Microbiological

Communication

Biosci. Biotech. Res. Comm. 9(4): 856-864 (2016)

Evaluation of phyto constituent and synergistic

antibacterial activity of

Ocimum sanctum

extract

against some gram-positive and gram-negative species

Fatema Shah*

, Ziaul Hasan

and Kamal Uddin Zaidi

Department of Microbiology, Sai a College of Science, Bhopal 462001, India

Biotechnology Pharmacology Laboratory, Centre for Scienti c Research & Development, People’s University,

Bhopal 462037, India

ABSTRACT

Plant and plant extracts have been used in traditional medicine since time immemorial. O.sanctum has often been

cited as one of the main pillars of herbal medicine as it possesses greater medicinal value. It has been proved to be

effective against gram positive and gram negative bacteria. This study aimed to determine the in vitro antibacterial

activity of the medicinal plants O. sanctum against the bacterial strains associated with infectious diseases. Extracts

of O. sanctum were tested for their antibacterial activity against three bacterial species includes Staphylococcus

aureus, Escherichia coliandPseudomonas aeruginosa using the microdilution method. In the present study we found

that methanolic extract of leaves of O. sanctum was used in combination with ampicillin against E. coli, S. aureus

and P. aeruginosa showed fraction inhibitory concentration index (FICI ≤ 0.5). The synergistic antibacterial activ-

ity (FICI ≤ 0.5 ) was observed with combination of methanolic extract of stem and root of O. sanctum when used in

combination with antibiotic ampicillin against E. coli, S. aureus and P. aeruginosa showed indifferent antibacterial

activity (FICI = 1.0 - 4.0). Benzene extract of leaves, stem and root of O. sanctum when used in combination with

ampicillin against E. coli, S. aureus and P. aeruginosa showed partial synergistic antibacterial activity (FICI = 0.5 -

1.0, 0.5 - 4.0, 1.0 - 4.0) respectively. The synergistic antibacterial activity (FICI ≤ 0.5) was observed with combination

of aqueous extract of leaves of O.sanctum with ampicillin. While, the aqueous extract of stems and roots of O. sanc-

tum when used in combination with antibiotic ampicillin against E. coli, S. aureus and P. aeruginosa showed partial

synergistic antibacterial activity and indifferent antibacterial activity (FICI = 0.5 - 4.0, 1.0 - 4.0) respectively. present

investigation indicates clear evidence supporting the traditional use of O. sanctum in treating infectious diseases.

KEY WORDS: TULSI, SYNERGISTIC EFFECT, DISEASES, EXTRACTION, FRACTION INHIBITORY CONCENTRATION

856

ARTICLE INFORMATION:

*Corresponding Author: me_fatema3@yahoo.com

Received 7

Nov, 2016

Accepted after revision 24

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Shah, Hasan and Zaidi

INTRODUCTION

In modern complementary and alternative medical prac-

tice, plants are the primary source of therapeutics and

each part of the plant, including the seeds, root, stem,

leaves, and fruit, potentially contains bioactive com-

ponents. The search for alternative antimicrobial com-

pounds is an urgent area of biomedical research and

extracts derived from plants have long held interest as

potential sources of new therapeutic agents. The medici-

nal use of plants is probably as old as mankind. Plants

have continued to be a valuable source of natural prod-

ucts for maintaining human health. Various medicinal

plants have been used for years in daily life to treat

disease all over the world. One of the remotest works in

traditional herbal medicine is Viriksh Ayurveda, com-

piled even before the beginning of Christian era (Himal

et al., 2008, Mandave et al., 2014. Jiang et al., 2015 and

Sarah et al., 2016).

In the ancient Ayurvedic text, the Charaka Samhita,

Tulsi has been documented to be of immense use in

the treatment of headaches, rhinitis, stomach disorders,

in ammation, heart diseases, various forms of poison-

ing and malaria (Gupta et al., 2002). Each part of the

plant has proven to offer protection against various

diseases; the aqueous and alcoholic extract from the

leaves have various pharmacological activities such as

anti-in ammatory, antipyretic, analgesic, antiasthmatic,

antiemetic, antidiabetic, hepatoprotective, hypotensive,

hypolipidemic, and antistress agents. Further, distilla-

tion of the leaves yields oil of the plant which is known

to possess antibacterial, antioxidant, and anti-in am-

matory properties and is used extensively in the phar-

maceutical industry mainly for skin cream preparations

(Watson et al., 2012). Different parts of Ocimum sanc-

tum Linn (known as Tulsi), a small herb seen throughout

India, have been used for various medicinal purposes.

