Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(2): 359-364 (2017)

Antibacterial and antioxidant activity of leaf organic

extracts of local cultivars of

Murraya koenigii

(L.)

Spreng from Tamilnadu

M. Kavitha

School of Biosciences and Technology, VIT University, Vellore-632014, Tamilnadu, India

ABSTRACT

The aim of the present study was to evaluate the antibacterial activity against  ve bacterial pathogens and anti-

oxidant activity of three organic extracts prepared from the leaves of local cultivars of Murraya koenigii. Agar well

diffusion was utilized to determine the zone of inhibition. Broth microdilution was employed to  nd out the minimal

inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Anti-oxidant activity was analysed

using 2,2-Diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging assay. The organic extracts displayed promis-

ing antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Ethyl alcohol

extract presented highest inhibitory activity when compared to hexane and chloroform extracts. In relation to agar

diffusion method, the ethyl alcohol extract displayed lowest MIC and MBC value of 12.5 and 25 mg ml

-1

against

Staphylococcus aureus and same MIC and MBC value of 25 and 50 mg ml

-1

against Escherichia coli and Pseudomonas

aeruginosa. The radical-scavenging activity of ethyl alcohol extract (IC

= 30 μg ml

-1

) is very close to that of the

standard compound, gallic acid (IC

value of 35 μg ml

-1

). The results suggested that the local cultivars of M. koenigii

carry bene cial health effects in terms of antimicrobial and anti-oxidant activities and could be used as a promising

dietary supplement.

KEY WORDS: MURRAYA KOENIGII; ETHYL ALCOHOL EXTRACT; CARBAZOLE ALKALOID; ANTIBACTERIAL ACTIVITY; ANTIOXIDANT ACTIVITY

359

ARTICLE INFORMATION:

*Corresponding Author: mkavitha1972@gmail.com

Received 27

Nov, 2017

Accepted after revision 26

Sep, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.3/4

360 ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LEAF ORGANIC EXTRACTS OF LOCAL CULTIVARS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

M. Kavitha

INTRODUCTION

Spices and herbs which are used in food preparations

traditionally are well known to contain several health

promoting bioactive substances. Herbal medicines are

used alternatively in many countries to cure many ail-

ments (Kamboj, 2000). Herb based medicines are now

marketed as nutraceuticals (health foods) (Brower,

1998). Indian Ayurvedic medicine is believed to be more

than 5000 years old and it was well practiced and well

recorded over the years (Garodia et al., 2007). Herbal

drugs are generally used to prevent and treat various

diseases and ailments or to support health and healing

(De Smet, 1997). The World Health Organization (WHO)

has also recognized the importance of traditional medi-

cine and has set precise guidelines for the evaluation

of the safety, ef cacy, and quality of herbal medicines.

In recent years the research on the medicinal proper-

ties of various traditionally used medicinal plants has

gained considerable interest in order to ascertain their

true medicinal values and also to assess their toxicity

(Newman and Crag, 2007). Some drugs have been dis-

continued due to their toxicity, while others have been

modi ed or combined with additional herbs to counter-

balance side effects (Harish, 2001).

Bioactive principles of plants, also known as secondary

metabolites exert a range of pharmacological activities in

human beings besides serving as defense barriers in plants

(Paul et al., 2015). They are responsible for therapeutic

activities like hypoglycemic, anti-diabetic, anti-oxidant,

anti-microbial, anti-in ammatory, anti-carcinogenic,

anti-malarial, anti-cholinergic, anti-leprosy activities

etc. (Makri and Kintzios, 2007; Negi etal., 2011). Com-

mon bioactive principles present in plants are, alkaloids,

 avonoids, polyphenols, terpenoids, coumarins, tannins,

saponins, anthocyanins and glycosides (Paul etal., 2015).

Additionally secondary metabolites also prevent

spoilage and deterioration of food products Panghal

et al., 2011). Murraya koenigii (L.) Spreng popularly

known as curry leaf plant is a familiar spice used in

India for its characteristic  avour and aroma. According

to Wealth of India, it is also used in traditional Indian

medicine as analgesic, febrifuge, stomachic, carminative

and for the treatment of dysentery and skin eruptions.

