Anti-Inflammatory Effects of The Chloroform Extract of Annona muricata Leaves on Phospholipase A2 and Prostaglandin Synthase Activities

Oyekachukwu AR, Elijah JP, Eshu OV* and Nwodo OFC

Faculty of Biological Sciences, Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

*Corresponding Author:
Okpashi Victor Eshu
Faculty of Biological Sciences
Department of Biochemistry, University of Nigeria
Nsukka, Enugu State, Nigeria
Tel: +2348037636808
E-mail: vic2reshu@gmail.com

Received date: March 14, 2016; Accepted date: April 19, 2016; Published date: April 22, 2016

Citation: Oyekachukwu AR, Elijah JP, Eshu OV, Nwodo OFC (2017) Anti-Inflammatory Effects of The Chloroform Extract of Annona muricata Leaves on Phospholipase A2 and Prostaglandin Synthase Activities. Transl Biomed. Vol.8 No.4:137. doi:10.21767/2172-0479.100137

 
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Abstract

This study ascertained the mechanisms of the anti-inflammatory activity of the total lipid (chloroform) extract of Annona muricata leaves. The plant material was extracted with a mixture of chloroform and methanol (2:1) and partitioned with 0.2 volume water. The chloroform extract was investigated for its effect on the in vitro activities of phospholipase A2, prostaglandin synthase and membrane stabilization. The extract significantly (p < 0.05) inhibited phospholipase A2 activity in a concentration-related manner compared to the control, with a range of 0.2 - 0.6 mg/ml inhibiting the enzyme activity by 23.91 - 43.48%. Effect of the extract on prostaglandin synthase activity showed a significant (p < 0.05) inhibition of enzyme activity at the doses 0.1, 0.5 and 1.0 mg/ml compared to the control. The highest percentage inhibition (87.46%) attained at 0.5 mg/ml was comparable to that of 1.0 mg/ml indomethacin. At various concentrations (0.1-0.8 mg/ml), the chloroform extract also significantly (p < 0.05) inhibited heat and hypotonicityinduced haemolysis of human red blood cells (HRBCs) compared to the control. The highest percentage inhibition of heat-induced haemolysis (53.03%) was obtained at 0.4 mg/ml of the extract while the highest percentage inhibition of hypotonicity-induced haemolysis (77.91%) was obtained at 0.8 mg/ml. This study thus confirmed that the mode of action of this extract of Annona muricata leaves on inflammation could be through the inhibition of phospholipase A2 and prostaglandin synthase activities and by membrane stabilization.

Keywords

Annona muricata; Anti-inflammatory; Phospholipase A2; Prostaglandin synthase; Membrane stabilization; HRBCs: Human Red Blood Cell Membranes

Introduction

Inflammation is a pervasive phenomenon that operates during severe perturbations of homeostasis, such as infection, injury and exposure to contaminants [1]. The triggering of inflammation leads to the release of inflammatory mediators which can either be cell-derived or plasma-derived. Cell-derived mediators include: vasoactive amines, cytokines, nitric oxide, prostaglandins, thromboxane A2 (TXA2), prostacyclin, leukotrienes and plateletactivating factor. They are released from inflammatory cells such as leukocytes, platelets and vascular endothelial cells. Mediatorproducing systems in plasma include the complement, clotting, fibrinolytic and kinin systems. Acute inflammatory response is defined as a series of tissue responses that occur within the first few hours following injury [2]. It is initiated by resident tissue macrophages, mast cells and endothelial cells (Anosike [3]. Vascular responses in the acute inflammatory response include vasodilatation and increased vascular permeability which leads to the leakage of plasma protein-rich fluid from the vascular compartment into the extravascular space. Acute inflammation also involves the emigration of leukocytes from the vascular to the extravascular compartment. There is also activation of platelets and their aggregation and the release of proteases and oxidants from phagocytes during acute inflammation [2]. The outcomes of acute inflammation are complete resolution, healing by connective tissue replacement, abscess formation or progression to chronic inflammation. The resolution of inflammation is important to protect the host from overt tissue damage and amplification of the acute inflammatory response towards chronicity. Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids are classically used to alleviate inflammation. Long term use of NSAIDs causes side effects including gastric ulceration and renal toxicity [4] since they concurrently inhibit both isoforms of prostaglandin synthase or cyclooxygenase (COX). The development of NSAIDs which are selective COX inhibitors still have side-effects as they have been connected with an increased risk of heart attack and stroke [5]. There is therefore, a need for potent anti-inflammatory drugs with fewer side effects. Annona muricata Linn (Soursop) has potentials in curbing inflammation as it is a rich source of active phytoconstituents that provide medicinal benefits against various ailments and diseases.

