IN VITRO STUDIES OF IMMUNOMODULATORY AND FREE RADICAL SCAVENGING ACTIVITIES OF FLAVONOID ISOLATED FROM MIMOSA PUDICA

IN-VITRO STUDIES OF IMMUNOMODULATORY AND FREE RADICAL SCAVENGING ACTIVITIES OF FLAVONOID ISOLATED FROM MIMOSA PUDICA

Joby Jose ¹, Sudheesh Sudhakaran ^{* 1}, A. T. Dhanya ¹, T. M. Sumesh Kumar ², Sony Jayaraman ² and E. Jayadevi Variyar ²

Department of Chemistry ¹, School of Chemical Sciences, Swami Anandatheertha Campus, Kannur University, Payyanur - 670327, Kerala, India.

Department of Biotechnology and Microbiology ², Palayad Campus, Kannur University, Kerala, India.

ABSTRACT: Flavonoid isolated from Mimosa pudica was evaluated for its radical scavenging and immunomodulatory activities in-vitro. Aim of this study is to isolate flavonoid from Mimosa pudica and to determine its antioxidant and immunomodulatory properties. Flavonoid was isolated from dried samples of Mimosa pudica using soxhlet extraction in 80% methanol and purified by column chromatography. Various tests were done for the presence of flavonoid, and the isolate was confirmed as flavonoid by Thin Layer Chromatography (TLC) and Fourier Transform Infrared (FT-IR) analysis. Antioxidant activity of the isolated flavonoid was evaluated using assays such as the 1, 1‑diphenyl‑2‑picryl‑hydrazil (DPPH) radical scav­enging assay and hydroxyl radical scavenging assay. In-vitro assay of immunomodulatory activity of flavonoid isolated from Mimosa pudica was done by using lymphocytes isolated from a human blood sample. The isolated flavonoid exhibited a dose-dependent enhancement in activity in DPPH assay and hydroxyl radical scavenging assays. In DPPH assay 10μg of ascorbic acid, equivalent activity was shown by 30μg of isolated flavonoid. The isolate exhibited strong DPPH radical scavenging activity with an IC₅₀ value of 56.32 μg/ml as compared to the IC₅₀ value of the reference standard, ascorbic acid 21.11μg/ml. In hydroxyl radical scavenging assay 100μg of quercetin equivalent activity was shown by 373.77μg of isolated flavonoid. Flavonoid isolated from Mimosa pudica showed in-vitro growth stimulatory effect on isolated normal lymphocytes, which was estimated by MTT assay. Percentages of the proliferation of lymphocytes at different concentrations were obtained. The results of the present study indicate that the flavonoid isolated from Mimosa pudica is a compound having potent antioxidant and immunomodulatory activities.

Flavonoids, Immunomodulation, Antioxidant, Mimosa pudica, MTT assay, Lymphocytes

INTRODUCTION: The Immune System is the most complex biological systems in the body designed to protect the host from invading pathogens.

Several herbal preparations that can enhance the immune system are extensively used, as they are free from side effects. The effect of immuno-modulators can be classified into three, which are; stimulation, suppression, and restoration of the immune response ^3-6.

Immunomodulators are used in clinical practices to stimulate and normalize an immune system activity. Immunomodulation using medicinal plants can provide an alternative to conventional chemotherapy for a variety of diseases particularly when host defense mechanism has to be activated under the conditions of the impaired immune response or when selective immunosuppression is desired in a situation like autoimmune disorders. The immunocorrective properties of immuno-modulators also can be successfully applied in the treatment of oncological diseases ^7-9.

Flavonoids are bioactive secondary metabolites present in plants and are reported to have various pharmacological activities. Flavonoids are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones ^{10, 11}. Biological activities of flavonoids include antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor, antithrombotic, hypolipidemic, and hypoglycemic activities ^12-14. Flavonoids have been reported to have potent antioxidant activities. Flavonoid helps to reduce oxidative stress that has been linked to cancer, aging, atherosclerosis, ischemic injury, inflammation, and neuro-degenerative diseases (Parkinson’s and Alzheimer’s) ^15-17. The role of flavonoids includes the inhibition of activation of pro-carcinogens, inhibition of proliferation of cancer cells, selective death of cancer cells by apoptosis, inhibition of metastasis and angiogenesis, activation of the immune response against cancer cells, modulation of the inflammatory cascade and the modulation of drug resistance ^18-20.

