Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 5, 2006, pp. 336-340

Indian Journal of Pharmacology, Vol. 38, No. 5, September-October, 2006, pp. 336-340

Research Paper

Stimulation of immune function activity by the alcoholic root extract of Heracleum nepalense D. Don

Dash S, Nath LK, Bhise S, Kar P, Bhattacharya S

Himalayan Pharmacy Institute, Majhitar, Rangpo, East Sikkim-737132
Correspondence Address:Himalayan Pharmacy Institute, Majhitar, Rangpo, East Sikkim-737132, sdash777@sify.com

Date of Submission: 15-Feb-2006
Date of Decision: 15-May-2006
Date of Acceptance: 23-Jun-2006

Code Number: ph06094

Abstract

Objective: To assess the immunostimulatory activity of H. nepalense, using different in vitro and in vivo experimental models.
Materials and Methods: The immunostimulatory potential of the test compound was investigated by in vitro , phagocytic index and lymphocyte viability tests, using interferon a-2b, a known immunostimulant drug, as the standard. Other tests such as carbon clearance, antibody titer and delayed type hypersensitivity were studied in mice, using levimasole as the standard.
Results: The dried root extract (1000 µg/ml) and isolated quercetin glycoside (50 µg/ml) significantly increased the i n vitro phagocytic index and lymphocyte viability in all assays. They also showed a significant increase in antibody titer, carbon clearance and delayed type hypersensitivity in mice.
Conclusion: H. nepalense exhibited a dose-dependent immunostimulant effect, which could be attributed to the flavonoid content or due to the combination with other component(s).

Keywords: Cell proliferation, immunostimulation.

Introduction

Heracleum nepalense D. Don (Apiaceae) is a small shrub which grows in Nepal and Sikkim. It is used in veterinary medicine. It exhibits stimulant property and increases the rate of respiration and blood pressure in goats.[1] The root of the plant is used as a digestive, an aphrodisiac, a carminative and an antidiarrheal in folk medicine.[2] We have earlier reported the antioxidant and antimicrobial properties of the plant.[3] H. nepalense was studied for its potential immunomodulatory activity, driven by the presence of its antimicrobial property, and its usage in folk medicine.

Immunostimulatory therapy is now being recognised as an alternative to conventional chemotherapy for a variety of disease conditions, involving the impaired immune response of the host.[4] Immunostimulators have been known to support T-cell function, activate macrophages and granulocytes, and complement natural killer cells apart from affecting the production of various effector molecules generated by activated cells (Paraimmunity).[5] It is expected that these non-specific effects offer protection against different pathogens, including bacteria, fungi, viruses and so on, and constitute an alternative to conventional chemotherapy.[6] In view of the above, the present investigation was undertaken to evaluate the immunostimulatory potential of H. nepalense roots, using in vitro and in vivo models.

Materials and Methods

Plant material and preparation of extract

The fresh roots (9 kg) of H. nepalense were collected from the southern district of Sikkim between September-November, 2003. The roots were authenticated by the Botanical Survey of India, Gangtok, Sikkim. A voucher specimen was preserved in our laboratory for future reference. The shade-dried root was ground and yielded 2.5 kg of powder. It was stored in an air-tight, hard polyethylene container with silica pouch up to 10-12 days. One kg of powdered plant (40 mesh size) was extracted by cold percolation with 3 liters of 70% methanol in a percolator for 72 h, at room temperature. The residue was removed by filtration. The solvent was then evaporated to dryness, under reduced pressure, in a rotary evaporator at 42-45°C and yielded 400 mg of extract. The concentrated methanol extract was kept in a dessicator for further use.

Phytochemical studies

The chemical constituents of the methanol extract were identified by qualitative chemical tests for the presence of flavones, tannins, sterols, triterpenoids and saponins (Data not presented).[7] The methanol extract was concentrated, suspended in hot distilled water, cooled and the blast precipitate was filtered. The water-soluble component was fractionated by extracting it successively with petroleum ether, ethyl acetate and acetone. The ethyl acetate soluble fraction was subjected to column chromatography on Sephadex LH-20 column using benzene:ethyl acetate as eluent with increasing polarity. Fractions 32-46 were combined, evaporated under reduced pressure, dissolved in MeOH and purified, on a silica gel (60-120) column. A known flavonol glycoside, namely quercetin-3-O-b-D-glucopyranoside, was isolated along with some minor compounds.[8]

Test compound formulations

Oral suspensions of the extract and isolated compound quercetin-3-O-b-D-glucopyranoside were prepared by suspending them separately in 1% solution of sodium carboxy methyl cellulose to prepare suitable dosage forms.

