Free Radical Scavenging Activity of Leaves of Memecylon edule Roxb

 

S Sheela*, N Dora Babu and K Ilango

SRM College of Pharmacy, SRM University, Kattankulathur-603203, Tamil Nadu.

 

ABSTRACT

Free radical scavenging potential of leaves of Memecylon edule. Roxb was studied using superoxide radical scavenging effect, hydroxyl radical scavenging effect, lipid peroxide assay, nitric oxide scavenging effect. The content of superoxide radicals was studied by NBT reduction method, hydroxyl radical by Deoxy-ribose degradation method, lipid peroxide level by estimating the thiobarbituric acid releasing substances (TBARS) and the nitric oxide radical by Griess method. The ethanolic and aqueous leaf extracts were used for the study with reference to the ascorbic acid as standard drug. It was found that the aqueous extract showed significant free radical scavenging potential compared to the ethanolic extract. The IC50 for aqueous extract was found to be 502.9g/ml, 493.78g/ml, 469.6g/ml and 227.8 g/ml for superoxide radical scavenging effect, hydroxyl radical scavenging effect, lipid peroxide assay and nitric oxide scavenging effect respectively. The extracts studied can provide efficient antioxidant protection by complementary mechanisms, such as free radical scavenging and metal ions reduction with the non polar extracts.

 

KEYWORDS: Memecylon edule, superoxide radical scavenging effect, hydroxyl radical scavenging effect, lipid peroxide assay, nitric oxide scavenging effect.

 

INTRODUCTION:

Free radicals having broad ranges of effects in biological systems have drawn on the attention of many experimental works. Highly reactive free radicals, especially oxygen-derived radicals, which are formed by exogenous chemicals or endogenous metabolic processes in the human body, are capable of oxidizing biomolecules, resulting in cell death and tissue damage. Oxidative damage plays a significantly pathological role in human diseases. Cancer, emphysema, cirrhosis, atherosclerosis and arthritis have all been correlated with oxidative damage 1.Almost all organisms are well protected against free radical damage by enzymes such as superoxide dismutase and catalase or compounds such as ascorbic acid, tocopherol and glutathione 2. When the mechanism of antioxidant protection becomes unbalanced by factors such as ageing, deterioration of physiological functions may occur resulting in diseases and accelerating ageing. However, antioxidant supplements may be used to help the human body to reduce oxidative damage4. The safety of natural antioxidants have been much focused for its use to inhibit lipid peroxidation or to protect the human body from the oxidative damage by free radicals.Therefore our aim in this study is to evaluate the scavenging potential of extracts of Memecylon edule Roxb leaf.

 

Memecylon edule Roxb (Melastomataceae) is a small evergreen tree native to India, especially the Deccan Plateau, including most of Karnataka, Andhra Pradesh, and parts of Tamil Nadu. The common names include kaayam, delek bangas, delek air, miat, and nemaaru. Leaves and roots are used as medicine for dysentery and as astringent. The decoction of the wood is a powerful astringent. It is stated that the leaves of the plant Memecylon edule Roxb is used as powerful astringent., emmengogue. It contains tartaric and malic acids. Tonic for diarrhea4, 5.

 


The anti-inflammatory, analgesic and antioxidant activities of the leaves of Memecylon edule Roxb. was determined using in vitro for their interleukin-10 production; the most active fraction was further studied in vivo for its anti-inflammatory and analgesic activities using the ethylphenylpropiolate (EPP)-induced mouse ear edema and the writhing test with mice. All fractions except Hex were tested for their radical scavenging activity towards 1′-diphenyl-2-picrylhydrazyl radical (DPPH). The results provided support for the traditional claim of Memecylon edule Roxb leaves in relieving inflammation and pain6.