Mimosa pudica (Family: Leguminosae) is a small or middle sizes tree about 1.5m in height with leaves are very sensitive, both pinnae and leaflets folding when touched. It is reported to contain alkaloid, glycoside, flavonoids, and tannins. All parts of the plant are considered to possess medicinal properties. The plant has anti-microbial, anti-convulsant, hyperglycemic, anti-venom, diuretic, anti-cancer, antidiabetic, antiulcer, anti-fertility and anti-histaminic activities ^21-24. The medicinal plants have been the object of research in both systematic and advanced areas of plant sciences. Plant-derived natural products such as flavonoids, terpenoids, and steroids, etc. have received considerable attention in recent years due to their diverse medicinal benefits.

The aim and subject of many researchers is the discovery and development of isolating a new efficient, active, and less toxic molecule having medicinal benefits ^{25, 26}. The present study was designed to isolate flavonoid from Mimosa pudica and evaluate its immunomodulatory and free radical scavenging activities.

MATERIALS AND METHODS:

Collection of Plant Material: The whole plant Mimosa pudica was collected from Kannur district of Kerala, India. After selection, plants were taxonomically identified by Dr. Sujanapal P, Scientist, Kerala Forest Research Institute (KFRI), Thrissur, India.

Sample Preparation: The whole plant Mimosa pudica was thoroughly washed with water. They were chopped into small pieces, dried in the shade, grinded into powder form and stored in an airtight container.

Extraction and Isolation Procedure: The dried samples were Soxhlet extracted in 80% methanol (100ml/g dry weight) for 24hrs. The extracts were concentrated and reconcentrated in petroleum ether (40-60 ºC), ethyl ether and ethyl acetate. The ethyl acetate fractions, which contained the highest amount of flavonoids were subjected to Column Chromatography over silica gel (60-120 mesh). Gradient elution was conducted initially with n-hexane and gradually enriched with benzene, chloroform, ethyl ether, acetone, ethyl acetate, ethanol, methanol, and water successively in the order of increasing polarity ²⁷. Fractions were collected and combined based on their TLC patterns. The fractions were then dried and analyzed for flavonoids.

Test for Flavonoids: Shinoda Test: To the extract, added 5 ml of 95% ethanol and few drops of concentrated HCl. To this solution, 0.5g of magnesium turnings were added. Observance of pink coloration indicated the presence of flavonoids ²⁸. A small quantity of the extract was heated with 10 ml of ethyl acetate in boiling water for 3 min, and the mixture was filtered. The filtrate was then shaken with 1 ml of 1% aluminum chloride solution and observed for light yellow color. It indicated the presence of flavonoids. The yellow solution turns colorless on adding diluted NaOH and HCl, which confirmed the presence of flavonoids ²⁹.

Identification of Flavonoids by TLC: TLC was performed for the identification of flavonoids. The concentrated extracts were spotted on the lower side of the TLC plate (20 × 20 cm) precoated with silica gel G. the diameter of each spot was about 5mm. Then TLC was run one-dimensionally in the mobile phase solvent (ethyl acetate - methanol-water, 5:1:5, v/v/v) at room temperature ³⁰. The plates were developed and visualized under UV light ³¹.

FT-IR Analysis: The column purified samples were mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The sample pellet was placed into the sample holder, and FT-IR spectra were recorded (FT-IR Spectrometer-8400S Shimadzu).

In-vitro Antioxidant Activity:

DPPH Radical Scavenging Activity: Antioxidants react with 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), a stable free radical which is reduced to DPPH‐H and as a consequence the absorbance is decreased from the DPPH radical to the DPPH‐H form. On accepting hydrogen from a corresponding donor, the solution loses the characteristic deep purple color, and the discoloration was proportional to the concentration and scavenging activity of the compound³².