The main bioactive components in medicinal plants are

considered to be combinations of secondary metabolites

(Wu et al., 2016).

Recently Hanaa et al (2016) have been demonstrated

that antimicrobial activity of Ocimum tenui orum essen-

tial oil and their major constituents against three species

of bacteria. This study was designed at authenticated

the traditional use of Ocimum sanctum medicinal plants

against human pathogenic bacteria, causing a number of

human disease includingEscherichia coli,Pseudomonas

aeruginosa andStaphylococcus aureus by assess their in

vitro antibacterial activity.Due to insuf cient screening

of the natural compounds and the limited understanding

of their mechanism of action against the microorganism

the need of the hour is to identify more and more natural

compounds which exhibit synergistic behavior with the

antibiotics.

MATERIAL AND METHODS

Plant materials Ocimum sanctum (Tulsi) were purchased

from local market and were authenticated by Dr. S.S.

Khan of Botany, Department Sai a Science College Bho-

pal. The voucher specimen no (J/R201) was deposited at

the Herbarium of the Faculty of Botany Department, Sai-

 a Science College Bhopal (M.P.) India. Human disease

causing bacteria; Escherichia coli (MTCC 739), Staphy-

lococcus aureus (MTCC 96) Pseudomonas aeruginosa

(MTCC 74) were procured from Institute of Microbial

Technology, Chandigarh (IMTECH), India. These bacte-

rial strains were then used for studying the antimicrobial

ef ciency.

The phytochemical constituents of the Ocimum sanc-

tum plant parts (leaf, stem, and root) were extracted in

soxhlet apparatus using various solvents Kokate, 1991;

Trease and Evans, 1989). Soxhlet extraction is used for

separating components based on the difference in the

solubility in the solvent. The powdered plant material

(50 gm) was placed in the soxhlet extractor  ask. 500

ml of the organic solvent was taken in the round bot-

tom  ask. The soxhlet extraction was carried out con-

tinuously at an appropriate temperature for 6-8 hrs, till

colorless extract is collected in the extractor  ask. The

extract thus obtained was collected in collection bottles

and was further subjected to concentration using Rotary

vacuum evaporator. After soxhlet extraction the extracts

obtained were  ltered and then each of the extract was

concentrated using rotary vacuum evaporator. The indi-

vidual extracts were taken in round bottom  ask which

was heated at appropriate temperature on a water bath.

The vapors of the solvent rise in the condenser and after

condensation the solvent droplets was collected in the

collecting  ask.

The resultant sticky mass was collected in the cruci-

ble. It was dried at a low temperature in the oven. The

solid mass obtained was stored in a suitable volume of

10% dimethyl sulphoxide (DMSO) with a drop of Tween-

20. Aqueous extract of the individual plant parts was

prepared by decoction method. Filter paper packets of

50 gm of the individual plant parts were prepared. These

packets were place separately in 200 ml of hot water

contained in bottles. The extraction was carried out for

C with intermittent shaking. The extracts obtained

were concentrated and dried. The dried mass obtained

was stored in a suitable volume of 10% dimethyl sul-

phate (DMSO) with a drop of tween-20.

Phytochemical analysis

Chemical test were carried out to identify various con-

stituents using standard method of (Trease and Evans

1989; Harbone, 1973). Mayer reagent was prepared by

dissolving 1.36 grams of mercuric chloride in 60 ml of

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distilled water and 5 grams of potassium iodide in 20 ml

of distilled water. Both the above solution was mixed

and volume of the reagent adjusted to 100 ml by dis-

tilled water. 1 ml of the plant extract was taken and few

drops of mayer reagents were added. Formation of cream

colour precipitate was con rms the presence of alkaloid.

