Evaluation of Abelmoschus moschatus extracts for
antioxidant, free radical scavenging, antimicrobial
and antiproliferative activities using in vitro assays
Mir Z Gul1, Lepakshi M Bhakshu1, Farhan Ahmad2, Anand K
Kondapi2, Insaf A Qureshi2 and Irfan A Ghazi1*
Department of Plant Sciences, School of Life Sciences, University of
Hyderabad, Gachibowli, Hyderabad 500 046, India
BMC Complementary and Alternative Medicine
2011
Impact factor-2.24
Introduction:
Abelmoschus moschatus, leaves and seeds are used as valuable traditional
medicine used in treatment of intestinal problems and check queasiness.
The free radicals and reactive oxygen species are produces through
physiological and biochemical processes in the human body as byproduct.
These ROS leads to oxidative damage of biomolecules in the body that can
initiate number of diseases like atherosclerosis, diabetes mellitus, cancer,
heart and neurodegenerative diseases etc.
These free radicals can be blocked by the antioxidants
produced by plants such as phenolic compounds like phenolic
acids, flavonoids, quinines and coumarins and nitrogen
compounds like alkaloids and amines, terpenoids etc.
The study was aimed to investigate the antioxidant, anti free
radical, antimicrobial and antiproliferative activities of
different extracts of A.moschatus, which belongs to Malvaceae
family and is popularly known as Mushkdana/ Kasturi bhendi
Methods:
1. Preparation of plant extracts.
2. Determination of total phenolic content
3. Determination of total flavonoid content
4. Evaluation of antioxidant capacity
a) Detemination of total antioxidant activity (TAA)
b) Determination of reducing antioxidant power (FRAP)
c) DPPH radical scavenging activity
d) Hydrogen peroxide scavenging activity
e) Superoxide radical scavenging activity
f) Hydroxyl radical scavenging activity
g) Inhibition of Fenton’s reagent-induced strand breaks in
plasmid DNA.
h) Determination of inhibition of Lipid peroxidation.
5. Antiproliferative activity.
6. Antimicrobial Activity.
Plant materials: Seeds and healthy leaves of A.moschatus
were collected from the Central Institute of Medicinal and
Aromatic Plants (CIMAP), Regional Centre Hyderabad,
India during the month of September – October.
Microbial Cultures: Bacterial reference strains Bacillus
subtilis, Staphylococcus aureus, E.Coli, Pesudomonas
aeroginosa, Proteus vulgaris, Salmonella enterica paratyphi
and Candida albicans were obtained from Central Research
Institute of Unani Medicine, Hyderabad, India.
Preparation of Plant extracts:
1) The seed and leaf powder of A.moschatus were subjected to different modes of extraction
using ethanol and water.
2) Aqueous extracts of A.moschatus seed (AMS-I) and leaf (AML-I) were prepared by soaking 1
g of dried powder in 4ml of distilled water for 24 hrs at RT.
3) Second aqueous extract (AMS-II and AML-II) were prepared by dried powder of seed and
leaf through slow evaporation at 30-40 oC.
4) The third extract (AMS-III and AML-III) was prepared by mixing 1g each of the sample
with 20mL distilled water for 3-4hrs at 80 – 90 oC.
5) Hydrolic extracts were prepared by dissolving 1g of the dried powder of both
the samples in 20mL of 80% ethanol for 3-4hrs at 40- 50 oC and was evaporated to 4mL and
labeled as (AMS-IV and AML-IV) .
Note: The dried extracts of 20mg/ mL stock solution is used.
1) Determination of total phenolic content: FC method
10μl of extracts from stock + 100μL of FC reagent
10 mins of incubation
300μl of 20% Na2CO3 solution is added & volume is made up to 1mL by
using distilled water.
The mixture is incubated in dark for 2hrs and absorbance at 765nm is checked
using UV Vis-Spectrophotometer against blank sample.
Note: the total phenolic content was measured as gallic acid equivalents
2) Determination of total flavonoid content: Calorimetric
method
20μL of each extract + mixed with 500μL Milli – Q water & 30μL of 5%
NaNO2 solution.
5mins incubation at RT
60μL of 10% AlCl3 solution + 350μL of 1M NaOH and 40μL of Milli-Q water
were added to make the final volume 1mL.
15 mins of incubation at RT
Absorbance of the samples was checked at 510nm.
Note : Total flavonoids were determined as qurecetin equivalents.
3) Evaluation of antioxidant capacity:
a) Determination of total antioxidant activity (TAA) by
Phosphomolybdenum method.
10μL of extracts in 90mL distilled water + 1mL reagent solution (0.6 M H2SO4,
28nM Sodium phosphate and 4nM ammonium molybdate) in 1.5mL capped
tubes.
Incubated in thermal block at 950C for
90min
After cooling to RT, absorbance of the solution was measured at 695nm
against blank samples.
Note: Ascorbic acid was used as a standard.
b) Determination of reducing antioxidant power: FRAP method.
Various extracts were mixed with 2.5mL phosphate buffer and 2.5mL of
Potassium ferricyanide.
Incubated at 500C for 20 min
2.5mL of Trichloroacetic acid was added and centrifuged at 1000 rpm for
10 min.
