issn: 0975-766x coden: ijptfi available online through ...in this study a comparative evaluation of...

Varish Ahmad * et al. /International Journal Of Pharmacy&Technology

IJPT | Sep-2011 | Vol. 3 | Issue No.3 | 3015-3028 Page 3015

ISSN: 0975-766X CODEN: IJPTFI

Available Online through Research Article www.ijptonline.com

COMPARATIVE EVALUATION OF ANTIMICROBIAL ACTIVITY OF METHANOLIC EXTRACTS OF LEAVES, STEM OF ECLIPTA ALBA AND NATURAL OILS

Varish Ahmad*, Lokesh Kumar, Q.M.Sajid Jamal, Gaurav Dhama, Tej Prakash and Ajeet Singh. Institute of Applied Medicines and Research, Ghaziabad, U.P., India. 201206.

Email:[email protected]

Received on 06-07-2011 Accepted on 28-07-2011

Abstract

Plants are the oldest source of pharmacologically active compounds, and have provided humankind with many

medically useful compounds for centuries. In this study a comparative evaluation of methanolic extract of leaves,

stem of Eclipta alba and natural oils namely Clove oil, Olive oil, peppermint oil and eucalyptus were tested for

antimicrobial activity by disc diffusion method against E. coli, E. cloacae, E. faecalis, P. vulgaris, S. aureus, K.

pneumoniae, S. saprophyticus. It was observed that methnolic extract of leaf, stem and natural oils shown reliable

Zone of inhibition (ZOI) against all tested microbes. The variable inhibition effect were observed with methanolic

extract and natural oil with organisms to organism. Clove oil found most potent inhibitor among all tested agents.

Maximum inhibitory effect of methanolic extract of leaves was found against E. cloacae and lowest against E. coli

and S. aureus and methanolic extract of stem shown highest against S. saprophyticus and lowest against E. coli.

The highest ZOI of clove oil was found against K. pneumoniae and lowest against E. coli Olive oil was found to be

resistant against all microbes. Thus natural oils were found effective as positive control and were more effective as

compared to methanolic extract of Eclipta alba.

Keywords: Antimicrobial, Eclipta alba, methanolic extract, natural oils.



Introduction

The traditional use of plant derived medicinal compounds received much attention against the multifactorial

antibiotic resistance as they are well tested for their efficacy and being used to treat various microbial diseases (15).

Medicinal plants have been conventionally used as antimicrobial and anti-inflammatory agents. Eclipta alba

commonly known as False daisy, yerba de tago, and bhringraj, is a plant belonging to the family Asteraceae. The

leaf extract is considered as a powerful liver tonic, rejuvenative and especially good for the hair. It is used as anti-

venom memory disorders treatment, general tonic, edema, fevers and rheumatic joint pain treatment (15, 16).

The shoot extract of E. alba showed antimicrobial (2, 17), antifungal activity (3) and weak cytotoxicity against the

M-109 cell lines by alkaloids Verazine (9) antiviral activity against Ranikhet disease virus effective against internal

and external parasites (4). The herb has been used in the treatment of infective hepatitis in India (6) and snake

venom poisoning in Brazil (13). It has been reported that the leaves of this herb are used in the case of gastritis and

respiratory disorders like cough and asthma (10). digestion, hepatitis, enlarged Plant is bitter, hot, sharp, dry in taste

and is used in ayurveda & "siddha" for the treatment of Kapha and Vata imbalances (17). The herb Eclipta alba

contains mainly coumestans i.e. wedelolactone (I) and demethylwedelolactone (II) polypeptides, polyacetylenes,

thiophene-derivatives, steroids, triterpenes and flavonoids. Wedelolactone possesses a wide range of biological

activities and is used for the treatment of hepatitis and cirrhosis (6-16), as an antibacterial, anti-hemorrhagic and for

direct inhibition of IKK complex resulting in suppression of LPS-induced caspase-11 expression (10). The

antimicrobial assays were performed by MIC determination and diffusion disc method (12). The antimicrobial

activity of essential oils against Escherichia coli, Salmonella enteritidis, and Salmonella typhimurium was

conducted by (14) Methanol, ethyl acetate, and hexane extracts of Bridelia ferruginea leaves exhibit good

antimicrobial activity against various pathogenic bacteria (14).



