phytochemical screening and evaluation of the antimicrobial potential of coleus … ·...

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2811

Paul et al. World Journal of Pharmaceutical Research

PHYTOCHEMICAL SCREENING AND EVALUATION OF THE

ANTIMICROBIAL POTENTIAL OF COLEUS AMBOINICUS

Nivya Mariam Paul*, Anjali Mohan1, Elza John1, Nayomi John1,

Ajitha A. R. 2, Shanti A Avirah2

1Department of Microbiology, M A College, Kothamangalam, Kerala-686666

2 Department of Chemistry, M A College, Kothamangalam, Kerala-686666

ABSTRACT

Coleus amboinicus of the family Lamiaceae is a large succulent

aromatic herb used for flavoring drinks and medicine. Ethanolic extract

of fresh, shade dried and oven dried leaves were used for antibacterial

analysis. The antibacterial activity was evaluated against twelve

different bacterial strains by detecting minimum inhibitory

concentration and zone of inhibition. Phytochemicals present in the

leaf extracts were analyzed and these were separated by Thin Layer

Chromatography and are identified using spray reagents. The presence

of phenols, Terpenes, Flavanoids, Tannins and Steroids in fresh, shade

dried and oven dried leaves were revealed.

Keywords: Coleus amboinicus, Phytochemical analysis, Antibacterial activity, Minimum

Inhibitory Concentration, Thin Layer Chromatography.

INTRODUCTION

India is rich in indigenous herbal resources permitting the growth of more than 20,000 plant

species, of which about 2,500 are of medicinal value1 .The medicinal value of these plants

lies in some active chemical substances called phytochemicals that produce a definite

physiological action on human body. Alkaloids, flavanoids, phenolic compounds and tannins

are the major phytochemicals present in plants2. According to World Health Organization

medicinal plants would be the best source to obtain a variety of drugs. The use of plant

extracts and phytochemicals with known antimicrobial properties can be of great

significances in therapeutic treatment3.

World Journal of Pharmaceutical research

Volume 3, Issue 2, 2811-2826. Research Article ISSN 2277 – 7105

Article Received on 02 January 2014 Revised on 28 January2014, Accepted on 26 February 2014

*Correspondence for

Author

Nivya Mariam Paul

Department of

Microbiology, M A

college, Kothamangalam,

Kerala, India.


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According to Ayurveda, Coleous amboinicus, the plant under study is vatha and kapha

suppressant. It is a good pain killer. It is also very effective in convulsions paralysis and has a

small amount of necrotic effect. It is a good remedy in indigestion and is helpful in avoiding

tastelessness, diarrhoea, liver related problems and worm infestation. It is effective in

expelling out the extra amount of mucus present in the respiratory tract thus preventing

diseases like cough and asthma. It is also effective in treating kidney stones and renal

calculus as it is a diuretic.

MATERIALS AND METHODS

Collection of Plant study material: - fresh and dried leaves of Coleous amboinicus

collected from Aromatic Medicinal Plants Research station, Kothamangalam (Ernakulum

district).The plants were freshly collected and the leaves were separated from the stem. Then

washed under running tap water then with distilled water.

After air drying, a portion of the samples were shade dried until all the water molecules

evaporated and the leaves get dry. Another portion of leaves were dried in hot air oven at

40ºC. After drying, the plant leaves were ground well into fine powder and then transferred

into airtight containers. Another portion of washed fresh leaves (30g) were ground well. The

3 set of samples were then subjected to soxhlet apparatus for extraction

Preparation of plant extracts (soxhelt extraction)

10 g each of both shade dried and hot air oven dried leaves were taken and extracted with

ethanol and water as solvent for about 72 hours by soxhlet extractor. Similarly 30g of ground

fresh leaves were taken and extracted with ethanol and water as solvent for about 72 hours by

soxhlet extractor.Ethanol and water were evaporated off, and then concentrated aqueous and

ethanolic extracts were obtained as residues.

