INTRODUCTION:

Herbal remedies have been used for decades and centuries Plants have been used to treat or prevent illness since before recorded history. The sacred Vedas dating back between 3500 B.C and 800 B.C give many references of medicinal plants. Plants and plant-based medicaments are the basis of many of the modern pharmaceuticals¹. In the history of ancient civilizations, the use of medicinal herbs for curing diseases has been documented. Even today, herbal source is the most reliable foundation to discover the new chemical entities for the health care products. The evidences of the utilization of herbals in therapeutics are well documented in Indian system of medicine².

According to world health organization (WHO), more than 80% of the world’s population relies on traditional medicines for their primary health care needs. The purpose of this work is to identify the active ingredient in Mimosa pudica through scientific methods.

Mimosa pudica belongs to the family Mimosaceae, is a stout stragling prostrate shrubby plant with the compound leaves sensitive to touch, spinous stipules and globose pinkish flower heads, and grows as weed in almost all parts of the country³. Leaves and stems of the plant have been reported to contain an alkaloid mimosine, leaves also contain mucilage and root contains tannins. Mimosa pudica is used for its anti-hyperglycemic⁴, anti-diarrhoeal⁵, anti-convulsant⁶and cytotoxic properties⁷. The plant also contains turgorins, leaves and roots are used in the treatment of piles and fistula. Plant is also used in the treatment of sore gum and is used as a blood purifier³. In ayurvedic and unani system of medicine, this plant has been used in diseases arising from corrupted blood and bile, bilious fever, piles, jaundice, leprosy, ulcers, small pox.

The medicinal value of plants lies in some chemical substances that produce a definite physiologic action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds. The phytochemical research based on ethno-pharmacological information is generally considered an effective approach in the discovery of new anti-infective agents from higher plants. Knowledge of the chemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information may be of value in disclosing new sources of such economic materials as tannins, oils, gums, precursors for the synthesis of complex chemical substances. In addition, the knowledge of the chemical constituents of plants would further be valuable in discovering the actual value of folkloric remedies⁸. Chemically constituents may be therapeutically active or inactive. The ones which are active are called active constituents and the inactive ones are called inert chemical constituents⁹. In this view, the present study deals with chemical profile and antioxidant activity to authenticate the medicinal properties of Mimosa pudica.

MATERIALS AND METHIOD:

Preparation of Extract:

The whole plants of Mimosa pudica were procured in Western Ghats of Siruvani hills, Coimbatore. The aerial parts of the plants are shade dried and coarsely powdered. The powdered plant material was defatted with petroleum ether (60-80®C) by Soxhlet extractor. The marc was further extracted with ethanol for 72 hours. Extract was filtered and concentrated under reduced pressure. The crude extract was kept in sample bottles and stored in refrigerator.

Chemicals:

L- Ascorbic acid, Gallic acid, Folin ciocalteu reagent 1, 1 ‐ diphenyl‐2‐picryl hydrazyl (DPPH) was obtained from Sigma chemicals. All other reagents including solvents used were of analytical grade.

Phytochemial Screening:

A qualitative phytochemical tests were carried out on the ethanol extract of Mimosa pudica using standard procedures to identify the presence of phenols, Flavonoids, Glycosides, Tannins, Resins, Carbohydrates and steroids as described by Sofowora¹⁰ and Trease¹¹ .

Estimation of total Phenols:

Total phenolic content of whole plant of Mimosa pudica was measured based on Folin- Ciocalteu assay as described by kahkonen¹². Briefly, 1.2ml of sodium carbonate (7.5% w/v) was added to the 5 gm of Ethanolic extract of plant. After 30 min, absorbance was measured at 765nm with UV/Vis spectrophotometer. Total phenolic content was expressed as mg Gallic acid equivalents (GAE) /g fresh weight. The total content of phenolic compounds in the extract in Gallic acid equivalents (GAE) was calculated by the following formula:

T = C. V / M

Where, T = total content of phenolic compounds, milligram per gram plant extract, in GAE; C = the concentration of Gallic acid established from the calibration curve (mg/ ml) V = the volume of extract (ml), M = the weight of ethanolic plant extract (gm)

Determination of Antioxidant Activity:

DPPH Radical Scavenging Assay:

