Anti-inflammatory, analgesic and antipyretic properties of ethanolic extracts of three plants of Benin pharmacopoeia: Euphorbia hirta, Citrus aurantifolia and Heterotis rotundifolia CURRENT STATUS: POSTED

Objective Euphorbia hirta , Citrus aurantifolia and Heterotis rotundifolia are commonly used in Benin in the treatment of infectious diseases. The aim of this study is to evaluate the anti-inflammatory, analgesic and antipyretic properties of ethanolic extracts of these plants. The study was performed on wistar rats with 200 mg/kg b.w.Results Administration of these extracts prevents edema inflammatory induced by 2% formalin in rat paw. Inhibition percentages of edema vary between 23.67% and 86.76% for the three extracts. These extracts have similar anti-inflammatory activity (p> 0.05) to that of diclofenac at 50 mg/kg. Analgesic activity show that these extracts inhibit very significantly (p<0.001) the pain induced by 3% acetic acid and the highest inhibition percentage is 60.34% (Citrus aurantifolia ). This percentage is like to that of acetylsalicylic acid (67.35%) administered at the same dose. Likewise, these extracts attenuate significantly (p <0.05) the thermal pain induced by tail immersion of each rat in hot water at 50°C. Indeed, these extracts, reduces very significantly (p <0.001) pyrexia induced by 20% beer yeast suspension in rats and they have similar effect (p> 0.05) to that of acetylsalicylic acid at the fourth hour. These results prove, some traditional therapeutic indications of these plants.


Background
Inflammation is a reaction implemented by organism whenever its morphological and biological constants are threatened [1]. It may have an infectious origin or caused by physical or chemical agent [2]. Usually, inflammatory response is a beneficial process. Its purpose is to eliminate the pathogen and repair tissue damage. However, inflammation can be harmful, because of the aggressiveness of the pathogen, its persistence, by abnormal regulation of the inflammatory process, or by quantitative or qualitative abnormalities of cells involved in inflammation [3]. It can be manifested by various 3 symptoms such as edema, pain and heat usually accompanied by fever.
Nowadays, anti-inflammatory and/or analgesic potential molecules are used in the treatment of inflammatory diseases. They are steroidal and non-steroidal antiinflammatory drugs [4,5]. These molecules, despite their efficiencies, most often have side effects, which can cause other annoyances as a result of prolonged use [6,7]. Indeed, the use of phytochemicals compound is useful and without side effects [8]. Besides, herbal medicine has been used for centuries to treat several diseases. Thus, herbal teas, decoctions, macerations, infusions and plasters are used successfully [9].
In the rich and diverse Benin flora, Euphorbia hirta, Citrus aurantifolia, and Heterotis rotundifolia are known and used as having effects on conditions that cause inflammatory reactions [10,11]. Especially that inflammation and pain are a common feature of many diseases [12]. Due to the wide use of these plants in traditional medicine, their extracts have been the subject of several phytochemical studies. The richness of these plants in polyphenols and flavonoids gives them several pharmacological activities [13].
Nevertheless, very few studies have focused on their anti-inflammatory activity. That's why this study has the aim to evaluate anti-inflammatory, analgesic and antipyretic properties of the ethanolic extracts of these plants (Euphorbia hirta, Citrus aurantifolia, Heterotis rotundifolia). The results of this study can be used to enhance and improve their use.

Animals used
Male and female Wistar rats having 8 to 12 weeks old and weighing between 100 and 150 g were used. These animals came from the Applied Biomedical Sciences Institute of University of Abomey-Calavi. These animals were acclimatized to the conditions of the animal house for a week before experiments.

Plants used
The whole plants of Euphorbia hirta ( E. hirta) and Heterotis rotundifolia (H. rotundifolia) and the leaves of Citrus aurantifolia ( C. aurantifolia) were used. They were harvested and confirmed at the national herbarium of University of Abomey-Calavi. These plants were dried and powdered in laboratory. The powders were used to make ethanolic extracts.

