Toxicological profile, phytochemical analysis and anti-inflammatory properties of leaves of Vitex doniana Sweet. (Verbenaceae).

Background: Natural herbal compounds are a source and alternative for new compounds with anti-inflammatory properties. Therefore, in-depth studies should be carried out to assess their safety and efficacy in order to avoid or prevent toxicological risks of these natural plant-based substances. V. doniana is a species of food plant used for the treatment of inflammatory diseases in Burkina Faso. This study aimed to estimate the anti-inflammatory properties in vitro and in vivo of the ethyl acetate fraction of the leaves of V. doniana.
Methods: To do this, acute toxicity was first evaluated on the animal model. Biological activities (carrageenan test, formalin and xylene induced oedemas) were used to assess the anti-inflammatory power.
Results: Regarding the dietary toxicology of the extracts, visual and clinical signs showed that the extracts are not at the end of the 15-day (acute toxicity) and 45-day (subacute toxicity) tests. In terms of anti-inflammatory activity in vivo, it was observed that the bioactive fraction (50, 100 and 200 mg/kg.b.w) had effective neurogenic anti-inflammatory properties. All of these effects observed were all dose-dependent.
Conclusion: The results of this present research have proven that V. donania extracts can be used as anti-inflammatories.

Introduction

Since time immemorial, plants have been used by human beings for food and medicinal purposes [1]. Many studies have shown a number of benefits of using medicinal plants on the health of populations and their ability to prevent certain diseases [2]. In Africa and Asia, many people depend only on phytomedicine because of its effectiveness [3]. Currently, much research has shown that medicinal plants possess a great number various biochemical compounds and biological properties, including anti-inflammatory activity [4]. Indeed, inflammation is a protective reaction of the body to certain tissue damage or damage to the body, which is often caused by certain agents, harmful substances, live or dead pathogens [5]. For the treatment of inflammatory diseases, the drugs usually used are nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids [6], but it should be noted that both can cause serious adverse side effects to the patient. Therefore, the development of new safe anti-inflammatory compounds would be necessary and hence the usefulness of medicinal plants. Plants used in traditional medicine to treat inflammatory conditions are believed to have biologically active compounds [7], as the synthetic molecules used are very expensive for often poor patients. Due to the pharmacological importance and low cost of natural substances of plant origin, great attention would be required for the discovery of new plant-based bioactive molecules with anti-inflammatory properties [1]. Vitex doniana (V. doniana) is a perennial forest plant widely distributed in tropical West Africa. In ethnomedicine, the leaves of V. doniana are used in the treatment of stomach and rheumatic pain, inflammatory disorders, diarrhea and dysentery [8]. According to recent literature, this Verbenaceae is an alicament for the forest and dry savannah regions of West and Central Africa. Africa's dependence on medicinal plants as food and medicine is well documented in the literature [9]. The leaves and fruits of V. doniana, are widely used in herbal medicine in many parts of Africa. Its wood is intended for light construction work and gum is considered an antidote against snakebites and arrow poison, leaves and fruits are also used in the treatment of diarrhea, aromatic bark serves as a tonic [10]. V. doniana Sweet., is also thought to be used in treatments for jaundice, anemia, dysentery, mental illness, rheumatism, gastrointestinal disorders and urinary conditions, etc [11]. In addition, the bark of the plant's stem has antibacterial properties [12]. In Burkina Faso, V. doniana Sweet., is a food plant used to treat inflammatory diseases and certains cancers [8]. Some studies show that phytochemical analysis of the different parts of the plant extract revealed the presence of certain secondary metabolites such as: saponin, tannins, phenols, cardiac glycosides, flavonoids, sterols and triterpenes as well as a high concentration of sodium, potassium, calcium, iron, phosphorus and sulfur [10, 11, 12, 13]. In order to find a new remedy for inflammation, this work aims to determine the scientific basis for the traditional use of V. doniana. Despite the intensive use of this plant in traditional health care, the literature provides little information about their toxicity, so the toxic effect of its use is unknown. In doing so, our aim is to study the anti-inflammatory properties of the leaves fractions of V. doniana Sweet., in model animals.

Materials and methods

Collection of plant materials

The plant material whose leaves of V. doniana were collected in August 2019 in the Hauts Bassins region, more precisely in Faramana (12°02′53″ N and 4°40′02″W) located 120 km from Bobo-Dioulasso, Burkina Faso's economic capital. The identification and authentication were carried out thanks to the services of Dr. Lassina TRAORE, botanist at the Laboratory of Biology and Ecology of the University of Ouagadougou. For this purpose, a reference specimen bearing the number 20/08/2019/PRB has been deposited in the herbarium of the Life and Earth Sciences Unit of the Joseph KI ZERBO University.