Fehling solution was prepared by dissolving 4.36 gram

of copper sulphate in 50 ml of distilled water and by

dissolving 17.3 grams of sodium potassium tartarate and

5 gram of sodium hydroxide in 50 ml of distilled water.

Both the solution were mixed prior to use.1 ml of the

extract were taken and few drops of fehling solution

was added. Formation of red precipitate con rms the

presence of carbohydrates and glycosides. Ferric chlo-

ride solution was prepared by dissolving 5 grams of fer-

ric chloride in 100 ml of 90% ethanol. 1 ml of extract

was taken and few drops of ferric chloride solution were

added. Formation of bluish black precipitate con rms

the presence of phenolic compounds and tannins.

Ninhydrin solution was prepared by dissolving 0.3

grams of ninhydrin in 100 ml of ethanol. 1 ml of extract

was taken and few drops of ninhydrin solution were

added and purplish pink colour con rms the presence of

proteins and amino acids in extracts. Alkaline reagent

was prepared by dissolving 10 grams of sodium hydrox-

ide in 100 ml of distilled water. 1 ml of extract was taken

and few drops of sodium hydroxide solution were added.

Intense yellow colour con rms the presence of  avo-

noids. 1 ml of the extract was taken and mixed with few

drops of chloroform and few drops of sulphuric acids.

A reddish brown colour con rms the presence of ter-

penoids.1 ml of the extract was taken and diluted with

distilled water to 10 ml. Formation of stable foam con-

 rms the presence of saponins.1 ml of the extract was

mixed with 5 ml of distilled water mixture was heated

and to it was added 5 ml of 1% HCl. Formation of red

precipitate con rms the presence of phlobatanins.1 ml

of the extract was taken and to it was added in 2 ml

of chloroform and 2 ml of concentrated sulphuric acid.

Formation of reddish brown layer at the interface con-

 rms the presence of steroids.

Assay of antimicrobial activity using disc diffusion

method

Disc diffusion method for antimicrobial susceptibil-

ity testing was carried out according to the standard

method by Bauer et al., 1996 to assess the presence of

antibacterial activities of the various samples. A bacte-

rial suspension was prepared for each of bacteria used

for the study. 1 ml of the bacterial suspension was taken

in sterile petriplate. To it was added molten nutrient agar

media under aseptic conditions and mixed well. It was

allowed to solidify for 1 hour to allow the bacteria to

grow. These plates were used for sensitivity test. What-

man  lter paper disc were impregnated with the samples

and were placed on nutrient agar surface. Positive con-

trol plate was also prepared with standard antibiotic disc

and negative control plate was prepared using DMSO.

The plates were then incubated at 37

C for 24 hrs. After

the incubation the plates were examine for zone of inhi-

bition. The inhibition zones were measured using antibi-

otic zone reader scale

Determination of Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration of the plant

extracts, antibiotics and combination of plant extracts

and antibiotics was determined by diluting the extracts

in Nutrient broth to give concentration of 1024, 512,

256, 128, 64, 32, 16, 8, 4 and 2 μg /ml. 2 ml of plant

extracts, antibiotics and combination of plant extracts

and antibiotics was added to the  rst tube containing

2 ml of broth. The tube was shaken and 2 ml trans-

ferred aseptically to the next tube containing the same

quantity of broth. This was done until serial dilution was

achieved in the last tube that is the tenth tube. Then 0.1

ml of the MTCC bacterial culture suspension was inocu-

lated into each test tube and they were incubated at 37

C for 24 hours. The absorbance of the tubes was taken

in UV-VIS spectrophotometer. The minimum inhibitory

concentration was regarded as the lowest concentration

of the extract that did not permit any visible growth

when compared with the control tube.

Calculate FIC and FIC Index

A widely accepted method, to measure the effect of com-

bination of plant extract and antibiotic is the fractional

index. The fractional index is used to identify whether a

combination therapy is synergistic, additive or antago-

nistic. The Inhibitory Concentration is determined using

MIC measurements. The fractional Inhibitory Concentra-

tion Index (∑FIC) is the sum of the FICS of each of the

plant extract and antibiotics (

Rakholiya et al., 2015).