Materials and Methods

Sample collection

The plant samples were collected from North India region mainly from Duhai village locality, surrounding the

campus of Institute of Applied Medicines and Research, Duhai, Ghaziabad, U.P. Fresh plant leaves were washed

separately under running tap water as well as distilled water and shade drying of leaves was carried out at room

temperature and then, homogenized to very fine powder and stored at 40C till the extraction was carried out (7).

Preparation of extract

20-20 grams of shade dried plant leaves and stem powdered, was added to 100 ml methanol in 250 ml conical

flasks and kept at room temp on a rotators shaker at 180 rpm for three days. The extracts were filtered through

muslin cloth and then centrifuged at 10,000 rpm for 10 minutes .The supernatants obtained were filter sterilized The

filtrates were made conc. by vacuum drier and responded in DMSO to make a conc. of 400 mg/ ml, and stored at

4 0 C for further use (11).

Microorganism used

The antimicrobial activity was carried out by using the organism Escherichia coli (ATCC25923), Enterobacter

cloacae (ATCC10699), Enterococcus faecalis (ATCC10741), Proteus vulgaris (ATCC12454) Staphylococcus

aureus (ATCC25923), Klebsiella pneumonia (ATCC15380) and Staphylococcus saprophyticus (ATCC35552). The

organisms were obtained from the Department of Microbiology, Institute of Applied Medicines and Research,

Ghaziabad, U.P. India. The microorganisms were maintained by sub-culturing on nutrient agar medium and used at

regular intervals to carry out the antimicrobial activity.

Assay method

The antimicrobial activity of extract was evaluated by growing the broth of each tested microorganism till the late

log phase developed by taking the absorbance using UV-Visible spectrophotometer Model SL-159, Elico.(O.D.at

590nm=0.6 0 . DMSO was taken as negative control and Ampicillin, Cephotoxime, Streptomycin and Tetracycline



were taken as positive control. Using a sterile cotton swab, the nutrient broth cultures were swabbed on the surface

of sterile nutrient agar plates.

Preparation of assay media

All the chemicals used were chemically pure and AR grade .The media used for maintaining, culturing of

microorganism and to carry out the antimicrobial susceptibility testing was prepared as -Dissolved 5 gm of peptone

(Qualigens fine chemicals), 3 gm of beef extract (Qualigens fine chemicals), 5 gm of NaCl (Merck) and 20 gm of

agar (Blulux labs) in 0.5 L of distilled water. pH was adjusted to about 6.8-7.2 and final volume was adjusted to 1 L

and autoclaved at 121°C and 15 Lbs for 15 minutes.

Disc diffusion method for antimicrobial activity

Briefly, nutrient broth / or agar was used to culture bacteria. Fresh overnight cultures of inoculums (0.1 ml) of each

culture, was spread on agar plate. The plates were kept for 10 min and then the prepared sterilized discs (5 mm

diameter) soaked with the 20 µL with each stock. The discs with concentrated extract were impregnated over the

surface of the plate, inoculated with the test microorganisms and allowed to incubate at 37 0C for the 24 h. The zone

of inhibition in mm was determined after incubation period. The assays were carried out in triplicates and average

zone diameter was noted (8).

Determination of Minimum Inhibitory Concentration (MIC)

Some idea of the effectiveness of a chemotherapeutic agent against a pathogen can be obtained from the minimal

inhibitory concentration. The MIC is the lowest concentration of a drug that prevents growth of a particular

pathogen. For determination of MIC, Dilution susceptibility test was applied which was carried out by using the

extract as 100%, (400mg/ml) 75% (300mg per ml) (300mg/ml) and 50% (200mg per ml) and 20micro L of each

extract were applied on dics. The lowest concentration of plant extract resulting in no inhibition after required

incubation is determined as the MIC. (12).

Results

Antimicrobial susceptibility The result of antimicrobial activity were found as depicted in the tables. (1-6 and fig.)



