Anti-inflammatory property of the methanol leaf extract of Parinari kerstingii (ENGL) in rats

1 Department of Biochemistry, University of Nigeria, Nsukka 41000 Enugu State, Nigeria. 2 Department of Pharmacognosy and Environmental Medicine, University of Nigeria, Nsukka 410001 Enugu State, Nigeria. 3 International Centre for Ethnomedicine and Drug Development, Nsukka, Enugu State, Nigeria. 4 Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, Nsukka 410001 Enugu State, Nigeria.


INTRODUCTION
Inflammation is a complex physiological response of vascular tissues to harmful stimuli and the body's protective mechanism to eradicate noxious stimuli and hence promote tissue repairs (Ferrero-Miliani et al., 2007).Activation of inflammation results in the generation of inflammatory mediators such as histamine, kinin, and prostaglandin leading to increased blood flow, leakage of plasma proteins and fluids, and migration of neutrophils from blood vessels into sicken tissues (Chaitanya et al., 2011).Chronic inflammation has become the major challenge in the world today and its implications in virtually all human diseases have motivated a global scientific research (Morales et al., 2014).For instance, a lot of inflammatory processes have been revealed to induce insulin resistance, reduce insulin secretion and dysfunction of β-cells (Akash et al., 2013;Uzayisenga et al., 2014).Several herbal drugs contain certain principles with the ability to stabilize biological membranes when exposed to induced lyses (Oyedapo et al., 1997(Oyedapo et al., , 2004)).
Parinari kerstingii Engl. is an evergreen plant of the genus Parinari and family Rosaceae growing up to the height of 20 m with ovoid shaped fruit and is widely distributed in tropical West Africa (Burkill, 1997).It is variously called aramon (Ivory Coast), kakyiki (Ghana), ningelia (Togo), okpe (Yoruba-Ife of Togo), and i ayi (Nigeria: Hausa).The plant is sometimes harvested from the wild for local medicinal use.It is used traditionally, for the treatment of bronchopneumonia and feverish pains and also serves as emetic and purgative agent (Burkill, 1997).Presently, there is no scientific report on the folkloric use of P. kerstingii, thus this research documented the observed anti-inflammatory effect of the MCE of the leaves of the plant in rats.

MATERIALS AND METHODS
All chemicals used in this study were acquired from Sigma Aldrich, Germany.

Plant
Fresh leaf samples of P. kerstingii were obtained from Otuku town of Nsukka Local Government Area, Enugu State, Nigeria.These were authenticated by Mr. A. Ozioko of the Bioresources and Development Centre Programme (BDCP), Nsukka.A voucher specimen (Interceded 0615) was deposited at the same centre.The leaves were air-dried and ground using a mechanical grinder.The resulting powdered crude drug (5000 g) was macerated in methanol for 24 h, filtered with a Whatmann No. 1 filter paper and then concentrated using a rotary evaporator (IKA, Germany) at an optimum temperature of 40 to 50°C.

Animals
Swiss albino mice (18 to 28 g) and Wistar rats (110 to 170 g) of both sexes were gotten from the animal house in the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.They were acclimatized to laboratory condition in a seven day period; water and growers mash (Niger Feeds, Nigeria) was given ad libitum.Ethical approval on the use of laboratory animals was obtained from the committee of University of Nigeria on the care and use of laboratory animals, in accordance to the revised National Institute of Health Guide for Care and Use of Laboratory Animal (Pub No.85-23, revised 1985).

Acute toxicity test
The method according to Lorke (1983) was used for the study, which was in two phases.Firstly, twelve mice of were grouped into four: Group 1 was not fed with any drug and used as vehicular control.Groups 2, 3, and 4 received 10, 100, and 1000 mg/kg extracts orally in that order.The mice were then observed under 24 h for irregularity in behavior and/or death.The second phase was similarly designed but group 2 mice were administered 1500 mg/kg of the extract while groups 3 and 4 were given the crude extract at 2900 and 5000 mg/kg, respectively.These were also similarly observed for signs of toxicity, and the lethality dose (LD50) was calculated as the square root of the product of the lowest lethal dose and the highest non-lethal dose or the geometric mean of consecutive doses for which 0 and 100% survival rates were recorded.