Fractionation of extracts

A hydro-acetone extract (HAE) with (80:20) under agitation for 48h was performed. After evaporation at 45 °C, a bio-guided fractionation was performed to obtain fractions: n-hexane (n-HF), dichloromethane (DCMF), ethyl acetate (EAF) and n-butanol (n-BF) fraction which were respectively concentrated, freeze-dried and the extraction yield was calculated [14].R= (m/M) × 100Where, R = extraction yields; m = mass after extraction; M = mass before extraction.

Experimental animals

This study was conducted with respect for animal welfare, in accordance with the ethical principles and guidelines of the Institut Burkinabé de Recherche en Sciences de la Santé (IRSS-Burkina Faso). Therefore, international guidelines for the care and handling of laboratory animals were strictly adhered to [15, 16] and an approval number of the experimental protocol was associated: CE-IRSS/2021-08. All experiments were preceded by a 15-hour fasting period and then weighed.

Acute toxicity study in albinos Wistar (A.W.) rats

A.W. rats of both sexes and weighing (170–180 g) used for this study. Five batches of four animals (two females and two males) per batch were formed. Thus, three test batches (group 2, 3 and 4) and one control batch (group 1) were set up. Before the administration of the extract or distilled water with 10% DMSO by the intraperitoneal route, an adaptation period was observed. The control group received 10% DMSO distilled water and the test groups received increasing doses of HAE of 600 mg/kg.b.w; 1000 mg/kg.b.w; 2000 mg/kg.b.w and 5000 mg/kg.b.w respectively followed by immediate observation of general behaviour. Morbidity and mortality were then recorded for 14 days. Toxicity was assessed and expressed as a median lethal dose value of 50 or LD50 [15]. On the 15th day, all the remaining animals were anesthetized and sacrificed. A macroscopic examination of the internal vital organs such as the lungs, spleen, liver, heart, kidneys and stomach was performed followed by mass sampling. The relative organ weight ratio (W) was calculated according to the following formula:W (%) = (A/B) × 100Where, W Relative organ weight ratio, A = Weight organ, B = Body weight.

Sub-chronic toxicity study

The protocol according to the guidelines of the Organization of Economic and Community Communities (OECD) number 407 [17] was used for this experimental research, but with slight modifications. Twenty-four A.W. rats of both sexes weighing (170–180 g) were divided into 4 groups of 6 rats per group, i.e. three males and three females. The test groups received the HAE of different doses via a gastric cannula daily: 50 mg/kg, 100 mg/kg and 200 mg/kg body weight, while the control rats were given distilled water at 10% DMSO for 49 days. At the end of the experiment, the rats were anesthetized under chloroform vapor samples and blood sampling was performed by cardiac puncture in vials of ordinary samples and containing EDTA for biochemical and hematological analyses respectively. The organs (liver, kidneys, heart, lungs, spleen, liver and stomach) were removed and weighed.

Phytochemical analysis

Evaluation of total alkaloid contents

Total alkaloid content of fraction extracts was evaluated by [18] with some modifications. Thus, 1 ml of 1 mg/ml of the solution of each fraction of extract one summer mixed with 5 ml of green bromocresol solution and 5 ml of phosphate buffer of 4.7 pH. The mixture was vortexed pendant 3–5 min with 1; 2; 3 and 4 ml of chloroform. Further, the whole mixture was collected in a 10 ml volumetric bottle. A calibration curve of atropine used as standard (20–100 mg/ml) was designed. The absorbance for standard solutions and test solutions was determined on the reagent blank at 470 nm with an UV/Visible spectrophotometer. The experiments were carried out in triplicate. The total content of alkaloids was expressed as mg Atropine Equivalent per 100 mg of dry fraction extract (mg AE/100 mg). Reagent blank was prepared in the same manner but without fraction extracts.

Evaluation of total phenolic contents

For this evaluation, the FCR (Folin-Ciocalteu) method was used to evaluate the total phenolic content of our fractionated extracts against the gallic acid used as standard [19] with minor modifications. The aliquot of 125μl of solution of each fraction extract in methanol (10 mg/mL) was used with 1.5 mL of sodium carbonate solution (7.5%). To the reaction mixture, 1.25 mL of FCR reagent (0.2 N), was added and vortexed for 2–3 min followed by incubation at room temperature for 60 min in the dark. After this incubation period, the absorbencies were measured at 760 nm against a blank on a UV/visible light spectrophotometer. The experiments were carried out in triplicate. The contents were expressed as mg gallic acid equivalent (standard) per 100 mg dry fractionated extract (mg GAE/100 mg).