Annona muricata is a lowland tropical fruit-bearing tree in the Annonaceae family, which is a family of flowering plants. Annonaceae is the largest family of the Magnoliales order and comprises of approximately 130 genera and 2,300 species [6]. Annona muricata is a species of the genus Annona, derived from the Latin word ‘anon’ which means ‘yearly produce’ or ‘annual harvest’. As the name suggests, various species in this genus generally have annual fruit production [7]. Other common species include Annona squamosa (sweetsop), Annona senegalensis (wild soursop), Annona reticulata (custard apple), Annona cherimoya (cherimoya) and the hybrid, Annona atemoya (cross between cherimoya and sweetsop). Annona muricata is the most tropical of the group and bears the largest fruits. It is commonly known as Graviola, Soursop or Gunbanana. In Nigeria, it is known in Igbo, Yoruba and Hausa as Sawonsop, Ebo and Tuwon Biri respectively. It is an underutilized [8] tree grown from sea level to 1000 m and adapted to areas of high humidity and relatively warm winters. It has alternate, large, glossy, leaves oval to lanceolate in shape, which are dark green on the upper surface and lighter green beneath. They have short petioles, 14 to 16 cm in length and 5 to 7 cm in width [9]. Temperatures below 5°C will cause damage to the leaves and small branches and temperatures below 3°C can be disastrous. All parts of Annona muricata are extensively used as traditional medicines against an array of human ailments and diseases. Previous reports on Annona muricata reveal it possesses anti-oxidant effect Baskar [10] anti-proliferative effect [11,12] and anti-trypanosomal effects [13], anti-bacterial effect [14,15], anti-hepatotoxic activity [16,17], wound healing activity [18]. The antinociceptive and anti-inflammatory effect of the ethanol extract of the plant in animal models has also been reported [19-21]. Despite studies on the anti-inflammatory activity of Annona muricata leaves, there is little or no empirical evidence on its anti-inflammatory mechanisms of action. This study thus aimed at determining the mechanisms of the anti-inflammatory action of the chloroform extract of the leaves of A. muricata.

Materials and Methods

Plant materials

Fresh leaves of Annona muricata were collected from Onuiyi- Nsukka in Nsukka Local Government Area of Enugu State, Nigeria atLongitude- 7.4093°E and Latitude- 6.8683°N. The leaves were authenticated by Mr. Alfred Ozioko of the Bioresources Development and Conservation Programme (BDCP) Research Centre, Nsukka, Enugu State. The plant Voucher is number- InterCEDD/16089. Bambara seed used as source of bambara oil was purchased from Orba market, Enugu State.

Blood samples

The blood samples were collected by an experienced phlebotomy, from Two (2) female volunteers, between 33 and 28 years with their consent and were free from drug treatments for at least two weeks. The blood was collected into EDTA bottles to prevent coagulation.

Bacterial organism

The bacterial organism used for the study was Bacillus cereus. The organism was cultured and obtained from the Department of Microbiology, University of Nigeria, Nsukka.

Bovine seminal vesicle

Bovine seminal vesicle used for this study was obtained fresh from the abattoir (Ikpa Market, Nsukka).

Chemicals

All the chemicals used were of analytical grade. They include Chloroform (Kermel), methanol (Emsure), distilled water (Lavans, Nigeria), sodium hydroxide, tween 80 (Qualikems), MaCconkey agar, calcium chloride, petroleum ether, gluthathione, hydroquinone (Kermel, China), citric acid, ethylacetate (JHD, China), potassium hydroxide, indomethacin (Elble Pharma Veranhandles, Germany) and prednisolone.

