Toxicological evaluation of Picralima nitida in rodents

Journal of Ethnopharmacology 236 (2019) 205–219

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Toxicological evaluation of Picralima nitida in rodents a

a,b,∗

Olufunsho Awodele , Abdul Gafar Victoir Coulidiaty Sunday Agagua, Bunmi Omoseyindemic,d, Kofi Busiae

a

, Gbenga Oluyemi Afolayan ,

T

a

Department of Pharmacology, Therapeutics and Toxicology of the College of Medicine, University of Lagos, Nigeria Centre MURAZ, Health Research Institute, Burkina Faso Past Chairman of the Lagos Traditional Medicine Board, Nigeria d Traditional Medicine Member Expert Committee of WHO and WAHO, Burkina Faso e Traditional Medicine Officer, West African Health Organization (WAHO), Burkina Faso b c

ARTICLE INFO

ABSTRACT

Keywords: Picralima nitida Teratogenic effects Toxicity Pregnancy Traditional medicine

Picralima nitida (Stapf) T. Durand and H. Durand (Apocynaceae), over the years has shown wide range of usage in African folk medicine and its safety profile in instances of prolonged use and pregnancy are major concerns. The study aimed to extensively investigate the toxicological effects of Picralima nitida in albino rodents and make appropriate extrapolations to humans. In the first phase of the experiment which evaluated the genotoxicity and subchronic toxicity of P. nitida, a total of 40 albino rats (male and female) were randomized into 4 groups of 10 animals per group. Group 1 (control group) was orally administered with 10 ml/kg of distilled water. Animals in Groups 2 to 4 were administered with aqueous seed extract of the plant at 100, 200, 400 mg/kg body weight/day, respectively. Oral administration at the designated doses was continued for 90 days after which they were sacrificed by cervical dislocation for subchronic toxicological assessment. In the genotoxicity phase, 30 female mice were randomized into 5 groups, the control group was treated with 10 ml/kg of distilled water, groups 2 to 4, treated with 100 mg/ kg, 200 mg/kg and 400 mg/kg doses of extract, and the 5th group had cyclophosphamide (0.1 mg/kg). The mice were sacrificed on the 28th day for bone marrow sampling for genotoxicity testing. In the second phase of the experiment which evaluated the teratogenicity of P. nitida, graded doses of the extract were administered to pregnant rats from day 1–19. Three groups of 6 female rats per group were administered 75, 150 and 300 mg/kg aqueous extract of P. nitida and a fourth group of 6 rats used as control was administered distilled water at 10 ml/kg. On day 20, 3 dams from each group were sacrificed and the foetuses were harvested through abdominal incision for physical examination. The 3 remaining dams were allowed to litter. The litters were sacrificed at 6 weeks for biochemical, haematological and histological analyses. The LD50 determined was 707.107 mg/kg. The aqueous seed extract of P. nitida was found to be genotoxic at all the test doses. There were no significant alterations in haematologic and renal parameters following subchronic administration. Notable dynamics were observed in hormonal characteristics: there was a significant dose-dependent reduction in FSH while oestradiol and progesterone showed dose-dependent increase. Furthermore, P. nitida may cause hepatopathy as shown by hepatic venous and sinusoidal congestion on hepatic histology. Also, there is non-significant reduction in total cholesterol and LDL. No significant alteration in glucose level. Furthermore, the extract produced a statistically significant decrease in birth weight (p < 0.0001). The extract induced a significant (p < 0.05) increase in creatinine and transaminase levels in the first filial of group 150 mg/kg. The platelet count was increased in all treated group (p < 0.005). All the histology of kidney in 150 mg/kg group showed vascular congestion. In conclusion, the aqueous seed extract of P. nitida has teratogenic effects and should not be used in pregnant women. Also, P. nitida is highly genotoxic and may cause hepatic damage and depletion of glutathione pool on chronic use, thereby causing oxidative stress and its potential sequelae.

∗ Corresponding author. Department of Pharmacology, Therapeutics and Toxicology of the college of medicine, University of Lagos, Nigeria and Centre MURAZ, Health Research Institute, Burkina Faso, Centre MURAZ 2054 Avenue Mamadou KONATE, 01 B.P. 390 Bobo-Dioulasso 01, Burkina Faso. E-mail addresses: [email protected] (O. Awodele), [email protected] (G.O. Afolayan), [email protected] (S. Agagu), [email protected] (B. Omoseyindemi), [email protected] (K. Busia). URL: http://[email protected] (A.G.V. Coulidiaty).

https://doi.org/10.1016/j.jep.2019.03.008 Received 29 October 2018; Received in revised form 17 February 2019; Accepted 4 March 2019 Available online 07 March 2019 0378-8741/ © 2019 Published by Elsevier B.V.

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1. Introduction

2.2. Housing and feeding of animal

Picralima nitida (Stapf) T. Durand & H. Durand is a West African plant which has great usefulness in African non- orthodox medicineespecially in the rainforest regions of Nigeria, Ghana, Cote d’Ivoire, Gabon and Cameroon. It belongs to the hunterieae tribe of the Apocynaceae family (Erharuyi et al., 2014). Preparations from different parts of the plant are employed as crude drug or crude herbal extract as remedy for various kinds of human diseases. The seeds are widely used in West Africa especially in Nigeria, Cote d’Ivoire and Ghana for the management of erectile dysfunction, fever, malaria, pneumonia and other chest-conditions. In Gabon the seeds are applied externally for the treatment of abscesses. In Ghana, the seed-decoction is given as an enema while the crushed seed is taken by mouth for chest-comp (Erharuyi et al., 2014). In central Africa the seeds are used in rheumatic fever and as an antipyretic (Olajide et al., 2014). The plant has been shown to be widely used in female infertility in Ghana and Nigeria (Diame, 2010; Solmonet et al., 2014). The leaves are used as vermifuge and the leaf sap is dripped into the ears for otitis (Raponda-Walker and Sillans, 1961). The bark is used as laxatives and purgative, anthelmintic, treatment of venereal diseases, as febrifuges and also, to treat hernia. In Ivory Coast, a decoction of the bark is drunk in draught for jaundice and ‘yellow fever’ (Erharuyi et al., 2014). The root is used as vermifuge, aphrodisiac, for fevers, malaria, pneumonia and gastrointestinal disorder. In Lagos, Nigeria, Picralima nitida ‘s seeds have been reported to be used in female fertility problems and to treat nausea and vomiting during first trimester of pregnancy by the traditional medicine board and from a popular traditional midwife (Erharuyi et al., 2014). Despite the widespread abundance and traditional use of P. nitida extracts, there is paucity of data on the toxicological effects of this herb hence limiting its acceptability (Olajide et al., 2014). The previous study by Sunmonu et al., 2014, was on acute toxicological effect of this ethnomedicinal plant species. Data on the long-term exposure to this agent is limited. In addition, we could not find data on teratologic evaluation. Due to its usefulness, in chronic condition such as diabetes, hypertension, it would necessitate the long-term usage of the plant and owing to the fact that herbal medicine is not usually appropriately standardised as regards dosing there is a grave possibility of toxicity following long term/chronic use. Hence, a study designed to evaluate the safety profile (systemic and genomic toxicity) and teratogenic effects following subchronic exposure to P. nitida will be a welcome ideation going by its widespread usage in large parts of African continent of which Nigeria is inclusive. The aim of this study was to investigate the safety profile of the aqueous seed extract of Picralima nitida in rodents.

