Phytochemical Analysis and In-Vitro Antioxidant Activity of Mimosa pudica Lin., Leaves

 

Rekha Rajendran*, R Hemachander, T Ezhilarasan, C Keerthana, DL Saroja, KV Saichand and Mohamed Gasim Abdullah

Department of Pharmacognosy and Phytochemistry, Mohamed Sathak A. J. College of Pharmacy, Medavakkam Road, Sholinganallur, Chennai – 600 119, Tamil Nadu, India.

 *Corresponding Author E-mail:  rekhacognosy@rediffmail.com

 

ABSTRACT:

The chloroform extract of Mimosa pudica Lin., leaves was screened for invitro antioxidant activity against free radical scavenging by DPPH, nitric oxide, super oxide dismutase and reducing power ability. The chloroform extract was also subjected to preliminary phytochemical analysis, high performance thin layer chromatographic analysis and fluorescence analysis. Mimosa pudica Lin., is traditionally used for its anti-hyperglycemic, antidiarrhoeal and cytotoxic properties. The results of the present study reveals that the chloroform extract of Mimosa pudica Lin., leaves showed a significant antioxidant activity against free radical scavenging by DPPH, nitric oxide, superoxide dismutase and reducing power ability.  The effects were comparable with the standard antioxidant ascorbic acid and these antioxidant properties were concentration dependent.  Preliminary phytochemical analysis revealed the presence of phytoconstituents such as steroids, flavonoids, glycosides, alkaloids, phenolic compounds, which is further confirmed by the thin layer chromatography and high performance thin layer chromatography and the results suggests that the chloroform extract of Mimosa pudica Lin., is a potential source of natural antioxidants and the extract have constituents which were capable of showing antioxidant activity and the said invitro antioxidant activity is due to antioxidant principle phenolic compounds and flavonoids. The significant antioxidant activity of chloroform extract of Mimosa pudica Lin., leaves was encouraging enough to pursue isolation and characterization of the active constituents which is responsible for the observed significant invitro antioxidant activity.

 

KEYWORDS: Mimosa pudica Lin., Antioxidant activity, free radical scavenging, chloroform extract.

 


INTRODUCTION:

In living systems, free radicals are generated as part of the body's normal metabolic process, and the free radical chain reactions are usually produced in the mitochondrial respiratory chain, liver mixed function oxidases, through xanthine oxidase activity, atmospheric pollutants and from transitional metal catalysts, drugs and xenobiotics. In addition, chemical mobilization of fat stores under various conditions such as lactation, exercise, fever, infection and even fasting, can result in increased radical activity and damage. Free radicals or oxidative injury now appears the fundamental mechanism underlying a number of human neurological and other disorders. Oxygen free radical can initiate peroxidation of lipids, which in turn stimulates glycation of protein, inactivation of enzymes and alteration in the structure and function of collagen basement and other membranes, and play a role in the long-term complication of diabetes 1.

 