Extraction procedure

The fresh leaves of A. muricata were cleaned and shade-dried for three weeks. The dried leaves were pulverised into coarse form using a mechanical grinder after which a known weight (1,200 g) of the pulverised leaves was macerated in chloroform - methanol (2:1). The suspension was left for 48 hr with occasional stirring and subsequently filtered using a muslin cloth. Further filtration was achieved with Whatman no.4 filter paper so as to remove the fine residue. Distilled water which measured up to 20% of the volume of the filtrate was added to the filtrate and the mixture shaken vigorously so as to enable the development of two layers. The upper layer (aqueous-methanol layer) was separated from the lower layer (chloroform layer) using a separation funnel. The chloroform layer was concentrated in a rotary evaporator to obtain the crude dark-green, slurry-like extract which was weighed and stored in the refrigerator until used. The method for extraction was a modification of the method described by Odo [22].

Determination of the effect of the extract on phospholipase A2 activity

The effect of the extract on phospholipase A2 activity was determined using modifications of the methods of [23,24]. Fresh human blood samples were centrifuged at 3,000 rpm for 10 min and the supernatant (plasma) discarded. The red cells were washed three times with equal volume of normal saline, measured and reconstituted as a 40% (v/v) suspension with normal saline. This served as the substrate for phospholipase A2.

Bacterial enzyme preparation was obtained from Bacillus cereus strain culture. The organism was cultured in MaCconkey agar for three days and the culture transferred into a test tube containing normal saline. This was centrifuged at 3,000 rpm for 10 min after which the bacterial cells settled at the bottom of the test-tube while the supernatant contained the enzyme preparation that was used for enzyme assay.

Aliquots (0.1 ml) of re-suspended erythrocytes (substrate), normal saline (1.2 ml), 2 mM calcium chloride (0.2 ml), 1 ml varying concentration of extract (0.2, 0.4 and 0.6 mg/ml) in 3% tween 80 (dissolved in normal saline) and 0.2 ml of the enzyme preparation were incubated at 37°C for 1 hr. Control tubes contained 0.1 ml of the erythrocytes, 1.2 ml normal saline, 0.2 ml calcium chloride, 1 ml 3% tween 80 and 0.2 ml of the enzyme preparation. The blanks contained everything in the test except the enzyme. After incubation, the reaction mixture was centrifuged at 3,000 g for 10 min and the absorption of the supernatant read against the blank at 418 nm. Prednisolone (0.4 mg/ml), a known inhibitor of the enzyme was used as the standard control. The percentage inhibition of enzyme activity was calculated thus:

equation

Determination of the effect of the extract on prostaglandin synthase activity

This was assayed using the methods of [25,26]. The method of Nugteren [27] was used in the isolation of the enzyme from bovine seminal vesicle while the method of Harold [28] was used in the preparation of the substrate. The bovine seminal vesicle was frozen, partially thawed and freed of fat and connective tissues. A 50g quantity of the tissue was weighed out, sliced, allowed to chill in a freezer and homogenised in 40 ml of 0.02 M Tris-HCl buffer, pH 7.6 for 2 min at 4°C with a blender. The homogenate was centrifuged at 6,000 rpm for 10 min after which the supernatant was decanted and centrifuged again at 15,000 rpm for 10 min at 4°C. The supernatant was again decanted and centrifuged at 18,000 rpm for 10 min. The resulting supernatant was used as the crude enzyme preparation. Bambara nut oil was used as the source of the substrate (arachidonic acid). The Bambara oil was extracted using petroleum ether by sohxlet method and filtered. The filtrate was concentrated at 60°C to obtain the substrate.

The crude enzyme preparation (7 mg) was weighed into each set of test tubes followed by the addition of 1.5 ml of cofactor solution (33 mM hydroquinone, 2 mM glutathione and 40 μM haemoglobin in the ratio 1:1:8 respectively). The mixture was allowed to preincubate for 2 min at 37°C. The reaction was started by adding 0.2 ml of the substrate and allowed to proceed for 2 min at 37°C. Varying concentrations of the extract and buffer were also added to make the volume up to 2.5 ml. Only buffer was added in the control tube. The reaction mixture was incubated for 2 min after which the reaction was terminated by the addition of 0.5 ml of 0.2 M citric acid. The reaction mixture was then extracted twice with 5 ml ethyl acetate and centrifuged at 2,500 rpm for 10 min. For each extraction, the top organic layer was pipetted out into a clean test tube. The combined ethyl acetate extract was evaporated to dryness on a sand bath. The residue was dissolved in 2 ml methanol. Then, 0.5 ml of 3 M potassium hydroxide solution was added to the solution and allowed to stand for 15 min. The absorbance of the tests against blanks at 37°C was read at 278 nm. The blanks contained exactly the same substances as in the test solution except that, the enzyme was boiled (denatured) in cofactor solution and cooled before use (Nwodo). Duplicate determinations were made with the assay mixture containing 0.1, 0.5 and 1.0 mg/ml of the extract. Indomethacin (1.0 mg/ml) was used as the standard drug. Enzyme activity and percentage inhibition of enzyme activity was calculated using the relations:

equation

Determination of the effect of the extract on membrane stabilization

The effect of the chloroform extract of A. muricata leaves on membrane stabilization was determined using both heat and hypotonicity induced haemolysis of HRBCs.