Ethanolic extracts preparation
The ethanolic extracts are obtained by maceration of 50 g of powder of each plant in 500 ml of 96% ethanol for 48 h with continuous stirring. The mixture was then filtered twice on hydrophilic cotton and once on N o 1 Whatman paper before being concentrated in rotavapor at 50°C. The concentrates were deposited in an oven at 50°C until obtained a dry mass which constitutes the ethanolic extract [14].

In vivo anti-inflammatory activity of extracts
The method used here has been inspired by that of Sy et al. [15] with some modifications.
Thus, 5 groups of 6 rats each were formed and fasted 15 hours before experimentation.
The treatment was done orally as follows : The first animals group were used as controls and they received only physiological water at 1 ml/100 g body weight (b.w.) ; The second animals group were used as a reference and they received diclofenac at 50 mg/kg b.w ; The other three animal's groups received ethanolic extracts of hirta (EE-Eh), C. aurantifolia (EE-Ca) and H. rotundifolia (EE-Hr) at 200 mg/kg b.w. respectively.
One hour after treatments, 0.1 ml of 2% formalin solution was injected into each rat under foot pad of the right hind paw. The paw diameter at the arch was measured every hour until fifth hour. Edema increase (EAP) and inhibition (EIP) percentages were calculated from the following formulas:

5
Where Dt: Mean diameter of the right hind paw at time t; Do: M ean diameter of the right hind paw at time 0 (before treatment).

Writhing test
The method proposed by Koster et al. [16] and taken over by Sy et al. [15] was used.
Thus, 5 groups of 6 rats each were formed and treated in the same way as before. But the reference animals group used here received acetylsalicylic acid (ASA) at 200 mg/kg b.w.
Thirty minutes after treatments, 0.1 ml of 3% acetic acid solution was injected intraperitoneally to all rats and the twisting for each rat was counted over 30 minutes.
Cramping inhibition percentage (CIP) was calculated according formula : Where TCc : Mean of twisting count of control group ; TCt : Mean of twisting count of treated groups

Tail immersion method
To evaluate analgesic effect of extracts by this approach, the method described by Gbenou et al. [17] was used with some modifications. The rats were arranged and treated as in the writhing test. Thirty minutes after treatments, the tail of each animal was immersed in hot water at 50°C and the reaction time was recorded.

Antipyretic effect of extracts
Pyrexia was induced in 5 groups of 6 rats each with a 20% beer yeast suspension. 24 hours after this induction, rats that showed an increase in temperature were treated with extracts and ASA as described above. The anal temperature of each rat was recorded every hour for 4 hours [18]. Pyrexia inhibition percentage (PIP) was calculeted ccording formula : Where T°o : Temperature before pyrexia induction ; T°t : Temperature after pyrexia induction and treatment at time t

Euthanasia
At the end of the experiments, all rats were euthanized in a specially designed enclosure.
Euthanasia by inhalation of CO 2 with progressive filling (80%) of the enclosure was used under ethics committee control.

Statistical data processing
GraphPad Prism 7 software was used to perform graphs and statistical analyzes. A comparison of mean was made with analysis of variances (ANOVA) followed by tukey's multiple comparison test. The differences are considered significant if p-value < 0.05 and very significant if p-value < 0.001. Figure 1 show, percentages of edema increase (a) and inhibition (b) of right hind paw of rats. The extracts prevent very significantly (p<0.001) from third to fifth hour edema in rats compared to Control. This prevention is similar to that of rats treated with Diclofenac at the same time intervals. The increase percentages of edema are steady (between 45.72 and 49.95%) in control rats, whereas in treated rats (Diclofenac and Extracts), these percentages decrease from the first to the fifth hour (Figure 1a). We also note that the 7 inhibition percentages of edema increase with time and there is no significant difference (p>0.05) between the anti-inflammatory effect of extracts at 200 mg/kg and that of Diclofenac at 50 mg/kg from first to fifth hour. However, the highest inhibition percentage of inflammatory edema were observed at the fifth hour in all treated rats. So we have: 79.80%, 86.75%, 83.07% and 87.94% respectively for E. hirta, C. aurantifolia, H. rotundifolia and Diclofenac (Figure 1b). Table 1 and Figure S1 show analgesic activity of extracts. The administration of ASA and extracts has very significantly (p <0.001) prevented pain to the rats. Which's noted by a clear decrease twisting count in treated rats (Extracts and ASA). Inhibition percentages ranges from 35.39% to 60.34% in extracts-treated rats ( Table 1). The reaction time is 4.66 seconds in control rats after immersion of their tail in hot water at 50°C. This time has significantly (p<0,05) increased and ranges from 10.66 to 14 seconds in rats treated with extracts ( Figure S1). Figure 2 show, the evolution of temperature of control and treated rats (a) and Pyrexia inhibition percentages (b). In control rats, the temperature remained high (between 38.43°C and 39.06°C) while in treated rats (ASA and extracts) there was a decrease until the fourth hour when it was very significant (p<0.001) compared to the control ( Figure   2a). Indeed, pyrexia inhibition percentages vary between 70.89% and 80.61% for extractstreated rats at fourth hour. These percentages are similar (p>0.05) to that of the ASA at the same hour.