Evaluation of total flavonoid contents

We had used by [20] method with some modifications. The aluminum chloride test was used to measure the total flavonoid content of the fractions. Then, a 30 min incubation at room temperature of the reaction mixture of 0.5 ml of the methanoic fraction (0.1 mg/mL) and 1.5 ml of AlCl3 (2%) is performed. The optical densities were read against a blank at 415 nm using a spectrophotometer. The different contents expressed in mg of quercetin equivalent per 100 mg of dry fractionated extract were obtained using a standard curve of quercetin (mg QE/100 mg).

In vivo anti-inflammatory potential of bioactive fraction

Assessment of carrageenan-induced paw oedema

For this method, carrageenan was used to induce oedema in the right hind foot of the rat according to [21]. Thus, five groups of four A.W. rats of both sexes were formed and treated by gavage. Two control groups (positive and negative batches) received prednisolone (10 mg/kg.b.w) and tween-80 (1%; 10 ml/kg.b.w) respectively, whereas the test batches received increasing concentrations of EAF (50 mg/kg.b.w, 100 mg/kg.b.w, and 200 mg/kg.b.w). Next, edema formation was initiated by subcutaneous injection of 90 μL of carrageenan (1% in 0.9% NaCl) into the right hind foot. For this purpose, the inhibition of edema formation is estimated by the foot area before carrageenan injection and after 1 h; 2 h; 3 h and 4 h after injection. The formula below makes it possible to estimate the inhibition of edema induced by carrageenan:(PWhere, (PI%) = Percentage of inhibition of edema, A = mean for the control group, B = mean for the test group.

Assessment of formalin-induced paw oedema

The method was carried out according to [22] with slight modifications. For this, five groups of four A.W. rats and weight (170–180 g) of both sexes were also formed and treated by gavage. Also, two control groups (positive and negative batches) received prednisolone (10 mg/kg.b.w) and tween-80 (1%; 10 ml/kg.b.w) respectively, whereas the test batches received increasing concentrations of EAF (50 mg/kg.b.w, 100 mg/kg.b.w, and 200 mg/kg.b.w). After a respective observation of 30 min (for control batches) and 1 h (for test batches), a subcutaneous injection of the mouse paw was performed with 45 μL of formalin (3%). Inhibition of edema formation is estimated by the surface area of the foot before formalin injection and after 1h; 2h; 3h and 4h after injection. The formula below is used to estimate the inhibition of edema induced by formalin:(PWhere, (PI %) = Percentage of inhibition of edema, C = paw edema of control at different time interval, D = paw edema of treatment at different time interval.

Assessment of xylene-induced ear oedema

For this method, xylene-induced ear edema was used according to [23] with slight modifications. For this purpose, five groups of four A.W. rats of both sexes were orally administered the different fractions of EAF (50, 100 and 200 mg/kg.b.w.) and the negative (Tween 80: 1%; 10 mL/kg.b.w.) and positive controls (prednisolone: 10 mg/kg.b.w.) respectively. After half an hour of observation, 25 μL of xylene was applied to the inner surface of the right ear of the rats using a syringe for the formation of an oedema. 30 min after the induction of oedema, both ears were excised and weighed and the sizes of the oedema measured. The percentage of oedema inhibition was assessed taking into account the differences in ear weight of negative and positive controls according to the above formula.

Statistical analysis

The analyses of the different tests were expressed as means ± standard deviation (SD) with (n = 3). Statistical analyses were performed using two-way or one-way analysis of variance (ANOVA) followed by Bonferroni test and Tukey test to determine significant differences between treatment groups using XLSAT version 2016 and graph pad prism version 8.4.3. The significance level was considered at P ≤ 0.05.

Results

Extraction yields

The extraction yields of HAE and the different fractions extracted: n-HF, DCMF, EAF and n-BF were respectively evaluated. HAE has the highest extraction yield followed by n-EAF. The different yields are arranged respectively: 21.48% (HAE); 12.42% (DCMF); 16.64% (EAF); 18.23% (n-BF) and 6.36% (n-HF). The EAF fraction (bioactive fraction) was used for biological activities through in vivo anti-inflammatory tests.

