Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 39, Num. 4, 2007, pp. 180-183

Indian Journal of Pharmacology, Vol. 39, No. 4, July-August, 2007, pp. 180-183

Research Paper

Immunosuppressive effect of medicinal plants of Kolli hills on mitogen-stimulated proliferation of the human peripheral blood mononuclear cells in vitro

S. Arokiyaraj, K. Perinbam, P. Agastian, K. Balaraju

Correspondence Address: PG and Research, Sathyabama University, Chennai - 600 119
sistperinbam@yahoo.com

Date of Submission: 07-Jul-2007
Date of Decision: 10-Aug-2007
Date of Acceptance: 30-Aug-2007

Code Number: ph07044

Abstract

Keywords: Cytotoxic, immune system, mitogen, secondary metabolites

Modulation of immune response, as a possible therapeutic measure, by using medicinal plant products has become a subject of scientific investigation. Indian medicinal plants are commonly used for the treatment of various ailments and are considered, by many, to have advantages over the conventionally used drugs, which are expensive and known to have harmful side effects. ^[1] Our immune system plays a vital role in protecting us from various infections. Its integrity and efficiency is important during the treatment of many diseases. ^[2] Besides primary metabolites with immunomodualtory activity, several plant have secondary metabolites that have been found to interfere with different immune system functions, including the activation of cell-mediated immunity. ^[3],[4] Several studies have previously demonstrated the immunomodulating effects of medicinal plants on lymphocyte proliferation in the presence of mitogens, allogenic cells, and specific antigens. ^[5],[6],[7] Knowing the potential of plant extracts as immunomodulators, the medicinal plants of the Kolli hills of Tamil Nadu were selected for this study. Although the local traditional healers have ethnomedical knowledge of the value of these plants, there have been no biological studies on the immunomodulatory activity of these plants. The present study was undertaken to test the extracts of four different medicinal plants for their immunomodulatory property on mitogen-induced lymphocyte proliferation in vitro.

Material and Methods

Local traditional healers

Ten local traditional healers who had practical experience with medicinal plants were interviewed in Kolli hills, Tamil Nadu, India, in the month of October 2006. They were interviewed separately and asked to collect the plants that they traditionally used for treating ailments such as rheumatism, cough, inflammation, diabetes, ulcers, and diarrhea. Of the collected plants, four were selected by the interviewer for the study. These plants were taxonomically identified and deposited in the department herbarium. The voucher specimens in duplicate were deposited in the department herbarium, Loyola College, Chennai (India).

Preparation of crude extracts

Fresh plant materials were washed under running water and shade dried. The parts were coarsely powdered and extracted with methanol after 48 h. The solvent was removed by rotary evaporation under reduced pressure at < 45°C temperature. The resulting crude extracts were stored at 4°C until use. Subsequent extraction was carried out in a soxhlet extraction unit (Borosil, Mumbai) using n-hexane, ethyl acetate, chloroform, ethanol 96%, benzene, acetone, and water. Organic solvents were removed under reduced pressure. Water was removed by freeze drying. The resulting extracts were stored in vials and assayed.

High performance liquid chromatography (HPLC) analysis

A Shimadzu model LC-64 liquid chromatograph with C18 reversed-phase column was used to separate the major components of the aqueous extracts. After the column was washed to achieve the baseline, the gradient parameters were set and the run was started by injecting 250 µl of the extract. A flow rate of 1.5 ml/min was employed. Peaks were detected by monitoring their absorbance at 405 nm and 280 nm. The chromatogram was recorded on a Shimadzu-C-R 3 A chromatopac recording integrator. Chromatographic patterns were plotted for the extracts. Fractions were collected in a collector, evaporated in a rotary vacuum pump, and lyophilized.

Cell culture

Vero cells (African green monkey kidney cell) were obtained from the National Centre for Cell Science, Pune, India. Vero cells were grown in EMEM (Eagle′s minimum essential medium) supplemented with Earle′s salts and 10% heat-inactivated NBCS (newborn calf serum), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO ₂ and were subcultured twice a week.

Cytotoxicity assay^[8]

Each extract (crude methanol extract; the six different solvents such as n-hexane, benzene, ethyl acetate, chloroform, acetone, ethanol, and aqueous residues; and the 17 HPLC fractions) was dissolved separately in 1 ml of 20% DMSO (dimethyl sulphoxide), filter sterilized, and then further diluted to attain concentrations of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.062 mg/ml. Cell suspension (100 µl) containing 5 x10 ⁶ cells was seeded onto a 96-well microtiter plate. After 24 h of seeding, 100 µl of the different concentrations of the extracts (2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0.062 mg/ml.) were added. Control consisted of cells without extract and with DMSO. The microtiter plates were incubated at 37°C in a humidified incubator with 5% CO ₂ for period of 72 h. The morphology of the cells was inspected daily and observed for microscopically detectable alterations.

Isolation of peripheral blood mononuclear cells (PBMC)

PBMC were obtained from healthy adult volunteers by centrifugation of heparinized venous blood over Ficoll/Hypaque solution (Histopaque, Sigma, St. Louis MO). Mononuclear cells were collected in the interphase and washed three times in RPMI-1640. Their viability was determined by trypan blue exclusion test. ^[9] Cell suspensions were adjusted to 5 x 10 ⁶ cell/ml and suspended in RPMI-1640 supplemented with 10% FCS.

