In-Vivo and In-Vitro Anti-Inflammatory Activities of the Aqueous Extract of Di-Herbal Formulation (Euphorbia hirta and lactuca virosa)

Euphorbia hirta and Lactuca virosa are medicinal plants that have been used in the cure and treatment of various diseases and for health care. This study aims at evaluating the anti-inflammatory activities of the aqueous extracts of Euphorbia hirta and Lactuca virosa plants. Albumin and histamine induced inflammation in mice and xylene induced ear edema were used for the in-vivo anti-inflammatory studies. Erythrocyte membrane stabilization and inhibition of protein denaturation assays were used for the in-vitro anti-inflammatory studies. Combined doses of 100 mg/kg Euphorbia hirta and 50 mg/kg Lactuca virosa, 100 mg/kg Euphorbia hirta and 100 mg/kg Lactuca virosa, 100 mg/kg Lactuca virosa only and 10 mg/kg diclofenac significantly reduced inflamed paw in mice (P<0.05) compared to control in albumin and histamine induced inflammatory test. Combined doses of 50 mg/kg Euphorbia hirta and 100 mg/kg Lactuca virosa,100 mg/kg Euphorbia hirta, 100mg/kg Lactuca virosa significantly reduced xylene induced inflammation (P<0.001) compared to control. The extracts at 1 mg/ml, 2 mg/ml and 3 mg/ml significantly inhibited protein denaturation (P<0.001) and heat induced hemolysis of erythrocytes (P<0.0001). The plant extract of Euphorbia hirta and lactuca virosa possesses in-vivo and in-vivo anti-inflammatory effects. DOI: https://dx.doi.org/10.4314/jasem.v24i11.19 Copyright: Copyright © 2020 Uwaya et al. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dates: Received: 10 October 2020; Revised: 11 November 2020; Accepted: 20 November 2020

Plants play a leading role in the health care system globally. It remained the pillar of medicinal revolution for a long time (Dar et al., (2017). The background of traditional medicine for years was been gotten from plants through which have been used to produce and give human with new medical therapies (WHO, 1998). Euphorbia hirta belongs to family Euphorbiaceae and commonly called asthma weed plant (Pranabesh et al., (2019). It is native to Central America. The plant is distributed throughout the tropical countries and wildly grown in low jungle areas, along roadside (Sunil et al., (2010). The plant is widely used to treat various ailment in traditional medicine. It has antiallergic, analgesic, anti-anaphylactic, anti-diarrheal, spasmogenic, anti-inflammatory, antioxidant, diuretic, anti-tumor and anxiolytic properties (Pranabesh et al., (2019). Lactuca virosa called wild lettuce or opium lettuce is a two-yearly herb that grows on the banks of rivers and wastelands to a height of 6 feet. It is cultivated in diverse areas of the world, such as Austria, France, Germany, Scotland and Nigeria. Traditionally, it is used for insomnia, restlessness, irritable cough, priapism, dysmenorrhea, nymphomania and muscular or articular pains (Darkwa and Darkwa, (2013).
Therefore, the objective of this study is to evaluate the anti-inflammatory activities of the aqueous extracts of Euphorbia hirta and Lactuca virosa plants Experimental Animals: Eighty-four mice of either sex weighing 20-35 g were purchased from a commercial animal house in Ibadan. The animals were allowed two weeks acclimatization in the animal facility of the Department of animal and environmental biology, Faculty of Life Sciences, University of Benin, Benin City. They were allowed to have free access to pellets and tap water and were exposed to natural light-dark cycle and room temperature. All animals were handled according to standard protocols for the use of Laboratory animals.

MATERIALS AND METHOD
In-vivo anti-inflammatory procedure: The in-vivo anti-inflammatory studies were carried out using albumin and histamine induced paw inflammation in mice and xylene induced ear edema on mice (Okokon, 2012;Anosike et al., (2013);vetriselvan et al., (2013).

Egg albumin induced inflammation in mice:
Adult albino mice of 20 -30 g were denied of food for 24 hours. The mice were divided into groups of seven with four mice each. Group 1 received 10 ml/kg of distilled water, group 2 received 10 mg/kg diclofenac, groups 3,4 and 5 received the di-herbal extract at different doses of 50 mg/kg Euphorbia hirta (EH) and 100 mg/kg Lactuca virosa (WL), 100 mg/kg EH and 50 mg/kg WL, 100mg/kg EH and 100 mg/kg WL, respectively. Group 6 received 100 mg/kg EH; Group 7 received 100 mg/kg WL. The initial paw size of all the animals were measured with a Vernier caliper before introduction of 0.1 ml of egg albumin at the left hind paw. The diameter of inflammation was measured at 0, 30, 60 and 120 minutes respectively. The paw swelling at each time was calculated as the difference between the diameters at time ta and that of the initial paw diameter before induction t0.