Acute toxicity study

The HAE of V. donania leaves produced no lethality or visible signs of toxicity in animals up to the oral dose of 5000 mg/kg body weight, 24 h after treatment. Further monitoring for fourteen days has not yet revealed any mortality or visible toxic signs. Therefore, the LD50 was greater than 5000 mg/kg body weight. On day 15, all the remaining rats were anesthetized and sacrificed. The weights of the internal vital organs namely: lungs, spleen, liver, heart, kidneys and stomach were evaluated. The results are presented by Table 1.

Table 1

Effect of the HAE of leaves from V. donania on albino rats’ relative vital organs weight after 14 observation days.

Parameters	Doses (mg/kg.b.w)
	Control	600	1000	2000	5000
Liver	4.10 ± 1.33	4. 14 ± 1.02	4.13 ± 1.64	4.12 ± 1.71	4.12 ± 1.33
Kidneys	0.81 ± 1.71	0.83 ± 1.22	0.83 ± 1.33	0.82 ± 1.64	0.81 ± 1.45
Heart	0.43 ± 1.45	0.45 ± 1.34	0.44 ± 1.64	0.44 ± 1.71	0.42 ± 1.22
Lungs	2.38 ± 1.01	2.40 ± 1.71	2.39 ± 1.10	2.39 ± 1.22	2.37 ± 1.01
Spleen	1.23 ± 1.64	1.26 ± 1.45	1.24 ± 1.01	1.25 ± 1.33	1.24 ± 1.64
Stomach	5.46 ± 1.54	5.48 ± 1.64	5.47 ± 1.20	5.48 ± 1.64	5.47 ± 1.10

No statistical difference (P > 0.05) compared with control group.

Sub-chronic toxicity study in rats

Body weight

No significant difference in body weight gain between the control and test groups was observed during the first day of treatment (p > 0.05). However, during seven weeks, a significant decrease in body weight was observed between the test and control groups (p < 0.01). The results are presented by Table 2.

Table 2

Effect of the HAE of leaves of V. doniana on rats’ body weight.

HAE of leaves from V. doniana
Weeks	Control	50 mg/kg.b.w	100 mg/kg.b.w	200 mg/kg.b.w
1st day	171.33 ± 6.98	173.50 ± 5.24	173.33 ± 6.06	175.50 ± 4.64
1rst week	177.5 ± 7.56	179.50 ± 5.54	179.66 ± 7.84	180.00 ± 4.20
2 nd week	184.67 ± 6.65	183.33 ± 5.32	185.33 ± 7.39	186.5 ± 4.09
3rd week	190.80 ± 3.43	189.83 ± 4.02	188.50 ± 3.21	192.00 ± 2.23
4th week	195.33 ± 2.16	196.17 ± 1.47	195.00 ± 1.51	198.33 ± 2.23
5th week	200.67 ± 4.20	201.50 ± 7.56	202.66 ± 5.54	204.00 ± 5.32
6th week	204.17 ± 4.64	206. 00 ± 6.06	205.50 ± 1.47	208.33 ± 7.39
7th week	210.00 ± 5.32	212.50 ± 4.02	209.33 ± 3.21	213.00 ± 7.56

Values are means ± SEM (n = 6).

No statistical difference (P > 0.05) compared with control group.

Effects on relative organ weight of rats

There was no significant difference in the rats’ relative vital organs weights between test and control group (Table 3).

Table 3

Effect of the HAE of leaves from V. doniana on organs relative weight.

HAE of leaves from V. doniana
Organs	Control	50 mg/kg.b.w	100 mg/kg.b.w	200 mg/kg.b.w
Heart	0.422 ± 0.021	0.419 ± 0.024	0.416 ± 0.014	0.410 ± 0.024
Liver	3.282 ± 0.139	3.288 ± 0.162	3.284 ± 0.223	3.281 ± 0.125
Lungs	0.57 ± 0.54	0.58 ± 0.82	0.57 ± 1.045	0.58 ± 1.54
Spleen	0.288 ± 0.03	0.262 ± 0.043	0.274 ± 0.050	0.278 ± 0.018
Left kidney	0.653 ± 0.03	0.652 ± 0.05	0.646 ± 0.044	0.652 ± 0.04
Right kidney	0.658 ± 0.021	0.654 ± 0.027	0.645 ± 0.028	0.650 ± 0.028
Stomach	4.63 ± 0.30	4.51 ± 0.23	4.64 ± 0.34	4.68 ± 0.22

Values are means ± SEM (n = 6).

No statistical difference (P > 0.05) compared with control group.

