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Antibacterial Potential of Clerodendrum inerme Crude Extracts Against
Some Human Pathogenic Bacteria
Abdul Viqar Khan and Athar Ali Khan
Department of Botany, Faculty of Life Sciences, Aligarh Muslim University,
Aligarh , 202002, India
Summary
This communication emphasized upon the sensitivity of the crude extracts of Clerodendrum
inerme (L.) Gaertn. [Verbenaceae] against some of the human pathogenic bacteria. Five plant extracts
(Petrol, Benzene, Methanol, Ethly acetate and Aqueous) under six different concentrations(500mg/ml,
1mg/ml, 2mg/ml, 5mg/ml,10mg/ml and 15mg/ml) were tested by disk diffusion method . Methanol,
Ethyl acetate and Aqueous extracts of the plant showed significant inhibition against fifteen of the
eighteen bacteria tested. No earlier report on antibacterial activity of this taxon could be found in
literature.
Key words : Clerodendrum inerme, sensitivity, inhibition.
Plants have been an integral part of human society since the start of civilization. India is rich in
its plants diversity, a number of plants have been documented for their medicinal potential which are in
use by the traditional healers, herbals folklorists and in Indian systems of medicine namely, Ayurveda,
Unani, Siddha apart from a Homeopathy and Electropathy. These plant species play major role in the
health care of the nations population.
Different national and international pharmaceutical companies are utilizing such plant based
formulations in treatment of various diseases and disorders world around (Chandel et al., 1997; Singh
& Gautam, 1997; Satyavati et al.,1987; Pulliah, 2002; Jain, 1991; Khan et al.,2002 ; Kirtikar & Basu ,
1935)
Many of the plant species have been documented pharmacologically and clinically which are
endowed in phytochemicals with marked activity on human pathogenic bacteria. (Anonymous, 1976;
Ray & Majumdar , 1976 ; Khan, 2002; Cox, 1994; Khan et al., 2002; Asolkar et al.,1992; Rastigi &
Mehrotra, 1991,1993; Rastogi, 1998 ; Perry & Metzer, 1998 ; Fransworth , 1988).
An attempt was made to study the possible anti bacterial potential of the plant Clerodendrum inerme
(L.) Gaertn. [Verbenaceae]. It is a straggling shrub, leaves obovate to elliptical oblong, and glabrous.
Plant is commonly grown as hedged. Locally the plant is known as Lanjai , its leaves are used in
chronic pyrexia (Khan , 2002).
Chemical constituents: 3- epicaryoptin, neolignan.
Pharmacology: Alcoholic extract of the plant proved to be hypotensive. While essential oil possess anti
fungal properties( Asolkar et al.,1992; Rastigi & Mehrotra, 1991,1993; Rastogi, 1998).
Materials and methods
Plant material
Clerodendrum inerme (L.) Gaertn. [Verbenaceae], leaves of the plant were collected from the
university campus, Aligarh Muslim University, Aligarh , India.
Preparation of extracts
Crude plant extracts; were prepared following Robinson (1963), the protocol is
described below:
i. Freshly dried and healthy plant material is ground into fine powder in an electric grinder. Powder so
obtained is stored in dessicator.
ii. Five hundred g plant powder is refluxed with 95% methyl alcohol (MeOH) in a round bottom flask
on a water bath for 10 hours. Mother liquor (Crude MeOH extract) is filtered out and residual plant
material is again refluxed with 95% methyl alcohol for 10 hours. The process is repeated four times to
obtain maximum yield of MeOH extract. The extract is evaporated to dryness at 50°C under reduced
pressure.
iii. Dried methanol extract is refluxed with light petrol (60-80°C) for five hours. After filtration, the
residual methanol extract is again refluxed with petrol for five hours and filtered. This process is
repeated five times. Petrol is evaporated under reduced pressure to obtain petrol soluble extract.
iv. Petrol insoluble fraction of methanol extract obtained in step (iii) is refluxed with benzene for five
hours. Thereafter, it was filtered and refluxed again with benzene for five hours and filtered. The
process was repeated five times. Benzene is evaporated under reduced pressure to obtain benzene
soluble extract.
v. Benzene insoluble fraction obtained in step (iv) is refluxed with ethyl acetate for five hours.