Histamine induced inflammation in mice:
Adult albino mice of 20 -30 g were denied of food for 24 hours. The mice were divided into groups of seven with four mice each. Group 1 received 10 ml/kg of distilled water, group 2 received 10 mg/kg diclofenac, groups 3,4 and 5 received the di-herbal extract at different doses of 50 mg/kg Euphorbia hirta (EH) and 100 mg/kg Lactuca virosa (WL),100 mg/kg EH and 50 mg/kg WL, 100mg/kg EH and 100mg/kg WL, respectively. Group 6 received 100 mg/kg EH; Group 7 received 100 mg/kg WL. The initial paw size of all the animals were measured with a Vernier caliper before introducing of 0.1 ml of 1% histamine into the sub plantar surface of the right hind paw. The diameter of inflammation was measured at 0, 30, 60 and 120 minutes respectively. The paw swelling at each time was calculated as the difference between the diameter at time ta and that of the initial paw diameter before induction t0.

Xylene induced inflammation on mice:
The mice of 20 -30 g were divided into seven groups of four animals each. Group 1 received ml/kg of distilled water, Group 2 received 2 mg/kg dexamethasone, Groups 3,4 and 5 received the di-herbal extract at different doses of 50mg/kg Euphorbia hirta(EH) and 100 mg/kg Lactuca virosa (WL),100 mg/kg EH and 50 mg/kg WL, 100 mg/kg EH and 100 mg/kg WL, respectively. Group 6 received 100 mg/kg EH; Group 7 received 100 mg/kg WL. One hour later, 30 µl of xylene was applied to the inner and outer surface of the right ear using a microliter pipette to induced edema in each mouse in each group. The animals were sacrificed by cervical dislocation and two ears copped off, sized and weighed after 3 hours of xylene application. The antiinflammatory activity (AIA) was stated as percentage inhibition Where MwtC = Mean weight of control MwtT = mean weight of test In-vitro experimental model: The in-vitro antiinflammatory studies were carried out using erythrocyte membrane stabilization assay by heat induced hemolysis and inhibition of protein denaturation assay (Sakat et al., (2010); Jayasuriaya et al., (2017).

Erythrocyte Membrane stabilization:
The combination containing 1 ml of extract of different concentration (1-3 mg/ml) and 1 ml of the erythrocyte suspension prepared. Aspirin was used as standard (1 -3 mg/ml), the mixture as that of the extract. The combination mixed gently and incubated, 30 minutes in a water bath at 60 0 C. Tubes were cooled below running water after incubation. The combination was centrifuged at 2500 rpm for 10 minutes and the absorbance of the supernatant was taken at 560nm with spectrophotometer. Percentage inhibition of hemolysis was calculated as follows: Where IH = Percentage inhibition of hemolysis Inhibition of protein denaturation assay: The reaction combination comprising of various concentrations of plant extract (1 -3 mg/ml), 2 ml of egg albumin and 10 ml of phosphate buffered saline was added to the reaction mixture. Aspirin (1 -3 mg/ml) is used as the standard. The combination was incubated at 37 0 C for 15 minutes and heated at 70 0 C for 5 minutes again. The reaction combinations were cooled with running water and their absorbance were measured at 660nm using a spectrophotometer. Percentage inhibition was calculated.

% = AC − AT AC × 100
Where IPD = Percentage inhibition of protein denaturation; AC = Absorbance of control; AT= Absorbance of test Histology of the inflamed ears: The inflamed ears from the xylene induced ear oedema were chopped off. One ear was taken from each group and one ear from a non-inflamed mouse.
The ears were fixed in 10% (V/V) formal saline, routinely pressed and embedded in paraffin wax, Paraffin sections of 5.0-micron thickness were cut using a rotary microtome, fixed unto glass slides and stained with hematoxylin and eosin for histological examination.
The slides were examined by a histologist under the compound light microscope provided with camera and image capture software.
Statistical analysis: Results are expressed as mean ± SEM. The differences between experimental groups were compared by one-way analysis of variance followed by multiple comparison tests, using the software GraphPad prism.