Phytochemical analysis of crude extract10

Ethanolic and aqueous extracts of Coleus amboinicus was taken for phytochemical analysis

of carbohydrates, alkaloids, flavanoids, saponins, proteins, phenolic compounds,

phlobotannins, cardiac glycosides, morphi c alkaloid, oil and gums using the following

methods

1) detection of carbohydrate and glycosides

a) Barfoed’s test


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To 1 ml of the filtrate, 1ml of Barfoed’s reagent was added and heated on a boiling water

bath for 2 minutes. Red precipitate indicated the presence of sugar.

b) Benedict’s test

To 0.5 ml of the filtrate, 1ml of Benedict’s reagent was added and this mixture was heated on

a boiling water bath for 2 minutes. A characteristics coloured precipitate indicated the

presence of sugar.

c) Molisch test

1g of extract was dissolved in 1ml water.Then add two drops of 1% alcoholic solution of

alpha naphthol. 1ml concentrated sulphhuric acid was added along the sides of the test tube.

A deep violet colour at the junction of two liquid indicated the presence of sugar.

d) Fehling test

1ml of the filtrate is boiled in water bath with 1ml of Fehling’s solution A and B .Red

precipitate indicated the presence of sugar.

2) Detection of alkaloids

a) Mayer’stest

To a few ml of the filtrate, one or two drops of Mayers reagent were added by the side of the

tube. A white creamy precipitate indicated the test as positive.

b) Wagners test

To a few ml of the filtrate, one or two drops of Wagners reagent were added by the side of

the tube. A reddish brown precipitate confirmed the test as positive

3) Detection of Phlobotannins

To 0.5 ml of sample mix 5ml of water and boil with 5ml 1% HCl. Red precipitate indicates

the presence of phlobotannins.

4) Detection of Flavanoids

1ml of extract was dissolved in dilute NaOH solution. A visible colour

observed indicated flavanoids.

5) Detection of Proteins and amino acids

a) Biuret test


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An aliquot of 2 ml of filtrate was treated with one drop of 2% copper sulphate solution. To

this 1 ml of ethanol was added, followed by excess of potassium hydroxide pellets. Pink

colour in the ethanolic layer indicated the presence of proteins.

6) Detection of phenolic compounds and tannins

a) Ferric chloride test

The extract was dissolved in (50mg) 5ml of distilled water. To this, few drops of neutral

ferric chloride solution was added. Dark green colours indicate the presence of phenolic

compounds.

b) Lead acetate test

The extract was dissolved in (50mg) 5ml of distilled water. To this, 3ml of 10% lead acetate

solution was added. A bulky white precipitate indicated the presence of phenolic compounds.

7) Detection of saponins

a) Frothing test

To 0.5 ml of sample added 5ml distilled water. Frothing persistence indicated the presence of

saponins.

8) Detection of cardiac glycosides

1ml glacial acetic acid containing trace of ferric chloride was added to 0.5g of extract.

Sulphuric acid was gently poured down the sides of the tube. Brown ring at the interphase

and violet ring beneath that layer and pale green upper layer was an indicative of deoxy

sugars.

9) Detection of morphine alkaloids

1ml of extract was evaporated to dryness and the residue was dissolved in 0.6ml of 1%

sulphuric acid. To this 2ml of distilled water and two drop sof 10% sodium nitrite was added.

The solution was then made alkaline with dilute ammonia solution. Reddish brown

precipitate indicated the presence of morphine alkaloids.

10) Detection of oil and fat

Small amount of extract was pressed between two filter paper. Oil stain on the paper

indicated the presence of oil.


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11) Detection of gum and mucilage

100 mg of extract was dissolved in 10ml of distilled water and to this 25ml of absolute

alcohol was added with continues stirring .White or cloudy precipitate indicates the presence

of gum and mucilage.

Test Organisms

Different samples of bacterial strains were collected from Mar Baselious Medical Mission

Hospital, Kothamangalam and are isolated and identified by different methods. These

organisms were further used against the plant extract to study its antimicrobial activity.

Identification of Bacterial Strains from Hospital

The selected isolates were subjected to gram’s staining4, Hanging Drop Method5 and then to

different biochemical procedures6 like: Sugar fermentation test, Oxidative fermentation test,

Mannitol motility test , Indole production test , Methyl red test, Voges – Proskauer test ,

Citrate utilization test, Urease test, Nitrate reduction test ,Triple sugar iron agar test,

Coagulase Test , Oxidase test ,Catalase test .