Calculations

The FIC was calculated for plant extract and antibiotic

as follows:

MIC of plant extractin combination

FIC for Plant extract =

MIC of plant extract

The FIC was calculated for plant extract and antibiotic

as follows:

MIC of plant extractin combination

FIC for Antibiotic =

MIC of plant extract

Calculated the summation of FIC (FIC) index for each

combination as follows:-

FIC = FIC of Plant Extracts + FIC of Antibiotics

858 EVALUATION OF PHYTO CONSTITUENT AND SYNERGISTIC ANTIBACTERIAL ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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RESULTS

Antibacterial activity of methanolic extracts of

sanctum

with ampicillin

The zone of inhibition of the leaf extract against E. coli,

P. aeruginosa and S. aureus was 18mm, 15mm and 23mm

respectively. The ZOI of the combination of leaf extract of

O. sanctum and ampicillin were 29mm, 27mm and 30mm.

The MIC of the leaf extract was 32μg/ml, 16μg/ml and

32μg/ml against E. coli, P. aeruginosa and S. aureus. The

MIC of the combination of leaf extract of O. sanctum and

ampicillin were 4 μg/ml, 4μg/ml and 4μg/ml respectively.

The ZOI of the stem extract against E. coli, P. aeruginosa

and S. aureus were 15mm, 10mm and 14mm respectively.

The ZOI of the combination of stem extract of O. sanctum

and ampicillin were 28mm, 21mm and 26mm (Table 1).

The minimum inhibitory concentration values of

the stem extract were 16μg/ml, 32μg/ml and 64μg/

ml against E. coli, P. aeruginosa and S. aureus. The

MIC of the combination of stem extract of O. sanc-

tum and ampicillin were 4μg/ml, 16μg/ml and 16μg/ml

respectively. The ZOI of the root extract against E. coli,

P. aeruginosa and S. aureus were 0mm, 0mm and 10mm

respectively. The ZOI of the combination of root extract

of O. sanctum and ampicillin were 24mm, 22mm and

23mm. The MIC of the root extract was 64μg/ml, 256μg/

ml and 128μg/ml against E. coli, P. aeruginosa and S.

aureus. The MIC of the combination of root extract of

O. sanctum and ampicillin were 32μg/ml, 32μg/ml and

16μg/ml respectively (Table 1).

Antibacterial activity of combined effect of

benzene extracts of

O. sanctum

with ampicillin

The zone of inhibition of the leaf extract against E. coli,

P. aeruginosa and S. aureus was 13mm, 8mm and 15mm

respectively. The ZOI of the combination of leaf extract

of O. sanctum and ampicillin were 26mm, 21mm and

28mm. The MIC of the leaf extract was 32μg/ml, 16μg/

ml and 32μg/ml against E. coli, P. aeruginosa and S.

aureus. The MIC of the combination of leaf extract of

O. sanctum and ampicillin were 8μg/ml. The ZOI of the

stem extract against E. coli, P. aeruginosa and S. aureus

was 11 mm, 0 mm and 10 mm respectively. The ZOI

of the combination of stem extract of O. sanctum and

ampicillin were 25 mm, 20 mm and 22mm (Table 2).The

minimum inhibitory concentration values of the stem

extract were 64μg/ml, 128μg/ml and 32μg/ml against

E. coli, P. aeruginosa and S. aureus. The MIC of the

combination of stem extract of O. sanctum and ampicil-

lin were 8μg/ml, 16μg/ml and 16μg/ml respectively. The

ZOI of the root extract against E. coli, P. aeruginosa and

S. aureus was 0mm, 0mm and 8mm respectively.The ZOI

of the combination of root extract of O. sanctum and

ampicillin were 24 mm, 22 mm and 23 mm. The MIC of

the root extract was 64 μg/ml, 32μg/ml and 64 μg/ml

against E. coli, P. aeruginosa and S. aureus. The MIC of

the combination of root extract of O. sanctum and ampi-

cillin were 16μg/ml, 16μg/ml and 32μg/ml respectively

(Table 2).

