In-vitro Anti Oxidant and Antimicrobial Activities of Ethyl Acetate Extract of Evodia lunu-Ankenda (Gaertn) Merr. Bark

 

 

T Venkatachalam*, V Kishor Kumar, P Satheesh Kumar, P Kalaiselvi, M Chitra and N Senthil Kumar

Department of Pharmaceutical Chemistry, JKKMMRF College of Pharmacy,  B. Komarapalayam, Tamil Nadu, India.

 

ABSTRACT

In this paper, in vitro antioxidant and antimicrobial activities of ethyl acetate extract of Evodia lunu-ankenda (Gaertn) Merr. bark was determined by four methods Diphenylpicrylhydrazyl (DPPH), Nitric Oxide, Super oxide disumatase, Hydrogen peroxide (H2O2)  and by agar disc diffusion methods. The crude ethyl acetate extract of Evodia lunuankenda bark inhibited the growth of both gram positive bacteria (Bacillus substilis, Staphylococcus aureus and micrococcus luters) and gram negative bacteria (Escharichia coil, Pseudomonas aeruglinosa and Salmonella typhlmurium) and also the crude ethyl acetate extract of Evodia lunuankenda bark inhibited the growth of fungi (candida albicans and Aspergillus niger). The ethyl acetate extract of Evodia lunuankenda bark showed dose dependent increase in reducing anti oxidant power that was comparable to standards. Antibacterial activity, highest concentration of ethyl acetate extract (5mg/ml) having good activity against gram positive and gram negative  organisms  as compared with the reference Ciprofloxacin. Antifungal activity, highest concentration of extract (5mg/ml) having good activity as compared with the reference Amphotericin-B. It can be concluded that the plant posses potent antioxidant and antimicrobial activity, responsible of the secondary metabolites like flavonoids, saponins, are likely observed for the plants.

 

KEYWORDS: Antioxidant activity, antimicrobial activity, agar disc diffusion method, Evodia lunu-ankenda (Gaertn) Merr. bark.

 

 

INTRODUCTION

Evodia lunu-ankenda (Gaertn) Merr. bark (Rutaceae) available throughout central and south India, in most dry stony hills and black cotton soil. Along the margin of evergreen forests upto 1400 m. Trees ca.10m tall. Bark grey, corky when mature; blaze Brownish. Leaves compound, trifoliate, opposite, decussate; rachis 3.5-11 cm long, minutely pubescent when  young, pulvinate; petiolule 0.6-11 cm long, canaliculate, slightely pubscent ; leaflets 7-20X3-8.5 cm (usually larger in saplings) elliptic to obovate, apex acuminae, base assymetric, or slightly attenuate, argin entire chartaceous, glandular unctate, glabrous shining above; midrib slightly canalicualte; secondarynerves 7-16 pairs, straight or gradually curved; tertiary nerves slender, broadly reticulate. Inflorescence spreading panicled cyme. Flowers greenish white, sessile.  Follicles, 4-valved; 1- seeded cocci, black1-2. In the present study, we report the in vitro antioxidant and anti microbial activities of ethyl acetate extract of Evodia Lunu-ankenda. A review of the literature revealed that the in vitro antioxidant and anti microbial activities of the plant

 

 

EXPERIMENTAL:

Collection and authentication of plant:

The plant was collected from Tiruvandapuram, Kerala, India, in the month of March 2009.  The collected plants were identified by Department of Botany the American college Madurai.

 

 

 

TABLE NO: 1 ANTIOXIDANT ACTIVITY OF EVODIA LUNU-ANKENDA (Gaertn) Merr. BARK

EXTRCTS/

STANDARDS

DPPH

NO2

SO2

H2O2

Conc µg/ml

%Inhibition

Conc µg/ml

%Inhibition

Conc µg/ml

%Inhibition

Conc µg/ml

%Inhibition

Ethyl acetate extract of  Evodia Lunu-ankenda

125

75.00 ± 2.64

500

65.87 ± 2.50

125

83.58 ± 1.05

500

84.58 ± 0.98

62.5

64.25 ± 1.29

250

40.15 ± 1.25

62.5

75.16 ± 3.12

250

78.00 ± 2.74

31.25

59.50 ± 1.57

125

31.24 ± 3.56

31.25

40.50 ± 2.21

125

71.56 ± 1.04

15.63

47.35 ± 1.36

62.5

7.80 ± 0.65

15.63

27.54 ± 1.35

62.5

31.01 ± 1.15

7.81

17.88 ± 0.85

31.25

00

7.81

19.50 ± 1.65

31.25

6.50 ±  0.35

IC50 µg/ml

16.55 ± 0.89

345.52 ± 11.02

39.05 ± 1.33

97.82 ± 4.56

STANDARDS  IC50 µg/ml

Ascorbic acid

2.69 ± 0.05

-------

11.25 ±  0.49

187.33 ± 1.93

Rutin

-------

61.44 ±  2.56

0.51 ±  0.01

63.66 ±  0.22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE NO: 2 ANTI BACTERIAL ACTIVITY OF EVODIA LUNU-ANKENDA (Gaertn) Merr. BARK