Thereafter, it is filtered and refluxed again with ethyl acetate for five hours and filtered. The process is
repeated five times. Ethyl acetate is evaporated under reduced pressure to obtain ethyl acetate soluble
extract.
vi. Ethyl acetate insoluble fraction obtained in step (v) is refluxed with methyl alcohol (95%) for five
hours, filtered and is repeatedly refluxed for five times with methyl alcohol (Methanol). The methanolic
soluble fraction is evaporated under reduced pressure to obtain methanolic extract, while methanol
insoluble residue is discarded. The steps are graphically presented as a flow chart in Fig. 3B
Preparation of aqueous extract
Shade dried plant material (500 g) is ground to a fine powder, It is poured with distilled water, and
left for 72 hours at room temperature. The flask is then refluxed over hot water bath for 10 hours and
the mother liquor is filtered. The solute is again added with solvent (distilled water) that is again
refluxed and filtered; this process is repeated for 4 times. The filtrate, thus obtained, is evaporated to
complete dryness on a water bath. The residue thus obtained is aqueous plant extract.
Yields per 1000 g dry material: Petrol ~ 10.0 g, Benzene ~ 12.5 g, EtOAC ~ 8.0 g and MeOH ~ 13.0 g.
aqueous extract material (500 g) (yield ~ 40.0 g).
Microorganisms
The leaf extracts were tested for possible antibacterial activity in the disk assay using eighteen
(18) human pathogenic bacteria listed in table no. 1. The bacteria were obtained from the bacterial
stock, Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh, India. The bacterial
cultures were maintained at 40C on nutrient agar.
Anti microbial assay
The (Mueller& Hinton, 1941) agar plates were inoculated with inoculums of 106 size, a sterile
swab is dipped into diluted culture inoculums, the agar surface of the plates is streaked in three
directions turning the plates by 600 by each streak .The paper disk(what man filter paper no 1) with
500mg/ml, 1mg/ml, 2mg/ml, 5mg/ml,10mg/ml and 15mg/ml plant extracts were dried and placed at the
agar surface with the help of a sterile forceps. Finally press the sensitivity disc with forceps to make
complete contact with the surface of the medium. Allow the plates to stand at room temperature for 30
minutes. (Pre diffusion time). Inoculated petridishes were incubated at 370C over night and the
inhibition zone were recorded.(Bauer et al., 1966, Cruickshank, 1968 ; Colle & Marr, 1989). The
experiments were repeated thrice and the mean of the triplicate of the results is summarized in table no.
1.
Studied activity
Antibacterial activity by disc diffusion method (Bauer et al., 1966, Cruickshank, 1968). Diameters
of petri dish and disc 9.0 cm and 0.5 cm respectively.
Results
The petrol extract of the plant was found to be effective against four of the gram
positive and seven of the gram negative pathogenic bacteria. Benzene extract of the plant
inhibited the growth of five gram positive and six gram negative bacteria. Ethyl acetate
and Methanol fraction of the plant was found to be effective against all the tested gram
positive bacteria while they were found inactive against three of the gram negative
bacteria.(Pseudomonas aeruginos, Salmonella typhi, Shigella dysenteriae ).The crude leaf
extract (Aqueous extract) of the plant was effective against fifteen of the tested
pathogens.(Table no. 1)
Table – 1
Antibacterial activity of Clerodendrum inerme (Whole plant)a crude extracts
Inhibition zone (mm)
Gram Positive Bacteria Gram Negative Bacteria
1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11
PETROL
500 mg06 02 - 0 0 - - - 00 00 04 - - - - 05 06 -
1 mg 06 05 - 04 04 - - - 03 04 04 05 05 - - 06 06 -
2 mg 11 08 - 09 08 - - - 08 09 09 08 08 - - 09 09 -
5 mg 14 11 - 12 11 - - - 12 12 12 11 11 - - 12 13 -
10 mg 18 16 - 17 16 - - - 17 17 17 16 16 - - 18 17 -
15 mg 22 19 - 20 19 - - - 20 20 21 19 19 - - 20 19 -