Egg albumin-induced paw oedema in rats
The procedure by Niemegeer et al. (1964) was adopted.The Wistar rats were grouped into 5, of 5 rats each.They were respectively administered via oral route, distilled water (5 ml/kg) and the crude extract (at 100, 200 and 400 mg/kg).The last group (5) was given 10 mg/kg indomethacin.After 30 min, 0.1 ml of freshly prepared egg albumin was injected into the sub plantar region of the left hind paw of each rat.The paw diameter was measured with the aid of vernier caliper at 0, 1, 2, 3, and 4 h after the injection of egg albumin.

Assay for membrane stabilization
P. kerstingii crude extract was screened for inhibition of haemolysis of HRBC induced by heat; hypotonic solution (distilled water) was evaluated (Shinde et al., 1999) with some modifications.

Heat induced haemolysis
The MCE of the study plant was dissolved in an isotonic phosphate buffer solution.Five centrifuge tubes were each loaded with 5 ml of the extracts in the respective doses of 100, 200, 400, 800, and 1000 μg/ml nd arranged in sets of 4 per dose.Two sets of control tubes cont ined 5 ml of the vehicle nd 5 ml of 200 μg/ml of indomethacin, respectively.A 0.1 ml HRBC suspension was gently mixed with the content of each tube.A regulated water bath at 54°C was used to incubate one pair of the tubes, while a second pair was maintained at -10°C in a freezer for 20 min.Next, the tubes were centrifuged at 1300 g for 3 min, followed by the estimation of the haemoglobin content of the supernatant using a Spectronic Spectrophotometer (21D Milton Roy) at 540 nm.Percent inhibition of haemolysis was calculated, viz.: % Inhibition of hemolysis = where OD1 = absorbance of test sample unheated, OD2 = absorbance of test sample heated, and OD3 = absorbance of control sample heated.

Hypotonicity induced haemolysis
The MCE was reconstituted in distilled water to form a hypotonic solution.Similarly, 5 ml of the graded doses of the extracts (100, 200, 400, 600 nd 800 μg/ml), in centrifuge tubes were grouped in pairs per dose.An isotonic solution containing 5 ml of the MCE in the dose of 100 to 1000 μg/ml was also similarly paired (per dose).A 5 ml of the distilled water and another 5 ml of indomethacin (200 μg/ml) were loaded into separate capillary tubes as vehicle and drug control, respectively.A 0.1 ml of erythrocyte suspension was added to each of the tubes and mixed gently.The tubes and contents were then incubated for 1 h at 37°C, and centrifuged for 3 min at 1300 g.Absorbance (OD) of the haemoglobin content of the supernatant was estimated at 540 nm using the Spectronic spectrophotometer.The percentage heamolysis was thus determined by the mathematical expression: % Inhibition of haemolysis = where OD1 = absorbance of isotonic test solution, OD2 = absorbance of hypotonic test samples, and OD3 = absorbance of control hypotonic solution.Heamolysis produced in distilled water was taken to be 100%.

Phospholipase A2 activity test
The phospholipase A2 was prepared from Aspergillus niger and was used to evaluate the effect of the methanol crude extract (MCE) on its activity (Vane, 1971).