Antibacterial activity of combined effect of aqueous

extracts of

O. sanctum

with ampicillin

The zone of inhibition of the leaf extract against E. coli,

P. aeruginosa and S. aureus was 17mm, 14mm and

20mm respectively. The ZOI of the combination of leaf

extract of O. sanctum and ampicillin were 28mm, 26mm

and 29mm. The MIC of the leaf extract was 16 μg/ml,

64μg/ml and 32μg/ml against E. coli, P. aeruginosa and

S. aureus. The MIC of the combination of leaf extract of

O. sanctum and ampicillin were 2μg/ml, 16μg/ml and 8

Table 1: Zone of Inhibition, Minimum Inhibitory Concentration and Fractional Inhibitory Concentration of methanolic

extract of leaf, stem and root of O. sanctum.

Plants

material

Zone of Inhibition (mm) Minimum Inhibitory

Concentration in μg/ml (MIC)

Fractional Inhibitory Conc.

FIC &  FIC

E. coli P. aeruginosa S. aureus E. coli P. aeruginosa S. aureus E. coli P. aeruginosa S. aureus

Leaf 18 15 23 32 16 32 0.125 0.250 0.125

Ampicillin 23 21 22 16 16 32 0.250 0.250 0.125

Leaf with

Ampicillin

29 27 30 4 4 4 0.375 0.500 0.250

Stem 15 10 14 16 32 64 0.250 0.500 0.250

Ampicillin 23 21 22 16 32 32 0.250 0.500 0.500

Stem with

Ampicillin

28 21 26 4 16 16 0.500 1.000 0.750

Root - - 10 64 256 128 0.500 1.000 1.500

Ampicillin 23 21 22 32 32 16 0.125 1.000 1.125

Root with

Ampicillin

24 22 23 32 32 16 0.125 1.000 1.125

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μg/ml respectively. The ZOI of the stem extract against

E. coli, P. aeruginosa and S. aureus was 12 mm, 0 mm

and 16 mm respectively. The ZOI of the combination of

stem extract of O. sanctum and ampicillin were 25mm,

22mm and 28mm (Table 3).

The minimum inhibitory concentration values of the

stem extract was 32μg/ml, 32μg/ml and 32μg/ml against

E. coli, P. aeruginosa and S. aureus. The MIC of the

combination of stem extract of O. sanctum and ampicil-

lin were 8μg/ml, 16μg/ml and 8μg/ml respectively. The

ZOI of the root extract against E. coli, P. aeruginosa and

S. aureus was 9mm, 0mm and 8mm respectively. The

ZOI of the combination of root extract of O. sanctum

and ampicillin were 24mm, 22mm and 23mm. The MIC

of the root extract was 32μg/ml, 32μg/ml and 256μg/ml

against E. coli, P. aeruginosa and S. aureus. The MIC of

the combination of root extract of O. sanctum and ampi-

cillin were 16μg/ml, 16μg/ml and 32 μg/ml respectively

(Table 3).

Analysis of the Plant Extracts

O. sanctum

Pytochemical screening of the leaf, stem and root of Oci-

mum sanctum L. were conducted and its results showed

presence of different constituents in methanolic, ben-

zene and aqueous extracts. (Table 4). Methanolic extracts

showed the presence of alkaloids, carbohydrate, glyco-

sides, phenolic compounds, tannins, proteins, amino

acids,  avonoids, terpenoids, saponins and steroids. In

Table 2: Zone of Inhibition, Minimum Inhibitory Concentration and Fractional Inhibitory Concentration of Benzene

extract of leaf, stem and root of O. sanctum.

Plants

material

Zone of Inhibition (mm)

Minimum Inhibitory

Concentration in μg/ml (MIC)

Fractional Inhibitory Conc.

FIC &  FIC

E. coli

aeruginosa

aureus E. coli

aeruginosa

aureus E. coli

aeruginosa S. aureus

Leaf 13 8 15 32 16 32 0.250 0.500 0.250

Ampicillin 23 21 22 16 16 32 0.500 0.500 0.250

Leaf with

Ampicillin

26 21 28 8 8 8 0.750 0.1000 0.500

Stem 11 - 10 64 128 32 0.125 0.125 0.500

Ampicillin 23 21 22 16 16 32 0.500 0.1000 0.500

Stem with

Ampicillin

25 20 22 8 16 16 0.625 1.125 0.1000

Root - - 8 64 32 64 0.250 0.500 0.500

Ampicillin 23 21 22 16 16 32 1.000 1.000 1.000

Root with

Ampicillin

24 22 23 16 16 32 1.250 1.500 1.500

Table 3: Zone of Inhibition, Minimum Inhibitory Concentration and Fractional Inhibitory Concentration of Aqueous

extract of leaf, stem and root of O. sanctum.

