PHARMACOGNOSTICAL AND ANTIBACTERIAL STUDIES OF DIFFERENT EXTRACTS OF EUPHORBIA HIRTA L

Abstract

Leaves of Euphorbia hirta, traditionally practiced in the treatments of boils, dysentery, enteritis and various skin conditions, were extracted by soxhlet extraction in various extraction mediums. The disc diffusion method was used to determine the antibacterial activity against many Gram positive and Gram negative bacteria (Standard strains and clinical isolates). Antibacterial sensitivity test indicated that the methanolic extract inhibited the growth of S. aureus, E. coli, and B. subtilis to varying extents while K. pneumonia was the most resistive strain to these extracts. Minimum inhibitory concentration (MIC), of the extract against E. coli, S aureus, and S. entertidis were in the range of 0.1mg/ml. Phytochemical analysis indicates presence of terpenes, tannins, alkaloids and flavonoids which might be accountable for its antimicrobial properties, and these results validate the traditional uses of the plant in the treatment of various diseases. Bhuvaneshwar Upadhyay et al. Pharmacognostical and Antibacterial Studies of Leaf Extracts of Euphorbia hirta L. J Phytol 2/6 (2010) 55-60 *Corresponding Author,

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Journal of Phytology 2010, 2(6): 55–60 ISSN: 2075-6240
Pharmacology Available Online: www.journal-phytology.com
REGULAR ARTICLE
PHARMACOGNOSTICAL AND ANTIBACTERIAL STUDIES OF
DIFFERENT EXTRACTS OF EUPHORBIA HIRTA L.
Bhuvaneshwar Upadhyay*, K.P. Singh and Ashwani Kumar
Biotechnology Laboratory, P.G. School of Biotechnology and Department of Botany,
University of Rajasthan, Jaipur, India
SUMMARY
Leaves of Euphorbia hirta, traditionally practiced in the treatments of boils, dysentery,
enteritis and various skin conditions, were extracted by soxhlet extraction in various
extraction mediums. The disc diffusion method was used to determine the antibacterial
activity against many Gram positive and Gram negative bacteria (Standard strains and
clinical isolates). Antibacterial sensitivity test indicated that the methanolic extract
inhibited the growth of S. aureus, E. coli, and B. subtilis to varying extents while K.
pneumonia was the most resistive strain to these extracts. Minimum inhibitory
concentration (MIC), of the extract against E. coli, S aureus, and S. entertidis were in the
range of 0.1mg/ml. Phytochemical analysis indicates presence of terpenes, tannins,
alkaloids and flavonoids which might be accountable for its antimicrobial properties, and
these results validate the traditional uses of the plant in the treatment of various diseases.
Bhuvaneshwar Upadhyay et al. Pharmacognostical and Antibacterial Studies of Leaf Extracts of Euphorbia hirta L. J Phytol 2/6 (2010) 55-60
*Corresponding Author, Email: bhuvan.com@gmail.com
1. Introduction
The value of ethnomedicine and
traditional pharmacology is these days
achieving great appreciation in modern
medicine, as the search for new potential
medicinal plants is frequently based on an
ethnomedicinal basis (Muthu et al., 2006;
Parveen et al., 2007; Upadhyay et al., 2010).
Ethnobotanical studies of different areas of
Rajasthan state has been carried out by many
workers in this field (Singh and Pandey, 1998;
Mishra and Kumar, 2000, 2001; Katewa et al.,
2004; Parveen et al., 2007; Upadhyay et al.,
2010).
E. hirta is an Annual plant growing to
0.3m by 0.25m. The plant prefers light (sandy)
and medium (loamy) soils and requires well-
drained soil. According to survey, different
parts of E. hirta are used for curing various
ailments. The aerial parts of the plant are
harvested when in flower during the
summer and dried for later use. The stem is
used as a treatment for asthma, bronchitis
and various other lung complaints. The
whole plant is decocted and used in the
treatment of athlete's foot, dysentery,
enteritis, and skin conditions (Upadhyay et
al., 2010). It has been used in the treatment of
syphilis. The sap is applied to warts in order
to destroy them, and treatment needs to be
repeated 2 - 3 times a day over a period of
several weeks to be fully effective.
Along with common secondary
metabolites like; alkaloid, flavonoids,
coumarins and terpenes, a number of
substances, as; tannins, gallic acid, quercetin,
phenols, phyto-sterols, alcohols, etc. have
been reported in the plant (Kerharo and
Adam, 1974; Burkill, 1985). Blanc et al., (1972)
reported ellagic, Gallic, chlorogenic and
caffeic acids, kaempferol, quercitol,
quercitrin, and a number of amino acids. The
alcoholic extract of the whole plant had an
anticancer action in mice (Hartwell, 1967;
Sharma and Kumar, 2000). The plant has also
been shown to have anti-helminthic activity
(Ayensu, 1979; Sofowora, 1993; Adedapo et
al., 2005).
Interaction with some traditional medical
practitioners revealed that the plant is very
popular amongst them, thus there is need to
determine its antibacterial potentials. This
work was therefore undertaken to