The free radical scavenging capacity of the flavonoid isolated from Mimosa pudica was determined using DPPH. The reaction mixture contained 2.8 ml of 100µM DPPH dissolved in methanol and various concentrations of compounds in 0.2 ml distilled water. This mixture was incubated at room temperature for 30 min. The mixture was shaken, and absorbance was read at 517 nm. Ascorbic acid was used as standard and the percentage of the DPPH scavenging effect was expressed as the percentage of inhibition from the given formula:

% inhibition of DPPH radical = [A₀-A₁/A₀] ×100

Where A₀ was the absorbance of the control and A₁ was the absorbance of the sample/standard. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. All tests were performed in triplicates ³³.

Hydroxyl Radical Scavenging Assay: The hydroxyl radical scavenging assay is based on the ability of compounds to compete with salicylic acid for OH radicals. The reaction mixture contains 1 ml of 1.5 mM FeSO₄, 0.7 ml of 6 mM hydrogen peroxide, 0.3 ml of 20 mM sodium salicylate, and 1 ml of various concentrations of the test compounds. The mixture was incubated for 1 h at 37 °C, and the absorbance of the hydroxylated salicylate complex was measured at 562 nm. Quercetin was taken as the standard for the assay. Lower absorbance of the reaction mixture indicated higher OH radical scavenging activity. All tests were performed in triplicates. Percentage scavenging of the OH radical was measured using the following equation, OH radical scavenging activity;

(%) = [A₀-A₁/A₀] × 100

Where A₀ was the absorbance of the control and A₁ was the absorbance of the sample/standard ³⁴.

In-vitro Lymphocyte Proliferation Assay: Fresh human blood samples were layered on equal volumes of Hisep^TM LSM solution and centrifuged at 400 x g for 20 min at 18˚C. The quality of the separation depends upon the sharp interphase between the blood and the solution. Carefully discarded most of the plasma and platelet-containing supernatant above the interface band. The thin white middle lymphocyte layer was collected and washed with PBS twice by centrifugation at 250 × g for 10 min at 18 ºC.

The supernatant was discarded, and the cells were suspended in RPMI medium. Briefly, 1×10⁴ cells ml^-1 seeded in each well of a 24 well plate and extracted at different concentrations were added and kept for 72hrs incubation at 37 ºC in a humidified 5% CO₂/95% air atmosphere.

After incubation, 20μl MTT (5mg/ml) was added to each well and incubation was continued for an additional 2 h. The insoluble formazan crystals formed were solubilized by the addition of 100μl MTT lysis buffer (SDS and dimethyl formamide) followed by an incubation of 4 h and the absorbance was measured at 570 nm using a microplate spectrophotometer ^{35, 36}.

RESULTS AND DISCUSSION: The methanol extract of Mimosa pudica showed the presence of a large number of compounds, fractionated with petroleum ether (40-60 ºC), ethyl ether and ethyl acetate. The ethyl acetate fraction, which contained the highest amount of flavonoids, was subjected to Column Chromatography. Gradient elution was conducted using solvents according to the increasing order of their polarity. Fractions were collected and tested for flavonoids and were found to be positive in acetone: ethanol (2:1 ratio). The fraction was then dried and subjected to TLC, which confirmed the presence of a single compound Fig. 1.

Fourier Transform Infrared (FT-IR) Analysis: FT-IR was used for identifying the functional groups and thereby confirming the isolate from Mimosa pudica was flavonoid Fig. 4. The functional groups present in the analyte will make vibrations of specific wave numbers. Quercetin and Rutin were used as flavonoid standards Fig. 2 and 3. The spectral analysis is shown in Table 1.

