Cytotoxic, Antimicrobial and Anti-oxidant Screening of Psidium Guajava Leaves Grown in Oman-Juniper Publishers
JUNIPER PUBLISHERS-OPEN ACCESS GLOBAL JOURNAL OF PHARMACY & PHARMACEUTICAL SCIENCES
Psidium guajava (guava) belongs to the family
Myrtaceae, is a most important medicinal plant used in folk medicine to
treat gastrointestinal, respiratory disturbances and used as
anti-inflammatory medicine. The present study was carried out to
evaluate the antioxidant, antimicrobial and cytotoxicity capacities of
various crude extracts of the leaves of guava. Dried plant material
macerated in ethanol gave crude extract that was Kupchan’s partitioned
into hexane, chloroform, and ethyl acetate fractions. The cytotoxicity
activity was carried out by brine shrimp lethality bioassay and
antimicrobial activity was determined by agar disc diffusion method
against E. coli, P. aeruginosa and S. aurues using four different
concentrations of each extract including 250, 500, 1000, and2000μg/ml.
Cytotoxicity was estimated using brine shrimp test. 2,
2-Diphenyl-1-picrylhydrazyl (DPPH) assay was used for radical scavenging
analysis. All extracts did not show any cytotoxicity or antibacterial
activity at any of the tested concentration but all extracts showed
potent radical scavenging activity, the highest is seen with the
hydro-alcoholic and ethyl acetate extracts.
Keywords: Gujava Psidium; Crude extracts; Antioxidant; Antimicrobial; Cytotoxicactivity; OmanAbbreviations: GUAVA: Psidium Guajava; DDPH: 2, 2-Diphenyl-1-Picrylhydrazyl
Introduction
Guava (PsidiumguajavaL L inn.) is commonly known for
its food and nutritional values throughout the world. The medicinal
properties of guava fruit, leaf and other parts of the plant are also
well known in traditional system of medicine since each part of guava
tree possesses economic and medicinal value [1,2]. It is distributed in
some Arabic countries; Saudi Arabia, Oman and Egypt [3-10]. The
biochemical analyses of the crude extracts from leaves and fruits
indicated the presence of different group of compounds such as
flavonoids, tannins, phenols, triterpenes, saponins, carotenoids,
lectins, vitamins, fiber and fatty acids [4,11]. This plant also
contains sugar, resins and glycosides [12]. The literature survey
reveals that no research has been done on Omani guava. Therefore the
present study was to determine the antioxidant, antimicrobial and
cytotoxic activity of different leaves crude extracts of guava native to
Sultanate of Oman..
Experimental
a. Materials
Hexane, ethanol, chloroform and ethyl acetate were
used in this experiment obtained Sigma-Aldrich Company, UK. The other
necessary chemicals such as 2-diphenyl-1-picrylhydrazyl, (DPPH), sodium
sulfate etc. was obtained from BDH, Germany. Gram positive bacteria
(Staphylococcus aureus) and gram negative bacteria (Escherichia coli and
Pseudomonas,) were from Biological Department, College of Art and
Science, University of Nizwa. Filter paper discs of diameter 6 mm were
obtained from Whatmann Company, catalogue number: 8174900. Nutrient ager
and plastic Petri dishes were from SharlauChemie Company. Brine shrimp
eggs (ARTEMIA CYSTS) were purchased from GOAQUA, Taiwan. Sea salt was
obtained from Al-Qurum Muscat. All the glassware used in this present
experiment was from Borosil, India.
b. Instruments
UV spectra were recorded on Thermo spectronic
spectrophotometer (Great Britain, UK, Model No. Biomate) Ultra speck in
methanol (λmax in nm). Rotatory evaporator was used Yamato Rotary
Evaporator, Model RE 801, Japan. Incubator used in this experiment
obtained from Gen Lab Model: MINO/75F, Serial number: Y5K041.
c. Plant Material
The leaves samples were collected from Alsharqia region,
Sultanate of Oman in October 2012. The fresh leave samples
were packed instantly after harvesting. The samples were
washed with tap water to remove the dust and other foreign
particles. The plant was identified and confirmed by the Ministry
of Agriculture and Fisheries.
d. Extraction
The samples were dried under shade at room temperature
for 7 days. The dried samples (139.45g) were macerated in
absolute ethanol (2L) for 7days to give crude extract. The
residue was suspended in ethanol/water mixture of 1:1 ratio
then extracted successively with hexane, chloroform, and ethyl
acetate. All solvents were later removed under vacuum using
rotatory evaporator.