BENZENE
500 mg03 - 06 - 04 00 04 00 00 04 04 - - - 00 - - 05
1 mg 03 - 07 - 05 04 05 05 02 05 06 - - - 03 - - 06
2 mg 06 - 10 - 08 07 09 08 06 09 09 - - - 07 - - 09
5 mg 12 - 14 - 11 10 12 11 09 13 12 - - - 12 - - 11
10 mg 16 - 17 - 16 12 16 16 12 16 16 - - - 16 - - 16
15 mg 19 - 20 - 19 16 21 19 16 19 19 - - - 19 - - 19
ETHYLACETATE
500 mg04 04 04 05 01 03 04 - 04 04 - 04 - 01 03 02 02 03
1 mg 04 05 05 05 02 04 05 - 04 05 - 04 - 02 05 03 03 04
2 mg 08 08 07 08 04 06 09 - 08 07 - 06 - 04 09 06 06 07
5 mg 14 14 12 14 07 10 15 - 10 10 - 09 - 06 14 09 09 10
10 mg 17 16 17 19 09 12 19 - 13 13 - 14 - 09 17 11 11 12
15 mg 21 20 21 21 12 18 21 - 17 18 - 19 - 11 21 13 15 17
METHANOL
500 mg
1 mg
2 mg
5 mg
10 mg
15 mg
03
04
08
14
17
21
04
05
09
15
17
21
04
04
08
12
17
20
-
02
06
09
13
19
-
-
04
07
09
12
02
03
06
10
12
16
04
05
08
14
17
20
05
05
08
14
17
20
-
-
-
-
-
-
-
02
06
09
12
16
03
04
08
11
13
18
-
-
-
-
-
-
03
04
06
09
12
16
-
-
-
-
-
-
-
-
04
06
09
11
02
02
05
09
11
15
02
05
06
09
11
14
04
05
07
12
14
16
AQUEOUS
500 mg
1 mg
2 mg
5 mg
10 mg
15 mg
-
-
05
10
13
16
-
03
06
11
13
17
-
02
05
10
13
15
02
03
06
10
14
16
-
-
-
03
05
08
-
-
05
07
10
12
-
-
03
08
11
15
-
-
-
-
-
-
-
-
02
05
08
10
01
02
05
0810
13
-
-
-
-
-
-
01
02
05
07
09
11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
04
06
08
11
-
-
-
-
-
-
-
-
05
07
10
12
-
-
-
-
-
-
Chloramphenicol
10 mg/disc 18 18 16 - - - 16 18 16 - 16 18 - 16 17 19 18 20
Gram Positive Bacteria
1.Staphylococcus aureus 2. Staphylococcus aureus ATCC 25953 3. Staphylococcus albus 4. Streptococcus haemolyticus Group-A
5. Streptococcus haemolyticus Group-B 6. Streptococcus faecalis 7. Bacillus subtilis.
Gram Negative Bacteria
1.Escherichia coli 2. Edwardsiella tarda 3. Klebsiella pneumoniae 4. Proteus mirabilis 5. Proteus vulgaris 6. Pseudomonas aeruginosa 7. Salmonella typhi 8.
Shigella boydii 9. Shigella dysenteriae 10. Shigella flexneri 11. Plesiomonas shigelloides.
aValues are the mean of replication of three; -, no inhibition.
Discussion
Very interesting facts were recorded during the sensitivity test performed .In case
of petrol extract Staphylococcus aureus was the most affected bacteria (zone of inhibition
6mm/500mg/ml/disk). Followed by Shigella dysenteriae and higella flexneri (zone of
inhibition 5mm/500mg/ml/disk). Benzene extract inhibited the growth of eleven tested
bacteria and the maximum inhibition zone was recorded against Staphylococcus
albus(zone of inhibition 6mm/500mg/ml/disk).Fifteen microorganisms were found
sensitive to ethyl acetate fraction and the most affected bacteria were Staphylococcus
aureus, Klebsiella pneumoniae and Shigella boydii(zone of inhibition
8mm/500mg/ml/disk/each).While methanolic fraction was found to most effective against
Streptococcus faecalis and Bacillus subtilis gram positive and three of the gram negative
bacteria(Klebsiella pneumoniae, Proteus mirabilis and Shigella boydii) (zone of inhibition
7mm/500mg/ml/disk/each).
From the results it is clear that leaves of Clerodendrum inerme are effective
in controlling bacterial pathogens, particularly gram positive bacteria. In these
investigations it becomes certain that most effective crude extract was ethyl acetate for
which maximum zone of inhibition was recorded. Followed by methanol fraction that also
inhibited the growth of fifteen tested human pathogens. While petrol and benzene extracts
as compared to the methanol showed weak anti microbial action. This action may be
synergistic and not due to the efficacy of one single substance. It was also noticed that
methanol, ethyl acetate and aqueous extracts showed antibacterial activity against both
types of pathogens (Fig. 1). The above results revealed that plant extracts could be
effective antibiotics. Both in controlling gram positive and gram-negative human
pathogens. The results also confirm the utility of plant as a wound-healing agent.
