British Journal of Pharmacology and Toxicology 3(4): 177-180, 2012
ISSN: 2044-2467
© Maxwell Scientific Organization, 2012
Submitted: June 20, 2012
Accepted: July 28, 2012
Published: August 25, 2012
Anti-Microbial Activity of Ethanolic and Aqueous Extract of Cynanchum acutum
1
Mehdi Dehghani, 1Zohre Ganjali, 2Fereshteh Javadian, 1Jasem Estakhr and 1Abbas Heidari
1
Department of Biology, Faculty of Science, University of Zabol, Zabol, Iran
2
Department of Biology, Faculty of Science, Mashhad Branch, Islamic Azad University, Mashhad, Iran
Abstract: Cynanchum acutum is an important medicinal plant belonging to the family of Apocynaceae. It is a well
recognized plant in the traditional medicine and is used by people in rural areas. This study is aimed to evaluate the
anti-microbial effects of ethanolic extract of Cynanchum acutum. The antimicrobial property of the Cynanchum
acutum was studied against gram positive and gram negative microorganisms using the agar disc diffusion method,
in which ethanolic extract of Cynanchum acutum has shown bigger zone of inhibition than aqueous extract. These
results demonstrate that Cynanchum acutum possesses anti-microbial effects and it can be a good candidate for
producing of anti-microbial drugs.
Keywords: Anti-microbial, aqueous extract, Cynanchum acutum, ethanolic
tannins, carbohydrate and essential oils. Plant based
natural constituents can be derived from any part of the
plant like bark, leaves, flowers, roots, fruits, seeds, etc
(Gordon and David, 2001). Many plants possess
antimicrobial activities and are used for the treatment of
different diseases (Arora and Kaur, 1999). Cynanchum
acutum is an invasive plant species belonging to the
family Apocynaceae. It has been used as a purgative in
the French pharmaceutical Codex (Garnier et al., 1961)
and its milky latex is used for skin and eye problems in
Tunisian folk medicine (Sayed et al., 2003; Boukef,
1986) and its seeds are edible in some parts of Iran. The
photochemical investigations on Cynanchum acutum L.
have revealed the presence of several natural
compounds including β-sitosterol, lupeol, lupyl acetate
and α-amyrin (Halim et al., 1990), sarcostine, quercetin
and quercetin 3-O-β-D-galactoside (El Sayed et al.,
1994), four flavonoid glycosides: quercetin di-Ohexoside, quercetin 3-O-rhamnosyl (1→2) glycoside,
quercetin 3-O-galactoside and quercetin 3-O-xyloside
(Heneidak et al., 2006) and 2 simple coumarins:
scopoletin and scoparone (El-Demerdash et al., 2009)
as well as of 7 other flavonoids (Ghada et al., 2008).
Studies on other species of this genus which have close
affiliations with C. acutum also have been done and the
following products have been distinguished: steroidal
glycosides (Liu et al., 2007), carbohydrates (Yi-Bin
et al., 2004), alkaloids (Tian-Ying et al., 2001),
phenolic compounds (Lou et al., 1993) and triterpenes
(Konda et al., 1990). This close affiliation enables us to
predict the presence of these products in C. acutum,
since close genotypes ends to production of similar
compounds. Antidiabetic and antioxidant activity of
INTRODUCTION
According to world health organization 1 of the
main causes of morbidity and mortality is diseases
resulted from pathogenic bacteria and fungi (World
health organization, 1998). On the other hand the
increasing resistance of pathogens to drugs encourages
scientists to search for new antimicrobial sources like
medicinal plants (Karaman et al., 2003). Owing to less
side effects and economical reasons plant-based drugs
have got great attentions during past few decades.
