Immunomodulatory activity of methanolic extracts of fruits and bark of Ficus glomerata Roxb. in mice and on human neutrophils.

OBJECTIVE: To evaluate the immunomodulatory activity of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on cyclophosphamide-induced myelosuppression in mice and the phagocytic effect on human neutrophils.
MATERIALS AND METHODS: Methanolic extracts of fruits and bark of Ficus glomerata Roxb. at two dose levels of 250 and 500 mg/kg p.o. were administered for 13 days to albino mice and cyclophosphamide (30 mg/kg i.p.) was administered on 11th, 12th, and 13th days, 1 hour after the administration of the respective treatment. On 14th day blood was collected and the hematological parameters were evaluated. The two extracts in the concentration range 100, 50, 25, 12 and 6.25 μg were also tested for phagocytic effect on human neutrophils using the in vitro models-nitroblue tetrazolium (NBT) dye test, phagocytosis of Candida albicans, and chemotaxis assay.
RESULTS: Methanolic extracts of fruit and bark of Ficus glomerata Roxb. showed significant counteracting effect (P < 0.01) to cyclophosphamide-induced reduction in total WBC, differential leucocyte count, platelet counts, RBC counts, and hemoglobin levels. The extracts of the plant in the concentration range 100, 50, 25, 12, and 6.25 μg also showed significant (P < 0.01) phagocytic effect on human neutrophils in the parameters studied.
CONCLUSION: Methanolic extracts of fruits and bark of Ficus glomerata Roxb. exhibited immunomodulatory property in both in vivo and in vitro models.

Introduction

Traditional Ayurvedic system of medicine considers immunostimulation as an alternative to conventional chemotherapy under the concept of ‘Rasayana’, where the host defense mechanism has to be activated under conditions of impaired immune responsiveness.[1] True immunomodulation includes both stimulation and suppression of the immune system.[2] Certain drugs that normalize or modulate pathophysiological processes are called immunomodulatory agents.[3] A number of medicinal plants have been screened systematically for their immunomodulatory activity such as Tinospora cordifolia and Mangifera indica.[4]

Ficus glomerata Roxb. (Syn. Ficus racemosa, family–moraceae) is an evergreen tree with a height of 15-18 m. Young shoots are glabrous, pubescent, leaves ovate, and tapering. The tree bears a few aerial roots, figs are pubescent, and reddish when ripened.[5] A review of published literature shows the presence of glycosides, tannins, and wax.[6] Traditionally leaves are used in bilious affection and diarrhea. Fruit is edible and is given in menorrhagia, hemoptysis, and diabetes. Bark is used in the form of fine powder in dysentery, diabetes, and in combination with gingelly oil, it is applied to cancerous affections.[7] The present study was aimed at screening the fruit and bark of Ficus glomerata Roxb. for immunomodulatory activity in cyclophosphamide-induced immunosuppressed albino mice and the phagocytic effect on human neutrophils using the in vitro models–nitroblue tetrazolium (NBT) dye test, Phagocytosis of Candida albicans, and chemotaxis assay.

Materials and Methods

Plant Material

Fruits and bark of Ficus glomerata Roxb. were collected from local areas of north Karnataka and authenticated by Dr. Srinathrao, Professor, Department of Botany, Gulbarga University, Gulbarga. A voucher specimen (Ref. No. GUG/BOT/Herbarium/2008-09/09) has been deposited at the departmental herbarium. The mentioned parts of the plant were dried and pulverized to particle size #40 and then were first defatted with petroleum ether (40-600C) and extracted with methanol using Soxhlet apparatus for 48 h to obtain methanolic extracts of fruit and bark of the plant, respectively.

Phytochemical Studies

The methanolic extracts of fruit and bark of Ficus glomerata Roxb. were subjected to preliminary qualitative chemical tests and the presence of major phytoconstituents (steroids and saponins) was confirmed by thin layer chromatography (TLC) studies.[89] TLC profile for steroids was carried out using petroleum ether and acetone in the ratio of 7:3 as solvent system and anisaldehyde: Sulfuric acid reagent as spraying reagent. TLC profile for saponins was carried out using chloroform, glacial acetic acid, methanol, and water in the ratio of 6.4:3.2:1.2:0.8 as solvent system and anisaldehyde:sulfuric acid reagent as spraying reagent.

Experimental Animals

Swiss albino mice of either gender, weighing 25-30g housed in standard conditions of temperature, humidity, and light were used. They were fed with standard rodent diet and water ad libitum. The study was approved by Institutional Animal Ethics Committee, Ref. No. HKECOP/IAEC/45/2011-12.