Bhuvaneshwar Upadhyay et al./J Phytol 2/6 (2010) 55-60
substantiate the antibacterial potentials of E.
hirta.
2. Material and methods
Extraction of the plants
The leaves of E.hirta were collected from
many regional areas of Jaipur city, during
post monsoon period and were authenticated
by botanists at Deptt. of Botany, University
of Rajasthan, Jaipur, India and a specimen
sample is kept in our institution (herbarium
voucher numbers RUBL 20280). Shade dried
coarsely powdered leaves (44 g) of E.hirta
were subjected to successive extraction with
various extraction solvents (54-55.5°C) for 24-
36 hr using a soxhlet extractor. These crude
extracts were concentrated using vacuum
evaporator. The dried filtrate was
reconstituted with 100% dimethylsulfoxide
(DMSO).
Paper disks (diameter 6mm) were then
impregnated with 100µl of the final extract,
which is equivalent of 10mg/ml of dried
plant material. Once the DMSO had
evaporated, the disks were placed in a
refrigerator and stored in darkness for the
duration of the assays. 0.01ml of one of the 24
h broth cultures culture were spread on
sterilized nutrient agar media and
impregnated discs were placed on it and
incubated for 24 h at 37°C.
Preparation of micro-organism culture
In vitro antimicrobial activity of the
different extracts of E. hirta was studied by
disc diffusion method using different
concentrations on different microbial strains
such as Escherichia coli (ATCC 25922 and
Clinical isolate), Proteus vulgaris (ATCC
13315), Salmonella enteritidis (clinical isolate),
Bacillus subtilis (ATCC 6633), Staphylococcus
aureus (ATCC 6538P and clinical isolate),
Pseudomonas aeruginosa (ATCC 9027 and
clinical isolate), Klebsiella pneumoniae (ATCC
13883). The bacterial cultures were obtained
from, SMS Hospital, Jaipur.
All the bacteria were incubated at 30 ±
0.1°C for 24 hours by inoculation into
Nutrient Broth (Sigma). Sterilized Petri
dishes (9 cm diameter) were inoculated with
0.01 ml of one of the above culture media
(~105 bacteria per ml). Muller-Hinton agar
(Sigma), sterilized in a flask and cooled to
45–50°C, was distributed by pipette (15 ml)
into each inoculated Petri dish and swirled to
distribute the medium homogeneously. Discs
injected with extracts were applied on the
solid agar medium by pressing slightly. The
treated Petri dishes were placed at 4°C for 2
hours and then incubated at 35 ± 0.1°C for 24
hours.
At the end of the period, inhibition zones
formed on the medium were measured with
a transparent ruler in millimeters and
compared with the standard drugs prepared
by using standard antibiotics as Ampicilin
(10µg/ml), Streptomycin (10µg/ml), and
Tetracyclin (30µg/ml) in sterile distill water.
The experiment was performed in triplicate,
and average diameter of zone of inhibition
was obtained. 
Phytochemical investigation by TLC
The detection of active principles in
medicinal plants plays a strategic role in the
both; qualitative and quantitative
phytochemical investigation of crude plant
extracts (Pascual et al., 2002). TLC is a rapid
and economical procedure for the
determination of the main active principles
of medicinal plants e.g., alkaloids, cardiac
glycosides, coumarins, flavonoids, saponins,
tannins, etc. TLC is also used for
fractionation of the extract obtained by
extraction procedure by using different
solvent compositions.
The extent of the surface of the spot is a
measure for the quantity of the material
present (Pascual et al., 2002). The volume of
the spots applied on the chromatographic
plates was 5µl, corresponding to
approximately 300µg for each dry extract.
Chromatography was performed in the
following solvent systems: Nonpolar solvent:
toluene-acetone (8:2); semi-polar solvent:
toluene-chloroform-acetone (40:25:35); polar
solvent: n-butanol-glacial acetic acid-water
(50:10:40). The chromatograms were
observed first without chemical treatment,
under UV 254 nm and UV 365 nm light, and
then using the spray reagents.