Many commonly used drugs are of herbal origins and
over 80% of world’s people use medicinal plants for
their primary health care needs. The use of plants as
source of remedies for the treatment of many diseases
dated back to prehistory and people of all continents
have this old tradition. The search for agents to cure
infectious diseases began long before people were
aware of the existence of microbes. These early
attempts used natural substances, usually native plants
or their extracts and many of these herbal remedies
proved successful (Sofowora, 1982). The effective
substances of many plant species are isolated for direct
use as drugs, lead compounds or pharmacological
agents (Fabricant and Farnsworth, 2001). Nowadays,
medicinal plants receive attention to research centers
because of their special importance in safety of
communities. The curative properties of medicinal
plants are mainly due to the presence of various
complex chemical substances of different composition
which occur as secondary metabolites (Karthikeyan
et al., 2009; Lozoya and Lozoya, 1989). They are
grouped as alkaloids, glycosides, flavonoids, saponins,
Corresponding Author: Fereshteh Javadian, Department of Biology, Faculty of Science, Mashhad Branch, Islamic Azad
University, Mashhad, Iran, Tel.: +989153422059
177
Br. J. Pharmacol. Toxicol., 3(4): 177-180, 2012
metanolic extract of aerial parts of Cynancum acutum
has been reported and antiulcerogenic effects of
ethanolic extract of the plant also have been shown in
rats (Atta et al., 2005). The current study was
undertaken to evaluate the anti-microbial activity of
ethanolic and aqueous extract of Cynancum acutum by,
till now no pharmacological evaluation has been done
on the anti-microbial activity of Cynancum acutum.
MATERIALS AND METHODS
Plant material: Cynancum acutum was collected from
Sistan and Baluchestan province (Iran) in March 2011
and authenticated at Medicinal Plants and Drugs
Research Institute, Shahid-Beheshti University, Tehran,
Iran. Its leaves and fruits were dried, under shade and
powdered.
Preparation of the ethanolic and aqueous extract of
Cynancum acutum: Air dried powdered plant was
divided in to 2 equal parts (1 kg each); one part was
macerated with ethanol (90% v/v) in glass percolator
and allowed to stand at room temperature for about 24
h. Then the extract obtained was filtered, concentrated
by rotary vacuum pump to get the solid mass. The
percentage yield was 19.6%, 2nd part of powdered was
cold macerated with distilled water then the same
procedure was repeated as mentioned above. The
percentage yield was 10.5%.
Phytochemical screening: The freshly prepared
ethanol and aqueous extract of Cynancum acutum was
qualitatively tested for the presence of chemical
constituents. Phytochemical screening of the extract
was performed using the following reagents and
chemicals: Alkaloids with Mayer’s, Hager’s and
Dragendorffs reagent; Flavonoids with the use of
sodium acetate, ferric chloride, amyl alcohol; Phenolic
compounds and tannins with lead acetate and gelatin;
carbohydrate with Molish’s, Fehling’s and Benedict’s
reagent; proteins and amino acids with Millon’s, Biuret
and xanthoprotein test. Saponins was tested using
hemolysis method; Gum was tested using Molishs
reagent and Ruthenium red; Coumarin by 10% sodium
hydroxide and Quinones by Concentrated Sulphuric
acid. These were identified by characteristic color
changes using standard procedures (Trease and Evans,
1989). These were identified by characteristic color
changes using standard procedures. The screening
results were as follows: Alkaloids + ve; Carbohydrates
+ve; Proteins and amino acids +ve; Steroids -ve; Sterols
+ve; Phenols +ve; Flavonoids +ve; Gums and mucilage
+ve; Glycosides +ve; Saponins -ve; Terpenes +ve and
Tannins +ve. Where +ve and -ve indicates the presence
and absence of compounds.
Microorganisms:
Bacterial
(Bacillus
subtilis,
Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli and Proeus vulgaris) were procured
from Pastore Diagnostic Labs, Tehran, Iran.
Chemicals and drugs: Ethanol was obtained from
Bidestan Co. (Tehran, Iran). Cloxicillin, Amoxiclav,
Cefuroxime, Cefixim were from Tablets Iran P.vt,
Limited (Tehran, Iran) were used as Reference
Antibiotics (RA) against bacteria. The nutrient agar was
from Zist Iran (Tehran, Iran). Indomethacin was from
Merck.
Anti microbial activity:
Sensitivity test: agar disc diffusion assay: The disc
diffusion method was followed to evaluate antimicrobial activities using a range of microorganisms.