Plants

material

Zone of Inhibition ( mm ) Minimum Inhibitory

Concentration in μg/ml ( MIC )

Fractional Inhibitory Conc.

FIC &  FIC

E. coli P. aeruginosa S. aureus E. coli P. aeruginosa S. aureus E. coli P. aeruginosa S. aureus

Leaf 17 14 20 16 64 32 0.125 0.250 0.250

Ampicillin 23 21 22 16 32 32 0.125 0.500 0.250

Leaf with

Ampicillin

28 26 29 2 16 8 0.250 0.750 0.500

Stem 12 - 16 32 32 32 0.250 0.500 0.250

Ampicillin 23 21 22 16 32 32 0.500 0.500 0.250

Stem with

Ampicillin

25 22 28 8 16 8 0.750 0.1000 0.500

Root 9 - 8 32 32 256 0.500 0.500 0.125

Ampicillin 23 21 22 16 32 32 1.000 0.500 1.000

Root with

Ampicillin

24 22 23 16 16 32 1.500 1.000 1.125

860 EVALUATION OF PHYTO CONSTITUENT AND SYNERGISTIC ANTIBACTERIAL ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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benzene extract carbohydrate, glycosides, phenols, tan-

nins, proteins and amino acids were present. In aqueous

extract along with above components terpenoids and

saponins were also present. Stem extract of O. sanctum

in methanol showed presence of alkaloids, phenols, tan-

nins,  avonoids, terpenoids, saponins and phlobatannin

while extraction with benzene solvent showed presence

of  avonoids only. In aqueous extract phenols, tan-

nins,  avonoids, terpenoids, saponins and phlobatan-

nins were present. Methanolic extracts of root showed

the presence of the different phytochemical constituents

viz. alkaloids, carbohydrates, glycosides, phenols, tan-

nins, saponins, phlobatannins and steroids. Extraction

with benzene showed presence of carbohydrate, gly-

cosides, phenols and tannins. Aqueous extracts of the

roots showed the presence of alkaloids, carbohydrates,

glycosides, phenols, taninns,saponins and phlobatanins

(Table 4).

DISCUSSION

In this study, we attempted to obtain information on

the antimicrobial ef cacy of Ocimum sanc, particularly

against pathogens namely E. coli, S. aureus and P. aerug-

inosa, as these microbes are more commonly associated

with initiation and progression of various pathogenic

diseases, especially aggressive periodontitis. Synergism

was found when methanolic extract of leaves of Oci-

mum sanctum was used in combination with antibiotic

ampicillin against E. coli, S. aureus and P. aeruginosa

bacterial species ( FICI ≤ 0.5 ). The results showed that

ocimum leaves extract showed good inhibition against

the bacterial strains. This observed antimicrobial activ-

ity could be explained by the fact that plant extract may

attach to the surface of the cell membrane disturbing

permeability and respiration functions of the cell. The

interaction of plant extract with microbial cytoplasmic

components and nucleic acids can inhibit the respiratory

chain enzymes and interferes with the membrane per-

meability limiting the development of bacteria. It is also

possible that extract not only interact with the surface

of membrane but can also penetrate inside the bacte-

ria. The synergistic antibacterial activity ( FICI ≤ 0.5 )

was observed with combination of methanolic extract of

stem of Ocimum sanctum with ampicillin showed par-

tial synergistic antibacterial activity (FICI = 0.5 - 1.0) .

Methanolic extract of root of Ocimum sanctum when

used in combination with antibiotic ampicillin, against

E. coli , S. aureus and P. aeruginosa showed indifferent

antibacterial activity (FICI = 1.0 - 4.0).