Minimum inhibitory concentration: Minimum inhibitory conc. was determined by dilution susceptibility testing.

The MIC of methanolic extract of leaves and stem were found at the dilution of 25% or the dilution of 100 mg/ml

(20µg per disc). Similarly the natural oils were also effective at the concentration of less 25% of dilution, from the

original stock bought from local market.

Table No.1: Antimicrobial activity of leaf and stem methanolic extract of E.alba.

S.NO. Name of

Microorganism

Leaves ext. Stem ext. M control

ZOI

(mm)

ZOI

(mm)

ZOI

(mm)

1. E. coli S 7 S 7 R -

2. E. clcae S 15 S 11 R -

3. E. faecalis S 9 S 8 R -

4. P. vulgaris S 8 S 8 R -

5. Staphylococcus

aureus

S 7 S 9 R -

6. K. Pneumoniae S 9 S 8 R -

7. S.Saprophyticus S 11 S 12 R -

Table No. 2: Antimicrobial activity of leaf methanolic extract of E. alba at different concentration.

S.NO. Name of

Microorganism

Methanolic leaves extract

100%

(400mg/ml)

ZOI (mm)

75%

(300mg/ml)

ZOI (mm)

50%

(200mg/ml)

ZOI (mm)

1. E. coli S 7 S 6 R -



2. E. clocae S 15 S 12 R -

3. E. faecalis S 9 S 7 S 6

4. Pseudomonas

vulgaris

S 8 S 6.5 S 5.5

5. Staphylococcus

aureus

S 7 S 6 S 5.5

6. K. Pneumoniae S 9 S 8 S 6.8

7. S.

Saprophyticus

S 11 S 9.5 S 7.4

Table No. 3: Antimicrobial activity of stem methanolic extract of E. alba at different concentration.

S.NO. Name of

Microorganism

Stem Methanolic extract

ZOI

100%

(mm)

ZOI

75%

(mm)

ZOI

50%

(mm)

1. E. coli S 7 S 6 S 5.5

2. E. cloacae S 11 S 10 S 9.5

3. E. faecalis S 8 S 7.5 S 6.2

4. P.vulgaris S 8 S 7.2 S 6.5

5. S.aureus S 9 S 8 S 7.5

6. K. Pneumoniae S 8 S 7 S 6.5

7. S.

Saprophyticus

S 12 S 10 S 8.5



Table No.4: Antimicrobial activity of Clove oil & Olive oil.

S.NO. Name of

Microorganism

Clover oil Olive oil

ZOI

50%

(mm)

ZOI

100%

(mm)

ZOI

50%

(mm)

ZOI

100%

(mm)

1. E. coli S 8 S 10 R - R -

2. E. cloacae S 14 S 16 R - R -

3. E. faecalis S 17 S 20 R - R -

4. P.vulgaris S 11 S 14 R - R -

5. S. aureus S 16 S 20 R - R -

6. K. Pneumoniae S 20 S 21 R - R -

7. S.Saprophyticus S 18 S 20 R - R -

Table No.5: Antimicrobial susceptibility of peppermint oil and eucalyptus.

S.NO. Name of

Microorganism

Pepperment Oil Eucalyptus oil

ZOI

50%

(mm)

ZOI

100%

(mm)

ZOI

50%

(mm)

ZOI

100%

(mm)

1. E. coli S 12 S 14 S 7 S 9

2. E. cloacae S 12 S 15 S 12 S 15

3. E. faecalis S 13 S 15 S 6 S 7

4. P.vulgaris S 14 S 16 R - R -

IJPT | Sep-2011 | Vol. 3 | Issue No.3 | 301

5. S. aureus

6. K. Pneumoniae

7. S.Saprophyticus

Table No. 6: Antimicrobial activity of different antibiotic

Microbes

Bacteria Cephotoxime

S.No. Name Inf.

1. E.coli S

2. E.clocae S

3. E.faecalis S

4. P.vulgaris S

5. S.aureus S

6. k.pneumoniae S

7. S.saprophyticus R

Fig.1


15-3028

S 7 S 9 S 7 S

S 7 S 8 S 17 S

Saprophyticus S 7 S 8.5 S 12 S

Antimicrobial activity of different antibiotic.