The animals were allowed to acclimatize for two weeks before the commencement of the experiment. The animals were housed in wellventilated plastic cages under standard room condition temperature, with 12 h natural light and alternating with 12 h darkness per day, in the Laboratory Animal care centre of the College of Medicine, University of Lagos. The animals were fed twice a day with Livestock Balanced Rations Growers Mash, which was purchased at the Mushin market, Lagos. Clean tap water was provided ad libitum. 2.3. Methods 2.3.1. Extraction The seeds were air dried until constant weight was obtained. Then the seeds were peeled and further air dried until constant weight was gotten. It was then grounded with an electric grinder at the Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos. The grounded seeds were macerated in water for 72 h (with mixture stirred every 24hrs while in the refrigerator). After 72hrs, the mixture was decanted and the supernatant filtrated using Whatman filter paper No.1. The obtained liquid was dried in an oven (Gallenhamp, England) at 40 °C. The resulting extract were placed in sterile sample bottles and stored in a refrigerator till required. The extract was reconstituted daily with the use of the vehicle (Distilled water). 2.3.2. Oral acute toxicity for LD50 determination Mice were randomly divided into five groups of five animals per group. Graded doses of the Picralima nitida seed extract (250, 500, 1000, 2000 and 4000 mg/kg) were administered orally to separate groups. The control group was administered 0.1 ml of distilled water orally. The mice were observed for 24 h post-treatment for mortality, behavioural changes (restlessness, dullness, and agitation) and other signs of toxicity (Dapaah et al., 2016; Erharuyi et al., 2014). The animals were further observed for 14 days for any signs of delayed toxic effects. 2.3.3. Teratogenic evaluation 2.3.3.1. Cycle determination. After two weeks of acclimatization, every morning, by 8 a.m., the oestrous cycle of female rats of proven fertility was accessed by doing a vaginal smear. Vaginal secretion was collected (in the morning between 8:00 and 9:00 a.m.) with a plastic pipette filled with 0.2 ml of normal saline by inserting the tip into the rat vagina, but not deeply (2–5 mm deep) the pipette is gently squeezed and release till a whitish is obtained. The (vaginal) fluid was then placed on glass slides to observe under a light microscope (×10 magnification) according to the method of the team of Marcondes (Marcondes et al., 2002). Female rats in the oestrous stage were housed together with adult male rats overnight in a ratio of 2:1 (female: male). The vaginal smears of mated female rats were assessed for the presence of sperm. The first day sperm is seen was taken as day 0 of pregnancy.

2. Materials and methods 2.1. Mateials 2.1.1. Plant source Picralima nitida dry seeds were obtained from the traditional medicine materials market situated in Agege, Lagos State, Nigeria. The seed was identified and authenticated by Mr. Nodza George, a Taxonomist at the Department of Botany, Faculty of Science, of the University of Lagos. A sample of the plant (seeds and leaves) were deposited in the herbarium with the voucher number LUH7731.

2.3.3.2. Experimental design. After confirmation of sperm in the vagina smear, pregnant rats were divided into four (4) groups of six (6) animals each as follows; ⁃ ⁃ ⁃ ⁃

2.1.2. Experimental animals Sexually matured adult Albino rats and mice of both sexes with an average weight of 160 g and 20 g respectively were obtained from the Laboratory Animal Centre of College of Medicine, University of Lagos, Nigeria.

Group Group Group Group

1: 2: 3: 4:

received received received received

distilled water (10 ml/kg), 300 mg/kg of the P. nitida extract, 150 mg/kg of the P. nitida extract, 75 mg/kg of the P. nitida extract.

The administration started on day 1 of pregnancy and continued to day 19.

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Grunwald and Giemsa stains. At least 2000 erythrocytes per mouse were scored at × 1000 for MN in polychromatic erythrocytes (MNPCE) and normochromatic erythrocytes (MNNCE) (Bakare et al., 2016; Schmid, 1975).

Three dams in each group were randomly selected, subjected to light ether anaesthesia and were sacrificed on day 20 of pregnancy by cervical dislocation prior to day 21 of gestation and the foetuses were harvested through abdominal incision for physical examination. This physical examination carried out are: measurement of tail length, crown-rump length, umbilical cord length, total weight (includes weight of foetus and placenta) and weight of foetus. After the physical examination, the following morphological anomalies were also assessed: formation of digital rays, neural tube defects, cleft palate and general growth abnormalities. The three (3) other dams in each group were allowed to litter and the litters were allowed to grow until they were approximately 4 weeks old after which, six litters were randomly selected from each of the group. Blood samples were collected from three randomly selected litters in each group for biochemical and haematological examination. Liver and kidney were collected from 3 animals of the first filial generation for histology analysis.

2.3.3.5. Determination of biochemical and haematological parameters. On the 91st day after termination of administration of the extract, the rats were anesthetized and sacrificed by cervical dislocation. Blood samples were collected through the retro-orbital plexus vein of the eye for biochemical and haematological parameters. The fully automated clinical chemistry analyser (Hitachi 912, Boehringer Mannheim, Germany) was used to determine the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, albumin, total protein, bilirubin, cholesterol, triglyceride, serum catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and the fully automated clinical haematological analyser (Pentra-XL 80, Horiba ABX, USA) was used to determine the levels of white blood cells, red blood cells, haemoglobin, haematocrit (packed cell volume), platelet, mean cell haemoglobin concentration (MCHC) and mean cell haemoglobin (MCH). The serum levels of Creatinine, Urea, Electrolytes, Alkaline phosphatase (ALP) Aspartate transaminase (AST) and Alanine transaminase (ALT), Lipids (Cholesterol and triglycerides were determined using a fully automated clinical chemistry analyser (Hitashi 912, Boehringer Mannheim, Germany).