Effects on hematological indices

Table 4 represents the effects of HAE on hematological indices. There were insignificant alterations in hematological parameters in rats between test and exposed to all control groups. No statistical difference (P > 0.05) compared with control group.

Table 4

Effect of the HAE of leaves from V. doniana on hematological parameters.

Parameters	Doses
	Control	50 mg/kg.b.w	100 mg/kg.b.w	200 mg/kg.b.w
WBC (x109/L)	11.93 ± 0.03	11.38 ± 1.215	11.51 ± 1.43	11.56 ± 1.65
RBC (x1012/L)	8.89 ± 0.86	8.64 ± 0.37	8.76 ± 1.19	8.79 ± 1.20
HGB (g/dL)	16.79 ± 0.79	16.48 ± 1.05	16.64 ± 1.21	16.71 ± 0.52
PCV (%)	53.46 ± 0.51	53.18 ± 1.61	53.41 ± 0.54	53.57 ± 1.01
MCV (fl)	36.32 ± 1.20	36.16 ± 0.71	36.02 ± 0.58	36.12 ± 0.89
MCHC(g/dL)	32.26 ± 0.21	31.98 ± 1.01	32.22 ± 1.38	32.16 ± 1.46
PLT (103/L)	953.11 ± 1.91	948.70 ± 0.88	950.61 ± 1.91	952.88 ± 2.01
Basophil (%)	0.15 ± 0.38	0.13 ± 0.24	0.14 ± 1.15	0.16 ± 0.80
Neutrophils (%)	25.96 ± 1.25	25.28 ± 1.03	25.15 ± 1.49	25.68 ± 1.84
Lymphocytes (%)	71.90 ± 1.39	71.45 ± 1.07	71.34 ± 0.86	71.94 ± 0.88
Monocytes (%)	1.36 ± 1.07	1.19 ± 0.58	1.27 ± 0.21	1.41 ± 1.08
Eosinophil (%)	1.43 ± 0.48	1.42 ± 0.90	1.63 ± 1.07	1.76 ± 0.72

Values are means ± SEM (n = 6). RBC: red blood cells; HGB: hemoglobin; PCV: packed cell volume; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; WBC: white blood cells; PLT: platelets.

No statistical difference (P > 0.05) compared with control group.

Effects on biochemical parameters

The effects of HAE assessment on biochemical parameters in rats are shown in Table 5. The extract produced non-significant effects on the plasma levels of liver enzymes (ALT, AST and ALP), total proteins, albumin and glucose of treated rats compared to the control group.

Table 5

Effect of the HAE of leaves from V. doniana on biochemical parameters of rats.

Parameters	Doses
	Control	50 mg/kg.b.w	100 mg/kg.b.w	200 mg/kg.b.w
Glucose (mmol/L)	6.52 ± 1.41	5.18 ± 1.33	4.96 ± 1.04	4.67 ± 1.54
AST (IU/L)	128.86 ± 1.04	129.64 ± 1.64	130.93 ± 1.38	131.16 ± 1.54
ALT (IU/L)	50.86 ± 0.75	51.13 ± 0.21	51.44 ± 0.81	51.52 ± 1.21
ALP (IU/L)	151.79 ± 1.75	151.25 ± 1.84	151.71 ± 1.15	151.51 ± 1.39
TP (g/dL)	6.32 ± 1.41	6.17 ± 1.54	6.37 ± 1.38	6.53 ± 1.54
Alb (g/dL)	2.63 ± 1.71	2.77 ± 1.33	2.68 ± 0.54	2.82 ± 1.01
Total bilirubin (mg/dL)	0.36 ± 1.04	0.38 ± 1.75	0.37 ± 1.21	0.420 ± 1.54
Direct bilirubin (mg/dL)	0.27 ± 0.75	0.26 ± 1.64	0.28 ± 1.38	0.30 ± 1.04
Total cholesterol (mmol/L)	1.82 ± 0.33	1.80 ± 1.21	1.87 ± 1.39	1.88 ± 1.54
HDL (mmol/L)	0.38 ± 1.41	0.37 ± 0.54	0.36 ± 1.33	0.35 ± 1.21
LDL (mmol/L)	1.17 ± 1.54	1.16 ± 1.33	1.14 ± 1.71	1.12 ± 1.01
Triglycerides (mmol/L)	0.65 ± 1.41	0.66 ± 1.04	0.68 ± 1.33	0.70 ± 1.15

Values are means ± SEM (n = 6). AST, Aspartate aminotransferase, ALT: alanine aminotransferase; ALP: alkaline phosphatase; TP: total protein; Alb, albumint; HDL: high density lipoprotein; LDL: low density lipoproteins; VLDL: very low-density lipoprotein.