The antioxidant activity of Mimosa pudica ethanolic extract was determined using the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay as described by Lee¹³. The free radical scavenging capacity of the ethanol extract of Mimosa pudica was determined using DPPH. DPPH solution (0.004% w/v) was prepared in 95% methanol. The crude ethanol extract of Mimosa pudica was mixed with 95% methanol to prepare the stock solution (10 mg/100ml). The concentration of plant extract solution was 10 mg /100 ml or 100μg/ml. From stock solution 2ml, 4ml, 6ml, 8ml and 10ml of this solution were taken in five test tubes and by serial dilution with methanol and was made the final volume of each test tube up to 10ml whose concentration was then 20μg/ml, 40μg/ml, 60μg/ml, 80μg/ml and 100μg/ml respectively. Freshly prepared DPPH solution (0.004% w/v) was added in each of these test tubes containing Mimosa pudica extract (20μg/ml, 40μg/ml, 60μg/ml, 80μg/ml and 100μg/ml) and after 10 min, the absorbance was taken at 517 nm using a spectrophotometer. Ascorbic acid was used as a reference standard and dissolved in distilled water to make the stock solution with the same concentration (10 mg/100ml or 100μg/ml) of extract of Mimosa pudica. Control sample was prepared containing the same volume without any extract and reference ascorbic acid. 95% methanol was used as blank.

Scavenging activity (%) =

(Control _OD – Control _Sample / Control _OD) X 100

A percentage inhibition versus concentration curve was plotted and the concentration of the extract required for 50% inhibition of radicals was expressed as IC 50 values (μg/ ml).

Reducing power determination:

The reducing power of Mimosa pudica was determined according to the method of Nabavi¹⁴. 2.5 ml of extract (50-800 mg /ml) in water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN) 6] (2.5 ml, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture to stop the reaction, which was then centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Vitamin C was used as positive control.

HPTLC Analysis for Flavonoids:

A densitometric HPTL:C analysis was performed for the development of characteristic finger printing profile as described by Sasikumar et al¹⁵. The ethanolic extract of M. pudica was centrifuged at 3000rpm for 2min. This solution was used as test solution for HPTLC analysis. 0.2µl of test solution and 3µl of standard solution (3mg in 10ml methanol) was loaded as 6mm band length in the 3 x 10 Silica gel 60F₂₅₄TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument. The samples loaded plate was kept in TLC twin trough developing chamber (after saturated with Solvent vapour) with respective mobile phase (Flavonoids) and the plate was developed in the respective mobile phase (Ethyl Acetate-Butanone- formic Acid- water 5:3:3:1) up to 90mm. The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at White light, UV 254nm and UV 366nm. The developed plate was sprayed with respective spray reagent (1% ethanolo Aluminium chloride) and dried at 100°C in Hot air oven. The plate was photo-documented in Day light and UV 366nm mode using Photo-documentation (CAMAG REPROSTAR 3) chamber. Before derivatization, the plate was fixed in scanner stage and scanning was done at UV 254nm. The Peak table, Peak display and Peak densitogram were noted

Statistical Analysis:

All the assay such as total phenolic content, DPPH and reducing power determination were done in triplicate in the ethanolic extract. The data obtained was subjected to one way analysis variance (ANNOVA) using the SPSS (Version 16.0 Tokyo, Japan)

Result and Discussion:

Preliminary phytochemical Screening:

Table 1: Phytochemical Screening of M. pudica

S. No

Phytochemical Tests

Ethanolic extract

10.

Proteins

Biuret Test

Millon’s Test

Carbohydrates

Fehling’s Test

Benedict’s Test

Alkaloids

Wagner’s Test

Meyer’s Test

Flavonoids

Dilute Hcl

Phenols

Ferric Chloride Test

Lead Acetate test

Steroids

Libermann’s Test

Saponins

Sodium Bicarbonate

Glycosides

Resins

Tannins

Ferric Chloride Test

Lead Acetate Test

+ Present; - Absent

The preliminary phytochemical screening showed the presence of Saponins, phenols, Glycosides, Resins, Tannins, Thiols, Flavonoids and Carbohydrates (Table 1). It has been mentioned that antioxidant activity of plants might be due to their phenolic compounds¹⁶.The presence of polyphenolic compound in ethanolic extract of Mimosa pudica may be responsible for the free radical scavenging activity.

Total Phenolic content:

Phenols are the very important plant constituents because of their scavenging ability. It may contribute directly to antioxidant activity. Total phenol compounds, as determined by Folin Ciocalteu method, are reported as Gallic acid equivalents by reference to standard curve. The total phenolic content of Mimosa pudica ethanolic extract was found to be 7.24± 2.5 µg/ ml compared to Gallic acid equivalents

DPPH Radical Quenching Activity:

DPPH (2,2-diphenyl-1-picrylhydrazyl) is an easy, rapid and sensitive method for the antioxidant screening of plant extract. This assay is based on the ability of DPPH a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron, which is responsible for the absorbance at 517 nm and also for visible deep purple colour. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance¹⁷.