2.3.3.3. Subchronic toxicity study. A total of 40 albino rats of either sexes were randomized into 4 groups of 10 animals. Group 1 (control group) was orally administered with 10 ml/kg of distilled water. Animals in Groups 2 to 4 were administered daily with aqueous seed extract of P. nitida seeds at 100, 200, 400 mg/kg, respectively. Oral administration at the designated doses was continued for 90 days. The rats were sacrificed after 90 days by cervical dislocation. An aliquot (2 ml) of the blood was collected into ethylene diamine tetraacetic acid (EDTA) embedded sample bottles (BD Diagnostics, preanalytical systems, Midrand, USA) for haematological analysis. Another 3 ml of the blood was collected and centrifuged at 2000 rev/min × 5 min and the serum carefully aspirated with a Pasteur pipette into Lithium heparin sample bottles and used within 12 h for the biochemical assays. The rats were quickly dissected and the abdominal organs were excised, freed of fat, blotted with clean tissue paper and then weighed. The organ to body weight ratio was determined by comparing the weight of each organ with the final body weight of each rat. Determined weights of the ovary, liver and kidney were cut and chopped into pieces then homogenized with ice cold 0.25 M sucrose solution (1 in 5 dilution) using precooled pestle and mortar in a bowl of ice-cubes. The supernatant was carefully collected for enzyme assay.

2.3.3.6. Histological examination of organs. Portions of selected abdominal organs; the liver, ovary and kidney were fixed immediately on removal from the animals in 10% Buffered Neutral Formalin (BNF) for 72 h at room temperature for histological analysis using the method described by Krause (2004). 3. Results 3.1. Phytochemical screening Table 1 shows that the constituent of the extract were flavonoids, steroids, alkaloids, saponins, terpenoids, cardiac glycosides and anthraquinones. The most important being saponins (37.06 mg/dg) followed by alkaloids (28.76 mg/dg).

2.3.3.4. Genotoxicity study. 30 female mice were randomized into 5 groups (6 animals per group), the control group 1 was treated with 10 ml/kg of distilled water, groups 2 to 4, treated with 100 mg/kg, 200 mg/kg and 400 mg/kg of P. nitida seed extract, and the 5th group had cyclophosphamide (0.10 mg/kg) daily. The mice were sacrificed following 28 days of administration and the bone marrow was harvested for genotoxicity testing. Following 28 days of administration, the mice were sacrificed (via cervical dislocation) and the bone marrow collected using a slight modification of the procedure of Schimdt (1975), we substituted the Foetal bovine serum, FBS used for Phosphate buffered saline, PBS. The femurs were removed and bone marrow flushed with PBS. Cells were centrifuged at 2000 rpm for 5 min and slides were stained with May-

3.2. Potential of hydrogen (pH) The extract is acidic, with a pH value equal to 4.9. 3.3. Estimation of the LD50 of the aqueous extract of Picralima nitida The LD50 was determined using Probit analysis method of Finney (1971). The LD50 is 707.107 mg/kg, 95% of Fiducial confident interval (426.293, 1172.902) as shown in Fig. 1.

Table 1 Qualitative and quantitative phyto-chemical screening. Phytochemical

Flavonoid

Steroid

Alkaloid

Saponin

Terpenoid

Cardiac glycoside

Tannin

Phenol

Anthraquinone

Phlobatanin

Qualitative Quantity in mg/100 mg

+ 23.89

+ 12.03

+ 28.7

+ 37.06

+ 10.40

+ 21.98

– –

– –

+ –

– –

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Fig. 1. Graph showing Probit against log10 dose for LD50 determination.

3.4. Teratogenic evaluation results

3.4.2. Biochemical parameters Table 4 shows the results from the biochemical analysis. There were statistically significant differences (p < 0.05) in the creatinine, ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in 150 mg/kg extract administered group when compared to the control group. The 75 mg/kg group also showed statistically significant difference (p < 0.05) in ALT compared to control. Table 5 shows the lipid profile parameters in the first filial generation. None of the parameters was statistically different (p > 0.05)

3.4.1. Morphological parameters Table 2 gives and overview of the outcomes of our intervention and Table 3 shows the morphological parameters in control and 75 mg extract administered group during pregnancy. Fig. 2 shows live foetuses after morphological assessment and Figs. 3 and 4 shows early and late resorption at various doses of P. nitida.

Table 2 Overview of the experimental outcomes after oral administration of Picralima nitida to treated groups. Group

Sacrificed dams number

Control 75 mg/kg 150 mg/kg 300 mg/kg

3 3 3 3

Dams that littered Sacrificed animals Percentage live foetuses (%)

% resorption (%)

100 33.33 0 0

0% 66.66% 100% 100%

number

Percentage live foetuses (%)

% resorption (%)

3 3 3 3

100 66.66 66.66 0

0% 33.33% 66.66% 100%

Table 3 Distribution of morphological parameters in control and 75 mg extract administered group during pregnancy. Parameters

TW

FW

PW

UCL

CRL

TL

Control 75 mg/kg

4.46 ± 0.06 3.83 ± 0.07****

3.98 ± 0.06 3.23 ± 0.06****

0.48 ± 0.01 0.60 ± 0.02****

2.92 ± 0.2 2.96 ± 0.22

6.04 ± 0.13 5.54 ± 0.18*

1.58 ± 0.04 1.32 ± 0.06**

Values are presented as MEAN ± SEM (standard error of mean) (n = 15). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 as compared to control. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc Pairwise Multiple Comparison test. TW: Total weight in g; FW: Foetus Weight in g; PW: Placenta Weight in g; UCL: Umbilical Cord Length in cm; CRL: Crown-Rump Length in cm; TL: Tail length in cm (see Fig. 1).

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Fig. 2. Live foetuses after morphological assessment.

Fig. 3. Pictorial representation of resorption sites following administration of 150 and 300 mg/kg of Picralima nitida.

Fig. 4. Showing late resorption following administration of 75 mg/kg P. nitida. 209

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Table 4 The effects of P. nitida on hepatic and renal function parameters of animals in 1st filial generation.

Control 75 mg/kg 150 mg/kg

Creatinine (in μmol/L)

Urea (in mmol/L)

ALT (in U/L)

AST (in U/L)

Albumin (in g/L)

24.00 ± 1.42 21.53 ± 0.94 28.95 ± 1.31*

6.90 ± 0.29 7.28 ± 0.25 5.95 ± 0.27

67.68 ± 7.49 35.88 ± 8.37* 30.88 ± 5.08*

215.15 ± 19.34 165.83 ± 25.07 126.00 ± 8.93*

41.33 ± 0.46 35.60 ± 2.54 42.83 ± 0.75

Table 5 The effects of Picralima nitida on lipid profile of litters in first filial generation.

