Antioxidant, Antimicrobial Activity and
Antioxidant, Antimicrobial Activity and
Antioxidant, Antimicrobial Activity and
Research Article
Antioxidant, Antimicrobial Activity and
Toxicity Test of Pilea microphylla
1 1 2
Amir Modarresi Chahardehi, Darah Ibrahim, and Shaida Fariza Sulaiman
1 Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains
Malaysia, Penang, 11800 Minden, Malaysia
2 Phytochemistry laboratory, School of Biological Sciences, Universiti Sains Malaysia, Penang, 11800 Minden, Malaysia
Copyright © 2010 Amir Modarresi Chahardehi et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A total of 9 plant extracts were tested, using two different kinds of extracting methods to evaluate the antioxidant and
antimicrobial activities from Pilea microphylla (Urticaceae family) and including toxicity test. Antioxidant activity were tested
by using DPPH free radical scavenging, also total phenolic contents and total flavonoid contents were determined. Toxicity
assay carried out by using brine shrimps. Methanol extract of method I (ME I) showed the highest antioxidant activity at 69 .51
±1.03. Chloroform extract of method I (CE I) showed the highest total phenolic contents at 72 .10±0.71 and chloroform extract
of method II (CE II) showed the highest total flavonoid contents at 60.14 ± 0.33. The antimicrobial activity of Pilea
microphylla extract was tested in vitro by using disc diffusion method and minimum inhibitory concentration (MIC). The Pilea
microphylla extract showed antibacterial activity against some Gram negative and positive bacteria. The extracts did not exhibit
antifungal and antiyeast activity. The hexane extract of method I (HE I) was not toxic against brine shrimp (LC50 value was
3880 μg/ml). Therefore, the extracts could be suitable as antimicrobial and antioxidative agents in food industry.
specimens have been deposited at the Herbarium of the School 1.0 mL of 10% dilution of Folin-Ciocalteu reagent and after
of Biological Sciences, Universiti Sains Malaysia in April 3 minutes, 3 mL of Na2CO3 (1%, w/v) were added and the
2008. The plant materials were washed, dried, and grounded to resulting mixture was incubated at room temperature for 2
small pieces. The first method (Method I) of extraction hours. The absorbance of all samples was measured at 760 nm
included the using of four solvents by following nonpolar to using a spectrophotometer(Model U-1900 Spec-trophotometer
polar solvents (by using Soxhlet apparatus). In this method, Hitachi High Technology Corporation 2006). The standard
dried powdered plant was extracted. The solvents used were curve was prepared using 0, 50, 100, 150, 200, and 250 mg/l.
hexane, chloroform, ethyl acetate, and methanol. The second Results were expressed as milligrams of gallic acid equivalent
method (Method II) included 5 solvents system (by using per gram of dry weight (mg GAE/g dry weight), which is a
partition technique). For Method II, the dried materials were common reference compound.
extracted by using soxhlet extractor with methanol as a solvent
◦
for 72 hours at room temperature (30 C). The methanolic 2.4. Determination of Total Flavonoid Content. The total
extracts were further partitioned by adding distilled water in a flavonoid content was determined using the Dowd method
separating funnel and then followed using chloroform, diethyl
[13]; 5 mL of 2% aluminium trichloride (AlCl3) in
ether, ethyl acetate, and butanol as described by Mellidis et al.
(1993), with a slight modification [11]. The dried extracts
methanol was mixed with the same volume of the extract
◦ solu-tion (0.4 mg/mL). Absorption readings at 415 nm
were then weighed using microbalance and were kept at 4 C. using PerkinElmer UV-VIS lambda 25 spectrophotometer
Abbreviations for crude extract used in this paper, namely, HE were taken after 10 minutes against a blank sample
I (hexane extract of method I), CE I (chloroform extract of consisting of a 5 mL extract solution with 5 mL methanol
method I), EAE I (ethyl acetate extract of method I), ME I without AlCl3. The total flavonoid content was determined
(methanol extract of method I), ME II (methanol extract of using a standard curve with quercetin (0–100 mg/l) as the
method II), CE II (chloroform extract of method II), DEE standard. Total flavonoid content is expressed as mg of
quercetin equivalents (QE)/g of extract.
