Evaluation of the Immunomodulatory Activity of the Methanol- Methylene Chloride Extract of Parkia Biglobosa Jacq Benth. (Fabaceae) Stem Bark in Mice

Drugs

Cyclophosphamide 500 mg/vial (Olphamide®, Kwality Pharmaceuticals Ltd, India) and Levamisole 40 mg tablets (Retrax®, Ecomed Pharmaceuticals Ltd, Nigeria).

Chemicals, Solvents and Reagents

Analytical grades of methanol (Sigma-Aldrich, Germany), dichloromethane (Sigma-Aldrich, Germany), WBC diluting fluid, Leishman’s stain, zinc sulphate, barium chloride.

Animals

Adult Swiss albino mice (20-35g) of either sex were used. The animals were obtained from the Laboratory Animal Facility of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka. Animals were housed in steel cages within the facility under standard conditions and allowed free access to standard pellets and water. Prior to their use, they were allowed two weeks for acclimatization within the work area environment. All animal experiments were in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animal (Pub No. 85-23, revised 1985).

Collection and Preparation of Plant Material

The fresh stem bark of Parkia biglobosa was collected in May 2018 from Ede-Oballa in Enugu state. The plant was identified and authenticated at the International Centre for Ethnomedicines and Drug Development (InterCEDD) Nsukka. The plant material was cleaned, sun-dried and reduced to a coarse powder using milling machine.

Extraction of Plant Material

The powdered material (1.57 kg) was extracted by cold maceration in 6 L of a 1:1 mixture of methanol-methylene chloride for 72 hours with intermittent vigorous shaking every six hours. The extract was strained with a muslin cloth and filtered with Whatman No. 1 filter paper. The extract was concentrated in an electric rotary evaporator set at 400C to obtain the methanol-Methylene extract (MME).

Phytochemical Analysis of the Extract

The extract was subjected to phytochemical analysis for the identification of constituents using the methods of Harborne [14].

Acute Toxicity Test

The acute toxicity of the methanol-methylene chloride was evaluated using the method described by Lorke [15].

Neutrophil Adhesion Test

Adult Swiss albino mice were divided into five groups of five mice per group. Group 1 and 2 were used as the negative and positive controls with the animals receiving orally 2 ml/kg of the vehicle (distilled water) and Levamisole respectively. Groups 3, 4 and 5 received orally different concentrations of Parkia biglobosa extract (250, 500 and 750 mg/kg) respectively for 10 days. On day 11, blood samples were collected from the retro-orbital plexus into heparinized vials and differential leukocyte count (DLC) was determined after fixing the blood smear and staining with the Leishman’s stain. After the initial counts, blood samples were incubated with 40 mg/ml of nylon fibres for 15 minutes at 37°C. The incubated blood samples were again analyzed for DLC and TLC (total leukocyte count). The difference in the neutrophil count before and after incubation of blood samples with nylon fibres was determined [16].

Cyclophosphamide Induced Neutropenia

Adult Swiss albino mice were divided into five groups of five mice per group. Group 1 and 2 were used as the negative and positive controls with the animals receiving orally 2 ml/kg of vehicle and 50 mg/kg of Levamisole respectively. Groups 3, 4 and 5 received orally, different concentrations of Parkia biglobosa extract (250, 500 and 750 mg/kg) respectively for 10 days. On the 11th day, a neutropenic dose of cyclophosphamide (200 mg/kg, s.c) was administered and this day was labelled as day zero. Blood samples were collected through retro-orbital vein of the mice and the total leukocyte count (TLC) and differential counts (DLC) were performed on day 0 prior to and on day 3 after injection of cyclophosphamide. The TLC and DLC in treated groups were compared with the values of the control group [17].

Non-Specific Immunity Determined by Survival Rate Against Fungal Infection (Mice Lethality Test)

Adult Swiss albino mice were divided into five groups of five mice per group. Group 1 and 2 were used as the negative and positive controls with the animals receiving orally 2 ml/ kg of the vehicle and 50 mg/kg of Levamisole respectively. Groups 3, 4 and 5 received orally, different concentrations of Parkia biglobosa extract (250, 500 and 750 mg/kg) respectively for 10 days. On the 11th day that is the day of the challenge, all groups were injected with 5 x107 viable Candida albicans cells and observed daily for a period of 10 days and the number of deaths per group recorded [18].

Statistical Analysis

Data obtained was analyzed by one-way ANOVA followed

by Dunnett’s multiple comparisons post-hoc test using Graphpad Prism version 5.0. Differences between means were accepted significant at P<0.05. Results were presented as Mean ± SEM (standard error of mean).

Extractive Yield

The extraction process yielded 132.22 g of the methanol/

methylene chloride extract (MME) giving a percentage extractive yield of 8.42% w/w.

Phytochemical Constituents of MME

Preliminary phytochemical analysis showed that MME possibly contains tannins, tested positive to saponins, terpenoids, steroids, flavonoids, resins, tannins, carbohydrates, reducing sugar, proteins, glycosides, alkaloids and carbohydrate (Table 1).

Key: MME = Methanol/methylene chloride extract; ++++ = abundantly present; +++ = present in very high concentration; ++ = present in moderately high concentration; + = present in small concentration; - = not present. Table 1: Phytochemical constituents of MME.

Effect of Extract on Neutrophil Adhesion Test

The percentage neutrophil adhesion in the control group was 44.99±6.54%, in the levamisole- treated group it was 46.18 ± 1.97 whilst for MME-treated group at 250 mg/kg, it was 51.14±5.05% (Table 2). There was a significant (P < 0.05) increase in neutrophil count and an increase in neutrophil adhesion at this dose which was higher than that observed with the standard immunostimulatory agent, levamisole (Table 2). However, at 500 and 750 mg/kg doses, there was no increase rather a decrease in neutrophil adhesion compared to the control (Table 2).

* P < 0.05 compared to control (One Way ANOVA; LSD post hoc). Values shown are as Mean ± SEM; MME = Methanol/methylene chloride extract. n = 5. UnB = untreated blood, FTB= nylon fiber- treated blood. Table 2: Effect of MME on neutrophil adhesion.

Effect of Extract on Cyclophosphamide-Induced Immunosuppression

Administration of the methanol/methylene chloride extract did not give a significant increase in the total and differential leucocyte counts after cyclophosphamide administration. The percentage reduction in these parameters as a result of the suppressive effect of cyclophosphamide treatment was lower in the MME-treated groups especially at 750 mg/kg than in both the negative and positive control groups (Table 3).

Values shown are as Mean ± SEM; MME = Methanol/methylene chloride extract; n = 5; TLC=total leucocyte count. Table 3: Effect of MME on cyclophosphamide induced neutropenia.

However, MME treatment offered no restoration of the suppressed values observed after cyclophosphamide treatment.

Effect of Extract on Non-Specific Immunity Determined by Survival Rate against Fungal Infection (Mice Lethality Test)

From the mice lethality test conducted, no deaths were recorded in all treatment groups. However, exfoliation and death were observed in the negative control. Animals treated with MME and levamisole did not show such exfoliation (Table 4).

MME = Methanol/methylene chloride extract; n = 5 Table 4: Effect of MME on the non-specific immune system.