Control 75 mg/kg 150 mg/kg

LDL (g/L)

HDL (g/L)

TC (g/L)

Triglycerides (g/L)

0.08 ± 0.04 0.15 ± 0.03 0.04 ± 0.01

1.63 ± 0.08 1.54 ± 0.02 1.58 ± 0.07

1.87 ± 0.13 1.86 ± 0.06 1.81 ± 0.06

0.79 ± 0.06 0.82 ± 0.09 0.94 ± 0.1

when compared with the control group. Values are presented as Means ± SEM *p-value < 0.05 Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. ALT: alanine aminotransferase and AST: aspartate aminotransferase. Values are presented as Means ± SEM Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. HDL: High-density lipoproteins; LDL: Lowdensity lipoproteins and TC: Total Cholesterols.

corpuscular haemoglobin concentration in g/dL; PLT: Platelet count per μL and PCT: plateletcrit in percentage.

3.4.3. Haematological parameters Table 6 represents the Full Blood Count parameters in the 3 treatment groups (control, 75 and 150 mg/kg) of first filial generation (F1). There was a statistically significant elevation (p < 0.05) in the platelet levels in the 75 and 150 mg/kg treated group. The platelet count (PLT) and the plateletcrit (PCT) were significantly lower (p > 0.05) in 150 mg group compared to control group (see Table 6). Values are presented as Means ± SEM *p-value < 0.05 Statistical level of significance analysed by one-way ANOVA followed by Turkey post hoc pairwise comparison test. WBC: White blood cells per μL; Lymph: Lymphocytes per μL (absolute); GRAN: Granulocytes cell per μL (absolute); LYMPH% percentage of lymphocytes in the WBC; GRAN%: percentage of granulocytes in the WBC; HGB: Haemoglobin, RBC: Red blood cell count per μL; HCT%: Haematocrit; MCV: Mean corpuscular volume in fL; MCH: Mean corpuscular haemoglobin in pg; MCHC: Mean

3.5. Genotoxicity results

3.4.4. Histology All histological analyses performed on the liver samples of the first filial generation (F1) were normal (Fig. 5). The histological analyses performed on first filial generation (F1) on the kidney revealed that control and 75 mg/kg were normal. However, there were congested blood vessels in the 150 mg/kg group (Fig. 6).

Membrane blebbing, cytoplasmic and membrane degeneration were most marked in the positive control group (cyclophosmide) with a notable reduction in the number and size of polychromatic and normochromatic erythrocytes (PCE and NCE) (Fig. 8 A, B and C). There was also more micronucleus formation when compared to the P. nitida aqueous extract treated groups (Fig. 8C). There was increased frequency of micronucleus, ablative phenomena membrane and cyto-degeneration in mice at all the tested concentrations of Picralima nitida aqueous seed extract (Figs. 8, 9A and 9B, 10A and 10B) compared to the negative control group (Fig. 7) and this occurred in a dose-dependent manner for the exposure periods, though less so, when compared to the cyclophosphamide group (positive control) (Fig. 11). At the respective tested concentrations, MPCE increased by 3.5, 8.5, and 14.3 folds after

Table 6 The effects of Picralima nitida on Full blood count of the F1 generation. Parameters

Control

75 mg/kg

150 mg/kg

WBC Lymphocytes Granulocytes Lymphocytes % Granulocytes % HBG (Haemoglobin) Haematocrit % MCV MCH MCHC Platelet count Plateletcrit

7175 ± 912.3 3675 ± 478.5 2675 ± 421.06 51.33 ± 2.41 37.08 ± 2.27 12.28 ± 0.23 38.8 ± 0.62 66.13 ± 1.02 20.83 ± 0.36 31.58 ± 0.14 756250 ± 13634.36 0.5 ± 0.01

7125 ± 1240.55 3800 ± 719.95 2275 ± 311.92 52.28 ± 2.01 33.53 ± 3.38 11.75 ± 0.38 37.65 ± 0.99 66.1 ± 1.09 20.55 ± 0.1 31.2 ± 0.37 772750 ± 23023.09 0.54 ± 0.01

7100 ± 1183.92 3375 ± 295.45 3175 ± 846.93 48.58 ± 4.11 41.7 ± 4.49 12.93 ± 0.14 39.9 ± 0.44 64.6 ± 0.71 20.88 ± 0.31 32.33 ± 0.15 921500 ± 62817.86* 0.59 ± 0.04*

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Fig. 5. Histological section of the liver of first filial generation rats. Showing radial plates of hepatocytes. No cytoplasmic fat vacuoles or areas of necrosis were seen: normal hepatocytes.

Fig. 6. Histological section of the kidney of first filial generation rats. Showing normocellular glomerular tufts disposed on a background containing viable tubules corresponding to a normal study for control and 75 mg/kg groups. While in 150 mg/kg group, the histologic section of kidney tissue shows normocellular glomerular tufts disposed on a background containing viable tubules. Congested blood vessels were seen, diagnosed as vascular congestion.

Fig. 7. Plate showing frequency of MPCE and PCE in the bone marrow of mice exposed to distilled water. Polychromatic erythrocytes (PCE) (black arrow) and normochromatic erythrocytes (NCE) (red arrows). A micronucleated PCE (MPCE) is also seen in this field of view (thick black arrow head). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

the 28 days exposure period. Significant difference (p-value= < 0.0001, < 0.0001 and 0.0063 at 100 mg/kg, 200 mg/kg and 400 mg/ kg doses respectively) is observed (Table 8). Observed PCE/1000 erythrocytes reduces in a dose-dependent manner in the Picralima nitida treatment groups, more severe reduction occurred in the cyclophosphamide group (Fig. 10).

3.6. Chronic toxicity 3.6.1. Haematological results following sub chronic administration of Picralima nitida There is significant increase in the total white cell count, the granulocyte and lymphocyte count (P-value: < 0.05) at 200 mg/kg

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Fig. 8. Plates showing frequency of MPCE and PCE in the bone marrow of mice exposed to cyclophosphamide. 8C, 8 B revealed extreme cytoplasmic and cell membrane degeneration alongside cellular ablation, very few and smaller PCE with micronucleus formation. PCEs were also fewer and smaller in size compared to controls and other treatment groups 10C). Membrane blebs and degeneration (10 A).

Fig. 9. Plate showing membrane blebs, some cytoplasmic membrane degeneration and MNNCE with 400 mg/kg dose.