Animals used

Swiss albino mice of either sex, weighing 17-25 g each, were used. They were housed under standard conditions of temperature (23±10°C) and relative humidity (55±10%), 12/12 h light/dark cycle, and fed with standard pellets and water ad libitum . The Institutional Animal Ethics Committee reviewed the animal protocols prior to the experiments.

Drugs and chemicals

EDTA, RPMI-1640, Hank's balanced salt solution (HBSS), dextran, phosphate buffered saline, fetal calf serum, streptomycin, penicillin, amphotericin, and Trypan blue were purchased from Himedia. Phytohaemagglutinin, ficoll hypaque and L-glutamine were purchased from Sigma Diagnostic, USA. Interferon a-2b and levimasole were obtained as gift samples from Fulford (I) Ltd. and Khandelwal Laboratories Ltd., Mumbai, respectively.

Antigen

Fresh blood was collected from a healthy sheep from the local slaughter house. Sheep red blood cells (SRBCs) were washed thrice with normal saline and adjusted to a concentration of 0.1 ml containing 1X10 ⁸ cells for immunisation and challenge.

In vivo carbon clearance test

The mice were divided into 8 groups, each consisting of 10 animals. Group I (Control) was given 1% sodium carboxy methyl cellulose in water (0.3 ml/mouse, i.p.) for 7 days, Group II-VIII were given different concentrations of methanol extract (250-1000 mg/kg, p.o.), isolated compound (25, 50 mg/kg, p.o.) and standard drug (Levimasole 50 mg/kg, p.o.) for 7 days. At the end of 7 days, the mice were injected, via the tail vein, with carbon ink suspension (10 µl/g, body weight). Blood samples were drawn (in EDTA solution 5 µl), from the retroorbital vein, at intervals of 0 and 15 min, a 25 µl sample was mixed with 0.1% sodium carbonate solution (2 ml) and the absorbance measured at 660 nm. The carbon clearance was calculated using the following equation: (Loge OD ₁ - Loge OD ₂ )/15, where OD ₁ and OD ₂ are the optical densities at 0 and 15 min, respectively.[9]

In vivo humoral antibody (HA) titer and delayed type hypersensitivity (DTH) response

Humoral antibody (HA)

The mice were divided into 8 groups, each consisting of 6 mice. Group I (Control) was given 1% sodium carboxy methyl cellulose in water (0.3 ml/mouse) for 7 days, Group II-VIII were given drug treatment which was exactly the same as with the carbon clearance test.

The animals were immunised by injecting 0.1 ml of SRBCs suspension, containing 1X 10 ⁸ cells, intraperitoneally, on day 0. Blood samples were collected in microcentrifuge tubes from individual animals of all the groups by retroorbital vein puncture on day 8. The blood samples were centrifuged and the serum separated. Antibody levels were determined by the haemagglutination technique.[9] Briefly, equal volumes of 50 µl individual serum samples of each group were pooled. Serial two-fold dilutions of pooled serum samples were made in 50 µl volumes of RPMI-1640 in microtitration plates. To this 50 µl of 1% suspension of SRBC in RPMI-1640 was added. After mixing, the plates were incubated at 37°C for 1 h and examined for haemagglutination under the microscope (button formation). The reciprocal of highest dilution, just before the button formation, was observed and titre values were calculated.

Delayed type hypersensitivity test (DTH)

On Day 8, the thickness of the right hind footpad was measured using a Vernier calliper. The mice were then challenged by injection of 1 X 10 ⁸sub SRBCs in the right hind footpad. The footpad thickness was measured again after 24 h of challenge. The difference between the pre- and post challenge footpad thickness, expressed in mm, was taken as a measure of the DTH response.[10]

In vitro phagocytic index

Preparation of microorganism

Escherichia coli 832 (E. coli) was grown and kept on a slope of solid agar medium. Before use, the microorganism was cultured in 100 ml of 2.5% nutrient broth (oxoid) for about 18 h at 37°C. The culture was then washed twice with phosphate buffer saline and re-suspended in gelatin-HBSS to a concentration of 1 X 10 ⁷sub cells/ml. During each experiment, the number of viable microorganisms was determined microbiologically by counting colony forming units (cfu), using nutrient agar plates after incubation, at 37°C for 18 h.[11]

Preparation of human polymorphonuclear leukocytes (PMNCs)

Human blood was collected from a local blood bank and the RBCs removed by sedimentation in 5% (w/v) solution of dextran in buffered saline (m.w. 200,000; 3 ml of solution to 10 ml of blood) for 30 min at 37°C. The PMNC-rich supernatant layer was washed twice with heparin-saline, concentrated by centrifugation (10 min at 110 g), counted with a hemocytometer, and suspended in gelatin-HBSS to make up a concentration of 1 X 10 ⁷cells/ml.