No statistical difference (P > 0.05) compared with control group.

Effects on kidneys function parameters

Analysis of renal function parameters in treated rats showed a non-significant decrease in the different doses used compared to those in the control group. In other words, there was no statistical difference (P > 0.05) compared to the control group (Figure 1).

Figure 1

: Effects of HAE of leaves from V. doniana on rats kidney indices. No statistical difference (P > 0.05) compared with control group.

Total phytochemical contents

Total alkaloid contents (TAC)

The total content of alkaloids was expressed as atropine equivalent (mg AE/100 mg of dried fraction extract). As a result, the n-BF extract had a higher TAC content (12.56 ± 1.71 mg AE/100 mg) compared to the extract of the other solvent fraction, while the n-HF had the lowest total alkaloid content (3.22 ± 1.45 mg AE/100 mg). According to Figure 2, there was statistically significant difference between groups as demonstrated by one-way ANOVA, values are Mean ± SD (n = 3) (p < 0.05) for our different fraction extracts.

Figure 2

Phytochemical contents of fractions of leaves from V. doniana. mg GAE/100mg fraction: mg equivalent Gallic acid for 100mg dried fraction; mgQE/100mg fraction: mg equivalent Quercetin for 100mg dried fraction; mg AE/100 mg fraction: mg equivalent Atropine for 100 mg dried fraction. Values are Mean ± SD (n = 3). Different in the same column indicate significant difference (P < 0.05) for our different extracts.

Evaluation of total phenolic and flavonoid contents

The total phenolic profile of the different fractions (n-HF; DCMF; EAF and n-BF) gave respectively: 10.12 ± 1.01; 39.32 ± 1.45; 51.22 ± 1.54; 45.46 ± 2.00 mg GAE/100 mg. Total flavonoid content 4.44 ± 1.54; 13.18 ± 1.01; 18.73 ± 1.22 and 12.53 ± 1.71 mg QE/100 mg were obtained with different fraction extracts (n-HF; DCMF; EAF and n-BF), respectively. The results of total phenolics and flavonoids contents are presented by Figure 2. It was indicated statistically significant difference between groups as demonstrated by one-way ANOVA, values are Mean ± SD (n = 3) (p < 0.05) for the different fraction extracts.

In the current study, there is generally a dose-dependent relationship for the different doses used. Prednisolone, a positive control, slightly inhibited paw oedema and started to inhibit paw oedema from the 3rd hour by 71.22% and the 4th hour by 81.36% significantly compared to the test groups. The bioactive fraction at 200 mg/kg.b.w showed an excellent effect in this method which inhibited paw oedema from 69.71% to 75.67% at 200 mg/kg.b.w after its administration up to hour 3 and 4 (Figure 3). No significant difference (P > 0.05) when comparing this doses (200 mg/kg.b.w) with the control.

Figure 3

Anti-inflammatory activity of bioactive fraction of leaves from V. doniana (Verbenaceae) by using carrageenan-induced paw edema test.

In the formalin-induced paw oedema test, the bioactive fraction also showed inhibition of paw oedema from hour 3 to hour 4 which ranged from 61.28% to 73.44% (200 mg/kg.b.w), after its administration up to hour 3 and 4 at 200 mg/kg.b.w compared to prednisolone (positive control). The results of the test groups were significant at ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 and no statistical difference (P > 0.05) when compared to the Prednisolone group (Figure 4).

Figure 4

: Anti-inflammatory activity of bioactive fraction of leaves from V. doniana (Verbenaceae) by using formalin-induced paw edema test.

The potential of the bioactive fractions to inhibit xylene-induced ear oedema was significant compared to the negative control at ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. Indeed, the bioactive fraction reduced oedema by 49.40% at low dose and 67.04% at high dose compared to the negative control group showed a strong increase in ear oedema. On the other hand, the positive control, prednisolone, reduced oedema by 75.48% (Table 6).

Table 6

Anti-inflammatory activity of EAF of V. doniana (Verbenaceae) leaves using xylene to induce edema.

Tests		Mean weight difference of the ears	Percentage of inhibition (%)
Negative control (Tween-80)	10 mL/kg	38.62	-
Positive control (Prednisolone)	10	9.47∗∗∗	75.48
Bioactive fraction	50	19.54∗∗∗	49.40∗∗∗
	100	16.18∗∗∗	58.10∗∗
	200	12.73∗∗∗	67.04∗

∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.