Preparation of enzyme
Nutrient agar plates were prepared by dissolving 28.0 g of nutrient agar in 1000 ml of distilled water.The nutrient agar solution was homogenized in a water bath, 100°C, for 10 min and dispensed into sterilized conical flasks.The agar was autoclaved at 121°C for 15 min and then distributed into sterilized petri dishes.Aspergillus niger was then inoculated on the plates and left to grow for 3 days.The nutrient broth was prepared by dissolving 15 g of Sabouraud dextrose agar in 1000 ml of distilled water, homogenized in a water bath for 10 min and dispensed into 250 ml conical flasks.The conical flasks were sealed with cotton wool and foil paper.The broth was then autoclaved at 121°C for 15 min.The broth was allowed to cool to room temperature and then the organism in the Petri dishes were aseptically inoculated into the broth and incubated for 72 h at room temperature.The culture was transferred into test tubes containing 3 ml of phosphate-buffered saline, and centrifuged at 3000 g for 10 min.The supernatant was used as the crude enzyme preparation.5 ml of blood was drawn out from a healthy volunteer and dispensed into plastic tubes containing 0.01 ml of 1% EDTA as an anticoagulant.They were also similarly centrifuged and the supernatant (plasma) was discarded.The erythrocytes were re-suspended in a volume of normal saline and plasma (1:1) and centrifuged to discard the supernatant so formed.This solution of red cells was reformed to 40% (v/v) suspension with phosphate buffered saline and this served as the substrate for phospholipase.0.2 ml of CaCl2 (2 mM), 0.2 ml of human red blood cell (HRBC), 0.2 ml of the crude enzyme preparation and varying concentrations of normal saline and the test sample or reference drug were incubated in test-tubes for 1 h.The control contained the human red blood cell suspension, CaCl2 and free enzyme.The blanks were treated with 0.2 ml of boiled enzyme separately.The incubation reaction mixtures were centrifuged at a speed of 3000 g for 10 min.Samples of the supernatant (1.5 ml) were diluted with 10 ml of normal saline and the absorbance of the solutions read at 418 nm.Prednisolone was used as the reference drug.The percentage of maximum enzyme activity and percentage inhibition was determined as follows: % Maximum enzyme activity = % Inhibition =

Leukocyte mobilization test in rats
The method outlined by Ribeiro et al. (1991) was used to check the effect of the MCE P. kerstingii on in vivo leukocyte mobilization induced by an inflammatory stimulus.Twenty five adult Wistar rats of both sexes (110 to 150 g) divided into five groups of five rats each were used for the test.Groups 3, 4 and 5 were administered varied doses of the extract (100, 200 and 400 mg/kg), while groups 1 (vehicle control) and 2 (treatment control) received distilled water and indomethacin (10 mg/kg), respectively.Three hours after oral administration of the extracts, distilled water or reference drug, each animal in the respective groups received intraperitoneal injection (i.p) of 0.5 ml of 3% w/v agar suspension in normal saline.Four hours later, the animals were sacrificed and the peritoneal cavities washed with 5 ml of a 5% solution of EDTA in phosphate buffered saline (PBS).The peritoneal fluid was recovered and both total and differential leukocyte counts (TLC and DLC) were performed on the perfus tes using m nu l cell counter fter st ining with Wright's stain.The percent inhibition of leukoctye migration was calculated using the formula: where T represents the leukocyte count of the treated groups.

Vascular permeability test in rats
The effect of the extract on acetic acid induced vascular permeability was assessed by a modification of the method of Whittles (1964).Twenty five adult Wistar rats of both sexes (120 to 170 g) divided into five groups of five rats each were used.The animals were fasted for 10 h prior to the experiment and were then administered with varied doses of the extract and drug as stated earlier.Three hours later, each animal received 0.5 ml intravenous injection of 1% Evans blue solution.Vascular permeability was induced 30 min afterwards, by (i.p) injection of 1 ml of 0.6% acetic acid.The animals were sacrificed 20 min later, and their peritoneum washed with 10 ml of normal saline.The recovered peritoneal fluid was centrifuged and the absorbance of the supernatant measured at 610 nm using a spectrophotometer.

Statistical analysis
The data was expressed as Mean ± standard error of mean (SEM).Analysis of variance (ANOVA) followed by post hoc and Dunnet-ttest was used to statistically analyze the data.P values less than 0.05 (P<0.05) were considered as significant.