FIG. 1: HPTLC PROFILE OF FLAVONOIDS ISOLATED FROM MIMOSA PUDICA [A] UNDER UV 366nm

FIG. 2: FTIR SPECTRA OF QUERCETIN

FIG. 3: FTIR SPECTRA OF RUTIN

FIG. 4: FTIR SPECTRA OF FLAVONOID ISOLATED FROM MIMOSA PUDICA

TABLE: 1 FTIR SPECTRA ANALYSIS OF QUERCETIN, RUTIN AND ISOLATE FROM MIMOSA PUDICA

DPPH Radical Scavenging Assay: The radical scavenging activity of the flavonoid isolated from Mimosa pudica against experimentally generated DPPH ions showed marked scavenging activity even at low concentrations. The scavenging activity increased in a concentration-dependent manner. Ascorbic acid is used as the standard. 10μg of ascorbic acid equivalent activity was shown by 30μg of isolated flavonoid. The isolate exhibited strong DPPH radical scavenging activity with an IC₅₀ value of 56.32μg/ml as compared to the IC₅₀ value of the reference standard, ascorbic acid 21.11μg/ml. The percentage of radical scavenging property of the ascorbic acid standard and the flavonoid isolated from Mimosa pudica are given in Fig. 5.

Hydroxyl Radical Scavenging Assay: This assay shows the abilities of the flavonoid isolated from Mimosa pudica and quercetin standard to scavenge the hydroxyl radical generated. The scavenging activity increased in a concentration-dependent manner. The radical scavenging activity of flavonoid isolated from Mimosa pudica is promising and may aid in the prevention or reduction of the progress of various oxidative stress-induced diseases, thereby benefiting human health. 100μg of quercetin equivalent activity was shown by 373.77μg of isolated flavonoid.

The percentage of radical scavenging property of the quercetin standard and the flavonoid isolated from Mimosa pudica are given in Fig. 6.

Lymphocyte Proliferative Activity in-vitro: The effect of the flavonoid isolated from Mimosa pudica at various concentrations on normal lymphocytes was evaluated using the MTT assay. The compound was found to enhance the in-vitro growth of lymphocytes. The percentage of the proliferation of lymphocytes at different concentrations was given in Fig. 7. The results of this experiment show that the flavonoid isolated from Mimosa pudica exhibited higher growth stimulations even at low concentrations, indicating its potential as an effective immu­nomodulatory compound.

The FT-IR fingerprinting provided the presence of OH stretched phenol, C=O aryl ketone and C---C aromatic ring in an isolate from Mimosa pudica which confirmed the isolate was a flavonoid.

FIG. 5: DPPH RADICAL SCAVENGING ACTIVITY OF ASCORBIC ACID STANDARD AND FLAVONOID ISOLATED FROM MIMOSA PUDICA

FIG. 6: HYDROXYL RADICAL SCAVENGING ASSAY OF QUERCETIN STANDARD AND FLAVONOID ISOLATED FROM MIMOSA PUDICA

FIG. 7: LYMPHOCYTE PROLIFERATIVE ACTIVITY ASSAY OF FLAVONOID ISOLATED FROM MIMOSA PUDICA IN VITRO AFTER 72 h OF INCUBATION

CONCLUSION: The isolated flavonoid exhibited strong DPPH radical scavenging activity, hydroxyl radical scavenging activity and potent stimulatory action on lymphocytes. So this isolated antioxidant ngredient can be therapeutically used as a natural immunomodulator in disorders of immunological origin. Further investigations are under progress to find out the effect the isolated flavonoid on other immune parameters such as macrophage activity and NK cell activity and to characterize and elucidate the structure of the active principle behind the activity.

ACKNOWLEDGEMENT: The first author is greatly acknowledging Kannur University, Kerala, India for providing financial assistance (JRF Fellowship).

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Jose J, Sudhakaran S, Dhanya AT, Kumar TMS, Jayaraman S and Variyar EJ: In-vitro studies of immunomodulatory and free radical scavenging activities of flavonoid isolated from Mimosa pudica. Int J Pharm Sci & Res 2014; 5(10): 4254-61. doi: 10.13040/IJPSR.0975-8232.5(10).4254-61.

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