Heat-induced haemolysis of human red blood cells

This was evaluated using modifications of the method of Shinde et al. [29]. Fresh blood samples (3 ml each) were collected from healthy adult volunteers into EDTA bottles and centrifuged at 3,000 rpm for 10 min and the supernatant discarded. A volume of normal saline equivalent to that of the supernatant was used to dissolve the red blood pellets. The volume of the dissolved red blood pellets was measured and reconstituted as a 40% (v/v) suspension with normal saline. The reconstituted red blood cells were used as such.

Samples of the extract and indomethacin used were dissolved in 3% tween 80 in normal saline. A set of five test tubes containing respectively, 5 ml graded doses of the extracts (0.1, 0.2, 0.4, 0.6 and 0.8 mg/ml) were arranged in quadruplicates (4 sets per dose). Control tubes contained 5 ml of the vehicle and 5 ml of 0.2 mg/ml indomethacin respectively. HRBC suspension (0.1 ml) was added to each of the tubes and mixed gently. A pair of the tubes was incubated in a regulated water bath at 54°C for 20 min while the other pair was maintained in the freezer at -15°C for 20 min. afterwards, the tubes were centrifuged at 1300 rpm for 3 min and the haemoglobin content of the supernatant was estimated using a spectrophotometer at 540 nm. The blanks contained varying concentrations of the extract, indomethacin and vehicle (for control) without HRBC suspension. The percentage inhibition of haemolysis by the chloroform extract or indomethacin was calculated thus:

equation(1)

Where OD1 = Absorbance of test sample (extract or indomethacin) unheated.

OD2 = Absorbance of test sample heated.

OD3 = Absorbance of control sample heated.

Hypotonicity-induced haemolysis of human red blood cells

This was determined using modifications of the method of [30]. Blood samples (3 ml each) obtained from healthy volunteers were placed into EDTA bottles, centrifuged at 3,000 rpm for 10 min and washed three times with equal volumes of normal saline. For each, the blood volume was measured and reconstituted as a 40% (v/v) suspension with normal saline.

Samples of the extract and indomethacin used were dissolved in 3% tween 80 in distilled water which served as the hypotonic solution. Aliquots (1 ml) of varying concentrations of the extract (0.1, 0.2, 0.4, 0.6, 0.8 mg/ml) were respectively added to each of a set of five test-tubes. Another tube contained 1 ml of 0.4 mg/ml indomethacin. The contents of the respective tubes were made up to 4.9 ml with the vehicle (3% tween 80 dissolved in distilled water). Two control tubes were used for this test. A control tube contained 4.9 ml of the vehicle while another contained 4.9 ml of 3% tween 80 dissolved in normal saline (isotonic solution). HRBCs suspension (0.1 ml) was added to each tube, and after gentle mixing, the mixtures were incubated for 1 hr at 37°C. After incubation, the reaction mixture for each tube was centrifuged at 3,000 g for 10 min and the absorbance of the supernatant measured at 418 nm using a spectrophotometer. The tests were carried out in triplicates. Reaction media containing 1 ml varying concentrations of extract or indomethacin made up to 5 ml with 3% tween 80 in normal saline, without HRBCs suspension were used as the respective blank for the tests. The blank for the control tubes contained 3% tween 80 in normal saline also without HRBCs suspension. The percentage inhibition of haemolysis was calculated using the relation:

equation(2)

Where OD1 = Absorbance of control I (isotonic solution)

OD2 = Absorbance of test sample

OD3 = Absorbance of control II (hypotonic solution)

Statistical analysis

The data obtained were expressed as mean ± SD and were analysed using Statistical Product and Service Solutions (SPSS), version 18. Tests of statistical significance were carried out using one-way Analysis of Variance (ANOVA). Significant levels were at p < 0.05.