Qualitative phytochemical analysis
The paw volume of all the treated groups were signific ntly (P≤0.05)reduced from the first hour fter oedema induction as compared to the control group (Table 2).The extent of oedema inhibition for the treated groups increased with time.P. kerstingii extracts at test doses of 100, 200 and 400 mg/kg reduced the egg albumin induced-oedema by 53, 46.7 and 47%, respectively at the 4th hour as compared to the standard drug (51%).In the control group, egg albumin-induced paw oedema was sustained for 2 h after which it reduces signific ntly (P≤0.05) when compared with the 4th hour.
Table 5 shows the effect of P. kerstingii extract on phospholipase activity.The result shows that phospholipase activity of the extract at different doses was non-signific nt (P≤0.05)s comp red to the standard drug, prednisolone.
The extract of P. kerstingii leaves produced a significant increase (p≤0.05) in agar induced leukocyte mobilization into the peritoneal cavity.The proportion of neutrophils in the perfusate was higher than lymphocytes and other cells in all the groups.The total leukocyte count of the treated groups increased significantly (p≤0.05) when compared with that of the control.
Intraperitoneal injection of 0.6% acetic acid evoked an increased vasodilation and permeability of the blood vessel of the animals as indicated by the leakage of Evans blue across the epithelial walls of the blood vessel into the peritoneal cavity.Table 7 shows that the P. kerstingii extract elicited a significant and dose dependent reduction (p≤0.05) in vascular permeability.