Bhuvaneshwar Upadhyay et al./J Phytol 2/6 (2010) 55-60
Determination of Minimum Inhibitory
Concentration
For determination of Minimum
Inhibitory Concentration (MIC), the method
of Cheesbrough (2000) was used. Stock
solutions were prepared by dissolving the
extracts in DMSO. Two-fold serial dilutions
were employed to determine MIC values.
Each microorganism was incubated with an
extract in duplicate tubes containing a total
volume of 10 mL.
The final concentration of extract was in
the range 0.1 to 1.5 mg/mL. Control tubes
without extract were constituted similarly.
Antibiotics were included as positive control
in different tubes. The MIC was the lowest
concentration of extract with no visible
bacterial growth or no turbidity.

3. Result and Discussion
Table 1.Ethnopharmacological studies of E. hirta
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Table 2.Antibacterial screening of the different extract of Euphorbia hirta
*Ace.=Acetone, Eth.= Ethanol, Aq.=Aqueous extract, Chl.=Chloroform, Met.=Methanol
** Strepto= Streptomycin, Amp= Ampicilin, Tetra= Tetracycline
E
. hirta
Whole
plant
Amoebic
dysentery
Decoction of whole plant is taken internally with
milk for 5 days
athlete's foot Whole plant is decocted and taken internally and
paste is also applied on affected area. in the
treatment of athlete's foot
Skin
problems
Crushed whole plant is applied on the affected
area in skin conditions
Leaf
Asthma and
bronchitis
The drug is administered in the form of aqueous
extract with Grindelia or senega in the treatment
Leucorrhoea
About 20 leaves are crushed and the extract is
given orally with honey once a day in the
morning.
syphilis Decoction of leaves is taken internally with milk to
treat syphilis
Fresh
latex
Warts The fresh latex is applied to warts and The
treatment needs to be repeated 2 - 3 times a day.
Micro-organisms E. hirta Plant leaf extracts*
(10 mg/ml)
Standard Antibiotics**
Ace. Eth. Aq. Chl. Met. Strepto.
10 µg
Amp.
10µg
Tetra.
30µg
B. subtilis
(ATCC 6633)
6.5 11.3 9.4 7.6 12.3 19 15 25
E. coli
(ATCC 25922)
12.3 10.2 - 6.9 9.6 19.4 15 -
E. coli
(clinical isolate)
12.8 - 11.0 11.3 11.7 20.6 - 27
P. vulgaris
(ATCC 13315)
9.3 6.5 8.9 9.4 9.4 18.3 18 25
P. aeruginosa
(ATCC 9027)
8.1 7.1 8.9 - 11.2 22.5 - 20
P. aeruginosa
(clinical isolate)
8.0 6.5 8.6 - 9.6 20.0 14 12
S. aureus
(ATCC 6538P)
13.0 12.0 11.2 11.4 10.6 17.9 15 -
S. aureus
(clinical isolate)
12.5 11.6 - - 13.2 19.5 16 -
S. enteritidis
(clinical isolate)
11.3 5.6 10.3 - 8.9 18.6 - 24
K. pneumoniae
(ATCC 13883)
- - 6.5 - - 20.4 17 26
S.typhae
(clinical isolate)
11.0 12.1 9.8 - 8.6 18 19 24

Bhuvaneshwar Upadhyay et al./J Phytol 2/6 (2010) 55-60
Table 3.Minimum inhibitory concentrations of the ethanolic extract of Euphorbia hirta against test isolates
S.No. Microorganism E. hirta
1. B. subtilis (ATCC 6633) 0.2
2. E. coli (ATCC 25922) 0.1
3. E. coli(clinical isolate) 0.1
4. P. vulgaris (ATCC 13315) >0.5
5. P. aeruginosa(ATCC 9027) NA
6. P. aeruginosa(clinical isolate) NA
7. S. aureus(ATCC 6538P) 0.1
8. S. aureus(clinical isolate) 0.1
9. S. enteritidis(clinical isolate) 0.1
10. K. pneumonia(ATCC 13883) 1.0
11. S.typhae(clinical isolate) 0.2