Our  ndings are in agreement with Mishra and

Mishra, (2011) who studied antibacterial activity of

the aqueous, alcoholic, chloroform extract and oil

obtained from leaves of Ocimum sanctum against E.coli,

P.aeruginosa, S. typhimurium and S.aureus. Extract

obtained from Ocimum sanctum were observed equally

effective against pathogenic gram positive and gram

negative bacteria. Benzene extract of leaves of Oci-

mum sanctum when used in combination with antibiotic

ampicillin against E. coli, S. aureus and P. aeruginosa

showed partial synergistic antibacterial activity (FICI =

0.5-1.0). Benzene extract of stems of Ocimum sanctum

when used in combination with antibiotic ampicillin,

against E. coli , S. aureus and P. aeruginosa showed

partial synergistic antibacterial activity and indifferent

antibacterial activity (FICI = 0.5 - 4.0). Benzene extract

Table 4: Phytochemical constituents present in methanolic, benzene and aqueous extracts of leaf, stem and root of

O. sanctum.

Plant

Constituents

Leaf Stem Root

Methanol Benzene Aqueous Methanol Benzene Aqueous Methanol Benzene Aqueous

Alkaloids + - + + - - + - +

Carbohydrates

Glycosides

+ ++++++-+

Phenolics

compounds

Tannins

+-+------

Proteins

Amino acids

+ +++++ - -+

Flavonoids + - + - - - - - -

Terpenoids + - + - - - + - +

Saponins + - - + - + + - +

Phlobatannins - - - - - - - - -

Steroids + - - + - - + - -

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Shah, Hasan and Zaidi

of roots of Ocimum sanctum when used in combination

with antibiotic ampicillin against E. coli , S. aureus and

P. aeruginosa showed indifferent antibacterial activity

(FICI = 1.0 - 4.0) These results are broadly similar to

those of studies that used disk diffusion or optical den-

sity reduction methods; however, there are differences in

reported activity toward Gram positive and Gram nega-

tive bacteria, ( Helen et al 2011; Poole et al 2011 and

Hanaa et al 2016).

The synergistic antibacterial activity (FICI ≤ 0.5) was

observed with combination of aqueous extract of leaves

of Ocimum sanctum with ampicillin. Aqueous extract

of stems of Ocimum sanctum when used in combina-

tion with antibiotic ampicillin against E. coli, S. aureus

and P. aeruginosa showed partial synergistic antibacte-

rial activity and indifferent antibacterial activity (FICI

= 0.5 - 4.0). Aqueous extract of roots of Ocimum sanc-

tum when used in combination with antibiotic ampicil-

lin against E. coli, S. aureus and P. aeruginosa showed

indifferent antibacterial activity (FICI = 1.0 - 4.0). This is

in agreement with many literatures reporting of differ-

ences in the activities of extracts obtained from the same

morphological part of a plant using different solvents.

Similar  ndings have been reported by Ahmad and

Aqil, (2007) who found synergistic interaction between

crude extracts of Indian medicinal plants and antibiot-

ics against extended spectrum  lactamase producing

multidrug-resistant enteric bacteria. Similar observation

was found by Sajjanshetty et al., (2016) who reported

the antimicrobial ef cacy of Ocimum sanctum leaf

extract on periodontal pathogens it was observed Oci-

mum sanctum i extracts showed antimicrobial activity

against A. actinomycetemcomitans, similar to doxycy-

cline with similar inhibition zones (P > 0.05). P. gingi-

valis and P. intermedia, however, exhibited resistance to

Tulsi extract that showed signi cantly smaller inhibition

zones (P < 0.05).

Methanolic extracts of the leaf showed the pres-

ence of alkaloids, carbohydrate, glycosides, phenolic

compounds, tannins, proteins, amino acids,  avonoids,

terpenoids, saponins and steroids. In benzene extract

carbohydrate, glycosides, phenols, tannins, proteins

and amino acids were present. In aqueous extract along

with above components terpenoids and saponins were

also present. Phytochemical constituents such as ster-

oids, alkaloids,  avonoids, tannins, phenol, and several

other aromatic compounds are secondary metabolites of

plants that serve a defense mechanism against predic-

tion by many microorganisms, insects and other herbi-

vores (Bonjar et al., 2004). These secondary metabolites

exert antimicrobial activity through different mecha-

nisms. Secondary metabolite alkaloids are one of the

largest groups of phytochemicals in plants found in all

of extract of Ocimum sanctum. One of the most common

biological properties of alkaloids is their toxicity against

cells of foreign organisms like bacteria, tannins have

been found to form irreversible complexes with proli-

nerich protein resulting in the inhibition of cell protein

synthesis (Sibanda and Okoh 2007).