Antibiotic

Cephotoxime Chloramphenicol Tetracycline

Zone

(mm)

Inf. Zone

(mm)

Inf. Zone

(mm

2.6 S 18 R -

32.5 S 15 S 11

28.5 S 10 S 19.5

32 S 29 S 8

24 R - S 20

26.5 R - S 15

- S 11 S 11

et al. /International Journal Of Pharmacy&Technology

Page 3022

S 9

S 18

S 13

Tetracycline Streptomycin

Zone

(mm)

Inf. Zone

(mm)

S 11

R -

19.5 S 10

R -

R -

R -

R -



5. Antimicrobial activity of methanolic extract of leaf and stem of E.alba against S .aureus.

6. Antimicrobial activity of methanolic extract of leaf and stem of E.alba against E.coli.

7. Antimicrobial activity of methanolic extract of leaf and stem of E. alba against .cloacae.

8. Antimicrobial activity of methanolic extract of leaf and stem of E. alba against K. pneumoniae

Fig. 2. Antimicrobial activity of methanolic extract of leaves and stem

Fig. 3. Antimicrobial activity of methanolic extract of leaves at different conc. (MIC)

0

2

4

6

8

10

12

14

16

LM ZOI (mm)

SM ZOI (mm)

M control ZOI (mm)

0

2

4

6

8

10

12

14

16

LM ZOI 100%

LM ZOI 75%

LM ZOI 50%



LM- Leaves Methanolic.

Fig. 4. Antimicrobial activity of methanolic extract of stem at different conc.

SM- Stem Methanolic.

Fig.5. Antimicrobial activity of clove and olive oil at different conc.

0

2

4

6

8

10

12

14

SM ZOI 100%

SM ZOI 75%

SM ZOI 50%

0

5

10

15

20

25

100% clove oil

50% clove oil

100% olive oil

50% olive oil



Fig.6. Antimicrobial activity of pipperment oil and eucalyptus oil at different conc.

Fig.7. Antimicrobial activity of different antibiotics as positive control

0

2

4

6

8

10

12

14

16

18

20

100%Pipperment oil

50%Pipperment oil

100%Eucalyptus oil

50%Eucalyptus oil

0

5

10

15

20

25

30

35

Cephotoxime

Chloramphenicol

Tetracycline

Streptomycin



Discussion

In this work the antimicrobial activity of aerial parts of plants compared with standard drugs and natural oils were

carried out. The methanol was used as solvent for extraction of plant. The antimicrobial susceptibility was carried

out by disc diffusion method. (8-12). The methnolic extract of leaf also showed reliable ZOI against all microbes

and highest found against E. cloacae and lowest against E. coli and S. aureus. The methanolic extract of stem

showed reliable ZOI against all microbes and highest found against S. saprophyticus and lowest against E.coli.

The antimicrobial activity of clove oil, olive oil, peppermint oil and Eucalyptus oil was also checked against these

microbes. Clove oil showed reliable ZOI against all microbes. The highest ZOI was found against K. pneumoniae

and lowest against E. coli. Olive oil was found to be resistant against all tested microbes. The peppermint oil

showed ZOI against all microbes and highest against P. vulgaris and lowest against S. aureus, K. pneumoniae and

S. saprophyticus. The eucalyptus oil showed reliable ZOI against all microbes except P. vulgaris (14). In case of

antibiotics cephotoxime the maximum ZOI was observed against E. cloacae minimum ZOI was observed against S

.aureus .In case of chloramphenicol the maximum ZOI was observed against P. vulgaris and minimum against E.

faecalis. In case of Tetracycline the maximum ZOI was observed against S. aureus and minimum against

P.vulgaris. In case of Streptomycin the maximum ZOI was observed against E. coli and minimum ZOI against E.

faecalis.

Conclusion

From the results of present work it was concluded that Methanolic extract of plant extract shown significant

antimicrobial activity against all tested organism. Maximum zone of inhibition were shown by Methanolic extract

of stem against E. clocaeand and S. saprophyticus while other strains were shown almost equal zone of inhibitions.