Antimicrobial Sensitivity Testing

Antibiotic Sensitivity Test

Using a sterile wire loop, colonies of similar appearance were picked and emulsified in 3-4ml

of peptone water. The turbidity of the suspension was matched with that of the McFarland’s

Turbidity Standard7. Antibiotic sensitivity test was carried out using Kirby-Bauer technique8.

Antibiotic discs of Ampicillin, Neomycin, Vancomycin, Bacitracin, Gentamycin,

Streptomycin, Penicillin, Tobramycin and Chloramphenicol were placed under sterile

conditions. The plates were incubated overnight. Following incubation, the diameters of the

zones of inhibition of the growth including the diameter of the disc were measured. The

diameters of the zones were measured to the nearest millimeter and comparing with the

antibiogram, the culture was found to be sensitive, intermediate or resistant.

. Agar Well Diffusion Method

A sterile cotton swab was dipped into the bacterial suspension of McFarland’s Turbidity

Standard and was rubbed gently over the plate to obtain a confluent lawn of growth. The agar

was allowed to dry. The different diameters of wells are prepared by using sterile gel

puncture. The extracts were completely dissolved in suitable solvent and different


2816


concentrations of plant extracts were added to the well. The plates were incubated and the

diameter of the zone of inhibition was measured.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal Concentration (MBC) 9

The MIC is regarded as the lowest concentration of antimicrobial agent which completely

inhibits the growth. Prepared stock solution of extracts and labeled these concentrations as

stock C. A row of sterile test tubes was arranged and labels them C1-C10. To all the test tubes

2ml nutrient broth was added. Then 2ml of stock solution of extract was added to C1, after

well mixing transfer 2ml to the C2.This procedure is continued up to 10th tube. Inoculate one

drop of an overnight broth culture of the test organism and incubate tubes for 18-24 hours.

Inoculate a tube containing 2ml broth with organism and keep at 40C in the refrigerator

overnight, to be used as standard for the determination of complete inhibition. The minimum

inhibition concentration (MIC) value was determined as the lowest concentration of the crude

extract in the broth medium that inhibited the visible growth of the microorganism. The

highest dilution that yielded no single bacterial colony on solid medium was taken as MBC.

Antifungal Studies

The antifungal activity of aqueous and Ethanolic extracts of fresh, oven dried and shade dried

leaves of Coleus amboinicus was performed by cup plate method10. Pure cultures of Mucor

species, Aspergillus species, Aspergillus niger, Penicillium species and Fusarium species

were taken from stock cultures.

Spore suspension of each fungus was prepared by adding three loopful of spores collected

from seven to ten day old cultures into 10 ml of sterile distilled water. 10 ml of spore

suspension was poured into 250 ml Sabouraud’s Dextrose Agar and mixed and poured into

petri plates. After solidification, a well of 5mm diameter was made. 20 ml of selected extracts

was poured into the well using micropipette. Prepared plates were incubated at room

temperature for two to three days. Inhibition zones were measured and data were recorded.

Thin Layer Chromatography 11

The extract was dissolved in diethyl ether and was spotted over the silica gel TLC plate;

allow the solvent to completely evaporate from the spot. Pour the solvent (toluene and ethyl

acetate, 80:20) into a small bottle and place the the plate over the developing solvent. If the

substances being separated are coloured, the spots can be seen without any further effort. The


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Rf value for the substance was calculated. Phytochemical constituents can also be visualized

by using spray reagents12 such as anisaldehyde- H2SO4 reagent (phenols and terpenes),

Antimony (III) Chloride (Flavanoids), Lead acetate (Flavanoids and Tannins) etc.

Column chromatography

Using toluene, ethyl acetate solvent system column chromatography was done and the

separated components were tested for the presence of phytochemicals.

