity of transaminases, confirming the protective activity of E. senegalensis plant

several studies, destroys pancreatic islets and causes hyperglycemia and in the short run a reduction in insulin secretion, leading to diabetes (Szkudelski, 2001). The administration of E. senegalensis aqueous extract decreased the blood pressure, HR as well as blood glucose in HD rats. These findings suggest that E. senegalensis may have antihypertensive and antidiabetic activity. That activity might pass through the maintenance of endothelial integrity, relaxation of vascular smooth muscle cells, and an increase in insulin sensitivity (Han et al., 2007). The cardiovascular beneficial effects of E.senegalensis might also be involved (Nembo et al., 2015; Atsamo et al., 2013). Some flavanones with isoprenyl groups, erysenegalensein (D, O, N, E) identified in the extract have already proved to have a strong inhibitory activity against diacylglycerol acyltransferase that could explain the antidiabetic effect observed (Oh et al., 2009). Warangalone and senegalensin, compounds identified in the extract have already shown protein tyrosine phosphatase 1B inhibitory activity. In fact, that inhibition increases insulin sensitivity and obesity resistance. Furthermore, 6,8-diprenylgenistein present in the extract stimulates glucose-uptake in basal and insulinstimulated L6 myotubes. AMPK activation, GLUT4 and GLUT1 expressions and PTP1B inhibition by bioactive constituents appear to be involved in the mechanism of the stimulation of basal and insulin-responsive glucose-uptake (Njamen, 2006; Cheng-shi et al., 2012). Alkaloids, phenols, and flavonoids present in the extract, whose antihypertensive and antidiabetic effects have been demonstrated (Eka et al., 2011), can also explain the observed results. The lack of more significant changes in blood glucose and blood pressure at the higher doses observed in the present study may be due to antagonism. The extract may contain antagonistic molecules. Therefore, at low doses, the concentration of antagonistic molecule(s) is low, thus, offering no hindrance to the hypoglycaemic and hypotensive causative substance (s) (Dzeufiet et al., 2006). The weight loss observed in this study in HD rats is consistent with the work of Sadie and Screekumara (2010) who found a similar loss of weight after STZ injection to rats. Insulin plays an important role in the synthesis of proteins of several cell types in humans and in rats (Proud, 2006). This weight loss can be explained by a decrease in insulin secretion. This may also be due to sensitization of proteins, and the action of proteases that leads to their destruction and elimination (Levine, 2002). The administration of the extract significantly increased the weight of the treated animals. These results suggest that the aqueous extract of E. senegalensis by protecting the pancreas prevented the decrease in insulin secretion. The extract may also have an insulin-like effect or else stimulate the secretion of insulin from the remaining β cells. Lipid metabolism disorders involving hypercholesterolemia and dyslipidemia are very often associated with diabetes and hypertension (Dzeufiet et al., 2014; Aziz, 2017; Albai et al., 2017). In HD rats, serum triglycerides, LDL-cholesterol, total-cholesterol, atherogenic index increased while HDL-cholesterol decreased. Such results were obtained by many authors (Dzeufiet et al., 2014; Bilanda et al., 2017, 2018), confirming that hypertension is closely associated with dyslipidemia in rat regardless of the model of hypertension-induced, and even in human especially if the patient is also diabetic (Aziz, 2017). These changes could be explained by the mobilization of fatty acids from adipose tissue in these animals following the under-utilization of glucose. In fact, insulin resistance can promote the hepatic production of vLDL and their transformation into LDL-cholesterol through its antilipolytic action. Type 2 diabetes alone is associated with dyslipidemia which is in part responsible for its complications (Albai et al., 2017). The coexistence of the two pathologies in the present models worsens dyslipidemia and its consequences both in cardiovascular system and kidney (Albai et al., 2017). The treatment with our plant extract has almost normalized the lipid profile suggesting that E. senegalensis has beneficial effects on lipid metabolism. That effect could be explained by the presence of some isoflavonoids with pancreatic lipase inhibitory activity (Jo et al., 2017). Furthermore 6,8-diprenylgenistein identified in 8

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against lipid peroxidation. Moreover the treatment with 6,8-diprenylgenistein identified in E. senegalensis aqueous extract had proved to protect hepatic cells (Yang et al., 2015). The extract might be able to protect or repair the membrane of these cells, preserving the structural integrity of those organs. This protection is confirmed by the low level of bilirubin observed in the animals treated with the extract. Our finding is in agreement with some authors (Bilanda et al., 2017, 2018; Asuk et al., 2015). The low level of creatinine obtained in the animals treated with the extract confirms the protective action of the extract on the kidney. The bioactive compounds in the extract would have protected the liver and kidneys from the harmful effects of alcohol, sucrose, streptozotocin and free radicals. All these observations suggest that the decrease in arterial hypertension and blood glucose is achieved by improving insulin sensitivity and protecting the function of the organs involved in their regulation.

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5. Conclusion The aqueous extract of the bark of E. senegalensis significantly reversed the hyperglycemia, hypertension, dyslipidemia, weight loss, liver, and kidney dysfunction and oxidative stress induced in hypertensive diabetic rat models. The improvement of these parameters may be due at least in part to the presence of erysenegalensein (D, O, N, E), warangalone, senegalensin and 6,8-diprenylgenistein identified in the extract. The possible mode(s) of action include antioxidant, hepatonéphroprotective, improvement in endothelial dysfunction to release more nitric oxide pancreatic islets regeneration and/stimilation. These results provide many pharmacological arguments in favor of E. senegalensis and thus justify its empirical use in the treatment of metabolic disorders associated with diabetes and arterial hypertension. However, more studies are required to evaluate its safety and efficacy in clinical conditions in human. Conflicts of interest The Authors have no conflict of interest to declare. Authors'contributions All authors strived to accomplish the present work, from the experiments to the manuscript. DCB conducted pharmacological assays and data analyses under assistance of RGB, YBF, RFN and YT; SCNW performed HPLC and MS analyses; PDDD did relevant interpretation of the results; DCB, PDDD, TD and PK designed, supervised the experiments and reviewed the manuscript. Acknowledgment The authors are grateful to French association PCD (Pathology Cytologie Développement) for providing histological reagents. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.112200. References Al-awar, A., Kupai, K., Veszelka, M., Sucs, G., Attieh, Z., Murlasits, Z., Török, S., Pósa, A., Varga, C., 2016. Experimental diabetes mellitus in different animal models. J. Diab. Res. 1–12 2016. Albai, O., Timar, B., Roman, D.L., Timar, R., 2017. Characteristics of the lipid profile in patients with diabetes mellitus and chronic kidney disease. Rom J. Diabetes Nutr. Metab. Dis. 24 (3), 237–245. Asuk, A.A., Dasofunjo, K., Okafor, A.I., Mbina, F.A., 2015. Antidiabetic and Antioxidative Effects of Jatropha curcas extracts in streptozotocin-induced diabetic rats. Br. J. Med. Med. Res. 5 (3), 341–349. Atsamo, A., Néné-Bi, S., Kouakou, K., Fofié, K., Nyadjeu, P., Watcho, P., Datté, J.,

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