The antimicrobial susceptibility of natural oils eucalyptus oil, clove oil., olive oil, and peppermint oil tested against

isolated microorganism were shown very good antimicrobial activity except olive oil which did not shown any

significant activity. Clove oil shown maximum inhibition effect, peppermint oil shown moderate inhibition effect

and eucalyptus oil gave minimum significant inhibitory effect and all tested microbes were found resistant to olive



oil. The antimicrobial susceptibility testing carried out by using standard drugs which were shown that cephatoxime

shown maximum antimicrobial effect against all tested organism except S. saprophyticus which was found

resistant. Chloramphenicol and Tetracycline were shown moderate inhibitory effect while streptomycin shown only

moderate zone of inhibition against E.coli and E. faecalis and other strains were found resistant.

References

1. A. Cremer, Microbiological methods, 1991, Butterworth and Co., London, 6th Ed, 235.

2. Anonymous. The Wealth of India - Raw Materials, CSIR, New Delhi, 1952, Vol. III, pp.127.

3. B.R. Hoffman, H .Delas Atlas, K. Blanco, N. Wiederhold, R.E. Lewis, L. Williams, J. Pharm. Biol, 2004,Vol.

1(42), pp 13-17.

4. C. Lans, T. Harper, K. Georges, E. Bridgewater, BMC Compl, 2001.

5. C. Wiart, S. Mogana, S. Khalifah, M. Mahan, S .Ismail, M. Buckle, A.K. Narayana, M. Sulaiman, Fitoterapia,

2004, Vol.75, pp. 68-73.

6. H. Wagner, B. Geyer, Y. Kiso, G.S. Rao, Planta Med., 1986, Vol. 52, pp. 370-373.

7. J. Parekh, S. Chanda, African J. Micro.Res., 2007,Vol. 1(6), pp. 92-99.

8. K. M. Elizabeth, Indian J. Microbiol, 2001, Vol 41, pp 321-323.

9. K.M.S. Abdal, B.D.B. Malone, S. Werkhoven, M.C. Van, T.F. David, J.H. Wisse, I. Bursuker, K.M.

Neddermann, S.W. Mamber, and D.G. Kingston, J. Natl. Prod, 1998, Vol 61(10), 1202-1208.

10. M. Kobari, Z. Yan, D. Gong, V. Heissmeyer, H. Zhu, YK. Jung, M. Angelica, M. Gakidis, A. Rao, T. Sekine, F.

Ikegami, C. Yuan, J. Yuan, J. Cell Death and Differentiation, 2004, Vol .11(1), pp. 123-130.

11. M.H. Lee, H. A. Kwon, D.Y. Kwon , H. P, D. H. Sohn , Y.C. Kim , S.K. Eo , H.Y. Kang, S.W. Kim, J. H. Lee,

Int J Food Microbiol, 2006, Vol. 111, pp. 270 –275.

12. M.S. Ali-Shtayeh, M.R. Yaghmour, Y.R.Faidi, K. Salem, M.A. Al-Nurys, J. Ethnopharmacol, 1998, vol. 60,

pp. 265-270.



13. P. A. Melo, MC. Do Nascimento, W.B.Mors, G. Suarez-Kurtz, (1994). Toxicon, 1994, Vol. 32, pp. 595-603.

14. P. Penalver, B. Huerta, C. Borge, R. Astoraga, APMIS, 2005, pp. 113- 16.

15. R. M. Thorat, V.M. Jadhav, D. D. Gaikwad, S.L. Jadhav, IRJP, 2010, Vol. (1), pp.77-80.

16. S. Karthikumar, K. Vigneswari, K. Jegatheesan, Scientific Research and Essay, April 2007, Vol. 2 (4), pp. 101-

104.

17. W.B. Mors, M.C. Do Nascimento, J.P. Parente, M.H. Da Silva, P.A. Melo, G. Suarez-Kurtz (1989),

Toxicon.1989, Vol. 27, pp.1003-1009.

Corresponding Author:

Varish Ahmad*,

Email: [email protected]

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