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Fig. 10. At 200 mg/kg dose: Plates showing (A) micronucleus, cytoplasmic degeneration and blebs formation. (B) Membrane blebs and degeneration, fewer NCE, very scanty PCE (small-sized).

Fig. 11. (A) At 100 mg/kg: Numerous PCE with minimal cytoplasmic degeneration. (B) MCE with minimal blebs and membrane degeneration.

dose, Hb concentration decreased in a non-dose dependent manner (Pvalue: < 0.05), however the haematocrit, HCT, though appear to reduce but not statistically significant. Red cell indices (MCV, MCH) as well as the thrombocytes count are not significantly different from the control group (more details in Table 7).

3.6.2. Biochemical results following sub chronic administration of Picralima nitida Table 8 shows liver function, cholesterol and glucose results in rats treated with Picralima nitida aqueous seed extract and distilled water. All investigated variables ie, the total protein, albumin, liver

Table 7 Haematological results in rats treated with Picralima nitida aqueous seed extract and distilled water.

WBC LYMPH MID# GRAN# LYMPH% MID% GRAN% HGB RBC HCT MCV MCH PLT

Control

100 mg/kg

200 mg/kg

400 mg/kg

9.76 ± 7.53 5.24 ± 4.75 1.60 ± 1.06 2.92 ± 2.54 47.64 ± 26.80 22.32 ± 17.22 30.04 ± 12.32 15.24 ± 0.39 7.52 ± 1.52 61.72 ± 22.17 69.48 ± 4.76 18.58 ± 0.63 769.80 ± 83.65

14.02 ± 7.05 9.54 ± 4.47 1.08 ± 0.56 3.40 ± 2.06 69.22 ± 3.65 7.82 ± 0.16 22.96 ± 3.61 13.60 ± 1.47* 6.26 ± 2.50 49.00 ± 2.10 67.88 ± 3.90 27.64 ± 18.78 837.40 ± 50.43

24.80 ± 6.58* 15.88 ± 4.00* 1.90 ± 0.42 7.02 ± 2.18* 64.16 ± 1.23 7.74 ± 0.33 28.10 ± 1.30 13.90 ± 0.70 5.95 ± 1.80 40.34 ± 10.66 68.72 ± 3.23 25.02 ± 7.15 813.60 ± 36.77

11.40 ± 10.68 6.64 ± 7.76 1.76 ± 0.21 3.40 ± 2.62 40.44 ± 22.99 27.90 ± 19.76 31.54 ± 7.20 13.64 ± 0.93* 6.04 ± 2.63 49.38 ± 4.17 66.53 ± 4.45 18.85 ± 2.13 804.80 ± 55.09

Values are presented as Mean ± Standard deviation (N=5) *p < 0.05. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. (key: WBC: total white cell count; LYMPH: lymphocytes; MID: monocytes; GRAN: granulocytes; HBG: haemoglobin; HCT: haematocrit; RBC: red blood cell; PLT: platelet; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin).

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Table 8 Liver function, cholesterol and glucose results in rats treated with Picralima nitida aqueous seed extract and distilled water.

ALB GLU TP ALP ALT AST CHOL TRIG HDL LDL CREAT UREA

Control

100 mg/kg

200 mg/kg

400 mg/kg

39.96 ± 0.84 99.74 ± 6.96 62.94 ± 2.41 132.20 ± 21.75 22.80 ± 2.68 16.00 ± 2.55 225.50 ± 13.17 168.60 ± 6.49 65.78 ± 1.57 126.00 ± 14.78 1.44 ± 0.61 37.04 ± 3.11

39.52 ± 1.38 100.00 ± 8.21 60.58 ± 1.77 162.1 ± 6.53* 20.40 ± 2.19 16.00 ± 0.71 219.30 ± 23.13 166.70 ± 5.76 68.24 ± 3.40 117.80 ± 21.46 1.52 ± 0.52 36.14 ± 4.06

40.46 ± 2.48 97.94 ± 7.49 63.64 ± 2.67 145.30 ± 14.87 21.20 ± 3.90 15.60 ± 2.19 214.60 ± 16.84 170.70 ± 24.67 64.96 ± 3.65 115.50 ± 15.38 1.20 ± 0.28 36.94 ± 3.06

39.06 ± 0.68 97.02 ± 4.97 59.88 ± 1.66 147.20 ± 12.96 18.80 ± 1.10 16.20 ± 0.45 203.00 ± 5.09 170.00 ± 5.28 66.88 ± 0.63 102.10 ± 4.78 1.28 ± 0.18 36.14 ± 4.06

Values are presented as Mean ± Standard deviation (N=5) *p < 0.05. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. (Key: ALB: albumin; TP: total protein; AST: aspartate transaminase; ALT: alanine transaminase; ALP: alkaline phosphatase; CHOL: total cholesterol; TG: triglycerides; LDL: low density lipoprotein; HDL: high density lipoprotein; CREAT: creatinine; GLU: glucose).

Table 9 Hormone results in rats treated with Picralima nitida aqueous seed extract and distilled water.

LH FSH PROG E2

Control

100 mg/kg

200 mg/kg

400 mg/kg

0.61 ± 0.01 1.21 ± 0.03 29.70 ± 0.57 17.50 ± 0.71

0.65 ± 0.01 0.76 ± 0.02* 24.62 ± 0.35* 30.00 ± 1.41*

0.54 ± 0.01* 0.64 ± 0.01* 43.19 ± 0.33* 17.00 ± 1.41

0.62 ± 0.01 0.58 ± 0.01* 37.56 ± 0.42* 22.50 ± 0.71*

Values are presented as Mean ± Standard deviation (N=5) *p < 0.05. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. (key: LH: luteinizing hormone; FSH: follicle stimulating hormone; PROG: progesterone; E2: oestradiol).

transaminases (ALT, AST), the lipid profile (Tc, LDLc, HDLc and TG), indices of renal function (creatinine, urea) showed no significant alteration when compared to the control group. However, there is observed significant difference in the alkaline phosphatase (ALP) at the lowest tested dose of 100 mg/kg (p-value: < 0.05). Although differences also observed with the graded doses but not significantly different from the negative control. Table 9 shows the hormone results after administration of the seed extract of Picralima nitida and distilled water. There is a significant dose-dependent reduction in the gonadotrophins (FSH) (p-value < 0.05) observed with graded doses. LH was found to drop significantly at the 200 mg/kg dose. Oestradiol E2 and progesterone showed dosedependent rise with graded doses.