e. Anti-bacterial test
The antibacterial test was carried out by agar disc diffusion
method [13]. Each extract was subjected to serial dilution
technique, using dimethyl sulphoxide as a solvent to give
Concentrations of 2000, 1000, 500, and 250 μg/ml. Filter
paper discs (6 mm diameter) were Impregnated with each
concentration and placed on the agar plates inoculated with
the bacteria. Negative controls were prepared using the same
solvents employed to dissolve the samples. The plates were
incubated micro aerobically at 37ºC for 24 h. Anti-bacterial
activity was evaluated by measuring the diameter of the zones
of inhibition against the tested bacteria. Each assay was done in
triplicate.
f. Brine shrimp test
Brine shrimp (Artemiasalina Leach) larvae were used as
indicator animal for preliminary cytotoxicity assay as described
by McLaughlin and his group [14]. Shrimp larvae were hatched
in artificial sea water prepared by dissolving 38g of sea salt
in distilled water (IL). The sea salt was placed in a small tank
divided into two compartments by
Perforated polythene wall. About 50mg of GOAQUA
brine shrimp eggs were sprinkled at covered chamber of
duo compartment plastic container. The open compartment
was illuminated to attract the shrimp larvae from the dark
compartment once were hatched within 24 hours.
g. Brine shrimp lethality test
Solutions corresponding to 10, 100, 250, 500, 750 and 1000
mcg/ml were prepared in six vials by serial dilution of the stock
samples (10mg/ml).Each experiment was done in triplicate. A
total of 10 larvae were transferred in each vial and the solutions
were diluted to 5 ml by adding the artificial sea water. Mean
percent mortalities of the larvae were calculated after 24 hours
of exposure.
h. Radical scavenging activity using DPPH method
Free radical scavenging activity of different organic extracts
was estimated as described by Blois [15] with minor modification.
Four concentrations (12.5, 25, 50, 100 and 200 ppm equivalent
to 12.5, 25, 50, 100 and 200 μg/ml, respectively) were prepared
for each extract (hexane, chloroform, ethyl acetate and hydroethanol.
Four ml from each concentration were placed in a test
tube to which one milliliter of 0.1 mM methanol solution of
DPPH (2,2-diphenyl-1-picrylhydrazyl) was added and shaken
vigorously. After that all the test tubes were allowed to stand
at 27 ºC in dark place for 45 min. The control was prepared in
the same way but without adding extract. The absorbance of
the prepared samples was measured using UV spectroscopy at
517nm. Radical scavenging activity of the tested crude extracts
samples was estimated as the inhibition percentage and was
calculated by using the following formula,
Results and Discussion
a. Anti-microbial studies
Previous studies which were done by Gonclaves, et al. [4]
showed that there was some activity against S.aureus on the
hexane extract, ethyl acetate extract, and methanol extract, but
at high concentration of the three extract showed activity against
E. coli and only the methanolic extract showed activity against
Salmonella spp [4]. In a study that was done in Jordan, Psudium
guava acetone extract showed sensitivity to Provenciastearti,
Providenciarettgeri, Strepoccus group c, Staphylococcus
auress, Candidealbicans. There was no activity against Proteus
vulgaris, E. coli, Salmonella ssp, Pseudomonas aeruginosa,
Streptoccusfaecalis found [12].
Penecilla, et al. [13] in the Philippines showed that the
leaves acetone, ethanol and aqueous extracts had inhibitive
activity but not for the hexane extract [13]. The four extracts
hexane, chloroform, ethyl acetate and hydro alcoholic didn’t
show activity against gram +ve or gram -ve bacteria. This
variation from other studies might be attributed to variation
in environment, including day length, light intensity, ambient
temperature, rainfall, soil or season of collection. All the crude
extracts did not show any mortality at any concentration.
b. Anti-oxidant activity
The radical scavenging activity of the different crude extracts
of guava leaves are presented in Figure 1. All extracts showed
high radical scavenging activity at 50μg/ml concentrations,
the hydro-alcoholic and the chloroform extract exhibited more
scavenging effects on free radicals than did the hexane and
ethyl acetate extract. On the basis of the results obtained, guava
Hydro-alcoholic and chloroform extracts the leaf of Guava can
be used for a variety of beneficial chemo-preventive effects.
However, further studies on the antioxidative components of
guava extracts are required.
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