Recent studies on M. koenigii revealed its medicinal val-

ues like antidiarrhoeal, antimicrobial, hepatoprotective,

radical-scavenging, hypoglycemic, and immunomodu-

latory properties (Maa et al., 2016). Phytochemical

analysis of leaves, bark and root showed the presence of

several bioactive constituents like carbazole alkaloids,

-carotene, polyphenols, terpenoids and coumarins

(Malwal and Sarin, 2011; Gahlawat etal., 2014).

The pharmacological activities of M. koenigii as

reported in different studies seem to exhibit a certain

variation which is attributed mainly to the climatic dif-

ferences among locations (Onayade and Adebajo, 2000).

Therefore it has become a common practice to con-

 rm that the local cultivars of M. koenigii possess the

expected bene cial effects when consumed regularly.

There are a few reports on phytochemical analysis and

pharmacological activities of M. koenigii from India

(Ningappa et al., 2008; Panghal et al., 2011; Malwal

and Sarin, 2011; Biswas et al., 2012). But to my best

knowledge, in recent years there are no studies on the

antioxidant and antimicrobial activities of M. koenigii

from Tamilnadu, a southern state of India. Therefore the

present study on antibacterial and antioxidant activity

of leaf organic extracts of local cultivars of Murraya

koenigii from Tamilnadu was undertaken.

MATERIALS AND METHODS

2,2-Diphenyl-1-picrylhydrazyl (DPPH) was procured

from Sigma chemicals. Mueller Hinton agar and broth

were purchased from Hi-Media, Mumbai. All other

chemicals used were of analytical grade commercially

available in India.The antibacterial activity of the

organic extracts was tested against  ve bacterial strains.

The only Gram positive bacterium included in the study

was Staphylococcus aureus MTCC 96 (ATCC 9144).

Four Gram negative bacteria used were Escherichia coli

MTCC 901 (ATCC 13534), Klebsiella pneumoniae MTCC

109 (ATCC 15380), Salmonella paratyphi MTCC 735 and

Pseudomonas aeruginosa MTCC 8291. All these bacteria

were obtained from Microbial Type Culture Collection

(MTTC), Institute of Microbial Technology, Chandigarh,

India and they were maintained on nutrient agar. M.

koenigii leaves were collected locally from various loca-

tions of Tamil Nadu, India and authenticated by Bot-

anist. Fresh leaves were washed thoroughly and dried

under shade till constant weight. The dried leaves were

ground to powder in a mixer grinder.

100 g of shade dried M. koenigii leaves powder was

extracted with hexane, chloroform and ethanol sepa-

rately at room temperature for 2 days and then the sol-

vents were evaporated using vacuum rotary evaporator

at 50

C. The organic extracts thus obtained were dried at

room temperature till constant weight and then stored in

brown bottles at 4

C for further study.

Antibacterial activity of the three organic extracts

obtained from M. koenigii was evaluated by agar well

diffusion method on Mueller-Hinton agar (MHA). The

bacterial strains were grown on MHA and suspensions

were prepared to match 0.5 McFarland standard. 100 μl

of the suspension was spread on MHA plates. Wells of

6 mm diameter were punched in the seeded agar using

sterile borer. Test well were loaded with 50 μl extracts

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LEAF ORGANIC EXTRACTS OF LOCAL CULTIVARS 361

M. Kavitha

(100 mg ml

-1

) and negative control well loaded with

same volume of solvent. Ampicillin and streptomycin

were used as a positive control. The plates were incu-

bated at 37ºC for 24 h. The diameter of inhibition zones

were measured in mm and results were recorded.

Minhibitory concentration (MIC) of the organic

extracts was tested by the standard Clinical and labora-

tory standards institute (CLSI) methods (Wayne, 2008).

MIC was the lowest concentration of the extract which

gives no visible bacterial growth. Broth microdilution

technique was used to determine the MIC (Elansary

etal., 2012). The organic extracts from M. koenigii were

serially diluted to obtain the dilutions, 200, 100, 50, 25,

12.5, 6.25 and 3.12 mg ml

-1

. 100 μl from each dilution

was transferred to respective wells followed by 90 μl of

double strength Mueller-Hinton broth (MHB). Finally 10

μl respective bacterial suspensions were added to give

 nal dilution of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56

mg ml

-1

and the microtiter plate was incubated at 37

for 24 h. Blank, positive and negative controls were in

place. After incubation, the lowest concentrations of the

extract, which did not show any visual growth of test

organisms, were considered as MICs.