Discussion
After injection of formalin to animals, inflammatory reaction was almost immediate with appearance of classic signs of acute local inflammation (redness, pain, heat and edema) at the five experimental groups. This inflammatory reaction occurs in two phases. The first phase occurs between 0 and 2 hours after injection of phlogogenic agent and the second 8 phase, initiated after two hours extending to the fifth hour and even beyond [19,20].
The extracts, at 200 mg/kg, have edema inhibition percentages similar (p>0.05) to that of the standard (diclofenac) in the second phase of the inflammatory process ( Figure 1).
Many studies have reported that diclofenac acts at the 2nd phase of inflammation, by inhibiting prostaglandins releasing through Cyclooxygenase (COX2) inhibition [21,22]. It Furthermore, animals tail immersion in hot water at 50°C causes tail sudden movement and sometimes an overall animals body retreat in control rats. There was a significant (p <0.05) and very significant (p <0.001) increase in reaction time, respectively in extractstreated and standard-treated rats ( Figure S1). The reaction time varies between 10.66 (EE-Hr) and 14 seconds (EE-Ca) in extracts-treated rats. These times are greater than 4.47 ± 0.19 seconds and 5.48 ± 0.27 seconds obtained by Bose et al. [28] with Cleome Rutidosperma ethanolic extract at 200 mg/kg and 400 mg/kg respectively. The inhibition of this type of pain by plants studied extracts, had already been demonstrated with Paederia sandens extracts [29].
Indeed, same mediators that cause inflammation and pain often cause fever. This is why antipyretic effect of these extracts has been assessed. The results show that the extracts oppose fever installation by gradually lowering the temperature in extracts-treated rats.
Similar result was obtained with ASA with faster effect. It's therefore possible that extracts inhibits prostaglandins synthesis like ASA [30]. Antipyretic effect observed in this study is similar to that observed by Morabandza et al. [30] in their study with aqueous extracts of stem bark of Strychnos camptoneura at different doses.
As a conclusion, E. hirta, C. aurantifolia and H. rotundifolia, have anti-inflammatory, analgesic and antipyretic effects similar to nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac and Acetylsalicylic Acid. Our results confirm and justify the traditional use of these plants in the treatment of various diseases.

Limitations
The current study only investigated in vivo anti-inflammatory, analgesic and antipyretic effects of ethanolique extracts of E. hirta, C. aurantifolia and H. rotundifolia. It would be better to complete this study by measuring the mediators (histamine, prostaglandin, interleukins….) involved in these pathologies (before treatments and after observations) 10 and to explore the mechanism of action of these extracts.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests

Funding
This research was carried out through research funding no 487RG/BIO/AF/AC_G-FR3240293303 allocated to the research team by the World Academy of Sciences (TWAS) and the United Nations Educational Organization, science and culture (UNESCO). This research fund allowed us to acquire, the necessary materials/equipments as well as the animals used in this study.

Authors' contributions
VD and LB designed the project and supervised the work. OGR and JRK carry out the experiments, analyzed and interpreted the data. OGR wrote the manuscript. VD and LB edited the manuscript. All authors read and approved this manuscript