For correspondence:-  Vinod Rangari   Email: dr.rangarivinod@gmail.com   Tel:+9893939894

Received: 23 July 2015        Accepted: 5 August 2016        Published: 30 September 2016

Citation: Sharma A, Rangari V. Immunomodulatory activity of methanol extract of Adansonia digitata L. Trop J Pharm Res 2016; 15(9):1923-1927 doi: 10.4314/tjpr.v15i9.16

© 2016 The authors.
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Immunomodulation is a procedure that alters the immune system of an organism by interfering with its functions. Variety of plant extracts, pure phyto-constituents, natural adjuvants, synthetic agents, and antibody reagents are used as immune modulators which may work either as immunosuppressive and immune-stimulating agents. The use of these agents is often limited by factors such as increased risk of infection and generalized effects throughout the immune system [1,2]. Studies have shown that a number of Indian medicinal plants may possess immune-modulatory activities [3-5].

Adansonia digitata L., family Bombaceae, is known by various names such as Baobab, Dead-rat tree, Bottle tree, Monkey-bread tree, and Lemonade tree. It is native to Africa. In India it grows naturally in Mandu region of Madhya Pradesh and is very common in Andhra Pradesh, Uttar Pradesh, Bihar, Tamil Nadu and Maharashtra [6]. Different parts of A. digitata are widely used as food, medicine, clothing and shelter. In folk medicine, it is used as antipyretic, febrifuge, astringent in diarrhoea and dysentery, and as a substitute for cinchona in various systems of medicine [7]. The fruit pulp contains high levels of ascorbic acid, tartaric acid, and citric acid and is used in producing a refreshing drink, while the seeds are eaten fresh or dried. The leaves are said to be rich in vitamin C, sugars, potassium tartrate, and calcium. Various parts of A. digitata have shown anti-inflammatory, analgesic, antipyretic, antibacterial and antiviral activities [8]. A variety of phytochemical constituents such as terpenoids, flavonoids, sterols, vitamins, amino acids, carbohydrates, and lipids have been reported from A. digitata [9]. The fruit pulp contains sterols, triterpenes, saponins, tannins, carbohydrates, and glycosides [8,10].

The purpose of this study was to evaluate possible immune-modulatory activities of the leaves, fruits and bark of A. digitata.

Plant collection and authentication

Fresh leaves, fruits and bark of A. digitata L. were collected from Mandu, Madhya Pradesh, In-dia. The plant was authenticated by Dr. Pramod Patil, Professor, Department of Botany, M.L.B. Girls Postgraduate College, Bhopal, India. A voucher specimen, number 00919 was deposited in their laboratory for future reference.

Extraction

The plant materials were washed thoroughly with water and air-dried under a shade. They were then pulverized to coarse powder and 500 g of each sample was extracted with petroleum ether (60-80 ºC) for 24 h to remove fatty substances. The defatted, dried samples were further extracted with methanol and the extracts were evaporated to dryness under vacuum using a rotary evaporator at 40 °C.

Preliminary phytochemical screening 

The methanolic extracts were subjected to preliminary phytochemical screening using standard chemical tests for alkaloids, amino acids, glycosides, steroids triterpenoids, flavonoids and tannins [11]. Thin layer chromatographic analysis of the extracts was carried out on silica gel G adsorbent, using toluene: ethyl acetate: diethylamine (7:3:0.5 volume ratio) solvent system for the leaf and fruit pulp extracts while toluene: ethyl acetate (9:1) was solvent system for the root bark extract.  Vanillin-sulphuric acid was used as a visualizing agent.

Animals

This study was done as per the Standard Operating Procedures for Institutional Animal Ethics Committee of the CPCSEA [12] and ethical approval was obtained from the Committee for the purpose of Control and Supervision of Experimental Animals (CPCSEA), Chennai, India (1410/c/11/CPCSEA, IAEC approval no. DL/AMSR/2013/V/101). Wistar rats (150 - 200 g) maintained under standard environmental conditions (temperature 25 ± 1°C, relative humidity 55 ± 10 % and 12 h light/12 h dark cycle) were fed on standard laboratory feed and water ad libitum and allowed to acclimatize to the laboratory conditions. Groups of six animals each were used in all sets of the experiments. Animals in Group I (control) did not receive any drug or extract but those in group II (standard) receive dexamethasone (0.2 mg/kg). The extracts of the leaves, root bark and fruit pulp were given in two oral doses (250 mg/kg and 500 mg/kg) to two groups of animals namely Groups III and IV, groups V I and VI and groups VII and VIII, respectively.  The drug and extracts were administered orally for 5 days.

SRBC-induced delayed type hypersensitivity

The method of Lagrange et al [13] was used to analyze the effect of Delayed Type Hypersensitivity (DTH) responses in rats treated with Sheep Red Blood Cells (SRBC). Wistar rats were immunized with 0.1 ml 10 % SRBC subcutaneously, and administered test samples at 250 mg/kg or 500 mg per kg body weight. The control group received 25 µL of normal saline while dexamethasone (0.2 mg/kg) was used as standard immune-suppressive agent. Standard drug and all the extracts and were administered by oral route for 5 days. On the 4th day after immunization, the rats were administered 0.1 ml 1 % SRBC subcutaneously, and on the 5th day rats in all groups were administered 0.1 ml of 1 % SRBC in the paw. Increases in foot paw volume were measured after 24h using a digital vernier caliper (Mitutoyo JAPAN). Difference between the thickness of paw just before and 24 h after SRBC challenge (in mm) was taken as a measure of DTH [14].