II (diethyl ether extract of method II), EAE II (ethyl acetate
extract of method II) and BE II (butanol extract of method
II). 2.5. Antimicrobial Activity Test. Antimicrobial activity was
determined using Disc Diffusion following the method
described by National Committee for Clinical Laboratory
2.2. Determination of Antioxidant Activity: DPPH Radical Standard (NCCLS) [14]. All bacterial strains including clin-
Scavenging Assay. Free radical scavenging activity of Pilea ical and ATCC were used in the study. The test bacteria was
microphylla (MeOH, Chlorofom, Diethyl ether, Ethyl removed aseptically with an inoculating loop and transferred
acetate, n-hexane, and Butanol extracts) were tested using to a test tube containing 5 mL of sterile distilled water.
DPPH method with final concentration 1000 μg/mL Sufficient inocula were added until the turbidity equaled 0.5
(Etanolic DPPH) (Sigma Chemical Co., USA) (300 μM) 8
and was used in the reaction mixture. Freshly prepared test McFarland (10 cfu/mL) standards (bioMerieux, Marcy
samples (50 μL) were combined with DPPH solution (150 d’Etoile, France). The test tube suspension (1 mL) was added
μL) in a 96 well microtiter plate. DMSO was used as a to 15–20 mL of nutrient agar or Sabouraud dextrose agar
negative control. The reaction mixture were incubated for before setting aside the seeded agar plate (9 cm in diameter) to
solidify for 15 minutes. Three disks of Whatman’s No. 1 filter
30 minutes at 37◦C and the change in absorbance at 515 nm paper, 6 mm in diameter, were used to screen the antimicrobial
was measured using micro plate reader (Thermo Electron activity. Each sterile disk was impregnated with 20 μL of
Corporation, Finland). All determinations were performed extract (corresponding to 100 mg crude extract/mL),
in triplicate. The obtained absorbance values were amoxicillin (and vancomycin for Streptococcus sp.),
converted into the percentage of radical scavenging activity miconazole nitrate (30 μg/mL, as positive control for bacteria
using the following equation: and fungi, respectively), or 10% DMSO (v/v) (as negative
= − AC × control), before it was placed on the surface of the seeded
Radical scavenging activity (%) 100 100 , plates. The plates were incubated at 37◦C overnight and
AS
TABLE 1: List of different crude extract, depicting variable total phenolic content, total flavonoid content, antioxidant activity ∗, and EC50
values.
Name of crude Antioxidant activity EC50 values Total phenolic Total flavonoid Total
phenolic/Total
extract (%) (μg/mL) (mg GAE/g dw) (mg QE/g)
flavonoid
HE I 28.16 ± 4.76c ND 11.35 ± 0.26ab 0.45 ± 0.49a 25.22
CE I 0.46 ± 0.98a ND 72.10 ± 0.71g 53.44 ± 0.94e 1.35
EAE I 4.10 ± 0.14a ND 41.82 ± 1.34e 11.57 ± 1.07c 3.61
ME I 69.51 ± 1.03f 81.32 9.91 ± 0.33a ND ND
ME II 46.99 ± 4.69d 121.90 13.76 ± 1.52b ND ND
CE II 47.16 ± 4.98d 215.30 35.02 ± 0.11d 60.14 ± 0.33f 0.58
DE II 60.69 ± 2.46e 373.50 54.48 ± 1.65f 21.84 ± 1.65d 2.49
EAE II 8.62 ± 0.95a ND 40.76 ± 2.22e 7.58 ± 0.23b 5.38
BE II 18.34 ± 2.34b ND 22.47 ± 2.03c ND ND
Each value represented the mean ± SD of three replicates (n = 3). Values with different letters are significantly different(P < .05) based on One Way and
Tukey HSD test.
Data of total phenolic contents are expressed as milligrams of gallic acid (GAE) equivalents per microgram dry weight.
Quercetin and BHT were used as a positive control, EC50= 4.95 μg/mL and 50.31 μg/mL and the final concentration were 125
μg/mL. ND = Not determined
∗
Modarresi Chahardehi et al. [9]
the tested microorganism after macroscopic evaluation was Final concentration of extracts in each experiment were used
determined as the MIC. 5.000, 2.500, 1.250, 0.625, 0.312, 0.156, and 0.078 mg/mL.
0.5 mL of the plant extract was added to 4.5 mL of brine
2.7. Determination of the Minimum Bactericidal solution and maintained at room temperature for 24 hours
Concentra-tion (MBC). The minimum bactericidal under the light and surviving larvae were counted.
concentration of the plant extract on the clinical bacterial
isolates was done according to the method highlighted in 2.9. Statistical Analysis. Results were tested for statistical
National Committee for Clinical Laboratory Standard significance by GraphPad Prism. Differences were
(2002). Briefly, 1 mL that was pipetted from the mixture considered statistically significant at the P < .05 level and
obtained in the determination of MIC stage was streaked One Way ANOVA.
out on the nutrient broth for 24 hours. The least
concentration of the extract with no visible growth was
taken as the minimum bactericidal concentration.