Antioxidants may be defined as radical scavengers which protect the human body against free radicals that may cause pathological conditions such as ischemia, anaemia, asthma, arthritis, inflammation, neurodegeneration, Parkinson's diseases, mongolism, ageing process and perhaps dementias. Flavonoids and flavones are widely distributed secondary metabolites with antioxidant and antiradical properties 2, 3. Plants are potent biochemical factories and have been components of phytomedicine since times based natural constituents can be derived from any part of plant like bark, leaves, flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain active components . The beneficial medicinal effects of plant 2 materials typically result from the combinations of secondary products present in the plant. The medicinal actions of plants are unique to particular plant species or groups are consistent with this concept as the combination of secondary products in a particular plant is taxonomically distinct. Antioxidant based drugs/formulations for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer's disease, and cancer have appeared during the last three decades 4. This has attracted a great deal of research interest in natural antioxidants. Subsequently, a worldwide trend towards the use of natural phytochemicals present in berry crops, tea, herbs, oilseeds, beans, fruits, and vegetables have increased. Several herbs and spices have been reported to exhibit antioxidant activity, including rosemary, sage, thyme, nutmeg, turmeric, white pepper, chilli pepper, ginger, and several Chinese medicinal plants extracts. The majority of the active antioxidant compounds are flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins, and isocatechins. In addition to the above compounds found in natural foods, vitamins C and E, betacarotene are known to possess antioxidant activity. The importance of the reactive oxygen species (ROS) has attracted increasing attention over the last decade. Reactive oxygen species includes free radicals such as super oxide anion radicals (O 2 -•), hydroxyl radicals (OHŻ), non free radicals such as H2O2 and singlet oxygen (1O 2), along with various forms of activated oxygen. Several studies have reported that plants have potent antioxidant and represent an important source of natural antioxidants 5. Butyl hydroxyl anisol (BHA) and butyl hydroxyl toluene (BHT) are the most commonly used antioxidants, but both are suspected to cause liver damage 6. Many plants contain substantial amounts of antioxidants including Vitamin C and E, carotenoids, flavonoids, tannins and thus can be utilized to scavenge the excess free radicals from the human body.  Epidemiological studies have suggested the association between the consumption of antioxidant rich foods and beverages and the prevention of diseases. There is a lot of ongoing research on such plants for their potential usefulness as dietary supplements and as adjuvant for use in therapeutic management of stress related disorders.

 

Mimosa pudica Lin., (Mimosaceae) known as chue Mue, is a stout straggling prostrate shrubby plant with the compound leaves sensitive to touch, spinous stipules and globose pinkish flower heads, grows as weed in almost all parts of the country 7.  Leaves and stems of the plant have been reported to contain an alkaloid mimosaine, leaves also contain mucilage and root contains tannis7Mimosa pudica Lin., is used for its anti-hyperglycemic 8, anti-diarrhoeal 9, anti-convulsant 10 and cytotoxic properties 11.

 

The plant also contains turgorins, leaves and roots are used in the treatment of piles and fistula.  Paste of leaves is applied to hydrocele.  Cotton impregnated with juice of leaves is used for dressing sinus. Plants are also used in the treatment of sore gum and are used as a blood purifier7.  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.  Hence, the present studies were performed to screen invitro antioxidant activity of chloroform extract of Mimosa pudica Lin., leaves against various methods such as scavenging of DPPH radical, nitric oxide radical, superoxide anion radical and reducing power ability.

 

MATERIAL AND METHODS:

Plant material:

The leaves of Mimosa pudica Lin., were procured from the Thiruverkadu (near Poonamallee) in the month of October 2009.  The plant was identified by Prof. Jayaraman, Director, National Institute of Herbal Science, PARC, Chennai, Tamil Nadu, India. The voucher specimen (PARC/2009/403) was deposited at the department of pharmacognosy and phytochemistry M.S.A.J. College of Pharmacy, Medavakkam high road, Chennai - 600 119, Tamil Nadu, India.

 

Preparation of extract:

The coarsely powered leaves (300g) of Mimosa pudica Lin., was extracted to exhaustion in a soxhlet apparatus at 50°C with 500ml of chloroform.  The extract was filtered through a cotton plug, followed by Whatman filter paper (no.1) and then concentrated by using a rotary evaporator at low temperature (40-60°C) and reduced pressure to provide chloroform extractive of 22.8g.  The obtained extract was greenish brown in colour.

 

Preliminary phytochemical analysis:

The chloroform extract was then subjected to preliminary phytochemical 12 analysis to assess the presence of various phytoconstituents and it revealed the presence of phytoconstituents such as phenols, steroids, flavonoids, alkaloids and glycosides. Thin layer chromatographic studies also confirmed these phytoconstituents 13.