Sterile Discs (Whitman, 6 mm) were impregnated with
10 μL of reconstituted crude extracts (1 mg/mL) and
placed on the surface of Muller-Hilton agar dispersion
plates inoculated with microbes. Each extract was
tested in triplicate. Control discs contained pure DMSO
(100%). Standard antibiotics, Cloxicillin, Amoxiclav,
Cefuroxime and Cefixime (30 μg disc-1), were used to
eliminate variation between plates. Agar plates
containing bacteria were incubated at 37ºC for 24 h.
Inhibition zones were recorded as the diameter of
growth-free Zones (IZ), including the diameter of the
discs, in mm, at the end of the incubation period (Salie
et al., 1996).
Statistical analysis: All values are expressed as
mean±SEM. Data were analyzed by non-parametric
ANOVA followed by Dennett’s multiple comparison
tests and other data was evaluated using Graph Pad
PRISM software. A p-value <0.05 was considered
significantly different.
RESULTS
The ethanolic and aqueous extracts from
Cynancum acutum has shown inhibition effects on the
growth of all the organisms tested, but their efficiency
in inhibition was varied from one organism to another.
In almost all, the tested organisms’ growth was
inhibited by both ethanolic and aqueous extract has
shown higher range of Inhibition Diameter (IDZ) from
08 to 19 mm, where as ethanolic extract has shown
inhibition range of 15-20 mm. Staphylococcus aureus is
more sensitive and Escherichia coli is least sensitive to
ethanolic extract. Whereas aqueous extract has shown
inhibition range of 08-19 mm. Bacillus subtilis is more
sensitive and Escherichia coli are least sensitive to
aqueous extract. Cloxicillin, Amoxiclav, Cefuroxime,
Cefixime ranged from 20-28 mm at a concentration of
30 μg/zone. All IZD corresponding to test organisms
are tabulated in Table 1.
178 Br. J. Pharmacol. Toxicol., 3(4): 177-180, 2012
Table 1: Antimicrobial activity ethanolic and aqueous extract of Cynancum acutum on different microbes and their corresponding IZD
IZD (mm)
----------------------------------------------------------------------------------------------------------------------Microbe
Ethanolic extract
Aqueous extract
Standard
Staphylococcus aureus
25
16
26
Bacillus subtilis
20
19
27
Pseudomonas aeruginosa
17
12
22
Proteus vulgaris
15
13
23
Escherichia coli
15
09
18
Cloxicillin, Amoxiclav, Cefuroxime and Cefixime are the standards (values are mean±S.D)
DISCUSSION
Control of the spread of antibiotic-resistant bacteria
and the treatment of infections caused by them is a
major problem worldwide. According to the World
Health Organization (WHO), an infectious disease is
the first cause of death worldwide with more that 50%
of the death appearing in tropical countries. In the
developing countries, treatment of such diseases is
complicated not only because of the occurrence of
resistant microorganisms to the commonly used
antibiotics, but also because of the low income of the
population, which drastically reduce their accessibilities
to appropriate drugs. It is reported that about 80% of
the world population is dependent (wholly or partially)
on plant-based drugs (World health organization, 1996).
Results obtained in the present study have shown that
both the ethanolic extract and aqueous extract are active
against the growth of the microbes such as
Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa, Proteus vulgaris and Escherichia coli. Both
extracts possess promising anti-microbial activity.
Hence, the external application of these extracts on the
wound prevented the microbes to invade through the
wound, resulting protection of wound against the
infections of the various microorganisms, because the
presence of replicating microorganisms within a wound
that cause host injury includes Staphylococcus aureus,
Beta-hemolytic Streptococcus, E. coli, Klebsiella,
Pseudomonas, Acinetobacter, Stenotrophomonas etc.
The overall result of the antimicrobial indicates that
both the extracts can be useful in the development of
antimicrobial drugs. This could be confirmed by the
results of the IZD determination (Table 1). The
presence of antimicrobially active metabolites classes
such as flavonoids, phenols, terpenoids, alkaloids,
glycosides might explain the wide spectrum of activity
of the tested extracts. However, the isolation of the
active principles will confirm this hypothesis and
provide more explanation on mechanism of action of
these extracts. In conclusion, our results indicate that
Cynancum acutum has anti-microbial and antiinflammatory effects, which provide pharmacological
evidence for folk uses of it. Further molecular and
cellular experiments are warranted to explore its action
mechanisms.
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