Table 4.Preliminary phytochemical screening of ethanolic extract of Euphorbia hirta
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-= (negative result), += (small amount), ++ = (average), +++ = (high), nt= not tested
Plant extracts are generally rich in
antimicrobial agents after the flowering
(sexual) stage of their growth is complete,
and plants taken from stressful environments
were particularly active. Antibacterial
extracts from plants can be anticipated to be
S. No. Phytochemical E. hirta
1. Alkaloid ++
2. Flavonoids +
3. Saponin -
4. Coumarins +
5. Ployphenols ++
6. Cardiac glycosides +
7. Triterpenes +++
8. Cyanogenic glycosides -

Bhuvaneshwar Upadhyay et al./J Phytol 2/6 (2010) 55-60
useful in eliminating infectious diseases. The
infecting microorganisms are usually the
same as those infecting higher animals and
there is therefore compelling reason to
suppose that anti-infective agents could be
active against human or veterinary
pathogens. It is soothing to find, that the
spectrum of activity of these plant extracts is
broad enough to include human pathogens,
as was suggested by folkloric and historical
accounts.
During experiment this is noted that leaf
extract is more potent than any other extract.
Acetone extract was the second more potent
extract after methanolic extract. These results
are also according to the previous studies of
selection of extraction media (Eloff, 1998). As
evident by Table 2, the inhibition zone of S.
aureusby Methanolic extract of leaves of E.
hirta was 13.2mm, which is highest inhibition
zone, received. With observation of results
(table 2) it is clear that E. coli, S. aureus and B.
subtilis were the most susceptible bacteria to
almost all E. hirta extracts. On the contrary, K.
pneumoneae was the most resistant
microorganism, and very less number of the
extracts was active against K. pneumoneae.
Minimum inhibitory concentration of E.hirta
extracts were also recorded as 0.1 mg/ml in
case of B. cereus, S.aureus, and B. subtilis.
In this study, the results obtained
indicated that the Methanolic extract of the E.
hirta inhibited the growth of the test isolates
except K. pneumoniae. This, therefore, shows
that the extract contains substance(s) that can
inhibit the growth of some microorganisms.
Other workers have also shown that extracts
of some plants inhibited the growth of
various microorganisms at different
concentrations (Akujobi et al., 2004; Nweze et
al., 2004; Osadebe and Ukwueze, 2004). The
observed antibacterial effects on the isolates
is believed to be due to the presence of
alkaloids, tannins and flavonoids which have
been shown to posses antibacterial
properties(Cowan, 1999; Draughon, 2004).
Some workers have also attributed their
observed antimicrobial effects of plant
extracts to the presence of the sesecondary
metabolites (Nweze et al., 2004) and also
identified tannins, flavonoids and alkaloids
in the extracts of some medicinal plant
(Yoshida et al., 1990; Abo, 1990; Baslas and
Agarwal, 1980). The observed antibacterial
properties corroborate its use in traditional
medicine.
Traditionally, extracts of the plant are
used in sore and wound healing, as ear drop
for boils in the ear and treatment of boils.
They are also used in the control of diarrhea
and dysentery. The large zones of inhibition
exhibited by the extract against S. aureus and
P. aeruginosa justified their use by traditional
medical practitioners in the treatment of
sores, bores and open wounds. S. aureus and
P. aeruginosa have been implicated in cases
of boils, sores and wounds (Braude,1982).
Also the moderate growth inhibition against
E. coli justifiesits use in the control of
diarrhea and dysentery (Table 1). E. coli is the
common cause of traveler’s diarrhoea and
other diarrhea-genic infections in humans.
The low MIC exhibited by the extract against
S. aureus is of great significance in the health
care delivery system, since it could be used
as an alternative to orthodox antibiotics in
the treatment of infections caused by these
microbes, especially as they frequently
develop resistance to known antibiotics
(Singleton, 1999) (Table 3). Their use also will
reduce the cost of obtaining health care. The
relatively high zone of inhibition exhibited
by the extract against E. coli is also of
significance, since E. coli is a common cause
of diarrhea in developing countries.
On the basis of the results obtained, it
can be concluded that the crude extracts of E.
hirta exhibit significant antibacterial activity
and properties that support folkloric use in
the treatment of some diseases as broad-
spectrum antimicrobial agents. This probably
explains the use of extracts from these
species by the indigenous people of South
Africa against a number of infections for
generations. However, more work needs to
be carried out to determine the chemistry of
the particular active principle.
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