RESULT AND DISCUSSION

Phytochemical analysis of ethanolic extracts of fresh leaves showed the presence of

flavanoids, terpenoids, phenolic compounds, tannins and cardiac glycoside. Ethanolic

extracts of shade dried and Oven dried leaves showed the presence of terpenoids, phenolic

compounds, tannins and cardiac glycoside. Aqueous extracts of fresh leaves showed the

presence of alkaloids, flavanoids, phenolic compounds and tannins and gum. Aqueous

extracts of shade dried leaves showed the presence of alkaloids, phenolic compounds, tannins

and gum. Aqueous extracts of oven dried leaves showed the presence of phenolic

compounds, tannins, morphine alkaloid and gum.(Table 1). Roshan Patel13 detected the

presence of alkaloid, carbohydrates, glycosides, proteins aminoacids, terpenoids, quinine,

tannins and flavanoids.

The Bacterial strains used for the study were identified by various staining and biochemical

tests as Bacillus sp, Klebsiella sp., S.aureus, E.coli, Ps.aeruginosa, Salmonella paratyphi A,

S.typhi, Streptococcus sp, Vibrio sp., Serratia sp., Enterobacter sp.(Table 2). All the isolates

were found to be multidrug resistant (Table 3).

Antibacterial activity against all the strains was shown only by Ethanolic extracts while

Aqueous extracts showed no activity at all (Table 4). Alcoholic extracts from the leaves of

Coleus amboinicus exhibited antibacterial activity against Bacillus cereus, Bacillus subtilis,

S.aureus, Shigella flexneri and Salmonella paratyphi A14. Ethanolic exacts were selected for

further analysis. MIC and MBC values were determined (Table 6). The antifungal activity of

Ethanolic extract of fresh leaves showed zone of inhibition only for Fusarium species (Table

7). Essential oil extracted from leaf of Coleus aromaticus showed antifungal activity against

two fungal species Candida albicans and Aspergillus niger were investigated and maximum

zone of inhibition was seen against C. albicans.


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The components in the Ethanolic extract were separated by TLC using toluene: ethyl acetate

solvent and were visualized by spray reagents. Ethanolic extract of fresh leaves showed

presence of steroids, while shade dried showed phenols, flavanoids, terpenes and tannins,

oven dried showed phenols and tannins (Table 8, 9).

Table 1.Phytochemical analysis of aqueous and ethanolic extracts of Coleus amboinicus

SI No: Phytochemical test fresh leaves shade dried

leaves oven dried

leaves EE AE EE AE EE AE

1

Carbohydrate

a) Benedict’s Test

b) Molisch’s Test

c) Barfoed’s Test

d) Fehling Test

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

Alkaloid

a)Wagner’s Test

b)Mayer’s Test

-

-

+

-

-

-

+

-

-

-

-

-

3 Flavanoids + + - - - -

4 Saponins - - - - - -

5

Terpenoids

Salkowsk’s test

Diterpenoid test

+

+

-

-

+

+

-

-

+

+

-

-

6 Protein

a)Biuret Test

-

-

-

-

-

-

7

Phenolic compounds and

tannins

a)Ferric chloride test

b)Lead acetate test

+

+

-

+

+

-

+

-

+

-

-

+

8 Phlobotannins - - - - - -

9 Cardiac glycoside + - + - + -

10 Morphine alkaloid - - - - - +

11 Oil - - - - - -

12 Gum - + - + - +

EE- Ethanolic extract, AE -aqueous extract, + positive, - negative


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Table 2. Identification of bacterial strains

a acid, ag acid and gas,+ positive,- negative, F fermenting, FM fermenting and

motile, FN fermenting and non motile, A/A acid slant acid butt, K/A alkaline slant

acid butt, O oxidative, S1 = Bacillus sp., S2 = Klebsiella sp., S3 = E.coli, S4, S5 =

Pseudomonas aeruginosa (P1,P2), S6 = S.aureus, S7 = S.typhi, S8 = S. parathyphi A, S9 =

Streptococcus sp., S10 = Enterobacter sp., S11 = Serratia sp., S12= Vibrio sp.