Name of the Bacteria

Zone of inhibition in (mm)

ciprofloxacin 500µg/ml

1mg/ml

2mg/ml

3mg/ml

4mg/ml

5mg/ml

Staphylococcus aureus

20

4

6

8

10

16

Bacillus Subtilis

20

14

5

6

8

14

Micrococcus luteus

24

3

7

9

12

15

Escherichia coli

25

4

6

10

13

19

Pseudomonas aeruginosa

22

2

4

8

12

16

Salmonella typhi

26

4

8

10

14

18

 

 

TABLE NO: 3 ANTI FUNGAL ACTIVITY OF EVODIA LUNU-ANKENDA (Gaertn) Merr. BARK

Name of the Fungi

Zone of inhibition in (mm)

Amphotericin-B 500µg/ml

1mg/ml

2mg/ml

3mg/ml

4mg/ml

5mg/ml

Candida albicians

20

2

5

8

10

15

Aspergillus niger

22

3

5

8

11

14

 

 

 

 

 

 

 

Extraction procedure:

The barks of Evodia lunu-ankenda were shade dried under shade and then made in to a coarse powder with a mechanical grinder. The passed through sieve no 40 and stored in air tight container for further use. The bark (500mg) was first extracted with ethyl acetate in a soxhlet apparatus (48 hrs) the extract was concentrated  by distillation under reduced pressure using rotary flash evaporator to yield (5.12%w/w) a solid residue.

 

PRELIMINARY PHYTOCHEMICAL SCREENING:

Preliminary phytochemical screening of the ethyl acetate of Evodia lunu-ankenda showed the presence of Glycosides, Proteins, Phytosterols, Flavonoids, Phenolic Compounds, and Saponins.3-6

 

EVALUATION OF IN VITRO ANTI OXIDANT ACTIVITY7-13:

Diphenylpicrylhydrazyl (DPPH) radical scavenging activity, nitric oxide, superoxide dismutase, hydrogen peroxide (H2O2) radical scavenging activity was determined by following procedures

 

DPPH Radical scavenging activity:

0.1 mm solution of DPPH in ethanol was prepared and 1 ml of this solution was added to 0.3ml of extract solution in water at different concentration (10-1000μg/ml). After 30 minutes, the absorbance was measured at 517nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPH radical and the standard ascorbic acid and rutin was calculated using the following equation.

                                             Acont - Atest

Percentage inhibition  = ------------------   x 100
                                                  Acont

Where, Acont   is the absorbance of the control reaction and Atest is the absorbance in the presence of the sample of the extracts.

 

Nitric Oxide radical Scavenging activity:

The reaction mixture (6ml) containing sodium nitro prusside (10mM,4ml), phosphate buffer saline(PBS,pH7.4, 1ml)and extract in DMSO  at various concentrations or standard was incubated at 250°C for 150min. After incubation, 0.5 ml of the reaction mixture containing nitrite ion was removed, 1ml of sulphanillic acid reagent was added, mixed well and allowed to stand for 5min for completion of diazotization. Then, 1ml of NEDD was added, mixed and allowed to stand for 30min in diffused light. A pink colored chromophore was formed, ascorbic acid and rutin was used as the standard. The absorbance of these solutions was measured at 540nm. The % scavenging was calculated as mentioned under DPPH method.

 

Super Oxide disumatase radical Scavenging activity:

The plant extract dissolved in water were mixed. In the Tris-HCl buffer (3ml, 16mM, pH8.0) containing 1ml NBT (50um) solution, 1ml NADH (78um) solution. The super oxide radical generating reaction was started by the addition of 1ml of phenazine methosluphate (PMS) solution (10um) to the mixture. The reaction mixture was incubated at 250°C for 5min, and the absorbance was read at 560nm against corresponding blank samples, ascorbic acid and rutin was used as the standard. The % scavenging was calculated as mentioned under DPPH method..

 

H2O2 Radical scavenging activity:

A solution of H2O2 was prepared in phosphate buffer (pH 7.4). H2O2 concentration was determined spectroscopically measuring absorption with extinction coefficient for H2O2. Different concentrations of the extracts in distilled water were added to a H2O2 solution (0.6ml, 40mM). Absorbance of H2O2 at 230 nm was determined 10 min later against a blank solution containing the phosphate buffer without H2O2, ascorbic acid and rutin was used as the standard. The % scavenging was calculated as mentioned under DPPH method.