Microbiological assay for the phagocytosis

To assess phagocytosis, different concentrations of methanol extract (250-1000 µg/ml), its isolated compound (25, 50 µg/ml) and the standard drug, Interferon a-2b (0.5 million IU), in the final volume of 0.1ml, were incubated respectively with 2 ml of the PMNCs suspension (1 X 10 ⁷sub cells/ml), 2 ml of the suspended microorganisms (1 X 10 ⁷ cells/ml) and 0.4 ml of fetal calf serum at 37°C for 1 h in 5% CO ₂ atmosphere in a slanting position. At 30 min intervals up to 120 min, 0.5 ml aliquot of the suspension was removed and added to 1.5 ml of the ice-cooled gelatin-HBSS to stop phagocytosis. The control was run using gelatin-HBSS in place of the test compounds. These samples were centrifuged at 110 g for 4 min. Under this condition, the non-ingested microorganisms remained in the supernatant fluid. The viable count of the microorganisms was undertaken using the colony counter[11]. Phagocytosis was expressed as the percentage decrease in the initial number of viable extracellular bacteria according to the formula: P (t) = (1 - Nt /N ₀ ) x 100, where P (t) is the phagocytic index at time t = t, N ₀ and Nt are the number of viable extra cellular bacteria at time t = 0 and t = 30, 60, 90 and 120 min, respectively.[12]

In vitro cell proliferation assay

This test was performed with peripheral mononuclear blood cells, following their separation from the blood by using ficoll-hypaque gradient centrifugation, according to manufacturer's instructions (Sigma Diagnostic, USA). The rate of proliferation of mononuclear cells, under the influence of mitogens, was measured by the method of Sriwanthana,[13] with minor modification. Briefly, under sterile conditions, the cells were diluted to 1 X 10 ⁷sub cells/ml with RPMI-1640 (supplemented with 20% fetal calf serum). The cell suspension (2 ml) was transferred into a sterile culture tube and to each sample. Different concentrations of the plant extract (250-1000 µg/ml, filtered through 0.22 µ pore size filter), isolated compound (25, 50 µg/ml) and standard drug Interferon a-2b (0.5 million IU), in the final volume of 0.1 ml, were added, respectively. The proliferation of cells was induced by 50 µl phythaemagglutinin (PHA, 0.1 mg/ml). The prepared samples were incubated for 72 h at 37oC in a CO ₂ atmosphere, supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin and 0.25 µg/ml amphotericin. The control incubated with cells minus the plant extract. The viability of the cells was assessed after incubation with test compounds, using the Trypan blue dye exclusion method.[14] Briefly, 20 µl of the incubation mixture was mixed with 20 µl of Trypan blue dye. The total number of mononuclear cells and mononuclear stained blue (dead cells) were counted under an inverted microscope (Olympus, Japan), using the hemocytometer. The percentage of cell viability was taken as a measure of cell proliferation and calculated as per the following formula;[15]

Similarly, the percentage of cell stimulation was calculated as per the following formula:[5]

Statistical analysis

Statistical analysis was performed using one-way ANOVA, followed by Dunnett's test. The significance in difference was accepted at P < 0.05.

Results

The methanol extract, 250-1000 mg/kg, p.o. and its isolated compound, 25 and 50 mg/kg, p.o. exhibited a significant increase in carbon clearance from the blood in a dose-dependent manner. [Table - 1] The doses of test drugs, for which maximum carbon clearance were seen, are methanol extract (1000 mg/kg) and its isolated compound (50 mg/kg). The results [Table - 1] also indicate that animals treated with 250, 750 and 1000 mg/kg of methanol root extract produced a significant increase in HA titer (humoral immunity) as evident from hemagglutination after incubation of serum with SRBCs, while the isolated compound at 50 mg/kg and levimasole (50 mg/kg) showed 328.6±10.4 and 430.06±8.3 HA titer, respectively. In the DTH response (cell mediated immunity) test, the methanol extract at higher doses (750 and 1000 mg/kg) showed a statistically significant increase in mean paw edema in mice. The isolated compound at 50 mg/kg, on the other hand, exhibited the maximum DTH response of 0.50±0.22 compared with 0.57±0.21 for the standard drug, levimasole.

The effects of methanol extract (250-1000 µg/ml) and its isolated compound (25-50 µg/ml) on the phagocytic index model are presented in [Table - 2]. The phagocytic index was significantly increased in a dose-dependent manner after 30, 60, 90 and 120 min intervals with methanol extract as well as its isolated compound. Maximum phagocytic index was observed at 1000 µg/ml (97.86±1.67) of methanol extract and 50 µg/ml (97.24±1.23) of its isolated compound after 120 min of incubation; whereas the standard compound Interferon a-2b exhibited maximum phagocytic index (99.23±1.11) after 120 min. The results of the proliferative response, on the basis of the cell viability of mononuclear cells to the PHA mitogen, are presented in [Table - 3]. The percentage of viability of PH- activated mononuclear cells was significantly increased at 1000 µg/ml of the root extract, compared with the control. The maximum viability (60.4±4.58 %) was noticed at standard drug Interferon a-2b; whereas the isolated compound at 50 µg/ml concentration showed 58.2±4.53 % viability. Further, the methanol extract at 1000 µg/ml and its isolated compound at 50 µg/ml concentration showed 11.04 % and 14.79% cell stimulations, respectively, as compared with Interferon a-2b, for which it was observed as 19.13%.