Results

Phytochemical constituents of the chloroform extract of Annona muricata leaves

Phytochemical analysis showed that flavonoids and phenols were found to be present in very high concentration while terpenoids, alkaloids, tannins and soluble carbohydrates were found to be moderately present. The concentration of steroids, saponins, reducing sugars, hydrogen cyanides and glycosides were found to be minimal (Table 1).

Phytochemical constituents Relative abundance
Flavonoids +++
Terpenoids ++
Steroids +
Saponins +
Alkaloids ++
Tannins ++
Reducing sugars +
Soluble carbohydrates ++
Hydrogen cyanides +
Glycosides +
Phenols +++
+minimally present
++moderately present
+++highly present

Table 1: Phytochemical constituents of the chloroform extract of Annona muricata leaves.

Effect of the extract on phospholipase A2 activity

The chloroform extract of A. muricata leaves at 0.2, 0.4 and 0.6 mg/ml significantly (p<0.05) inhibited the activity of phospholipase (PLA2) in a concentration-related manner. This is shown by the progressive increase in the percentage inhibition of enzyme activity (Table 2).

Treatment Concentration (mg/ml) OD (nm) Percentage enzyme activity (%) Percentage inhibition of enzyme activity (%)
Control - 0.46 ± 0.04c 100 -
Extract 0.2 0.35 ± 0.04b 76.09 23.91
0.4 0.28 ± 0.01a 60.87 39.13
0.6 0.26 ± 0.01a 56.52 43.48
Prednisolone 0.4 0.29 ± 0.03a 63.04 36.96

Table 2: Effect of the chloroform extract of Annona muricata leaves on phospholipase A2 activity.

Effect of the extract on prostaglandin synthase activity

As shown in Table 3, the chloroform extract of A. muricata leaves at 0.1, 0.5 and 1.0 mg/ml significantly (p<0.05) inhibited prostaglandin synthase activity compared to the control. The inhibition of enzyme activity at 0.5 mg/ml of the extract (87.46%) was comparable to that of 1.0 mg/ml indomethacin.

Treatment Concentration (mg/ml) OD (nm) Enzyme activity (IU) Percentage enzyme activity Percentage inhibition of enzyme activity (%)
Control - 0.88 ± 0.03c 13.64 100 -
Extract 0.1 0.20 ± 0.01b 3.1 22.73 77.27
0.5 0.11 ± 0.00a 1.71 12.54 87.46
1 0.15 ± 0.00a 2.33 17.08 82.92
Indomethacin 1 0.11 ± 0.01a 1.71 12.54 87.46

Table 3: Effect of the chloroform extract of Annona muricata leaves on prostaglandin synthase activity.

Effect of the extract on membrane stabilization

Effect of the extract on heat-induced haemolysis of human red blood cells: At various concentrations (0.1, 0.2, 0.4, 0.6 and 0.8 mg/ml), the chloroform extract of A. muricata leaves significantly (p<0.05) inhibited heat-induced haemolysis of HRBCs compared to the control. This is shown by the reduced absorbance values of the heated solution relative to the control. The highest percentage inhibition (53.03%) of haemolysis was obtained at 0.4 mg/ml of the extract. The anti-haemolytic effect at 0.1 mg/ ml of the extract was comparable to that obtained for 0.2 mg/ml indomethacin (Table 4).

Treatment Concentration (mg/ml) Absorbance (nm)
Heated solution Unheated solution Percentage inhibition of haemolysis (%)
Control - 0.70 ± 0.08c 0.05 ± 0.01 -
Extract 0.1 0.52 ± 0.07b 0.06 ± 0.01 28.13
0.2 0.45 ± 0.10ab 0.04 ± 0.01 37.88
0.4 0.35 ± 0.01a 0.04 ± 0.00 53.03
0.6 0.37 ± 0.02a 0.02 ± 0.00 48.53
0.8 0.36 ± 0.00a 0.02 ± 0.01 50
Indomethacin 0.2 0.52 ± 0.00b 0.07 ± 0.01 28.57

Table 4: Effect of the chloroform extract of Annona muricata leaves on heat-induced haemolysis of human red blood cells.