SRBC-induced haemoglobin (HA) titre assay

The method described by Atal et al [3] was utilized for the SRBC-induced haemoglobin (HA) titer assay. As in SRBC-induced delayed type hypersensitivity, Wistar rats were immunized with 0.1 ml 10 % SRBC subcutaneously, and administered extract samples at 250 mg/kg or 500 mg per kg orally while animals in control group received 25 µL of normal saline. Dexamethasone (0.2 mg/kg) was used as standard immune-suppressant. On the 4th day after immunization, all the animals (groups I-VIII) were administered 0.1 ml 1 % SRBC subcutaneously. On the 5th day blood was withdrawn and serum was separated, diluted two-fold and incubated with 1 % SRBC in a 96 well plate. Antibody levels were determined by the method described by Shinde et al [15].  Serial two-fold dilutions of the serum were made using 25 µL of normal saline each time of transfer across the plate. To the 25 µL of diluted serum in each well was then added 25 µL of 1 % v/v SRBC suspension in normal saline. The microtiter plate was maintained at room temperature for 1 h and the well contents examined for hemagglutination. Each experiment was repeated twice (n=3).

Carbon clearance test

The method of Biozzi et al [16] was used to analyze phagocytic activity of the white blood cells in the rats. Carbon ink suspension was injected via the tail vein into the rats of each group 48 h after the five-day treatment. Blood samples (25 µL) were then withdrawn from the retro‐orbital plexus under mild ether anesthesia at 5 and 15 minutes after injection of colloidal carbon ink. The blood samples were lysed in 0.1 % sodium carbonate solution (3 ml), and optical density was measured at 660nm in a spectrophotometer.

Statistical analysis

Data were obtained in triplicate and expressed as mean ± SD (n ≥ 3). Comparison of means was per-formed by Dunnet-test to determine difference between groups. P < 0.05 was considered statistically significant.

The yield of methanol extracts of A. digitata leaves, bark and fruit pulp were 3, 2.3 and 1.4 %w/w, respectively. Preliminary phytochemical screening of all the extracts revealed the presence of flavonoids, steroids, tannins, glycosides and amino acids. Thin layer chromatographic study of the methanolic extract of the leaves revealed nine spots in the solvent system (toluene: ethyl acetate:diethyl-amine, 7:3:0.5) while the fruit pulp methanolic extract in the same solvent system showed five spots when sprayed with vanillin-sulphuric acid. However, the methanolic extract of the bark in the solvent system (toluene:ethyl acetate, 9:1) revealed one major and nine minor spots.

The results of delayed type hypersensitivity (DTH) response to SRBC, haemagglutinating anti-body titer and carbon clearance test are given in . Compared to the control, the extracts of the leaves, fruit pulp and root bark produced significant high levels of oedema, HA titre and phargocytic index at the high doses (500 mg/kg) of the extract. At the low doses of the extracts (250 mg/kg), other than HA titre values where significantly higher values than the controls were obtained, variable responses were obtained ().

DTH is antigen-specific and causes erythema and induction at the site of antigen infection in immunized animals; T-cells are required to initiate the reaction. Active phagocytosis is the major defense mechanism against infection. The clearance rate of granular foreign bodies from circulation reflects the phagocytic function of mononuclear macrophages. In this study, only animals treated with high dose (500 mg/kg) of test extracts showed increase in paw edema (). This reveals stimulatory effects of the methanolic extracts on T-lymphocytes.

Based on the  HA titre values, the interaction of B cells with antigen and their subsequent proliferation and differentiation into antibody secreting cells occurred at higher dose of test extracts. Even at a lower dose of fruit pulp extract (250 mg/kg), there was highly significant haemagglutination (p < 0.0001). The antibodies produced by the stimulatory effect of methanolic extracts on humoral immune response bind to antigens and neutralize them or facilitate their elimination by cross linking to form latex that is more readily ingested by the phagocytic cells. The immune stimulant activity might be attributed to the presence of flavonoids and vitamin C in the plants.

The effect of the extracts on reticulo-endothelial system (RES) was measured by the rate of removal of carbon particles from the blood circulation. This is a diffuse system comprising phagocytic cells, fixed tissue macrophages and mobile macrophages. The phagocytic cells in this system comprise mononuclear phagocytic system (MPS), and the macrophage is the major differentiating cell in the MPS. Cells of RES and MPS are known to be important in the clearance of particles from the blood stream. When colloidal ink containing carbon particles are injected directly into the systemic circulation, the rate of clearance of carbon from the blood by macrophage is governed by an exponential equation. Specifically, a faster removal of particles is correlated with an enhanced phagocytic activity of RES cellular components [17]. In this study, the immunosuppressant dexamethasone completely inhibited the carbon clearance while administration of leaf, bark and fruit pulp extracts significantly increased the rate of carbon clearance in a dose-dependent manner. More significant rate of carbon clearance in fruit pulp extract may be attributed to the presence of ascorbic acid in the fruits.

A. digitata L is widely used as food, beverage and traditional medicine in parts of Africa and India. The methanolic extracts of its leaves, bark and fruit pulp produce significant increases in delayed type hypersensitivity (DTH), humoral antibody (HA) titre and phagocytic index thereby indicating significant immune stimulant activities in the experimental animals. This present study provides scientific basis for the ethnomedical uses of A. digitata. Further studies might be required to determine detailed mechanisms and active phytochemicals responsible for immune-modulatory activity.