Acute Toxicity Studies

Acute toxicity studies were conducted as per OECD guideline by 425 method.[10] The animals did not show any mortality at the dose of 5000 mg/kg and hence its 1/10th dose i.e., 500 mg/kg and 1/20th dose i.e., 250 mg/kg were used as the therapeutic doses in the study.

Test Samples

Weighed quantities of test extracts were suspended in 1% sodium carboxy methyl cellulose (CMC) to prepare a suitable dosage form.[11] The control animals were given an equivalent volume of sodium CMC vehicle.

Drugs

Cyclophosphamide was used as a standard immunosuppressant, Cycloxan® (Biochem–Pharmaceutical Industries Ltd., Mumbai) containing 200 mg cyclosphosphamide, was procured from the market and dilutions were made using sterile water for injection as mentioned on the label of the marketed product.

Cyclophosphamide-induced myelosuppression[12]

The animals were divided into six groups of six mice each. The drugs were administered as shown below.

Group I - control group (1% sodium CMC vehicle for 13 days).

Group II (Cyclophosphamide group) - 1% sodium CMC for a period of 13 days and cyclophosphamide (30 mg/kg i.p.) injection on 11th, 12th, and 13th days.

Groups III and IV - methanolic extract of fruit of the plant daily for 13 days (250 and 500 mg/kg p.o., respectively).

Groups V and VI - methanolic extract of bark of the plant daily for 13 days, respectively, (250 and 500 mg/kg p.o. respectively).

The groups III, IV, V, and VI were injected with cyclophosphamide (30 mg/kg i.p.) on the 11th, 12th, and 13th days, 1 h after the administration of the respective oral treatment. Blood samples were collected on 14th day of experiment by retro-orbital puncture and hematological parameters were studied for hemoglobin levels, RBC, platelets, total WBC counts, and differential leucocytes counts (DLC).

In vitro screening methods[13–15]

Stock solutions for in vitro studies (100 μg/ml, 50 μg/ml, 25 μg/ml, 12.50 μg/ml, and 6.25 μg/ml) were prepared by dissolving the methanolic extract of fruit and bark of Ficus glomerata Roxb. in 0.5-ml dimethyl sulfoxide (DMSO) and with phosphate buffer salt solution.

Nitroblue Tetrazolium Dye Test

One part of 0.3% Nitroblue Tetrazolium (NBT) solution, prepared in 0.34% sucrose solution, was added to one part of phosphate buffer solution (PBS) and was used fresh. A suspension of leucocytes (5 × 106/ml) was also prepared in 0.5 ml PBS. The stock solutions of the methanolic extracts of fruit and bark of Ficus glomerata Roxb. in concentrations of 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.50 μg/ml, and 6.25 μg/ml were added individually to the 0.2-ml freshly prepared 0.15% NBT solution and suspension of leucocytes. In another test tube, 0.1 ml of endotoxin-activated plasma was added to the 0.15% NBT solution and leucocytes which served as a positive control (standard). A normal control was maintained in another test tube with only suspension of leucocytes and NBT solution.

All the test tubes were incubated separately at 37°C for 20 min and centrifuged gently at 400 g for 3-4 min. The supernatant was discarded. A drop of PBS was added and the cells were gently resuspended at the bottom of the test tube. A film was made by allowing a drop of this fluid to dry on a microscope slide, fixed gently by heating and counterstained with dilute carbol-fuchsin, washed, dried, and mounted using a 100X oil immersion objective; 200 neutrophils were counted and the percentage of NBT-positive cells containing blue deposits was determined.

Phagocytosis of Killed C. Albicans

0.25 ml of the concentrations of methanolic extracts of fruit and bark of Ficus glomerata Roxb. (100 μg/ml, 50 μg/ml, 25 μg/ml, 12.50 μg/ml, and 6.25 μg/ml) were taken in separate test tubes and 0.25-ml Hank's solution, 0.25-ml heat-killed C. albicans and 0.25-ml neutrophil suspension were added to each test tube. A standard (positive) and normal control were also maintained.

All the above test tubes were slightly shook for proper mixing and incubated at 37°C for 30 min and centrifuged at 200 g for 5 min. The supernatant was removed, leaving a droplet into which the sediment was resuspended. Smears were made, dried in air and stained with May-Grunwald Giemsa stain. About 200 neutrophils were examined, the number of ingested C. albicans associated with each cell was counted and the mean particle number (MPN) associated with each cell i.e., neutrophil was calculated.

Neutrophil Locomotion and Chemotaxis

Chemotaxis experiment can be carried out using a special apparatus arrangement called chemotaxis chamber, which consists of an upper chamber - in which the neutrophil cells suspension is placed separated by a micropore filter from a lower chamber - in which the chemotactic factor i.e., extract is placed. It is a self-constructed apparatus in which the lower chamber, a 5-ml beaker is placed in a sandwich box with holes bored in its lid and the upper chamber is a Sawnoff tuberculin syringe with the filter glued to its lower end. The photograph of chemotaxis chamber assembly is shown in Figure 1.