Acknowledgement
Thanks are due to Department of Science and Technology SERC Division, New Delhi for financial
support to the author Dr Abdul Viqar Khan. Authors are also grateful to the Chairman, Prof. Ainul Haq
Khan, Department of Botany, Aligarh Muslim University Aligarh for his cooperation and providing
space and other facilities.
BIBLIOGRAPHY
1. Anonymous (1976) Indian material medica, Vol.1, pp.283-284, Population Prakashan,
New Delhi, India.
2. Asolker LV, Kakkar KK & Chakra OJ. (1992) Second supplement to glossary of Indian
medicinal plants with active principles, part 1(A-K). XIVII+414, Pub. & Inf. Div.(CSIR),
New Delhi.
3. Bauer AW, Kirby WMM & Sherries T. (1996) Antibiotic susceptibility testing by a
standard single disc method. American Journal of clinical pathology 45,493
4. Colle JA & Marr W. (1989) Cultivation of Bacteria. In Mackie & Mc Cartney: Practical
microbiology 13thed. pp.121-140, Churchill livingston, USA.
5. Cox PA. (1994) The ethnobotanical approach to drug discovery: strengths and
limitations. In, ethnobotany and the search for the new drugs. pp. 25-36, John Wiley &
Sons. England.
6. Cruickshank R. (1968) Medical Microbiology : A guide to diagnosis and control of
infection, pp.888, Edinburgh and London : E & S Livingston Ltd.
7. Fransworth NR. (1988) Sereening plants for new medicines . Wilson EO(ed.).
Biodiversity, pp.83-97, National Academy Press, Washington.
8. Jain SK. (1991) Dictionary of Indian Folk medicine and ethnobotany, pp.XII+311, Deep
publication, N.Delhi.
9. Khan A.V. & Khan A.A. (2003) herbal abortifacients used by folk people of some
districts of Western Uttar Pradesh (India). Journal of Natural Remedies 3(1), 41- 44.
10. Khan AV, Parveen G,. Alam MM & Singh VK. (2002) Ethnomedicinal uses of Neem in
rural areas of Uttar Pradesh, India. Ethnomed & Pharmacog. II Rec. Prog. In Med. Plants
pp. 7, 319-326, (Sci. Tech. Pub. USA).
11. Khan AV, Alam MM & Singh VK. (2002). Ethnomedicinal uses of Citrullus colocynthis
(L.) Schard. In rural areas of Aligarh District of Uttar Pradesh, India Ethnomed. &
Pharmacog. II. Rec. Prog in Med. Plants. 7, 383-388, (Sci. Tech. Pub. USA).
12.
Khan AV. (2002) Ethnobotanical studies on plants with medicinal and anti-bacterial
properties pp. 1-293. (Thesis). Aligarh Muslim University, Aligarh.
13. Kirtikar KR & Basu BD. (1935) Indian Medicinal Plants. Vol 3, pp. 1841. Allahabad.
Lalit Mohan Publication.
14. Mueller JH & Hinton J. (1941) A protein free medium for primary isolation of the
gonococcus and meningococcus proceedings of society of exp. Biology and Medicine 48,
330-333.
15. Perry LM & Metzer J. (1998) Medicinal plants of east and south Asia, attributed
properties and uses. Massachisetts and London: MIT press Cambridge.
16. Rastogi RP & Mehrotra BN. (1991) Compendium of Indian Medicinal Plants, Vol. III 1:
497, CSIR Publication, India.
17. Rastogi RP & Mehrotra BN. (1993) Compendium of Indian Medicinal Plants, Vol. IV 1:
497 CSIR Publication, India.
18. Rastogi RP .(1998) Compendium of Indian Medicinal Plants, Vol. 1, 497, CSIR
Publication, India.
19. Ray PG & Majumdar SK. (1976) Antimicrobial activity of some Indian Plants. Economic
Botany 30(4), 317-329.
20. Satyavati. GV, Gupta AK & Tanabu N. (1987) Medicinal plants of India Vol.2 pp.
XI+557, CSIR Publication, Indian Council of Medical Research, Cambridge printing
worker, N. Delhi.
21. Singh SH & Gautam M. (1993) Bioresources of Med. & Aro. Plants of India. Their
conservation and related issues, Kurukshetra 56(3) 9-13.