3. Results
3.1. Antioxidant Activity. The results are shown in Table 1.
2.8. Toxicity Assay. The preliminary biological evaluation of ME I showed the highest antioxidant activity of 69.51 ±
the crude extract of Pilea microphylla was determined using 1.03% with the lowest EC50 of 81.32 μg/mL, if compared
brine shrimp lethality test according to Simionatto et al. with to other extracts. Thus, the extracts were used to screen for
some modification [15]. The brine shrimp (Artemia salina) the radical scavenging activity at different concentrations.
lethality assay is considered to be a useful tool for preliminary A narrow range of total phenolics content was found in
assessment of cytotoxicity [16]. Brine shrimp assays have also study. Their content ranged from 9.91 ± 0.33 to 72.10 ±
been used for the analysis of pesticides residues [17], to 0.71 mg gallic acid equivalent (GAE)/g of extracts, with an
monitor the toxicity of organic waste to marine organisms average of 41.00 mg/g. As shown in the Table 1, CE I had
[18] and active plant constituents [19]. The Brine shrimp eggs the highest phenolic at 72.10 ± 0.71 mg GAE/g and CE II
were provided by Muka Head Marine Research Station of the had the highest flavonoid contents at 60.14 ± 0.33 mg
Universiti Sains Malaysia, and were hatched in artificial sea QE/g, unless CE I showed higher flavonoid contents after
water (38 g salt per liter of water). After 24 hours, the hatched CE II at 53.44 ± 0.94 mg QE/g.
nauplii suspension was left to stand for 1 hour without CE I and EAE I showed the weakest DPPH radical
aeration, and then the nauplii were collected by pipetting from scavenging capability, at only 0.46 ± 0.98 and 4.10 ± 0.14,
middle layer of solution, in which most of nauplii were respectively. For further study, butanol extract of method
swimming. The crude extracts were dissolved in distilled II (BEPM II) was selected for get fractionations by paper
water to various concentrations and the shrimp larvae were chromatography (PC). Four bands were obtained. BEPMf 4
placed in to them (duplicate). Sea water without displayed the highest antioxidant activity than other frac-
extract was used as a negative control, while potassium tions (27.84 ± 1.27).
dichromate had a LC50 = 20 μg/mL as a positive control. Generally, if the ratio between total phenolic content and
Fifteen nauplii were withdrawn through a glass capillary total flavonoid as shown would be more than 1, it showed an
and placed in each vial containing 4.5 mL of brine solution. increase at in total phenolic content of the extract (as shown
4 International Journal of Microbiology
in last column). The relationship between total phenolic tested bacteria. PM extracts gave large inhibition zone on
content and antioxidant activity of PM was calculated in both Bacillus spizizenii ATCC 6633 and Micrococcus sp. by
methods of extraction. The results indicated that when all using CE I and CE II, respectively. According to Table 2,
extracts in method II were included in the statistical analysis, CE II was effective against on Escherichia coli and
2 methicillin resistant Staphylococcus aureus (MRSA).
there was a negative relationship between them (R = 0.051),
unless method I showed a little bit correlation between the All tested extracts showed no antifungal activity except
2 hexane extract against Aspergillus niger USM AI1 showed
antioxidant activity and total phenolic contents (R = 0.636).
reduced of UFC, but nor hypha.
3.2. Antimicrobial Activity. The antimicrobial effects of PM The MIC and MBC values that were tested on bacteria
extracts on pathogenic bacteria, fungi and yeasts are pre- are listed in Table 3. The MBCs were defined as the lowest
sented in Table 2. In addition, pure methanol (control) had no concentration that killed at least 99.9% of the cells in the
inhibitory effects on pathogenic microbes tested. The PM initial inoculum. The MBCs were tested for which has
extracts at 100 mg/mL concentration were effective on the shown high antimicrobial in MIC test.
International Journal of Microbiology 5
concentration
Bacillus cereus BE II 33.33 33.33
0
Bacillus spizizenii EAE I 8.33 8.33
ATCC 6633
0 20 40 60 80 100 120
−0.5 Lethality (%)
TABLE 4: The LC50 values of Percentage lethality of Artemia
log
salina (μg/mL). −1
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