Comparison of the antioxidant activity of the extract and the standard ascorbic acid is shown in Figure 1. The extract showed significant dose dependent inhibition of DPPH. The IC50 values were found to be 46 µg/ml and 64 µg/ml in M. pudica and Ascorbic Acid respectively.

The effect of antioxidants on DPPH is thought to be due to their hydrogen donating ability. Though the DPPH radical scavenging abilitiy of the ethanolic extract of Mimosa pudica was less than those of ascorbic acid, the study showed that the extract have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants

Figure 1: DPPH radical scavenging activity of ethanolic extract of Mimosa pudica as compared to standard Ascorbic acid.

Reducing Capacity:

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The reducing power of M. pudica ethanolic extract was very potent and it was increased with quantity of the sample. The extract caused significant elevation of reducing power when compared to the standard ascorbic acid, which is shown in Figure 2.

Figure 2: Reducing power of Mimosa pudica ethanloic extract as compared to Standard Ascorbic Acid.

In the reducing power assay, the presence of reductants (antioxidants) in the samples would result in the reduction of Fe³⁺ to Fe²⁺ by donating an electron. Amount of Fe²⁺ complex can then be monitored by measuring the formation of Perl's Prussian blue at 700 nm¹⁸. The ethanolic extract of Mimosa pudica could reduce the most Fe3+ ions, than the standard ascorbic acid. The absorbance of the reaction mixture was found to be increased which was an indication of increased reducing power of the ethanolic extract.

HPTLC Finger printing profile:

HPTLC finger print of the ethanolic extract gave eight spots at the following Rf values 0.02, 0.24, 0.28, 0.34, 0.48, 0.56, 0.67, 0.78, 0.95. R_f value, height and area of the peaks are presented in table 2. Among them peaks 2, 3, 4, 5, 6 and 9 were found as flavonoids. HPTLC is the quality assessment for the evaluation of broad number of biological compounds both efficiently and effectively. The corresponding HPTLC finger prints are shown in Figure 3. The extracts were run along with the standard flavonoid compounds (Rutin). Blue and yellow colour zones were detected in the chromatograph as well as in UV after derivatization (Figure 4 and 5) which indicates the presence of flavonoids.

Table 2: Peak table with R_f values, height, area of flavonoids and unknown compounds:

Track	Peak	R_fValue	Height	Area	Assigned substance
Sample A	1	0.02	364.6	6890.5	Unknown
Sample A	2	0.24	131.1	4515.1	Flavonoid 1
Sample A	3	0.28	96.9	3162.1	Flavonoid 2
Sample A	4	0.34	60.5	3747.8	Flavonoid 3
Sample A	5	0.48	71.6	4009.0	Flavonoid 4
Sample A	6	0.56	95.4	5183.7	Flavonoid 5

Sample A	7	0.67	25.8	463.3	Unknown
Sample A	8	0.78	23.8	732.6	Unknown
Sample A	9	0.95	168.0	6901.5	Flavonoid 6
RUT	1	0.32	287.0	8516.8	Rutin standard

Figure 3: HPTLC finger print profile of Mimosa pudica

Figure 4: Chromatograms of Mimosa pudica ethanolic extract in HPTLC analysis before derivatization

Figure 5: Chromatograms of Mimosa pudica ethanolic extract in HPTLC analysis after derivatization

CONCLUSION:

The result obtained in the present study shows that the ethanolic extract of whole plant of Mimosa pudica contains significant amount of phenolic compounds which exhibit higher antioxidant and free radical scavenging activities. This indicates that this plant is a source of natural antioxidants. However, the compounds responsible for this activity are still unknown. Therefore, further studies are needed to isolate the compounds responsible for antioxidant property. It is also observed that the ethanolic extract of Mimosa pudica has 8 peaks in HPTLC chromatogram which has been identified as flavonoids. HPTLC fingerprinting is proved to be a linear, precise, accurate method for herbal formulation. The developed HPTLC fingerprints will be useful for quality control and standardization of herbal drug formulation.

ACKNOWLEDGEMENT:

The authors are grateful to the Chancellor (Dr. Paul Dhinakaran), the vice chancellor (Dr. Paul P. Appasamy) and the Registrar (Dr. Anne Mary Fernandez) of Karunya University, Coimbatore, India, for giving encouragement and providing technical support to carry out this research.

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Received on 25.03.2011 Modified on 03.04.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1090-1094