Discussion

The present study established the immunostimulatory activity of the methanol extract and its isolated compound. Prophylactic treatment of H. nepalense and its isolated compound enhanced the rate of carbon clearance from the blood (more than a two-fold increase) when compared with the control group. The result is owing to a mechanism related to phagocytosis by macrophages. The process of phagocytosis by macrophages includes opsonisation of the foreign particulate matter with antibodies and complement C3b, leading to a more rapid clearance of foreign particulate matter from the blood[12]. H. nepalense was found to stimulate the phagocytic activity of the macrophages as evidenced by an increase in the rate of carbon clearance.

The methanol extract, at a dose of 1000 mg/kg, body weight showed almost a four-fold increase in HA titer, compared to untreated controls. The isolated compound also pronounced significant activity at a dose of 50 mg/kg body weight. This could be due to the presence of flavonoids which augment the humoral response, by stimulating the macrophages and B-lymphocytes subsets involved in antibody synthesis.[10] The DTH response, which directly correlates with cell-mediated immunity (CMI), was found to be the highest at the maximum dose tested in the root extract (1000 mg/kg). The mechanism behind this elevated DTH during the CMI responses could be due to sensitised T-lymphocytes. When challenged by the antigen, they are converted to lymphoblasts and secrete a variety of molecules including proinflammatory lymphokines, attracting more scavenger cells to the site of reaction.[16] The infiltrating cells are probably immobilised to promote defensive (inflammatory) reaction.[17] An increase in DTH response indicates that the root extract of H. nepalense and its isolated compound have a stimulatory effect on lymphocytes and accessory cell types required for the expression of the reaction.[18]

The in vitro immunostimulatory activity of the methanol extract and its isolated compound was tested on human polymorphonuclear and mononuclear cells. The phagocytosis and intracellular killing of microorganisms by polymorphonuclear phagocytes was determined by the direct measurement of the microbicidal activity.[12] Phagocytosis was expressed as the phagocytic index, in which the percentage decrease in the initial number of viable extracellular bacteria was determined microbiologically after incubation with polymorphonuclear leukocytes. In our study, the phagocytic index of H. nepalense root extract was found to be increased in a time- and dose-dependent manner. The isolated compound (50 µg/ml) and root extract (1000 µg/ml) showed significant phagocytic index as compared to control.

Further, the immunostimulatory effect of the extract and its isolated compound was tested in mitogen-activated cultured mononuclear cells. PHA was used to activate the mononuclear cells in the culture. The mitogenic PHA are polyclonal activators, in that they activate mononuclear cells including memory type cells, irrespective of their antigenic specificity.[19] The root extract (higher concentrations) and its isolated compound caused a significant stimulation of the mononuclear cells. This is attributed to the fact that the methanol extract and its isolated compound may stimulate the PHA-activated mononuclear cells and induce the release of cell proliferating factors such as interleukin and TNFa.[13]

Earlier reports on the phytochemistry of H. nepalense indicate the presence of compounds such as steroids and coumarins. We have isolated a known flavonoid, quercetin glycoside, from the plant.[8] However, there is no report on the pharmacological activity of the plant. Contemporary research revealed that quercetin glycoside, isolated from different herbal sources, has several pharmacological actions such as antioxidant, anticancer, antiulcer, antiinflammatory and antiviral.[20] Recent reports indicate that several types of flavonols stimulate human peripheral blood leukocyte proliferation. They significantly increase the activity of helper T cells, cytokines, interleukin 2, g-interferon and macrophages and are thereby useful in the treatment of several diseases caused by immune dysfunction.[21] It is thus apparent that the immunostimulatory effect produced by the methanol extract of H. nepalense, containing quercetin glycoside, may be due to cell mediated and humoral antibody mediated immune responses.

The present finding provides scientific evidence to the ethnomedicinal use of this plant by tribals in Sikkim. The plant H. nepalense has the potential for new therapeutic applications in the future.

Acknowledgment

The authors are thankful to the All India Council for Technical Education, Government of India, New Delhi for financial assistance under RPS with File no: 8022/RID/NPROJ/RPS-77/2003-04.

References

Copyright 2006 - Indian Journal of Pharmacology

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