3.6.3. Antioxidants results following sub chronic administration of Picralima nitida Table 10 showed non-significant dose-dependent rise in the SOD and CAT. MDA also showed non-significant increase compared to control, however, there is a significant reduction in the GSH level with dose increase (p-value: < 0.05). There is an observed increase in organ/weight ratio with liver and heart in a dose –dependent manner but statistically significant only at 200 m/kg and 400 mg/kg for the liver. It only become significant at the peak of the tested dose for the heart (p-value < 0.05). Significant increase in weight were also seen in the ovary and pancreas only at the dose of 200 mg/kg. All other tested organs showed no statistically significant alteration when compared to the control group (details in Table 11).

Table 10 Antioxidants results in rats treated with escalating Picralima nitida aqueous seed extract and distilled water.

GSH SOD CAT MDA

Control

100 mg/kg

200 mg/kg

400 mg/kg

52.35 ± 8.05 5.17 ± 1.46 19.73 ± 8.01 3.70 ± 0.71

42.03 ± 4.53 6.93 ± 1.58 21.81 ± 6.45 5.08 ± 1.96

38.91 ± 9.20* 6.05 ± 2.89 32.60 ± 17.08 4.56 ± 1.53

25.37 ± 5.02* 8.01 ± 1.18 34.47 ± 13.22 4.97 ± 1.39

Values are presented as Mean ± Standard deviation (N=5) *p < 0.05. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test. (Key: GSH: reduced glutathione; SOD: superoxide dismutase; MDA: malonyldialdehyde; CAT: catalase).

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Table 11 Organ weights relative to 100 g body weight of rats. Control LIVER KIDNEY OVARY SPLEEN BRAIN LUNGS HEART PANCREAS

4.23 0.88 0.15 0.43 0.96 0.78 0.48 0.18

± ± ± ± ± ± ± ±

0.23 0.13 0.04 0.04 0.70 0.23 0.04 0.09

100 mg/kg

200 mg/kg

400 mg/kg

4.97 0.91 0.16 0.41 1.32 1.01 0.47 0.20

5.34 1.05 0.25 0.46 1.30 1.02 0.54 0.43

5.50 0.99 0.19 0.43 1.31 0.91 0.58 0.28

± ± ± ± ± ± ± ±

0.68 0.15 0.08 0.07 0.21 0.35 0.09 0.04

Values are presented as Mean ± Standard deviation (N=5) *p < 0.05. Statistical level of significance analysed by one-way ANOVA followed by Tukey post hoc pairwise comparison test.

± 0.58* ± 0.09 ± 0.04* ± 0.11 ± 0.12 ± 0.20 ± 0.03 ± 0.14*

± 0.69* ± 0.06 ± 0.02 ± 0.04 ± 0.19 ± 0.20 ± 0.05* ± 0.06

Alkaloids, one of the constituents present in the P. nitida, have been reported to be fetotoxic in several studies (Green et al., 2013; Yakubu and Musa, 2012). Therefore, Picralima nitida alkaloids could be responsible for the teratogenic effects observed in this study. Picralima nitida has demonstrated oestrogenic induction proprieties (Maggi et al., 1993; Otoo et al., 2015). Some studies have shown that oestrogen tends to malformation in offspring when administered to mothers during pregnancy (Hemminki et al., 1999; Hussey, 1974). This could also explain the teratogenic effects of Picralima nitida observed in this study. Although, there were no external malformation observed in the foetuses, the foetal weights were significantly lower (p < 0.05) in the 75 mg/kg group compared to control. This may be due to the hypoglycaemic effect of Picralima nitida. Hypoglycaemic agents are reported to produce low birth weight (Ratnasooriya, 2003). Picralima nitida produces its hypoglycaemic effect by stimulation of influx of glucose into the cells for metabolism (Ajao et al., 2009; Shittu et al., 2010; Teugwa et al., 2013). This hypoglycaemic activity can explain the low birth weight induced by Picralima nitida in rats. This could also explain the significant reduction in weight, crown-rump length and tail length of the 75 mg/kg group when compared to the control group. Animals with low birth weight have been shown to be more susceptible to some diseases in their adulthood. These conditions include diabetes, obesity (Gillman et al., 2003; Rich-Edwards et al., 1999) and low intelligence (Matte et al., 2001). The creatine was significantly raised in 150 mg/kg compare with control group (p < 0.05). This showed that Picralima nitida may be nephrotoxic and this toxicity persisted in the first filial. This was further supported by the anomalies on histology sections for all the samples from 150 mg/kg. They all showed vascular congestions. If no treatment is provided, the renal impairment will inevitably progress to death (Neild, 2017). There was a decreased level of liver's aminotransferases enzymes namely, AST and ALT in all the treated groups in a dose dependent manner. There was a significant reduction in the level of liver AST (150 mg/kg) and ALT (75 and 150 mg/kg) (p < 0.05). The decrease in aminotransferases levels could be considered as an indicator of a protective effect (Olajide et al., 2014), alkaloids have been shown in some studies to be protective of the liver functions (Udoh et al., 2011; Kang, 2013; Zheng et al., 2014). This could be interesting for a new-born whose liver is not yet mature as the extract has protective effect. The only difference statistically significant (p < 0.05) in the parameters of the full blood count was the increase of platelet parameters: platelet count and the plateletcrit when compared with control. Picralima nitida has been shown to inhibit prostaglandins and to have anti-inflammatory actions (Olajide et al., 2014). When prostaglandin's inhibition has been shown to increase platelet parameters. This effect is explained by permanent inhibition of prostaglandin synthetase in platelets, the foetus' organism reacts by increasing the platelets count to compensate the low level of prostaglandin, resulting in thrombocythemia also known as thrombocytosis (Niebyl and Simpson, 2008). In thrombocythemia, there is a much higher risk of clotting or bleeding complications (Appleby and Angelov, 2017; Wille et al., 2017).