 

HPTLC analysis:

15µg of chloroform extract of Mimosa pudica Lin., was spotted on pre-coated silica gel TLC plate of dimension (10 x 6 cm size) (E. Merck) after activation at 105°C. Then the spotted plate was developed in a pre-saturated chamber containing the solvent system of Toluene: Ethylacetate (3:1) as the mobile phase conditions for separation. Developed plate was air dried and scanned under UV 254 nm using Camag densitometer and recorded the chromatogram. Then the plate was sprayed with spray reagent 1% vanillin sulphuric acid and heated at 105°C in hot air oven for 5 to 10 minutes to develop the color of the spots. After color development, the plate was again scanned under visible range at 550 nm and recorded the chromatogram. The retention factor was calculated by using the following formula,

 

                                         Migration distance of substance

Rf (Retention factor) = ------------------------------------------

                                          Migration distance of solvent

 

EVALUATION OF ANTIOXIDANT ACTIVITY:

Scavenging of DPPH radical14:

This assay is based on the measurement of the scavenging ability of antioxidant test extract towards the stable radical.  The free radical scavenging activity of the chloroform extract of leaves of Mimosa pudica Lin., was examined in-vitro using DPPH [1, 1-Diphenyl, 2-picryl-hydrazyl] radical. The test extract was treated with different concentration from a maximum of 1000µg/ml to minimum of 5µg/ml.  The reaction mixture consisted of 1ml of 0.1mMM DPPH in ethanol, 0.95ml of 0.05M Tris-HCl buffer (pHB-7.4), 1ml of ethanol and 0.05ml of the chloroform extract of leaves of Mimosa pudica Lin., The absorbance of the mixture was measured at 517nm exactly 30 sec after adding extract.  The experiment was performed in triplicate and percentage of scavenging activity was calculated using the formula 100-[100/blank absorbance x sample absorbance].  The blank was also carried out in similar manner, using distilled water in the place of extract.  The activity was compared with ascorbic acid, which was used as a standard anti-oxidant.

 

Figure - 1: TLC plates for chloroform extract of Mimosa pudica

  

Scavenging of nitric oxide15:

Sodium nitroprusside (5µM) in standard phosphate buffer solution was incubated with different concentration of the chloroform extract dissolved in standard 0.025M phosphate buffer (pH 7.4) and the tubes were incubated at 25°C for 5 hours. After 5 hours, 0.5ml of incubation solution was removed and diluted with 0.5ml of Griess reagent (prepared by mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in water). The absorbance of chromophore formed was read at 546nm. The experiment was performed in triplicate and percentage of scavenging activity was calculated using the formula 100-[100/blank absorbance x sample absorbance].  The blank was also carried out in similar manner, using distilled water in the place of extract.  The activity was compared with ascorbic acid, which was used as a standard anti-oxidant.

 

Scavenging of superoxide anion radical16:

The reaction mixture was prepared by dissolving Na 2 CO 3 (0.53 gm), EDTA (0.004 gm) and xanthine in 100 ml of distilled water. Ten ml of NBT solution (0.025 mM) was added to the reaction mixture. From this solution, 995 µl with xanthine was further added to chloroform extract and the reference compound, ascorbic acid in different concentrations in distilled water. After 15 minutes, the absorbance was measured using a spectrophotometer at 560 nm. The reaction mixture with xanthine oxidase was used as a control, whereas ascorbic acid was used as a reference compound. All tests were performed in triplicate. Percent inhibition was calculated by comparing the results of control and test sample.

 

Figure - 2: HPTLC profile for chloroform extract of Mimosa pudica at 260nm

 

Figure - 3: HPTLC profile for chloroform extract of Mimosa pudica at 550nm

 

Reducing power determination17:

Different amount of the chloroform extract was mixed with 2.5ml of 1% potassium ferriccyanate [K3Fe(CN)6].  The mixture was incubated at 50°C for 20minutes. 2.5ml of 10% trichloroacetic acid was added to the mixture, which was then centrifuged for 10 minutes at 1000rpm. 2.5ml upper layer of solution was mixed with 2.5ml of distilled water and 0.5ml of 0.1% ferric chloride.  The absorbance was measured at 700nm. The blank was also carried out in similar manner, using distilled water in the place of extracts. Increase in the absorbance of the reaction mixture indicated the increase in the reducing power.  The activity was compared with ascorbic acid, which was used as a standard antioxidant.