Discussion

The use of plants in traditional medicine around the world and in different societies dates back to ancient times. The use of traditional medicine is still relevant because of its medicinal value, the costs of often inexpensive drugs, cultural beliefs and especially the toxicity and side effects of synthetic molecules [24]. However, the issue of toxicity of some plants widely used as alicaments remains a concern for many researchers [25]. For this acute toxicity study, the results indicated that V. doniana extract shows no signs of toxicity. During the 14-day acute toxicity assessment observation period, there were also no signs of toxicity. . Recent studies have shown that when a substance has a 50% lethal dose (LD50) of less than 5 mg/kg. bw, the substance is said to be very toxic, when the substance has a 50% lethal dose of between 5 mg/kg. bw and 5000 mg/kg. bw, the substance is said to be moderately toxic, and finally, if its 50% lethal dose is greater than 5000 mg/kg. bw, the substance is said to be non-toxic to health [26]. In doing so, one could then say that V. doniana extract is non-toxic. Adjei et al., 2021 has been shown that extract ofV. doniana fruits have significant antioxidant and anti-inflammatory properties. The fruit extract of V. doniana was non-toxic under laboratory conditions [10]. In addition, about vital organs, after an observation of 15 days, there was no significant variation in weight between the control and test groups and (P > 0.05). This could be explained by the fact that the leaves of the plant are widely used for health care. Regarding the weight loss of animals in toxicology, according to [27], weight loss is a sure indicator of toxic effect after exposure to a toxic and a decrease in body weight could also be considered the effect of the toxic product [28]. For our study, it was found that the change in body weight of the test group animals compared to those in the control group could not show a significant difference after 45 days of experience (P ≥ 0.5). The relative weight of organs such as the lungs, spleen, liver, heart, kidneys and stomach did not also show a significant difference between the test and control groups (P ≥ 0.5). The extract administered in different doses namely: 50; 100 and 200 mg/kg could not induce adverse effects on the vital organs studied. For hematological parameters in toxicology, blood cells are parameters in the first rank, because they are the first cells to be exposed to the toxic during blood circulation. In doing so, the action of blood cells in toxicology gives a good appreciation of a toxic substance [29]. In general, in clinical toxicology, there has always been a decrease in the level of red blood cells in rats. This could be explained by the phenomenon of hemolysis probably due to the stress caused by the collection of blood from the animal. The blood samples were taken by cardiac puncture, which could explain the stress in the rats. In this work, the haematological indices of the different treatments did not show any significant difference compared to the control lot. HAE administered at different doses (50; 100 and 200 mg/kg) showed no signs of toxicity [30]. Regarding biochemical parameters, it should be noted that the liver is the essential organ involved in the metabolism of any foreign substance and because of this, it is very exposed to toxic effects. Among the biochemical parameters assessed, AST, ALT and ALP are the essential and fundamental markers of liver function [31]. The evaluation of these parameters is quite important in clinical toxicology since they are considered to be essential biomarkers of the level of toxicity [32]. It was described hepatocurative potential of V. doniana root bark, stem bark and leaves extracts against CCl4-induced liver damage in rats [33]. Some results show that AST and ALT are essential enzymes and their increase in serum would reflect liver dysfunction following various tissue alterations. ALP, an essential enzyme in liver function and increased serum levels may be due to biliary dysfunction [34]. In this study, there were no significant differences in these biomarkers between the treated groups compared to the control group (p < 0.05). Moreover, Kidney function as well as liver function is of metabolism par excellence and which ends with the purification and elimination of waste [35]. Kidney function is generally based on the dose of urea, creatinine and certain electrolytes considered biomarkers of kidney function. The kidneys are primarily involved in the excretion mechanism of metabolites and drugs. Therefore, the kidneys are highly exposed to the toxic risks of compounds found in drugs. For an analysis of the capacity of the kidney function, the primary measuring instruments are the determination of urine protein, urea and creatinine levels in serum and urine [36]. For the present, the values of urea and creatinine were not significant. This shows that the extract has no effect on the functioning of the kidneys. Our results show that the extract shows no signs of toxicity to kidney functions. The same applies to lipid biochemical parameters. The present study shows that the hydroacetonic extract does not negatively affect Lipid biochemical parameters including low-density lipoproteins (LDL) and mecanism of catabolic of LDL-cholesterol [36]. Repeated administration of the extract of V. doniana had no effect on the lipid profile of the animals. According to [37], excess of low-density lipoproteins (LDL) and lack of high density lipoproteins (HDL) are risk factors for atherosclerosis.