4. Discussion 4.1. Phytochemical constituents The phytochemical screening reveals that the main constituent of the aqueous extract of Picralima nitida seed were flavonoids, steroids, alkaloids, saponins, terpenoids, cardiac glycosides and anthraquinones. The results were partly confirmed by Erharuyi et al., they found the same constituents except from anthraquinones. But in addition to our similar constituents, they found tannins and phytophenol.(Erharuyi et al., 2014). The aforementioned study also found that the main constituents were alkaloids. Even though, we found alkaloids, it was much lesser than saponins the main constituent. Surprisingly, we did not find phenols neither tannins in our extract. Menzies et al., also found in 1998 that alkaloids were a major constituent of the seeds extract (Menzies et al., 1998). The present results are not in concordant with those of Erharuyi (2014) and Mabeku (2008), which both found polyphenols in the extract (Erharuyi et al., 2014; Mabeku et al., 2008). The difference in results may be due to the following: the plant: intra-species variations, localization, season of the seeds collection and age (Khattak and Rahman, 2015; Liu et al., 2015; Yang et al., 2014). It could also be explained by the extraction method, none of the earlier studies on the phytochemicals used aqueous extraction (which is the method of extraction in folk medicine). It has been shown that extraction method influences the phytochemical constituent quantity in the extract (Ghasemzadeh et al., 2015; Mahrous et al., 2017; Murugan and Parimelazhagan, 2014). 4.2. Acute toxicity LD50 of 707.107 mg/kg was obtained in this study. Erharuyi et al. (2014) got approximatively the same results (900 mg/kg) in a study where he used methanolic seeds (Erharuyi et al., 2014). However, Koffi et al., in Cote d’Ivoire found different results. The authors found that the LD50 was greater than 3000 mg/kg in mice (Koffi et al., 2014). In a study of acute toxicity conducted in Tanzania, the seeds extract of P. nitida presented moderate toxicity, with a LD50 estimated of 16.3 μg/ml (Moshi et al., 2010) These differences may be due to species variation of Picralima nitida and the specie of the mice. 4.3. Teratogenic study The administration of drugs or agents during pregnancy could produce congenital malformations in the unborn infants (Ujházy et al., 2012). Thus, use of agents during pregnancy should be cautious. These results showed that, there was a 100% resorption in the 300 and 150 mg/kg dose. Embryonic resorption is nowadays defined as prenatal death followed by subsequent degeneration and complete resorption of the conceptus (Jubb et al., 2007). 215

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4.4. Genotoxicity study

physical interaction with the components of the mitotic spindle during cell division or the interaction with proteins which are directly or indirectly involved in chromosome segregation may cause segregation failure. Interaction with the mitochondrial membranes may result in loss of the mitochondrial membrane potential, thus opening of the permeability transition pores leading to ROS production. This is another probable mechanism by which enhanced free radical –induced cellular damage occurs.

The frequency of micronucleus (MN) induction was directly proportional to the exposed doses (Fig. 13); the higher the dose; the greater the chromosomal damage induced by Picralima nitida. There was a dose dependent increase in genomic damage, in the form of micronuclei formation than those exposed to P. nitida. The numerical reduction (Fig. 12) in the PCE observed with increasing dose probably connotes dose –dependent cyto-ablation as shown by increasing membrane blebs, and cyto-degeneration. This suggests that Picralima nitida may have accumulated in a dose-dependent manner, thus affecting the bone marrow polychromatic erythrocyte (PCE) for a longer time. The agent may have interacted directly or indirectly with the genetic material of the bone marrow cells producing primary and/or secondary genotoxicity resulting in acentric chromosome fragments (increased micronucleus, MN) and chromosome loss. Several factors may account for the clastogenic and aneugenic characteristics of Picralima nitida. A direct interaction between Picralima nitida and the genetic material is a possibility. Access to the cell membrane, may have involved utilisation of specific transporter or penetration through the nuclear pore complex. Possible mechanisms of genomic damage may include generation of intracellular reactive oxygen species ROS evidenced by the dose-dependent depletion in the reduced glutathione pool (Table 11), of which the stable and diffusible forms such as hydrogen peroxide or lipid peroxidation intermediates could cause nuclear DNA damage. MDA increases with lipid peroxidation and this is observed in this study though increment was not statistically significant at the test doses. Depletion in GSH may have resulted in the observed increase in the CAT and SOD due to assumed free radical generation (Table 11). Also,

4.5. Subchronic toxicity study 4.5.1. Haematology . In contrast to what Otoo et al. (2015) found, there were insignificant changes in RBC count, haematocrit and red cell indices (MCV, MCH), this suggests that in our study Picralima nitida is unlikely to cause anaemia as pretended by these authors. This is in agreement with the study of Otoo et al. (2015) that failed to show anaemic effect of that P. nitida (Otoo et al., 2015). It could be that the extract has the potential to induce erythropoiesis by stimulating the kidney to release erythropoietin, the main humoral activator of bone marrow red blood cell formation. This may be linked to its androgenic effect as shown by Otoo et al. (2015). This is buttressed by association of anaemia with androgen deprivation (Strum et al., 1997). Administration of the aqueous extract of Picralima nitida on a long-term treatment of sub-chronic illnesses can therefore be done without fear of anaemia or marrow suppression. 4.5.2. Biochemistry In the liver function test, alanine transaminase (ALT), aspartate transaminase (AST), total proteins and albumin did not change significantly, while alkaline phosphates (ALP) was significantly elevated than those in the control group (p-value < 0.05) at the dose of 100 mg/ kg. Increase in serum ALP is associated with liver disease caused by intra or extra hepatic cholestasis and some destruction of the hepatic cell membrane, as well as extra hepatic and intra hepatic bile duct obstruction (Koffuor et al., 2011). However, there is no significant difference when compared to control group at subsequent doses thus showing no consistency and greater effect expected at higher doses. Hence, the observation may have resulted from release from other tissues such as bone, intestine, etc due to an external cause. Simultaneous assay for 5’ nucleotidase, gamma glutamyl transpeptidase would be needed to confirm possibility of hepatic source. However, despite the above observation, the observed increase ALP may be due to cholestatic effect of hepatic venous and sinusoidal congestion as revealed by histological sections of the liver in this study. This is in contrast with the study by Otoo et al. (2015), in which no statistically significant change was observed in the ALP on prolonged use. In this study, there is no statistically significant difference in AST, ALT levels when compared to the control arm even though slight elevation in AST was documented in previous study (Otoo et al., 2015). Notwithstanding, the safety of the liver cannot be assured following prolong exposure to P. nitida aqueous extract due to the observed cholestatic hepatic injury. This is buttressed by Sunmonu et al. (2014), who found that the aqueous seed extract at similar doses was hepatotoxic on acute toxicological evaluation. Lipogram reveals no significant difference in the Total cholesterol, Tc, Triglyceride TAG, LDLc and HDLc when compared to the control group. This was similar to observation by Otoo et al. (2015). Hence, there is no risk of drug-induced dyslipidaemia, a potential cardiovascular risk for atherosclerotic diseases such as angina, myocardial infarction, stroke and peripheral arterial disease. The total cholesterol and LDL levels appear to decline in a dose-dependent manner (though not statistically significant at the tested doses). This may become significant at much higher doses due to possibility of P. nitida extract to cause blockage of an enzyme system for steroidogenesis in the ovary and the capacity of the liver to store cholesterol due to general damage (Ganeshwade, 2012). Hence, there may be a potential for this P. nitida

Fig. 12. Relative frequency of polychromatic erythrocytes in the control groups and the test group.