BACTERIAL STRAINS

TEST

S1

S2

S3

S4 S5

S6

S7

S8

S9

S10

S11

S12

Gram’s staining + - - - + - - + - - -

Motility test M NM M M NM M M NM M M M

Carbohydrate test

Glucose Lactose Sucrose

a

ag

ag

a

a

a

ag

a a a ag

a ag ag - a - - a a - ag

a ag ag - a - - a a a ag

OF O F F O F F F F O O Mannitol motility FM FN FM FM FN FM FM F

N FM FM FM

IMViC --++ --++ ++-- ---+ -+-- -+-- -+++

---- --++ -+-+ +++

- Urease test - + + - + - - - - + - Nitrate test + + + + + + + - + + +

Coagulase test - - - - + - - - -

Oxidase test - - - + - - - - - - -

Catalase test + + + + + + + - + + -

TSI test A/A G-

H2S-

A/A G+

H2S-

A/A Gas+ H2S-

K/A G-

H2S-

A/A G-

H2S-

K/A H2S

+

K/A G+, H2S-

- A/A G -

H2S-

A/A G+

H2S-

A/A G-

-H2S


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Table 3. Antibiotic sensitivity test of different bacteria

Antibiotic

Van

com

ycin

Neo

myc

in

Peni

cilli

n G

Chl

oram

phen

icol

Bac

itrac

in

Gen

tam

ycin

Am

pici

llin

Toba

myc

in

Stre

ptom

ycin

Bacterial Strains

Bacillus sp. 15 19

9

23

19

24

10

23 24

Klebsiella sp. - 11

-

-

-

-

-

10 -

S.aureus 11

14

-

9

9

19

-

23

-

E.coli -

-

-

-

-

-

14

-

11

Ps. aeruginosa (P1) -

10

-

26

-

16

-

20

16

Ps. aeruginosa (P2) -

10

-

33

-

28

-

23

27

S.typhi 18

13

-

21 - 17

17

15

16

Salmonella paratyphi A -

13

9

26

-

18 17 19

18

Streptococcus sp. 24

22

-

25

35

28

34

25

32

Vibrio sp. -

10

-

20

-

12

-

13

-

Serratia sp. 12 12 - - 10 20 - 20 15

Enterobacter sp. -

14

-

30

-

17

-

16

20

Table 4. Antibacterial screening by agar well diffusion method

Bacterial

Strains

Amount of

extract (mg/µl)

Diameter of zone of inhibition(mm)± Standard deviation

Ethanolic extract Negative control Aqueous extract

Fresh Shade dried

Oven dried

Ethanol Water Fresh Shade

dried Oven dried

Bacillus Species

1 8.5±0.5 8 8.5±0.5 - - - - - 0.5 8.5±0.5 7.5±0.5 6.5±0.5 - - - - - 0.5 8 6.5±0.5 6.5±0.5 - - - - -

Klebsiella Species

1 8 8 7.5±0.5 - - - - - 0.5 8 7.5±0.5 6.5±0.5 - - - - - 0.25 8 7 6.5±0.5 - - - - -

S.aureus 1 9 8 8.5±0.5 - - - - -

0.5 8.5±0.5 8.5±0.5 7 - - - - - 0.25 8 7 6.5±0.5 - - - - -


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Table 5. Antibiotic sensitivity test of different bacteria against Ampicillin (positive control) Bacterial

Strains S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

Am

ount

of a

mpi

cilli

n (m

g/µl

)

0.04

Dia

met

er o

f zon

e of

in

hibi

tion(

mm

) ± S

D

11.5 ±0.5 - 12.5

±0.5 - - 11.5 ±0.5

24.5 ±0.5 24 12 14.5

±0.5 10 -

0.08 13 - 17 ±1 - - 12

±1 25.5 ±0.5

25.5 ±0.5

13±1 17 12 -

0.16 13 - 20 - - 17 28 26 ±1 47 19

±1 13±

1 -

S1 = Bacillus sp., S2 = Klebsiella sp., S3 = E.coli , S4, S5 = Pseudomonas aeruginosa (P1,P2), S6 = S.aureus, S7 = S.typhi, S7 = S. parathyphi A, S9 = Streptococcus sp., S10 = Enterobacter sp., S11 = Serratia sp., S12= Vibrio sp