Minimum bactericidal concentration (MBC) of the

organic extracts was also determined by the standard

CLSI methods (Wayne, 2008). MBC was the lowest con-

centration of the extract which kills the initial bacterial

inoculum. Broth microdilution technique was performed

to determine the MBC. The extracts from M. koenigii

were serially diluted to obtain the dilutions, 200, 100, 50,

25, 12.5, 6.25 and 3.12 mg ml

-1

. 100 μl from each dilu-

tion was added to corresponding wells followed by 90 μl

of double strength MHB. At last 10 μl respective bacte-

rial suspensions were transferred to provide  nal dilu-

tion of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 mg ml

-1

and

the plate was incubated at 37

C for 24 h. Blank, positive

and negative controls were also included. After incuba-

tion, the wells which did not show any visual growth

of test organisms were considered. 100 μl of the sam-

ple from those wells were transferred aseptically onto

MHA, spread uniformly and the plates were incubated

at 37

C for 24 h. After incubation, the concentrations

at which no visible growth was seen were  xed as the

MBC.

Free radical scavenging activity of the organic extracts

from M. koenigii was evaluated by its ability to scavenge

DPPH (Elansary etal., 2012). DPPH is a free radical, but

stable in nature. The DPPH solution is initially violet in

color which fades when antioxidants donate hydrogen.

The change in color is monitored using a spectrophotom-

eter and thus the DPPH free radical scavenging activity

is calculated (Molyneux, 2004). A stock solution of 0.1

mM DPPH in ethanol was prepared. Test concentrations

of organic extracts were made in the range of 50-250 μg

-1

in ethanol. Gallic acid was used as standard. 1 ml

of DPPH stock and 3 ml of test samples were mixed and

allowed to react at room temperature for 30 min. The

absorbance was measured at 517 nm using an ultravio-

let visible spectrophotometer (Shimadzu, Japan). Lower

absorbance of the reaction mixture indicated the higher

free radical scavenging activity (Saikia and Upadhyaya,

2011). The percentage of free radical scavenging was

calculated using the following Eq.

where A0 is the absorbance of the DPPH solution and AT

is the absorbance of the test or reference. The percentage

of free radical scavenging was then plotted against the

concentration and a regression equation was obtained to

calculate the IC

which is de ned as the total antioxi-

dants necessary to decrease the initial DPPH radical by

50% (Koleva etal., 2002).

All the experiments were done in triplicates. The

values in the tables represent the arithmetic mean and

standard deviation or mean. The standard error was

within experimental limits.

RESULTS AND DISCUSSION

The organic extracts obtained from M. koenigii were less

viscous in nature and brownish in colour. The yield of

the extracts was estimated to be 7, 6.5 and 9.2 % (v/w)

for hexane, chloroform and ethanol respectively. The

yield in the present study was comparable to the yield

reported in other investigations

(Ningappa etal., 2008;

Brind etal., 2014).

The results of antibacterial activity were presented in

Table 1. Based on the diameter of zone of inhibition,

the three organic extracts exhibited inhibitory activ-

ity against Staphylococcus aureus, Escherichia coli and

Pseudomonas aeruginosa (Figure 1). Klebsiella pneumo-

niae and Salmonella paratyphi were found to be resist-

ant except for chloroform extract which showed a very

faint zone of inhibition. The organic extracts presented a

good potential of antibacterial activity against Escheri-

chia coli and Pseudomonas aeruginosa with the zones of

inhibition well above standard antibiotic. Ethyl alcohol

extract displayed highest inhibitory activity when com-

pared to hexane and chloroform extracts. The results

were comparable to that of previous studies in which the

researchers reported that the various organic extracts of

M. koenigii exhibited signi cant antimicrobial activity

against both Gram positive and Gram negative bacteria

(Panghal etal., 2011; Baskaran etal., 2011). This prop-

erty is attributed to several carbazole alkaloids present

in the organic extracts (Malwal and Sarin, 2011).

362 ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LEAF ORGANIC EXTRACTS OF LOCAL CULTIVARS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

M. Kavitha

FIGURE 1. MHA plates exhibiting zone of inhibition of M. koenigiiorganic extracts

against (A) Escherichia coli, (B) Staphylococcus aureus, (C) Pseudomonas aeruginosa

and (D) Klebsiella pneumoniae.