 

MATERIALS AND METHODS:

Plant material:

The leaves of the plant Memecylon edule Roxb were collected from Chittoor district of Andhra Pradesh, India. The plant material was taxonomically identified by Prof. P.Jayaraman, Director, Plant Anatomy Research Center, Chennai, Tamil Nadu, India. A voucher specimen has been preserved in our laboratory for future reference. The collected plant material was dried under shade and then powdered with a mechanical grinder and stored in an air-tight container. The dried powder material of the leaves was defatted with petroleum ether and the marc thus obtained was then extracted separately with distilled water (by cold maceration) and 75% ethanol (using Soxhlet apparatus). The solvent was completely removed under reduced pressure and a semisolid mass was obtained. The dried MWE and MAE (water and ethanolic extracts of Memecylon leaves respectively) were dissolved in distilled water and used for the present study.

 

Chemicals:

Deoxy ribose, nitro blue tetrazolium, thiobarbituric acid, trichloro acetic acid, EDTA, was purchased from Sigma Chemical Co. Ltd, USA. All other unlabeled chemicals and reagents were of analytical grade and were used without further purification.

 

Superoxide radical scavenging effect:

Superoxide radical scavenging activity was studied by. Alkaline DMSO method7. The alkaline DMSO (1% in 5mM NaOH) was added to the reaction mixture containing nitro-blue tetrazolium (NBT 0.1mg) and MWE and MAE at various concentrations (100, 250, 500, 750, 1000μg). The absorption was determined at 560nm. The reduction of NBT by the superoxide radical generated was calculated in presence and absence of the test drugs. In this study, thiourea (20mM) was used as the reference standard.

 

Hydroxyl radical scavenging effect:

This study was conducted by measuring the inhibition of deoxyribose degradation in presence of the leaf extracts. Hydroxyl radical was generated by Fe-EDTA and H2O2 in presence of ascorbic acid. MWE and MAE were added in various concentrations (100, 250, 500, 750, 1000μg) to a reaction mixture containing deoxyribose (3mM); FeCl3 (0.1 mM), EDTA (0.1mM), ascorbic acid (0.1mM) H2O2 (2mM) in phosphate buffer(20 mM/pH- 7.4) to make a final volume of 3ml. To this mixture trichloroacetic acid and thiobarbituric acid (0.5ml each) were added and measured the absorbance at 532nm. The percentage of hydroxyl radical inhibition and IC50 were determined by the method of Halliwell et.al8. Catechol solution was used as a reference standard.

 

Lipid peroxide scavenging effect:

In this study the liver tissue homogenate was adjusted to 10mg/ml. The effect of leaf extracts on lipid peroxides was estimated as malondialdehyde by thiobarbituric acid (TBA) method9, 10. To the reaction mixture containing MWE and MAE at various concentrations (100, 250, 500, 750, 1000μg). 1ml of liver tissue homogenate and 1 ml of HCl- trichloroacetic acid thiobarbituric reagent was added. The mixture was heated for 5 minutes in a water bath at 370C, and after cooling, the flocculent precipitate was removed by centrifugation at 1000rpm for 10 min. The absorbance of the supernatant liquid was measured at 532nm against blank and the lipid peroxide content was determined using the extinction coefficient 1.5l 10-6m-1 cm-1. The final result was expressed as nanomoles of malondialehyde per mg of protein. Vitamin E at a concentration of 50mcg/ml was used for comparing the results.

 

Nitric oxide radical scavenging effect:

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions which were measured by the Griess reaction (11, 12). The reaction mixture (3mL) containing sodium nitroprusside (10 mM) in phosphate buffered saline (PBS) and the MWE and MAE in different concentrations (10, 25, 50, 75, 100 and 125μg) were incubated at 25oC for 150 minutes. Every 30 minutes, 0.5 ml of the incubated sample was removed and 0.5 ml of Griess reagent (1% sulphanilamide, 0.1% naphthylethylene diamine dihydrochloride in 2% H3PO4) was added. The absorbance of the chromophore formed was measured at 546 nm. All the analyses were performed in triplicate and the results were averaged. The percentage inhibition of nitric oxide generated was measured by comparing the absorbance values of control and test. Curcumin was used as a reference compound.