 

EVALUATION OF ANTIMICROBIAL ACTIVITY14,15:

Microorganisms and media:

The following bacterial strains used were Gram positive bacteria (Bacillus substilis, (ATCC 6633) Staphylococcus aureus (ATCC 25923) and micrococcus luters (ATCC 10240)) and Gram negative bacteria (Escharichia coil (ATCC 25922), Pseudomonas aeruglinosa (ATCC7853) and Salmonella typhlmurium (ATCC43579). The Fungal species used were Aspergillus niger (ATCC 16404) and candida albicans (ATCC 10231), for the present study. The bacterial and fungal cultures were maintained on Muller Hinton agar medium and sabouraud dextrose agar slants, respectively which were stored at 4ºC. Eight micro organism maintained on nutrient agar base were used to assess the antimicrobial activity of the plant extracts. The fungi were maintained on sabouraud dextrose agar, which is often used with antibiotic for the isolation of pathogenic fungi.

 

Antimicrobial screening:

Agar cultures of the test microorganisms were prepared as described as several; workers. Three to five similar colonies were selected and transferred  to 5 ml broth with a loop and the broth cultures were incubated for 24 hours at 37 ºC. The ethyl acetate extract of Evodia Lunu-ankenda were dissolved in dimethylsulfoxide with a magnetic stirrer. For screening sterile 6mm diameter filter paper disk were impregnated with 100-1000 µg of the ethyl acetate extract of Evodia Lunu-ankenda then placed Muller Hinton agar medium. The inoculum for each organism was prepared some broth cultures. The concentration of culture was 1x105 colony forming units per ml. The results were recorded by measuring the zones of growth inhibition surrounding the disc. Clear inhibition zones around the discs indicate the antimicrobial activity of extract. All data regarding antimicrobial activity are the average of triplicate analysis. The antibacterial ciprofloxacin (500µg/ml) and antifungal Amphotericin (B 500µg/ml) was used as standards as recommended by national committee for clinical laboratories standards.

 

RESULT AND DISCUSSION:

Anti oxidant activity:

The Evodia Lunu-ankenda (Gaertn) Merr. bark extract exhibited scavenging potential with IC 50 value of DPPH radicals (16.55 ± 0.89), NO2 radicals   (345.52 ± 11.02), SO2 radicals (39.05 ± 1.33), H2O2 radicals (97.82 ± 4.56) respectively. The values were found to be lesser than that standard Ascorbic acid (2.69 ± 0.05), (11.25 ± 0.49), (187.33 ± 1.93) and Rutin (61.44 ± 2.56), (0.51 ± 0.01), (63.66 ± 0.22) used as standards in respective assays in table no 1. The Evodia Lunu-ankenda (Gaertn) Merr. bark extract showed dose dependent increase in reducing antioxidant power that was comparable to standards.

 

Anti microbial activity:

Disc diffusion method is used extensively to investigate the antimicrobial activity of natural substances and plant extracts. These assays are based on the use of discs as reservoirs containing solutions of the substances to be examined. In the case of solutions with a low activity however a large concentration or volume is needed. Due to limited capacity of discs, holes or cylinders are preferably used.

 

Most of the bacteria species and fungi species were inhibited by the ethyl acetate extract of Evodia Lunu-ankenda (Gaertn) Merr. bark as shown in table no 2 and 3 is clearly demonstrates the antimicrobial activity against different microorganisms in different concentration like 1, 2, 3, 4 and 5mg/ml. The  highest concentration of ethyl acetate extract (5mg/ml) showed good activity against gram positive and gram negative  organisms (staphylococcus aureus, Bacillus Subtilis, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi) as compared with the reference Ciprofloxacin and also against fungus (candida albicians, aspergillus niger,) as compared with the reference Amphotericin-B.

 

CONCLUSION:

The Evodia Lunu-ankenda (Gaertn) Merr. bark belonging to family Rutaceae has been examined to gain an insight of its antioxidant and antimicrobial activity. The phytochemical investigation showed the presence of Glycosides, Proteins, Phytosterols, Flavonoids, Phenolic Compounds, and Saponins. The Evodia Lunu-ankenda (Gaertn) Merr. bark extract showed dose dependent increase in reducing antioxidant power that was comparable to standards. The highest concentrations of ethyl acetate extract Evodia Lunu-ankenda (Gaertn) Merr. bark (5mg/ml) showed good antibacterial and antifungal activity against different microorganism. As reported earlier, secondary metabolites like flavonoids, saponins, are likely responsible for the observed antimicrobial activity of plants. The data suggested that the extract containing compounds may be utilized as in vitro anti oxidant and antimicrobial agent. Further analysis including chemical characterization of isolated compounds, isolation of potent antioxidant marker from the extract is planned.

 

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Received on 11.09.2009

Accepted on 14.10.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(3): Nov. – Dec. 2009, 201-203