Figure 1

Chemotaxis assembly for assessing neutrophil locomotion and chemotaxis

Neutrophil cells were prepared in Hanks (buffer) solution in a concentration of about 106 cells/ml. The lower compartments of the chemotaxis chambers with appropriate chemotactic agent were pre-adjusted to a pH of 7.2.

Chamber 1: Hank's solution (Normal control)

Chamber 2: Casein 1mg/ml (positive control)

Chamber 3: Predetermined concentrations of extracts (100 μg/ml, 50 μg/ml, 25 μg/ml, 12.50 μg/ml, and 6.25mg/ml in separate chambers).

The upper compartments were filled with the neutrophil cells suspensions ensuring that the fluid level in the upper chamber was the same as in the lower to avoid gradient disturbance. The filters were allowed for wetting from the top before putting them in the lower compartments, when the contents of upper compartments were placed in the lower compartments. As soon as the filter was placed in chemotactic solution, the concentration gradient of chemotactic factor began to form the bottom of the filter. The arrangement was not disturbed until the end of the experiment. It was incubated for 3 hours at 37°C in air. After about 45 min the upper compartments were removed and inverted to empty their fluid which was fixed by immersing the filters in 70% ethanol or methanol. After few minutes in alcohol, the glue melted and the fluid became loose, this was picked off gently with dental packing forceps. It was stained with hematoxylin and xylol, respectively, mounted under microscope using mountant like glycerin and covered with a coverslip. The cell migration was measured microscopically by counting the number of cells which reached the lower surface of the filter after a given time interval. The count of lower surface was taken, as it is directly proportional to the number of cells placed on top of the filter at the start of the experiment.

Statistical Analysis

Data were expressed as mean±SEM and differences between the groups were statistically determined by analysis of variance (ANOVA) followed by Dunnett's test.

Results

The preliminary qualitative chemical screening of the methanolic extracts of fruits and barks indicated the presence of carbohydrates, glycosides, wax, steroids, saponins, and tannins but only the fruit extract showed the presence of proteins instead of wax.

The results of TLC studies were as follows- methanolic extract of fruits showed three pink to red-colored spots for TLC of steroids at Rf values – 0.62, 0.74, and 0.80 and two spots for TLC of saponins at Rf values – 0.51 and 0.84. Methanolic extract of bark showed two pink-colored spots for TLC of steroids at Rf values – 0.63 and 0.72 and two spots for TLC of saponins at Rf values – 0.81 and 0.93. The TLC results indicated the presence of major bioactive constituents, viz., steroids and saponins.

Cyclophosphamide-Induced Immunosuppression

Cyclophosphamide at the dose of 30 mg/kg. i.p. (group II) caused a significant reduction in total WBC count, DLC, and platelets along with marginal reduction in RBC and Hb % as compared to control group [Table 1]. Methanolic extracts of fruit and bark showed highly significant (P < 0.001) increase in total WBC, DLC, and platelets and significant (P < 0.01) increase in RBC and Hb% when compared with cyclophosphamide group (Group II) which was dose dependent. However, the increase in the hematological parameters was more with the bark extract as compared to the fruit extract at the mentioned doses, but both extracts showed similar levels of significance [Tables 1 and 2].

Table 1

Effect of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on cyclophosphamide-induced myelosuppression in mice

Table 2

Effect of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on differential leucocyte counts in cyclophosphamide-induced myelosuppression model

Nitroblue Tetrazolium (NBT) Dye Test

The results indicate that the methanolic extracts of fruits and bark of Ficus glomerata Roxb. Vent. have stimulated phagocytosis of NBT dye by the neutrophils at concentrations of 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.50 μg/ml, and 6.25 μg/ml. The methanolic extract of fruit and bark showed significant (P < 0.01) activity compared to the positive control i.e., endotoxin-activated plasma (86%). The activity of both the extracts was comparable at all concentrations [Table 3 and Figure 2].

Table 3

Nitroblue Tetrazolium (NBT) qualitative test on methanolic extracts of fruit and bark of Ficus glomerata Roxb. on human neutrophils

Figure 2

Photograph showing two neutrophils with reduced NBT, the other cells are normal neutrophils

Phagocytosis of killed C. albicans was determined as the MPN of C. albicans phagocytosed. The methanolic extracts of fruit and bark exhibited highly significant (P<0.001) activity compared to the positive control i.e., pooled serum [Table 4 and Figure 3].