 

RESULTS:

The Table: 01 shows the superoxide scavenging effect of MWE and MAE and Thiourea on the alkaline DMSO-NBT system. The decrease of absorbance at 560nm with antioxidants thus indicates the consumption of superoxide anion in the reaction mixture. MWE at concentration from 100-1000 μg/ml inhibited the production of superoxide anion radicals by 19.45 -63.75 %.. The IC50 value of MWE on superoxide radical scavenging activity was found to be 502.9μg/ml and for MAE was 910.5μg/ml. MWE had strong superoxide radical scavenging activity compared to that of MAE. Table: 02 shows the hydroxyl radical scavenging activity of MWE and MAE along with positive control Catechin. The concentration of MWE and MAE needed for 50% inhibition was found to be 493.78 and 963.2 μg/ml respectively. Table: 03 exhibits the reduction of lipid peroxide level in terms of thiobarbituric acid releasing substances by MWE and MAE with reference to ascorbic acid. It was found that the concentration required for reducing the lipid peroxide level for MWE and MAE are 469.6 and 982.7μg/ml respectively. Table: 04 represents the percentage inhibition of nitric oxide generation by MWE and MAE. Curcumin was used as a reference compound. The IC50 value of MWE was found to be 227.8 and 940.9 μg/ml respectively. The results reveal that MWE shows significant free radical scavenging effect and comparable with that of standard drugs, whereas the MAE does not have potential effect in scavenging the free radicals.

 

 


TABLE: 01 Superoxide radical scavenging effect of leaves of Memecylon edule Roxb.

SL.NO.

CONCENTRATION OF DRUG g/ml

PECENTAGE SCAVENGING EFFECT

IC50g/ml

MWE

MAE

MWE

MAE

1.                     

1000

63.75 0.31

54.91 0.33

502.9

910.5

2.                     

750

59.74 0.29

40.47 0.27

3.                     

500

49.71 0.22

27.90 0.26

4.                     

250

39.82 0.19

23.80 0.28

5.                     

100

19.45 0.09

17.76 0.15

6.                     

Thio urea (20 mM)

55.06 0.306

-

n= 5. MWE = Memecylon edule water extract. MAE= Memecylon edule ethanolic extract.

 

TABLE: 02 Hydroxyl radical scavenging effect of leaves of Memecylon edule Roxb.

SL.NO.

CONCENTRATION OF DRUG g/ml

PERCENTAGE SCAVENGING EFFECT

IC50g/ml

MWE

MAE

MWE

MAE

1.                     

1000

77.75 1.73

51.91 1.34

493.78

963.2

2.                     

750

56.74 2.12

36.47 0.98

3.                     

500

50.71 0.56

27.90 0.45

4.                     

250

39.82 0.12

23.80 0.36

5.                     

100

23.76 0.34

19.45 0.21

6.                     

Catechin (0.01mM)

54.46 1.63

-

n= 5. MWE = Memecylon edule water extract. MAE= Memecylon edule ethanolic extract.

 

TABLE: 03 Lipid peroxide scavenging effect of leaves of Memecylon edule Roxb.

SL.NO.

CONCENTRATION OF DRUG g/ml

TBARS(105 nano moles)

PERCENTAGE SCAVENGING EFFECT

IC50g/ml

 

 

 

MWE

MAE

MWE

MAE

MWE

MAE

1.                     

1000

1.680 0.21

1.208 0.49

57.62

50.88

 

 

469.6

 

 

982.7

2.                     

750

1.745 0.19

1.107 0.54

55.98

38.57

3.                     

500

1.854 0.14

1.098 0.43

53.23

21.27

4.                     

250

2.287 0.22

1.987 0.26

42.32

16.58

5.                     

100

3.008 0.15

2.487 0.21

24.13

14.96

6.                     

Control

3.956

-

-

7.                     

Ascorbic acid (50 g/ml)

1.91 0.27

51.82 0.92

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

n= 5. MWE = Memecylon edule water extract. MAE= Memecylon edule ethanolic extract.