 

RESULTS:

The preliminary phytochemical screening revealed the presence of phytoconstituents such as glycosides, alkaloids, flavonoids and phenolic compound in the chloroform extract of Mimosa pudica Lin., leaves.  TLC and HPTLC analysis also confirmed these phytoconstituents. HPTLC profile is shown in table – 1 and the TLC plate for the chloroform extract is shown in Figure 1 and HPTLC peaks were shown in figure 2 and 3 at 260nm and 550nm.  The HPTLC finger print for chloroform extract showed 6 peaks at the wavelength 260nm and at 550nm, the chloroform extract showed 7 peaks and the total height and the area of the peaks were shown in table – 1.

 

Table - 1: HPTLC Finger Print Data for Chloroform Extract of Mimosa pudica

Extract

Wavelength

No. of peaks

Total Height

Total area

Chloroform

260

6

782.8

32750.1

550

7

13670

85680.4

 

Table - 2: Invitro free radical scavenging of Mimosa pudica by DPPH method

Concentration

(µg/ml)

Percentage inhibition  (Mean ±SEM) of triplicates

Chloroform extract

Ascorbic acid

5

1.96±0.036

0.39±0.038

10

2.36 ±0.064

0.65±0.084

20

2.88±0.032

3.01 ±0.032

40

3.93±0.015

3.67±0.015

100

6.16±0.011

15.72±0.011

200

7.47±0.044

47.84±0.044

400

12.97±0.084

84.14±0.073

600

25.03±0.072

89.52±0.073

800

46.52±0.056

93.45±0.084

1000

64.48±0.037

96.07±0.052

IC 50

625

65

 

Table - 3: Invitro free radical scavenging of Mimosa pudica by nitric oxide scavenging

Concentration

(µg/ml)

Percentage inhibition (Mean ±SEM) of triplicates

Chloroform extract

Ascorbic acid

5

5.20±0.020

2.82±0.042

10

7.51±0.032

3.92±0.016

20

9.75±0.082

5.83±0.042

40

10.42±0.078

6.95±0.064

100

18.61±0.066

10.76±0.084

200

22.20±0.042

23.54±0.016

400

24.99±0.032

28.36±0.004

600

26.79±0.045

30.04±0.015

800

31.16±0.056

33.02±0.008

1000

33.63±0.024

41.14±0.058

IC 50

40

10

 

DPPH radical is considered to be a model of lipophilic radical. A chain reaction in lipophilic radicals was initiated by lipid autoxidation ascorbic acid. The DPPH antioxidant 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 color. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance. The scavenging effects of chloroform extract and ascorbic acid on the DPPH radical are shown in table. The extract had significant scavenging effects on the DPPH radical, which increased with increasing concentration from 5-1000 µg/ml. The extract possesses statistically significant DPPH free radical scavenging activity.

 

Nitric oxide is an important chemical mediator generated by endothelial cells, macrophages, neurons and involved in the regulation of various physiological processes. The percent inhibition of nitric oxide generation by chloroform extract and ascorbic acid is shown in table - 3. The antioxidant activity of chloroform extract was comparable with the standard antioxidant and the extract showed the significant antioxidant action.