Table 4

Phagocytosis of killed Candida albicans for methanolic extracts of fruit and bark of Ficus glomerata Roxb. on human neutrophils

Figure 3

Photograph showing neutrophil which has ingested three Candida and free extracellular Candida are also shown for comparison

The chemotactic activity of the methanolic extracts of fruit and bark of Ficus glomerata Roxb. was determined as the mean number of neutrophils attracted per field. The activity of both the extracts were highly significant (P < 0.001) compared to the positive control i.e., casein (200). The chemotactic activities of both extracts were concentration dependent i.e., higher at concentrations 100 μg/ml, 50 μg/ml, and 25mg/ml and lower at concentrations 12.50 μg/ml and 6.25 μg/ml [Table 5 and Figure 4].

Table 5

Neutrophil locomotion and chemotaxis of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on human neutrophils

Figure 4

Photograph showing enlarged view of the stained neutrophils seen during chemotactic assay

Discussion

Bone marrow, a site of continued proliferation of blood cells, is a source of cells involved in immune response. This high degree of cell proliferation renders it sensitive, particularly to cytotoxic drugs. Loss of stem cells and inability of bone marrow to regenerate new blood cells will result in thrombocytopenia and leucopenia.[14]

In the present study, immunomodulatory activity of methanolic extract of fruits and bark of Ficus glomerata Roxb. was explored by evaluating their effects on cyclophosphamide-induced myelosuppression in mice at 2 dose levels of 250 mg/kg and 500 mg/kg p.o. The results revealed the dose dependent effect of the extracts in counteracting the myelosuppression induced by cyclophosphamide as indicated by increase in RBC, total WBC, platelet counts, Hb%, and DLC in the extract treated groups (Group III, IV, V, and VI), when compared to cyclophosphamide-treated group (Group II). The bark extract was found to be more effective than the fruit extract at the mentioned doses, but both showed similar level of significance of activity. The potentiated activity of bark extract may be due to presence of excess number of tannins and steroids in the bark extract.

Ramesh A et.al. studied cyclophosphamide-induced immune inflammation wherein only WBC count was analyzed.[16] Vigila and Baskaran have also reported similar activity of analyzing the activity by measuring the hematological profile in cyclophosphamide-induced immune suppression.[17] The results indicate modulation of bone marrow activity, viz., suppression when cyclophosphamide used alone and stimulation in methanolic extract-treated groups.

Immunomodulatory activity of methanolic extract of fruit and bark of Ficus glomerata Roxb. was also evaluated on human neutrophils using the parameters, viz., NBT assay, phagocytosis of killed C. albicans assay and chemotaxis assay. Srikumar et al., reported the assessment of neutrophil functions, viz., NBT assay and Candida phagocytosis assay.[18] NBT assay is a qualitative test, in which stimulated neutrophils absorb NBT stain. Formazone granules can be seen in stimulated (opsonized) neutrophils. Mean percentage of reduced neutrophils was observed with extracts against positive control i.e., endotoxin-activated plasma. Phagocytosis of killed C. albicans assay is a method of measuring phagocytosis which relies on the uptake of particles by phagocytes over a brief period. In this the number of particles phagocytosed by neutrophil cells was counted under the microscope.

Neutrophil locomotion and chemotaxis is a process of chemoattraction i.e., attraction of neutrophils toward certain chemicals (chemoattractants). This is a micropore filter method, in which the extracts are tested to contain chemoattractants and the mean number of neutrophils attracted per field was noted against positive control - casein.

From these parameters, the process of immunomodulation (immunostimulation) of the methanolic extract groups of fruits and bark of Ficus glomerata Roxb. at various concentration range can be assessed by observing stimulation (opsonization) of neutrophils (NBT assay), particle ingestion (phagocytosis assay) and chemoattraction of neutrophils (chemotaxis). Both the methanolic extracts demonstrated potent in vitro immunomodulatoty activity with the bark extract showing more activity than the fruit extract.

From the phytochemical investigation, it was found that the major chemical constituents of the methanolic extracts of fruits and barks were steroids, saponins, tannins, proteins, wax, and carbohydrates. Saponins are proven to possess different pharmacological activities such as antiallergic, cytotoxic, antitumor, antiviral, immunomodulating, antihepatotoxic, and antifungal activities. Recently three diosgenyl saponins isolated from Paris polyphylla have been reported for immunostimulating activity.[19] Tannins are also known to possess immunostimulating activites, Triphala containing Terminalia chebula, Terminalia belerica, and Emblica officialis, which are rich in tannins, has been reported to have immunostimulating activity.[20-22] Hence, the immunomodulatory action of the extracts could be attributed to the collective presence of steroids, saponins, and tannins. It is concluded from the studies of in vivo and in vitro models that the methanolic extracts of fruit and bark of Ficus glomerata Roxb. possess immunomodulatory property.