E.coli 1 9.5±0.5 6 - - - - -

0.5 9 7.5±0.5 6 - - - - - 0.25 9 6 - - - - - -

Ps.aeruginosa (P1)

1 8±1 8 9±1 - - - - - 0.5 8 7 7.5±0.5 - - - - - 0.25 5 6.5±0.5 8.5±0.5 - - - - -

Ps.aeruginosa (P2)

1 8.5±0.5 8 - - - - - 0.5 7.5±0.5 7 - - - - - 0.25 6.5±0.5 6 - - - - -

S.typhi 1 7.5±0.5 7 - - - - -

0.5 8 8 5.5±0.5 - - - - - 0.25 8 6.5±0.5 5.5±0.5 - - - - -

Salmonella paratyphi A

1 9.5±0.5 9 8.5±0.5 - - - - - 0.5 8 7.5±0.5 7 - - - - - 0.25 7 7 7 - - - - -

Streptococcus species

1 9.5±0.5 7.5±0.5 6 - - - - - 0.5 7.5±0.5 6 5 - - - - - 0.25 6.5±0.5 6 5 - - - - -

Vibrio species

1 8 8 7.5±0.5 - - - - - 0.5 - 7 - - - - - - 0.25 - 6 - - - - - -

Serratia species

1 9.5±0.5 7 9±1 - - - - - 0.5 8.5±0.5 7±1 6.5±0.5 - - - - - 0.25 6±1 6 5.5±0.5 - - - - -

Enterobacter species

1 9.5±0.5 9.5±0.5 9.5±0.5 - - - - - 0.5 8 8 7.5±0.5 - - - - - 0.25 8 - 7.5±0.5 - - - - -


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Table 6. Determination of MIC and MBC of fresh, shade dried and oven dried

Ethanolic extract

organism FL SDL ODL Amount of extract mg/µl

Bacillus sp MBC 0.031 0.031 0.031 MIC 0.016 0.016 0.016

Klebsiella sp MBC 0.016 0.016 0.016 MIC 0.008 0.008 0.008

E.coli MBC 0.031 0.016 0.016 MIC 0.016 0.008 0.008

Pseudomonas aeruginosa (P1)

MBC 0.031 0.031 0.031 MIC 0.016 0.016 0.016

Pseudomonas aeruginosa (P2)

MBC 0.031 0.031 0.031 MIC 0.016 0.016 0.016

S.aureus MBC 0.063 0.063 0.063 MIC 0.031 0.031 0.031

S.typhi MBC 0.031 0.016 0.016 MIC 0.016 0.008 0.008

S. parathyphi A MBC 0.031 0.016 0.016 MIC 0.016 0.008 0.008

Streptococcus sp

MBC 0.031 0.031 0.031 MIC 0.016 0.016 0.016

Enterobacter sp MBC 0.031 0.031 0.031 MIC 0.016 0.016 0.016

Serratia sp MBC 0.031 0.016 0.031 MIC 0.016 0.008 0.016

Vibrio sp. MBC - 0.031 0.031 MIC - 0.016 0.016

Table 7.Antifungal Assay

Fresh leaves

extract of Coleus

amboinicus

Amount of

extract (mg)

Zone of diameter(mm)

Fusarium species

Penicillium species

Aspergillus species

Aspergillus niger

Mucor species

Ethanolic

25 + +++ +++ +++ +++

50 + +++ +++ +++ +++

100 * +++ +++ +++ +++


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Table 8. TLC Assay (toluene: ethyl acetate)

Fresh Extract Shade Dried Extract Oven dried Extract Spot Rf Spot Rf Spot Rf 0.5 0.03 0.5 0.03 0.4 0.024 0.8 0.048 0.8 0.05 0.7 0.04 1.2 0.073 1.1 0.07 1.0 0.06 1.9 0.115 1.5 0.09 1.5 0.09 2.2 0.133 2.1 0.13 7.7 0.47 3.0 0.182 2.9 0.176 9.8 0.59 4.5 0.273 7.8 0.472 11.2 0.68 6.1 0.37 8.9 0.54 15.2 092 7.3 0.442 9.7 0.59 16.1 0.98 7.9 0.479 11.4 0.69 - - 9.1 0.552 12.1 0.73 - - 9.8 0.59 12.7 0.77 - -