Inflammation is a complex biochemical mechanism generally regulated by the disruption of tissue homeostasis [38]. In effect, three methods have been used to assess anti-inflammatory activity. Among these mechanisms, the use of a carrageenan-based inflammatory model suggests that the interval necessary for the development of the inflammatory reaction leads to an up-regulation of cyclooxygenase (COX-2) either in the periphery or in the spinal cord [39]. In this study, the in vivo anti-inflammatory activity assessed by the carrageenan-induced paw oedema test, the EAF fraction (200 mg/kg.b.w) showed significant inhibition of paw oedema and compared to the positive control from hour 3 onwards. This anti-inflammatory mechanism of the bioactive fraction could be explained by the up-regulation of COX-2 and the inhibition of prostaglandin and nitric oxide synthesis [40]. These results corroborate previous findings of the good anti-inflammatory activity of the ethyl acetate fraction of V. doniana [8]. In this study, a formalin test was also performed to assess whether the action of V. doniana EAF is central or peripheral. Indeed, intraplantar administration of formalin generates biphasic nociceptive responses with two distinct mechanisms: an early phase in which the pain is neurogenic and a late phase reflecting inflammatory feedback through the action of mediators or stimulation of dorsal horn neurons [41]. The previous report indicated that neuropeptides, namely substance P and bradykinin, are released throughout the neurogenic phase, while mediators, including prostaglandins (PGs), serotonin, histamine and bradykinin, are associated with the inflammatory phase [42]. In this experiment, formalin-induced nociception in mice was significantly reduced in the late phases (Figure 4), i.e. 3–4 h after formalin injection. Indeed, only the bioactive fraction (200 mg/kg) showed an interesting anti-inflammatory effect. This proves that the fraction has a peripherally acting modulating effect as it only suppresses nociceptive mediators in the late phase [43].

Regarding the anti-inflammatory method by the xylene-induced ear oedema test, prednisolone (positive control) showed strong inhibitory activity on ear oedema formation and was significant compared to the negative control group which showed an increase in ear oedema. This could be explained by the fact that prednisolone acts as a steroidal agent that inhibits the production of phospholipase A2 [44]. Therefore, it is clear that this would inevitably reduce xylene-induced neurogenic inflammation. It also found that the bioactive fraction at the high dose (200 mg/kg) was more effective at reducing ear edema than the positive control group. Regarding the fraction showed a very slight effect and there is also the notion of a dose-dependent effect of concentration as an anti-inflammatory agent compared to control. Bioactive plant compounds (alkaloids and flavonoids) are thought to possess anti-inflammatory properties according to several previous studies [45]. Therefore, V. doniana, which contains good levels of these phytoconstituents, could be exploited as an excellent anti-inflammatory alternative. Several studies have reported that certain phenols and flavonoid compounds may exhibit anti-inflammatory activity under various inflammatory conditions [46]; the results therefore suggest that phytoconstituents of the experimental fraction can be responsible for the anti-inflammatory activity [47] of V. doniana. Indeed, some studies shown that phytochemical analysis of the different parts of the plant extract revealed the presence of saponin, tannins, phenols, cardiac glycosides, flavonoids, sterols and triterpenes [48]. Thus, ethyl acetate fractionation (EAF) contributes to an optimisation of the chemical profile (phenolic, flavonoids), antioxidants and correlates positively with anti-inflammatory activity [8]. Such phytonutrient aggregate would be an excellent nutraceutical against inflammation.

Conclusion

These results support the traditional use of the plant for painful inflammatory conditions, while encouraging its use in the development of new non-toxic anti-inflammatory agents. Therefore, further research is needed to characterise its active compounds that will surely be responsible for its anti-inflammatory properties. But already, V. doniana could be a very good candidate for the treatment of inflammatory pathologies.

Declarations

Author contribution statement

Poussian Raymond Barry, Abdoudramane Sanou and Kiessoun Konaté: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Oksana Sytar: Analyzed and interpreted the data; Wrote the paper.

Raissa RR Aworet-Samseny and Mamoudou Hama Dicko: Contributed reagents, materials, analysis tools or data.

Funding statement

This work was supported by the West African Biotechnology Network (RABIOTECH, ISP/IPICS project n°172 600 000) and financed the mobilities for the collection of samples, purchase of chemicals, and the acquisition of lab equipments.

Data availability statement

Data will be made available on request.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.