Table 1. Antibacterial activity of M. koenigii organic extracts by Agar well

diffusion method

Te st organism Zone of Inhibition (in mm)

 Hexane Chloroform Ethyl alcohol Standard

Staphylococcus aureus 15.5±0.5 16.23±0.87 16.33±0.57 23.16±0.76

Escherichia coli 11.5±0.86 12.83±0.28 15.83±0.28 11.83±0.76

Klebsiella pneumoniae 0 7.83±0.76 0 11.86±0.8

Salmonella paratyphi 0 7.5±0.5 0 14.83±076

Pseudomonas aeruginosa 13.66±0.76 13.66±0.76 14.83±0.76 12.9±0.85

The results of MIC and MBC of ethyl alcohol extract

were summarized in Table 2. Relative to the results of agar

diffusion method, the extract displayed lowest MIC and

MBC value of 12.5 and 25 mg ml

-1

respectively against

Staphylococcus aureus. The extract produced same MIC

and MBC value of 25 and 50 mg ml

-1

against Escherichia

coli and Pseudomonas aeruginosa. The results are equi-

table to the results presented by Panghal etal. in which

the organic extracts prepared from the leaves of M.

koenigii manifested slightly better MIC values against

the clinical isolates (Panghal et al., 2011). Zinc oxide

Table 2. Minimum Inhibitory Concentration and

Minimum Bactericidal Concentration of M. koenigii

organic extracts

Test organism MIC (mg ml

-1

) MBC (mg ml

-1

)



Test Standard Test Standard

Staphylococcus

aureus

12.5 3.12 25 6.25

Escherichia coli 25 25 50 50

Pseudomonas

aeruginosa

25 25 50 50

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LEAF ORGANIC EXTRACTS OF LOCAL CULTIVARS 363

M. Kavitha

nanoparticles synthesized using the leaf extract of M.

koenigii displayed almost similar results to the present

study (Elumalai et al., 2015). Antioxidant protein iso-

lated from the leaves of M. koenigii also exhibited com-

parable results (Ningappa etal., 2010). The variation in

the antimicrobial activity of M. koenigii organic extracts

speci ed in various studies may be due to the differences

in chemical composition which can result from the cli-

matic differences among locations (Paul, 2015). Though

several compounds like tocopherol, -carotene, lutein

and alkaloids (Khanum etal., 2000) are reported in the

leaf organic extracts, carbazole alkaloids are accounted

for the antimicrobial activity (Ramsewak et al.,

1999).

The results of free radical-scavenging ability of

organic extracts of M. koenigii measured by the DPPH

assay were given in Table 3. Ethyl alcohol extract dis-

played highest radical scavenging activity with the

value of 30 μg ml

-1

than the rest two extracts. The

radical-scavenging activity of ethyl alcohol extract is

very close to that of the standard, gallic acid exhibiting

the IC

value of 35 μg ml

-1

. This is in agreement with

other studies on anti-oxidant activity of leaf extracts

of M. koenigii (Rao etal., 2007; Ningappa etal., 2008;

Kusuma etal., 2011). The antioxidant activity of organic

extracts from M. koenigii is attributed to the presence of

polyphenolic compounds and carbazole alkaloids (Rao

etal., 2007; Garodia etal., 2007). Ethyl alcohol extract

exhibited greater radical-scavenging activity than the

other two extracts and this can be inferred as ethanol is

effective in extracting polyphenols (Singh etal., 2011).

CONCLUSION

As substantiated in the study, the ethyl alcohol extract

from the leaves of M. koenigii, grown locally to ful-

 l culinary needs of Indian cuisine displayed excel-

lent antimicrobial activity against both Gram positive

and negative bacteria and also exhibited a quality free

radical scavenging activity. Climate of the location and

growing season greatly in uence the chemical compo-

sition of

M. koenigii and thus have an impact on the

biological activities. The above study con rmed that the

local cultivars of M. koenigii possess bene cial health

effects in terms of antimicrobial and anti-oxidant activi-

ties. Consequently, local cultivars of M. koenigii could

be effectively used as spice and dietary supplement,

however further study on molecular mechanism behind

the role of various constituents on radical scavenging

and antimicrobial inhibition needs to be done.

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