DISCUSSION
Bioactive compounds present in plants exhibit varied biochemical and pharmacological actions in animals when ingested (Nwogu et al., 2008).The extract of P. kerstingii leaves revealed the presence of glycosides, reducing sugars, saponins, triterpenes, flavonoids, tannin, soluble carbohydrates, cyanide, steroids, phenols and alkaloids (Table 1).Some of the constituents of the extract have been documented to possess analgesic and anti-inflammatory activities (Park et al., 2001;Okoli et al, 2007).The presence of flavonoids in the leaf of P. kerstingii could account for its use as an antinflammatory agent as reported by Ekwueme et al. (2011), prevention of damage caused by free radicals in the body (Dweck and Mitchell, 2002), treatment of diarrhoea (Schuier et al., 2005), P. kerstingii leaves, due to its flavonoid content could also be used as antipyretic, analgesic and spasmolytic agents.Flavonoids exhibit dramatic effects on immune and inflammatory cells; these can be either immunosuppressant or immunostimulatory (Huang et al., 2010).The phytochemical analysis of the extract also revealed the presence of alkaloids and this indicates that the leaves could be used in hypertension treatment (Olaleye, 2007).Kumar and Subrahmanyam, (2013) reported that tannins possess immune-stimulating activities.Various plants that contain tannins are used for wound healing (Okwu and Josiah, 2006;Nayak et al., 2007), treatment of dysentery, diarrhoea and urinary tract infection (Okwu and Josiah, 2006;Fahey, 2005).This suggests the possible potential of P. kerstingii leaves in the treatment of dysentery, diarrhoea, urinary tract infection and in wound healing.Ekwueme et al. (2011), reported that the saponin content of senna might be responsible for its anti-inflammatory properties and for its immunomodulating effect and could as well be used to treat hyperglycaemia, to cleanse and purify blood, treat hypertension (Fahey, 2005), and might also have cholesterol binding properties, and haemolytic activities (Okwu, 2004).This also suggests the possible potential of P. kerstingii leaves in the treatment of inflammatory, hyperglycaemia, hypertension, cleansing and purification of blood and as an immunomodulating agent.
The egg albumin-induced inflammatory reactions have been shown to be due to the release of inflammatory mediators (Heller et al., 1998;Nunez-Guillen et al., 1997;Ndebia et al., 2007).Egg albumin causes inflammation of the rat paw similar to carrageenan and the extract inhibited the development of paw edema in the treated animals at 0.5 and 4.0 h post injection of irritant corresponding to the two phases of the inflammatory response.The extract of P. kerstingii produced significant acute anti-inflammatory effects on egg albumin-induced paw oedema, which is a model of acute inflammation used in the study of non-steroidal anti-inflammatory agents (Di Rosa et al., 1971).The effects of the extract was most pronounced at 3 h after induction of oedema, an action which was similar to that of the standard drug (indomethacin), suggesting its usefulness in the management of acute inflammation.
The inhibition of haemolysis was not dependent on the doses, increasing with decreased concentration of the extract in the medium and was comparable with that obtained for indomethacin.Membrane stabilization leads to the prevention of leakage of serum protein and fluids into the tissues during a period of increased permeability caused by inflammatory mediators (Chaitanya et al., 2011).It is well known that the vitality of cells depends on the integrity of their membranes (Ferrali et al., 1992).Exposure of red blood cell to injurious substances such as hypotonic medium, phenylhydrazine and heat results in lysis of its membrane and this is accompanied by haemolysis and oxidation of haemoglobin (Augusto et al., 1982;Ferrali et al., 1992).The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupturing of its membrane.Such injury to RBC membrane will further render the cell more susceptible to secondary damage through free radical-induced lipid peroxidation (Augusto et al., 1982;Ferrali et al., 1992).This conception is consistent with the observation that the breakdown of biomembranes leads to the formation of free radicals which in turn enhance cellular damage (Halliwell et al., 1988;Maxwell, 1995).The human erythrocyte membrane was protected by P. kerstingii extract against lysis induced by hypotonic solution and heat.Lyses of lysosomes occur during inflammation, thus releasing their component enzymes that result to various disorders.Non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the release of lysosomal enzymes or by stabilizing the lysosomal membranes (Mounnissamy et al., 2008).Since human red blood cell (HRBC) membranes are like lysosomal membrane components (Mounnissamy et al., 2008), the inhibition of hypotonicity and heat induced red blood cell membrane lysis was employed as a measure of the mechanism of anti-inflammatory activity of P. kerstingii extract ( tables 3 and 4 ).Compounds with membrane-stabilizing properties are well known for their ability to interfere with the early phase of inflammatory reactions, namely the prevention of the release of phospholipases that trigger the formation of inflammatory mediators (Aitadafoun et al., 1996) and according to Anosike et al. (2012), anti-inflammatory drugs usually offer protection of erythrocyte membrane against lysis induced by heat and water.These results indicate that the observed anti-inflammatory activities of this plant are related to their membrane stabilization activity and contained principles that protected the erythrocyte   (Middleton, 1996;El-Shabrany et al., 1997;Awe et al., 2009;Oyedapo et al., 2004).Inflammation, pain and fever are all associated with enhanced production of prostaglandins (Rang et al., 2003).Phospholipase A 2 cleaves free fatty acids from membrane phospholipids, for instance, from erythrocyte phospholipids.The phospholipase A 2 enzyme activity was carried out using its action on erythrocyte membrane which created a leakage that resulted in extravasation of hemoglobin into the medium.The enzyme activity is thus directly related to the amount of hemoglobin in the medium, hence the increase in absorbance since hemoglobin absorbs maximally at 418 nm.Inhibition of phospholipase A 2 suggests that the extract may suppress the synthesis of free fatty acids from membrane phospholipids and hence prostaglandin synthase is depriving of substrates for the production of prostaglandins (Iwueke et al., 2006).Anti-inflammatory and immunosuppressive steroids inhibit arachidonic acid and its metabolites (prostaglandins) by induction of lipocortin which inhibits phospholipase A 2 .The sequential inhibition of these two enzymes leads to potent suppression of prostaglandins synthesis and possible amplification of the anti-inflammatory activity of the extract.
Lymphocyte and neutrophils were mobilized than the other leukocytes.Neutrophil mobilization of the groups treated with different concentrations of extract increased in a concentration dependent manner.The increased neutrophil mobilization in our study is in agreement with the report of Vega and Corbi (2006) and Ekwueme et al. (2015) which state that, the number of phagocytic cell increases in the blood stream during injury and are responsible for increase in white blood cell count during infection.The extract triggered an increase mobilization of neutrophil which then fight and eradicate the harmful agent using myeloperoxidase present in the primary granules, lactoferrin and gelatinase present in the secondary granules, to degrade extracellular matrix, digest phagocytosed material, exert antimicrobial activity and initiate inflammation (Dale et al., 2008).The increase in neutrophil by the extract shows its ability to recruiting dendritic cells and monocyte that complete innate clearance of invading microbes and also initiate more specific adaptive immune responses (Vega and Corbi, 2006).Again, the mobilization of neutrophil by the extract( table 6) indicates that the extract stimulates the generation of respiratory burst, hence generates ROS used in killing microbes.This is consistent with the report of Puga et al. (2012) which states that the mobilization of neutrophil stimulates the generation of respiratory burst by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which generates ROS used in killing microbes.In addition, the extract can also mop up this ROS when they are produced in excess due to its antioxidant effect.The mobilization of lymphocyte decreased gradually as the concentration of the extract increased.
The inhibitory effect of P. kerstingii extract at various doses was comparable to that of indomethacin.Results demonstrated the effectiveness of the methanol extract of P. kerstingii against induced vascular permeability and infiltration of inflammatory cells to an injured area as was carried out using acetic acid induced vascular permeability test and agar induced leukocyte migration.Two major components of the inflammatory response mechanism are vascular changes leading to increased vascular permeability and emigration of leukocytes from the circulation to the site of inflammation (Ezike et al., 2015).Inhibition or suppression of any of these reactions could ultimately alleviate the extent and magnitude of the inflammatory response.In this study, intraperitoneal injection of 0.6% acetic acid resulted in an increased dilation and permeability of the blood vessels of the animals which was indicated by the increased leakage of fluids, including Evans blue across the blood vessel epithelial walls.Administration of the extract of P. kerstingii caused a significant and dose dependent reduction in vascular permeability of the extract treated animals.