11.5 0.70 13.4 0.81 - - 12.3 0.75 13.6 0.82 - - 13.4 0.81 15.2 0.92 - - 15.9 0.96 16.1 0.98 - -

Table 9. TLC Assay (spray reagents)

Fresh Extract Shade Dried Extract Oven dried Extract

Rf spot reagent Inter

pretation

Rf spot reagent Inter pretation Rf spot

reagent

Inter

pretatio

n

0.48 Green Anisaldehyde -H2SO4

steroids 0.36

Blue

Ani

sald

ehyd

e -H

2SO

4

phenol 0.06

Blue

Ani

sald

ehyd

e -H

2SO

4

phenol

0.66 Green steroids 0.49

Blue „ 0.3

5 Blu

e „

- - - - 0.60

Blue phenol 0.4

9 Blu

e phen

ol

- - - - 0.66 Red Terpenes 0.6

0 Blu

e „

- - - - 0.72

Blue phenol 0.7

2 Blu

e „

- - - - 0.76

Blue „ 0.8

0 Blu

e „

- - - - 0.80

Blue „ 0.8

4 Blu

e „

- - - - 0.84 Red Terpenes 0.9

2 Red „

- - - - 0.92 Red Terpenes - - „

- - - - 0.5 Blue

Antimony (III)

Flavanoids - - - -


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chloride

- - - - 0.93 Red

Lead

acetate

Tannins 0.93 Red

Lead acetate

Tannins

- - - - 0.96 Red Tannins 0.9

6 Red Tannins

CONCLUSION

Recently much attention has been focused towards the biologically active components

isolated from plant species. The use of medicinal plants plays a vital role in converting the

basic health needs and these plants may offer a new source of antibacterial agents with

significant activity against infective microorganisms.

Coleus amboinicus is a medically important plant. Investigations were carried out to evaluate

the antimicrobial activity of aqueous and ethanolic extracts of Coleus amboinicus against

multidrug resistant organisms. The results indicated that the crude extract of fresh, and oven

dried leaves of Coleus amboinicus at particular concentration showed significant antibacterial

effects against multi drug resistant bacteria such as Bacillus species, S.aureus, E.coli,

Serratia species, Streptococcus species, Klebsiella species, Vibrio species, S.typhi,

S.paratyphi A, Enterobacter species and Pseudomonas species(1&2). Oven dried leaves

show same result except for streptococcus.

The results of investigation indicates that ethanolic extracts of Coleus amboinicus possess

profound antibacterial activity. The finding reveals the medicinal property of Coleus

amboinicus as an antimicrobial agent against multi drug resistant pathogenic microorganisms.

Among the three types of leaves tested fresh leaves shows maximum activity than dried

leaves.

Coleus amboinicus was more effective against gram positive bacteria than gram negative

bacteria. They can be used to treat disease caused by multi drug resistant bacteria. Unlike

gram-positive bacteria, the lipopolysaccharide layer along with proteins and phospholipids

are the major components of the outer layer of gram-negative bacteria. So the outer

lipopolysaccharides layer may hinder access of antibacterial compounds to the peptidoglycan

layer of the cell wall. Plant extracts of fresh plant materials of Coleus amboinicus could

suppress the growth of both bacterial strains more than those of dried plant materials.

Inhibition of extracts of fresh plant materials was comparable with the extracts of dried plant


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materials. Ethanol extract of fresh leaf showed the highest antibacterial activity against

bacteria. Fresh plant materials may have more antimicrobial compounds than dried tissues

and probably during dryness antibacterial compounds may degrade due to temperature. Since

fresh materials give much higher antibacterial activities than did dried materials, fresh

materials should be used for the extraction in order to obtain maximum antibacterial

compounds.

FUNDING

The financial support from University Grants Commission, New Delhi is gratefully

acknowledged.

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