 

 

Table - 4: Invitro free radical scavenging of Mimosa pudica by superoxide anion radical scavenging

Concentration

(µg/ml)

Percentage inhibition (Mean ±SEM) of triplicates

Chloroform extract

Ascorbic acid

5

40.01±0.020

3±0.042

10

42.22±0.032

9±0.016

20

42.80±0.082

13±0.042

40

43.00±0.078

20±0.064

100

44.72±0.066

25±0.084

200

45.50±0.042

34±0.016

400

46.82±0.032

40±0.004

600

47.20±0.045

55±0.015

800

48.42±0.056

61±0.008

1000

49.65±0.024

75±0.058

IC 50

325

0.65

 

Table - 5: Invitro free radical scavenging of Mimosa pudica by reducing power ability determination

Concentration

(µg/ml)

Absorbance (Mean ±SEM) of triplicates

Chloroform extract

Ascorbic acid

5

0.054±0.001

0.088±0.002

10

0.117±0.001

0.162±0.001

20

0.230±0.001

0.280±0.003

40

0.310±0.002

0.390±0.001

100

0.404±0.002

0.496±0.002

200

0.512±0.001

0.564±0.001

400

0.602±0.003

0.694±0.001

600

0.714±0.001

0.760±0.001

800

0.820±0.001

0.898±0.001

1000

0.906±0.002

0.960±0.003

 

The chloroform extract was found to possess scavenging effects on superoxide anions, in a concentration dependent manner and the effects are shown in table – 4, thus showing strong superoxide radical scavenging activity, the activity was comparable with the standard antioxidant.

 

The reducing ability of a compound generally depends on the presence of reductants which have been exhibited antioxidative potential, breaking the free radical chain, donating a hydrogen atom. The presence of reductants (i.e. antioxidants) in the extract causes the reduction of the Fe 3+ /ferricyanide complex to the ferrous form. Therefore, the Fe 2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Table – 5 shows the reductive capabilities of the Mimosa pudica Lin., leaves and this effect was significant when compared to ascorbic acid. The reducing power of Mimosa pudica Lin., leaves extracts was very potent and the power of the extract was increased with quantity of sample.

 

DISCUSSION:

Most of the mammals have an inherent mechanism to prevent and neutralize the free radical induced damage. In biochemical system, superoxide radical and H 2 O2 react together to form a singlet oxygen and hydroxyl radical, this can attack and destroy almost all known biochemicals 18. The hydroxyl radical produced may cause sugar fragmentation, base loss and leakage of DNA strand 19. Hydroxyl radicals are the major ROS, causing lipid oxidation and enormous biological damage 20. It is apparent from the present study that the chloroform extract not only scavenges off the free radicals but also inhibits the generation of free radicals. It was already reported that naturally occurring phenolic compounds have free radical scavenging properties, due to their hydroxyl groups 21. Further, phenolic compounds are effective hydrogen donors, which make them antioxidant 22.

 

From the results it may be suggested that chloroform extract have hydrogen donor, thus scavenging the free radicals such as DPPH, Nitric oxide radical, superoxide anion radical and reducing power ability. Demonstration of the antioxidant potential of the plant extract, especially in view of the presence of rich spectrum of bio active molecules of therapeutic significance makes them likely candidates for bio activity guided fractionation of useful phytomolecules.  The result also supports the folklore claim of the usefulness of Mimosa pudica Lin., in various conditions such as hepatotoxicity, tumour, diabetes, microbial infections, inflammatory conditions etc.

 

It may be concluded that chloroform extract obtained from Mimosa pudica Lin., have significant antioxidant activity. The antioxidant potential may be attributed to the presence of Phytoconstituents such as flavonoids, glycosides, steroids, alkaloids and phenolic compounds. These results are encouraging enough to pursue isolation and characterization of chloroform extract of Mimosa pudica Lin., leaves.

 

ACKNOWLEDGEMENT:

Authors would like to thank the Department of Pharmacognosy and Phytochemistry, Mohamed Sathak A. J. College of Pharmacy, Chennai, for providing the necessary facilities for the completion of this study.

 

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Received on 30.11.2009       Modified on 28.01.2010

Accepted on 22.02.2010      © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 551-555