Stem extract of O. sanctum in methanol showed pres-

ence of phenols, tannins,  avonoids, terpenoids, sapo-

nins and phlobatannins while extraction with benzene

solvent showed presence of  avonoids only. In aqueous

extract phenols, tannins,  avonoids, terpenoids, saponins

and phlobatannins were present. Methanolic extracts of

root showed the presence of the different phytochemical

constituents viz. Alkaloids, carbohydrates , glycosides,

phenols, tannins, saponins, phlobatannins and steroids.

Extraction with benzene showed presence of carbohy-

drate, glycosides, phenols and tannins. Aqueous extracts

of the roots showed the presence of alkaloids, carbo-

hydrates, glycosides, phenols , tannins , saponins and

phlobatannins .

Eugenol (l-hydroxy-2-methoxy-4-al-

lylbenzene) the active constituent present in O. sanctum,

is mainly responsible for the therapeutic potential of the

plant (Prakash and Gupta, 2005) and the other important

constituents include carvacrol, linalool, methyl euge-

nol ,

caryophyllene , methyl chavicol and ursolic acid

as lead compounds in the composition of O. sanctum,

(Mohan et al., 2011).

As Ocimum is widespread in India, it can be recom-

mended as an easily available and renewal source of

antimicrobial agent instead of synthetic chemicals. The

present  ndings indicate that Ocimum possesses com-

pounds with antimicrobial properties against patho-

genic microorganisms. It is quit safer to use as an herbal

medicine as compare to chemically synthesized drug.

Prostanthera species, like many other australian plants,

have been shown to have essential oils with potent anti-

microbial activity. Essential oils from the desert species

P. centralis have been shown to be effective against

gram-positive bacteria with MICs against S. aureus of

approximately 0.1 mg/ml (Collins et al., 2014).

On the other hand, streptomycin sulfate and chlo-

ramphenicol used as positive controls showed strong

antibacterial activities against both Gram-positive and

Gram-negative bacteria like as the results of previous

studies (Khan et al., 2014). All the plants parts were

extracted with methanol, benzene and aqueous because

these considered as the best solvent for the extraction

of antimicrobial substances and may contain diverse

chemical compounds with biological activity (Robles

et al., 2013; Tekwu et al., 2012).

The alcoholic extract

has greater effect as compared to Benzene and aque-

ous extract which may be due to the fact that alcohol is

comparatively a better solvent as compared with water

and benzene for extraction of phytochemical (Levy and

Marshall, 2004). Plants antimicrobials have been found

862 EVALUATION OF PHYTO CONSTITUENT AND SYNERGISTIC ANTIBACTERIAL ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Shah, Hasan and Zaidi

to be synergistic enhancer in that they have little anti-

microbial property alone but when they are taken con-

currently with standard drug enhances the effect of anti-

biotics (Chanda and Rakholiya 2011).

CONCLUSION

In ancient and modern era, aerial parts of herbs have

been generally used for the cure of crucial health care

and variety of ailment across the world depends on

geographical cultivation. Leaves of O. sanctum play a

vital role in health care system due to containing of

certain phytochemical. Overall results of current study

re ect that highest antimicrobial activities were deter-

mined againstEscherichia coli,Pseudomonas aeruginosa

and Staphylococcus aureus. Among selected studied

medicinal plant material, O. sanctum leaf showed more

antibacterial activity.

ACKNOWLEDGEMENTS

The authors are grateful to Sai a College of Science

Bhopal, for providing laboratory facilities and for grant-

ing  nancial assistance to carry out this work, and Prin-

cipal and Secretary, Sai a College of Science, Bhopal,

for encouragement.

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