Conclusion
The study revealed that P. kerstingii is rich in flavonoids and other phenolic compounds have been associated with decreased risk of developing inflammatory and other related diseases and also has inhibitory effect of vascular changes that occurs during inflammation.It was reported that stimulation of leukocyte by the extract plays a significant role in immune response and that the antiinflammatory property of the extract is not at the level of leukocyte mobilization (Ekwueme et al., 2015).This is in line with our result which showed stimulatory effect on leukocyte mobilization.It can therefore be deduced from this study that the extract has anti-inflammatory and immunostimulatory effect and could be used in boosting immune response as revealed in the models studied.This study has membrane stabilizing and phospholipase A 2 inhibitory effects, and may offer some beneficial effects on haemolytic diseases and in the management of inflammatory conditions.

Table 1 .
Phytochemical content of methanol extract of Parinari kerstingii leaves.

Table 2 .
Effect of extract on egg albumin-induced rat paw oedema.

Table 3 .
Effect of P. kerstingii extract on heat induced haemolysis of HRBCs.Values represent mean ± standard deviation of triplicate sample.Percent inhibition of of haemolysis was calculated relative to control.Me n v lues, down the column, with different letters s superscripts re considered signific nt t p ˂ 0.05.

Table 4 .
Effect of P. kerstingii extract on hypotonicity induced haemolysis of HRBCs.

Table 5 .
Effect of P. kerstingii extract on phospholipase A2 activity.

Table 6 .
Effect of methanol extract of P.kerstingii on leukocyte mobilization.

Table 7 .
Effect of methanol extract of P. kerstingii on acetic acid-induced vascular permeability test in rats.