Lymphocyte proliferation assay by ³ H thymidine uptake

The in vitro cellular proliferation was performed as previously described by Lie-Chwen Lin. ^[10] The cell suspension (100 µl) was seeded in each well of a 96-well flat-bottomed microtiter plate (Nunc 167008, Nuncton, Roskilde, Denmark). The cells were incubated for 24 h. After incubation, 100 µl of the extracts at different concentrations (50, 100, 150 µg/ml) were added. The cultures were stimulated with 20 µg/ml of PHA (Gibco BRC, Gaithersberg, MD). Controls consisted of PBMC with PHA (100% stimulation activity), PBMC with RPMI (0% activity), and sample with methanol (0% activity) as background sample. The plates were incubated in 5% CO ₂ air humidified atmosphere at 37°C for 72 h. Lymphocyte proliferation was estimated by adding ³ H-thymidine (50 ci/mmol, 1 µci/well; Amershan USA). After 16 h of incubation, the cells were harvested on glass fiber filters. Radioactivity in the incorporated cells was measured by using a liquid scintillation counter (Packard TRI CARB 2100 TR USA).

Percentage of inhibition of lymphocyte proliferation for each extract was calculated by the following equation: ^[10]

Statistical analysis

Each experiment with PBMC was run in triplicate. Significance was calculated by Student′s t test, through the ANOVA model. P < 0.05 was taken to indicate statistical significance. Results are reported as mean ± S.D.

Results

Cytotoxicity assay

None of the tested extracts (crude methanol extract; six different solvents such as n-hexane, benzene, ethyl acetate, chloroform, acetone, ethanol and aqueous residues; and the 17 HPLC fractions) of the four different medicinal plants showed any signs of cytotoxicity on the Vero cell line, even at the highest concentration of 2 mg/ml, i.e., there was no loss of monolayer, granulation, or vacuolization in the cytoplasm [Figure - 1],[Figure - 2].

Lymphocyte proliferation assay by ³H thymidine uptake

Inhibition of lymphocyte proliferation: The methanol extract of the four medicinal plants (belonging to different families) used in the traditional system of medicine were tested for their ability to inhibit PBMC proliferation at a concentration between 50-150 µg/ml. After stimulation with PHA and incubation for 72 h, all the four species inhibited PBMC proliferation, as measured by ³H-thymidine incorporation by DNA from the cells. Extract of J. gendarussa showed the maximum inhibitory activity, when compared to P. indica, Aloe vera and. Aegle marmelos , at a concentration of 100 µg/ml [Table - 1].

Sequential extraction of J. gendarussa showed more inhibitory effect on lymphocyte proliferation in aqueous, chloroform, and ethanol than in n-hexane, ethyl acetate, and benzene at a concentration of 50 µg/ml [Table - 2].

HPLC analysis

Aqueous extract of J. gendarussa showed the maximum inhibitory activity on lymphocyte proliferation; HPLC fractionation of this extract was carried out. 17 HPLC fractions were collected and tested for inhibition of lymphocyte proliferation. Out of 17 fractions tested, fraction no.15 showed the maximum inhibition of 88% at a concentration of 10 µg/ml [Table - 3].

Discussion

On comparison of the cell proliferation in nontreated and extract-treated cultures, the methanol extracts of J. gendarussa, P. indica, Aloe vera , and A. marmelos showed the ability to cause significant decrease in mitogen (PHA)-induced lymphocyte proliferation at a concentration of 100 µg/ml, suggesting that they affected the T-cell proliferation in a dose-dependent manner. The results from this preliminary investigation provide evidence of the importance of ethnopharmacology as a guide to the screening of biologically active plant materials. ^[11] J. gendarussa , as a methanol extract, showed the maximum inhibition of 85% . Similarly, crude methanolic extract obtained from leaves of Meliacea were capable of inhibiting the in vitro proliferation of lymphocytes and in inhibiting several immune responses in which these cells are involved. ^[12] Rayward et al . ^[13] showed that the inhibitory effect of the alcoholic extract of the fern Polypodium leucotomos in his assay was related to a direct effect on T-lymphocytes and/or macrophages. Similarly, water and ethanol extracts of Cypreus rotundus and Eclipta alba strongly decreased PBMC proliferation in a dose-dependent manner. ^[14] Before performing the assay, trypan blue viability test was performed to check for 100% viability of PBMC. After 24, 48, and 72 h, all the extract-treated cells were checked for viability and in each case their viability was> 90%. Similarly, in the present study, we have observed that the inhibitory effects could not be considered as the toxic effect of the plants.

The present study indicated that proliferation of PHA-activated mononuclear cells was significantly inhibited by 100 µg/ml and 50 µg/ml; in comparison, concentrated lime juice extract inhibited PHA-stimulated MNC at 250 and 500 µg/ml. ^[15]

It is also important to note that a beneficial effect was observed; the traditional aqueous extract of J. gendarussa showed a strong immunosuppressive effect on mitogen-stimulated lymphocytes at a low concentration of 50 µg/ml.

J. gendarussa is common throughout India in waste places, hedges, and village shrubberies. It is an effective medicinal plant for chronic rheumatism, cephalalgia, cough, and bronchitis. The chemical constituents in the plant′s leaves includes 0-disubstiuted aromatic amines, 2-amino benzyl alcohol, 2(2′-amino benzyl amino) benzyl alchol, and their respective 0-methyl ethers, friedelin, lupeol, and β-sitosterol. ^[16]

This study revealed the inhibitory activity of J. gendarussa extract on nonspecific cellular immune response. Although the exact mechanism of this effect is not known, it may be mediated by the interaction between the active components of the extracts and cell-surface molecules or growth factors involving mitogen activation, and it is possible that identification and elucidation of the active constituents in this plant may provide useful leads to the development of new and effective immunosuppressant drugs.

Acknowledgment

References

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