Effect of the extract on hypotonicity-induced haemolysis of human red blood cells: From the data shown in Table 5, the chloroform extract of A. muricata leaves significantly (p<0.05) protected the human erythrocyte membrane against lyses induced by hypotonic solution compared to the control. This is indicated by the reduction in the absorbance values of the extract relative to the hypotonic control solution. The highest percentage inhibition (77.91%) of haemolysis was obtained at 0.8 mg/ml of the extract.

Treatment Concentration (mg/ml) Absorbance (nm) Percentage inhibition of haemolysis (%)
Control (hypotonic solution) - 0.99 ± 0.06e -
Control (isotonic solution) - 0.13 ± 0.04 -
Extract 0.1 0.73 ± 0.17d 30.23
0.2 0.57 ± 0.04cd 48.84
0.4 0.41 ± 0.06bc 67.44
0.6 0.45 ± 0.17bc 62.79
0.8 0.32 ± 0.17ab 77.91
Indomethacin 0.4 0.75 ± 0.14d 27.91

Table 5: Effect of the chloroform extract of Annona muricata leaves on hypotonicity-induced haemolysis of human red blood cells.

Discussion

This study revealed the mechanisms of the anti-inflammatory action of the chloroform extract of A. muricata leaves have been established using inhibition of phospholipase A2 and prostaglandin synthase activities, membrane stabilization and platelet aggregation. Phospholipase A2 is an enzyme that cleaves free fatty acids from membrane phospholipids. Arachidonic acid released from these phospholipids is acted upon by COX and lipoxygenase (LOX) both of which lead to the de novo synthesis of lipid mediators George [31]. The action of COX on arachidonic acid produces mediators such as TXA2, prostaglandin E2, D2 and I2, while the action of 5-LOX on arachidonic acid releases leukotrienes such as leukotriene B4 (LTB4). The chloroform extract of A. muricata leaves from 0.2 to 0.6 mg/ml exhibited significant (p<0.05) and concentration - dependent inhibition of PLA2 activity. This was shown by reduced absorbance as the inhibition prevented the action of PLA2 on erythrocyte membrane, causing reduced leakage of haemoglobin which absorbs maximally at 418 nm. This inhibition of PLA2 activity by the extract also implies that it was able to suppress the release of free fatty acids from red blood cell (RBC) membrane phospholipids and the consequent deprivation of COX and LOX precursors for the synthesis of inflammatory mediators, hence limiting their effects such as vasodilatation, vascular permeability, chemotaxis and pain, thus preventing inflammation. This inhibition also implies that the extract has potentials in curing atherosclerosis and cancer as PLA2 has been implicated in their aetiology Sato [32]. The mechanism of inhibition of PLA2 by the extract could be like that of corticosteroids which induce lipocortin [33]. This effect could be attributed to the presence in the extract of phytoconstituents such as flavonoids and tannins [34-36].

The chloroform extract also exhibited a significant (p<0.05) inhibition of prostaglandin synthase activity with the inhibition at 0.5 mg/ml comparing well with that of 1.0 mg/ml indomethacin. This effect was found to be concentration-independent, contrary to that obtained by previous researchers using different plant extracts [37-39] which established a concentration-dependent inhibition of the enzyme activity. This inhibition of COX by the extract implies that, it does not only prevent the release of free fatty acids but also prevents COX from acting on the fatty acids when released, thereby preventing the synthesis of inflammatory mediators. The extract could have possibly exerted this effect by competitively inhibiting COX like indomethacin [39]. This inhibitory effect could be due to the presence of flavonoids in the extract as they can inhibit arachidonic acid metabolizing enzymes such as PLA2, COX and 5-LOX [40,41]. The sequential inhibition of PLA2 and COX leads to potent suppression of the synthesis of inflammatory mediators, which implies amplification of the anti-inflammatory activity of the extract. This inhibition of PLA2 activity has previously been confirmed by Oliveira [42] using aqueous extract of Annona muricata. Thus, the action of PLA2 on the erythrocyte membrane causes the release of free fatty acids from membrane phospholipids thereby causing leakage. This allows haemoglobin to flow into the medium in the process. Decrease in absorbance values relative to the control is associated to decrease in the amount of haemoglobin in the medium. From Table 2, the chloroform extract of A. muricata leaves significantly (p<0.05) decreased the amount of haemoglobin in a concentration-related manner from 0.2 to 0.6 mg/ml, which corresponds to the activity of PLA2.