TABLE: 04
Nitric oxide radical scavenging effect of leaves of Memecylon edule Roxb.

SL.NO.

CONCENTRATION OF DRUG g/ml

PERCENTAGE SCAVENGING EFFECT

IC50 g/ml

MWE

MAE

MWE

MAE

1.                     

1000

68.95 0.39

53.14 1.96

227.8

940.9

2.                     

750

62.83 0.33

47.91 1.65

3.                     

500

61.29 0.25

45.91 0.87

4.                     

250

54.85 0.17

39.54 0.59

5.                     

100

51.82 0.013

37.85 0.34

6.                     

Curcumin (50g/ml)

78.5 0.036

-

n= 5. MWE = Memecylon edule water extract. MAE= Memecylon edule ethanolic extract.


 


 

DISCUSSION:

The present study has shown the confirmation of considerable free radical scavenging potential of the leaf extracts of Memecylon edule Roxb especially water extract. Superoxides are produced from molecular oxygen due to oxidative enzymes 13 of body as well as via non enzymatic reaction such as autoxidation by catecholamines 14. In the present study, superoxide radical reduces NBT to a blue colored formazan that is measured at 560 nm 15. Table 01 shows the superoxide scavenging effect of MWE and MAE and curcumin on the alkaline DMSO-NBT system. The decrease of absorbance at 560nm with antioxidants thus indicates the consumption of superoxide anion in the reaction mixture. MWE had strong superoxide radical scavenging activity compared to MAE.

 

Hydroxyl radicals are the major active species causing lipid oxidation and enormous biological damage 16, 17. The deoxyribose method is a simple assay to determine the rate constants for reactions of hydroxyl radicals 18. Ferric-EDTA was incubated with H2O2 and ascorbic acid at pH 7.4. Hydroxyl radicals were formed in free solution and were detected by their ability to degrade 2-deoxy-2-ribose into fragments that on heating with TBA at low pH form a pink chromogen 19, 20. Any hydroxyl radical scavenger added to the reaction would compete with Deoxy ribose for the availability of hydroxyl radicals, thus reducing the amount of MDA formation. In this study it was found that MWE had good scavenging effect compared to that of MAE and the results were comparable with the standard anti-oxidant.

 

Lipid peroxidation (LPO) is a free radical-related process that in biologic systems may occur under enzymatic control, e.g., for the generation of lipid-derived inflammatory mediators, or non enzymatically. This latter form is associated mostly with cellular damage as a result of oxidative stress, which also involves cellular antioxidants in this process. In this study, measurement of malondialdehyde (MDA) is a marker of free radical induced lipid peroxidation which may provide possible information on radicals accentuated structural membrane injury. The detection of MDA was measured as MDA-thiobarbituric acid adduct from the tissue homogenate used. This study shows that MWE had good effect on reducing lipid peroxidation on the tissue homogenate used. The results were comparable with the standard ascorbic acid.

 

Nitric oxide (NO) is an important chemical mediator generated by endothelial cells, macrophages, neurons, etc. and is involved in the regulation of various physiological processes21. Excess concentration of NO is associated with several diseases 22, 23. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxynitrite anions, which act as free radicals24, 25. In the present study, the extract competes with oxygen to react with nitric oxide and thus inhibits generation of the anions. Table 04 represents the percentage inhibition of nitric oxide generation by MWE and MAE. Curcumin was used as a reference compound. MWE showed significant scavenging effect on nitric oxide radicals than the MAE.

 

Our study reveals that the water extract of the leaves of Memecylon edule Roxb has potential capacity in eradicating the free radicals than the ethanolic extract. Further studies are needed to better characterize the active constituents of the plant responsible for eradicating free radical scavenging activity.

 

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Received on 12.07.2009

Accepted on 14.08.2009

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Research Journal of Pharmacognosy and Phytochemistry. 1(2): Sept. - Oct. 2009, 109-112