Albumin and histamine induced inflammation in mice:
This study shows that di-herbal formulation (Euphorbia hirta and Lactuca virosa) at all doses, Euphorbia hirta (100 mg/kg), Lactuca virosa (100 mg/kg) and the standard drug (10 mg/kg of Diclofenac) reduced the diameter of inflammation in both albumin and histamine induced paw inflammation (Table 1 and 2). Egg albumin and histamine in inflammatory model are well known animal model widely used to study anti-inflammatory activities of medicinal plants or anti-inflammatory agents (Benly, 2015). Egg albumin facilitates its inflammatory effect by initiating the discharging of histamine and 5-HT (5-hydroxytryptamine or serotonin) while histamine cause inflammation by activating increased vascular permeability, leading to amplified blood flow and vasodilation. This result to inflammatory signs such as redness and swelling (Benly, 2015). The anti-inflammatory effect of diherbal formulation (Euphorbia hirta and Lactuca virosa), Euphorbia hirta, and Lactuca virosa alone is comparable to diclofenac the standard drug (table 1and 2). Diclofenac is a non-steroidal anti-inflammatory drug that acts by inhibiting the enzyme cyclooxygenase which leads to the inhibition of prostaglandin synthesis responsible for inflammation (Kilci et al., (2016). The anti-inflammatory activities of the di-herbal formulation could be credited to the inhibition of cyclooxygenase an enzyme that lead to prostaglandin production.   Xylene induced ear inflammation on mice: In xylene induced ear inflammation, di-herbal formulation (Euphorbia hirta and Lactuca virosa), Euphorbia hirta, Lactuca virosa alone and the standard drug (2 mg/kg of Dexamethasone) and the di-herbal formulation reduced the weight of inflammation (Table 3). Xylene initiate inflammation by the release of histamine, kinin, fibrinolysin and phospholipase A2, these inflammatory mediators induce edema by vasodilation and increased vascular permeability (Zhang et al., (2015); Xu et al., (2014). The di-herbal formulation (Euphorbia hirta and Lactuca virosa) shows a better anti-inflammatory activity compared to the standard dexamethasone (Table  3). Dexamethasone is an agonist of the glucocorticoid receptor, it inhibits production of inflammatory cells and suppresses expression of inflammatory mediators (Vetriselvan et al., (2013). The anti-inflammatory effect of the di-herbal formulation may be due to the inhibition of the production of inflammatory cells, suppressing the expression of inflammatory mediators.

Histology of the inflamed ears in xylene induced ear inflammation on mice:
Histology slide of ear in mice showing normal stratified squamous epithelium with normal dermal tissue (Plate 1). Plate 2 shows histology of inflamed ear showing normal keratinized stratified squamous epithelium and some inflammatory cells in inflamed control. Plate 3 shows histology of inflamed ear with normal stratified squamous epithelium and normal dermal structures at 2 mg/kg of dexamethasone. Histology of inflamed ear with stratified squamous epithelium, presence of vacuoles and lymphocytic infiltrates at the di-herbal dose of 50 mg/kg Euphorbia hirta and 100mg/kg Lactuca virosa. Plate 5 shows the histology of inflamed ear with stratified squamous epithelium, lymphocytic infiltrates at the di-herbal dose of 100 mg/kg Euphorbia hirta and 50 mg/kg Lactuca virosa. Plate 6 and 7 shows histology of inflamed ear with stratified squamous epithelium, presence of vacuoles and few lymphocytic infiltrates in an edematous dermal layer at the di-herbal dose of 100 mg/kg Euphorbia hirta and 100mg/kg Lactuca virosa, 100 mg/kg of Euphorbia hirta and 100 mg/kg Lactuca virosa. The histology result shows that the standard drug (dexamethasone) and the aqueous extract of Euphorbia hirta and lactuca virosa were able to ameliorate the effect of inflammation caused by xylene.

Protein denaturation and heat induced hemolysis:
The in-vitro anti-inflammatory study revealed that of Euphorbia hirta and lactuca virosa has the ability to inhibit thermally induced protein denaturation compared aspirin (Figure 1 and 2). The denaturation of protein is a well-documented cause of inflammation and rheumatoid arthritis (Kirtikar and Basu, (1999). Aspirin as a non-steroidal anti-inflammatory drug has been shown to have the ability to inhibit thermally induced protein denaturation (Garg, 2001).
Euphorbia hirta and Lactuca virosa in erythrocyte membrane stabilization assay were able to stabilize the erythrocyte membrane by inhibiting heat which would have lysed the erythrocyte (Figure 3 and 4). Stabilization of lysosomal membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents which causes further tissue inflammation and damage upon extracellular release. Aspirin, as a non-steroidal anti-inflammatory drug acts by inhibiting the lysosomal enzyme and stabilizing the lysosomal enzyme (Rajendran and Lakshmi, (2001). The human red blood cell membrane stabilization has been used as a method to study the invitro anti-inflammatory activity because the erythrocyte membrane is analogous to the lysosome membrane (Shenoy et al., (2010). Its stabilization implies that the extract may well stabilize lysosomal membranes.