Fig. 13. Relative magnitude of micronucleus formation in the polychromatic erythrocytes in the test groups (100 mg/kg, 200 mg/kg and 400 mg/kg), the cyclophosphamide and negative control group (distilled water). 216

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to have lipid lowering effect at much higher doses. It is worthy of note that elevation of cholesterol, TAG, and LDL, and a decrease in HDL would increase the risk of cardiovascular disorders (Rame, 2012). Glucose estimation showed no significant difference compared to control group. At the tested doses, and duration of drug administration there is no risk of impaired glucose tolerance and drug –induced diabetes. The kidney function was also not affected as blood urea and creatinine levels did not change significantly with treatments compared to control, similar to observation by Lydia et al, (2015). BUN and creatinine are used to evaluate kidney function in the diagnosis of kidney disease, and to monitor acute or chronic kidney injury or failure. Elevation of these in blood suggests impaired kidney function which could be acute or chronic kidney disease, damage, or failure (Obici et al., 2008). Subchronic exposure to aqueous extract of Picralima nitida may not be associated with nephrotoxicity at the test doses used for this study. This is supported by normal kidney histology. However, kidney necrosis has been documented at 500 mg/kg dose by Mbegbu et al. (2015). The seed extract of Picralima nitida clearly demonstrated oestrogenic activity, similar to previous study by Otoo et al. (2015), It has been shown to possess alkaloids that have opioid binding activity (Duwiejua et al., 2002). Interestingly, more than five alkaloids isolated from Picralima nitida extract, have Mu receptor binding affinity (Maggi et al., 1993). Opioid-like effect of Picralima nitida may regulate gonadotrophin release via the Mu receptors and subsequently modulates testosterone and oestrogen secretion by the gonads (Maggi et al., 1993). Subsequently by influencing FSH and LH release from the anterior pituitary, Picralima nitida aqueous extract may possess estrogenic and androgenic effects as also shown by Otoo et al. (2015). This probably explain the initial aphrodisiac effect of this agent. However, chronic opioid administration tends to decrease serum testosterone and LH concentration (Yilmaz et al., 1999) which may explain why enhanced libido with acute administration become blunted with chronic use. This observation may have resulted in the dose-dependent decrease in the gonadotrophins (p-value < 0.05) in this study at the 200 mg/kg and 400 mg/ kg doses following chronic exposure. However, reduced gonadotrophin is expected to be accompanied by a decrease in oestrogen E2, and of course, testosterone due to loss of tropic effect on the gonads. Instead, E2 actually significantly rises with dose escalation suggesting a probable alternate mechanism in causing oestrogenic stimulation other than the Mu receptor-mediated modulatory effect of the gonadotrophins which are obviously down regulated on chronic use. Alternatively, increase in E2 may cause a negative feedback inhibition on Gonadotrophin release. Hence this agent may have acted via oestrogen receptors on the gonad to produce the observed rise in the E2 through agonist effect. The enhanced oestrogenic activity may be employed in providing contraception in males and females while levering on his relative safety profile in liver (except cholestasis), kidney, low cardiovascular risk and haematological considerations. Picralima nitida may also have a partial agonistic effect in this scenario by blocking testosterone at androgen receptors (Kemppainen et al., 1999). Significant rise in Progesterone level in a dose-dependent manner when compared to the control group may be an indication of induced formation of corpus luteum, an essential biologic index of prior ovulation. This suggest possible role in ovulation induction in an ovulatory infertility, activity which can be employed in disease condition such as Stein-leventhal syndrome (Polycystic ovary syndrome). At the test doses, there is no significant difference in the serum levels of superoxide dismutase (SOD), catalase (CAT) and Malonyldialdehyde (MDA). The only exception is seen in the reduced glutathione GSH concentration indicative of possible free radical generations such as superoxide anion, peroxides, causing increase

scavenging by the antioxidant defence system and depletion of the GSH pool. This might have resulted in non-significant rise in CAT and SOD levels observed at the test doses, and increase MDA production, indicating possible lipid peroxidation. The possibility of increased oxidative stress induced by subchronic/chronic exposure may be explained by the observed increase in MPCE in the micronucleus assay in which oxidative stress may play a significant role in lipid peroxidation, membrane and DNA damage. This is seen at all test doses ranging from 100 mg/kg to 400 mg/kg. The observed GSH reduction, other than those caused by free radical scavenging mechanism, may have also resulted from impaired recycling of the oxidized Glutathione to replenish the GSH pool probably inhibited by one or more of the constituent phytochemicals, causing a more rapid depletion in the GSH. MDA is a known by-products of lipid peroxidation, expected to rise with occurrence of ROS- induced/free radical mediated membrane lipid degradation. This is observed to increase, though not statistically significant at the tested doses of the extract. This may suggest that mechanism of membrane damage observed in this study might be related to lipid peroxidation. Furthermore, it is worth of note that the observed SOD and CAT levels might also be influenced by the potential antioxidant effect of phytoconstituents present in the extract as shown in previous studies (Fakeye et al., 2000; Nwankwo et al., 2017). 4.5.3. Weight The observed increase in the weight of the liver may be due to hepatic venous and sinusoidal congestion. This is reflected by the elevation in the ALP and hepatomegaly resulting from hepatic venous and sinusoidal congestion as revealed by histological section of the liver in this study. Observed weight alteration in the ovary may be related to follicular maturation and ovulation as earlier documented. 5. Conclusion Picralima nitida is a West African ethnomedicinal plant used in the treatment of wide range of acute and chronic diseases due to its wide geographical distribution and broad-spectrum pharmacologic actions. It can be concluded that Picralima nitida seeds extract possess teratogenic effects in rats. These effects are dose related. They include resorptions, low birth weight, renal impairment and platelet count elevation. It is highly genotoxic and also found to cause hepatic damage on chronic use, thereby exposes rodents to oxidative stress and its potential sequel. This may be extrapolated to humans even though similar toxicity may not be demonstrable due to genetic differences and dose considerations. Acknowledgments We wish to express our sincere appreciation to ECOWAS Nnamdi Azikiwe Academic Mobility Scheme (ENAAMS) for the MSc scholarship. We are also grateful to the Afrique One ASPIRE for the training in data analysis and the capacity building training. We equally thank all those who helped during the work, to the Traditional midwife, Tiwalade Fayemi. I declare that all the listed authors have read and approved the submitted manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.03.008. Declarations of interest None.

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Author contribution

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