The preponderance of polyunsaturated fatty acids in the RBC membrane makes the cells highly susceptible to oxidative damage [43] leading to haemolysis by which haemoglobin and other internal cell components are released into the surrounding fluids. Red blood cell membrane can be lysed due to its exposure to injurious substances such as hypotonic medium, heat and methyl salicylate. This study demonstrated the capability of the plant extract to inhibit the lyses of HRBCs membrane induced by hypotonic solution and heat. The chloroform extract of A. muricata leaves at concentrations of 0.1 to 0.8 mg/ml was able to stabilize significantly (p<0.05) HRBC membrane, which is an indication of the ability of the plant extract to prevent haemolysis. The anti-haemolytic effect of A. muricata leaves against Triton-X-100 induced lyses of HRBCs has been previously confirmed by Mounnissamy et al. [44] though a concentrationrelated inhibition was established in contrast to this research. The inhibition of RBC membrane lysis was used as a measure of the mechanism of anti-inflammatory activity of A. muricata leaves extract because HRBC membranes are analogous to lysosomal membrane components [45,46]. The stabilization of erythrocyte membranes by the extract thus implies that it may also stabilize lysosomal membranes. Stabilization of lysosomal membranes is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated leukocytes such as, bactericidal enzymes and proteases, which upon extracellular release cause further tissue inflammation and damage [47,48].

Prostaglandin synthase oxidizes the free fatty acid released from membrane phospholipids leading to the production of prostaglandins. Alkaline treatment (using KOH, from the methods) of the synthesized prostaglandin E2 results in the formation of prostaglandin B which absorbs maximally at 278 nm. Absorption at 278 nm thus corresponds to the enzyme activity. The lower the absorbance value relative to control, the lower prostaglandin B formed, the lower the enzyme activity and the higher the percentage inhibition of enzyme activity. The extract (0.5 mg/ml) produced the highest inhibition of enzyme activity while 0.1 mg/ml produced the least (Table 3).

Lyses of the red blood cell lead to the release of haemoglobin. The lower the quantity of haemoglobin released, the lower the absorbance value. At various concentrations (0.1, 0.2, 0.4, 0.6 and 0.8 mg/ml), the chloroform extract of A. muricata leaves significantly (p<0.05) reduced haemolysis of HRBCs induced by heat compared to the control. This is shown by the reduced absorbance values of the heated solution relative to the control. The highest percentage inhibition (53.03%) of haemolysis was obtained at 0.4 mg/ml of the extract (Table 4).

From the data shown in Table 5, the chloroform extract of A. muricata leaves significantly (p<0.05) protected the human erythrocyte membrane against lyses induced by hypotonic solution compared to the control. This leads to decrease in haemoglobin released, indicated by the reduction in the absorbance values of the extract relative to the control (hypotonic solution). The highest percentage inhibition (77.91%) of haemolysis was obtained at 0.8 mg/ml of the extract.

This result thus shows that membrane stabilization is part of the mechanisms of the anti-inflammatory effect of A. muricata. Both membrane stabilization and phospholipase A2 activity are related to low calcium availability. The haemolytic effect of hypotonic solution could be related to the uptake of water by the cells through osmosis, resulting in swelling and the subsequent rupturing of its membrane, releasing intracellular electrolyte and fluid components. This implies that the extract could have possibly stabilized the RBC membrane by inhibiting processes which enhance the efflux of these intracellular components, such as by preventing the release of lytic enzymes and mediators of inflammation and by inhibiting the activity of phospholipase A2. Stabilization of the membrane also prevents the leakage of serum protein and fluids into the tissues during a period of increased vascular permeability caused by inflammatory mediators [48].

Conclusion

This study indicates that the mechanisms of anti-inflammatory action of the chloroform extract of A. muricata leaves might be via the inhibition of the synthesis of inflammatory mediators, achieved by inhibiting the activities of phospholipase A2 and prostaglandin synthase. The extract also acts by stabilizing the RBC membrane, and by extension, the lysosomal membrane. The active agent might therefore, serve as an adjuvant to the conventional drugs. Further studies should be directed at determining if the extract inhibits the two isoforms of COX (COX- 1 and COX-2) or if it is a COX selective herb. The capability of the extract to inhibit 5-LOX should also be investigated. In addition, the active agent should be isolated and studied for further drug development.

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