The upper layer of the solution (2.5mL) mixed with deionised water (2.5mL)
and ferric chloride.
The absorbance was measured at 700nm at the reaction time of 30 mins.
Note: Ascorbic acid was used as a standard.
C) DPPH radical scavenging activity:
DPPH solution was prepared in 95% methanol and serial dilutions
were carried out with stock solutions of the extracts.
Various concentrations of extracts were mixed with DPPH solution 900μl
Incubated at dark for 30 min
Absorbance was measured at 517nm.
Note: Methanol, DPPH solution and Ascorbic acid were used as blank, control
and reference standard respectively.
d) Hydrogen peroxide scavenging activity: FOX assay method
Plant extracts of different concentrations were incubated with 10μL of 40nM
H2O2 for 10min at RT in dark
0.2mL FOX reagent was added and the volume was made up to 1mL with
distilled water.
mixture is vortex & incubated at RT for 30min
The absorbance of ferric – xylenol orange complex was measured at 560nm.
Note: The FOX reagent without extracts & H2O2 served as balnk and with
H2O2 served as control.
e) Superoxide radical scavenging activity:
1. 156μM nitroblue tetrazolium (NBT) dissolved in50nM phosphate buffer
(pH 7.4), 468μM nicotinamide adenine dinucleotide (NADH) and various
concentrations of extracts were mixed.
2. The reaction is started by addition of 100μL of 60μM phenazine
methosulfate (PMS) solution and final volume of the reaction was 3mL.
3. The reaction mixture was incubated at 250C for 5 min and absorbance at
560nm was observed against control samples (with NADH).
f) Hydrogen radical scavenging activity: Fenton’s reaction.
1)To 0.1mL Fenton’s reagent, Thiobarbituric acid in 25mM NaOH (1 mL)
and Trichloroacetic acid 1mL were added and volume was made up to
3mL by adding water.
Mixture is heated for 90 min on water bath at
800C
The amount of pink chromogen produced was considered as control and
measured spectrophotometrically at 532nm.
2) The protection of oxidation of D-ribose has been conducted by pre-incubation
with extracts in different concentrations and decrease in the formation of pink
color was considered as antioxidant property which is compared to standard
ascorbic acid.
g) Inhibition of Fenton’s reagent- induced strand breaks in
plasmid DNA: DNA nicking assay
1. The reaction mixture of 2.5ul of plasmid DNA(0.25ug) and 10ul Fenton’s
reagent + addition of 5ul of extracts and made the volume to 20ul with
distilled water.
2. The reaction mixture is incubated at 370C for 45 min.
3. Then analyzed on 0.9% agarose gel electrophoresis by staining with
ethidium bromide.
h) Determination of inhibition of Lipid peroxidation:
1. The rat liver was treated with 0.15 M KCl and homogenate was centrifuged at
800 g for 15min at 40C.The supernatant is used for triobarbutaric acid assay.
2. The extracts were mixed with the liver microsome preparation and the
mixture were incubated in the presence and absence of Fenton’s reagent in
phosphate and the final volume was made up to 1mL.
3. The reaction mixtures were incubated at 370C for 30 min.
4. 2mL of ice cold HCl containing 15% trichloroacetic acid,0.5% thiobarbutaric
acid and 0.5% butylated hydroxytoluene (BHT) was added to the mixture 7
heated at 1000C.
5. The mixture was kept in an ice-bath for 10min and centrifuged at 1000 g for
10min.
6. The presence of pink chromogen is monitored and measured
spectrophotometrically at 532 nm.
7. The decline in the color formation in the per-treated reactions was considered
as inhibition of lipid peroxidation.
4. Antiproliferative activity:
1. COLO-205 and Y79 cells were grown in DMEM and RPMI 1640
respectively, along with 10% Fetal Bovine serum in 96 well plates.
2. Increasing concentrations (25,50,100,200μg) of the samples dissolved in
10% Dimethyl sulfoxide were added to the cells and incubated at 370C
under 5% CO2 in a humidified incubator for 14h.
3. The cell suspension was centrifuged at 1000 g for 10 min and the medium
was aspirated.
4. The fresh growth medium containing 20μl of MTT solution of 5mg/mL
was added to each well & incubated for 4h.
5. The medium was removed by centrifugation at 1000 g for 10min and
200μl of DMSO was added to the wells to dissolve the MTT-formazan
crystals.
6. The plates were shaken and absorbance was determined by ELISA reader
at 570 nm.
Note: The conventional anticancer drug, ifosfamide was used as positive
control.
5) Antimicrobial activity:
The antimicrobial property of the extracts was evaluated against two gram
positive, four gram negative and one fungal strain.
The procedure was carried out in 96 microtitre plate containing different
concentrations of the extracts.
The culture suspension was added to each well having 105CFU/mL and made
up to 200 μL by adding LB broth.
The plates are incubated at 370C for 18h and then 10ul of MTT was added to
each well and examined by ELISA reader at 530nm.
The lowest concentration of the each extract which showed complete
inhibition was taken as its minimum inhibitory concentration.
Note: For control antibiotics such as ampicillin, kanamycin and nystatin were
used as positive control.
RESULTS:
1) Determination of total phenolic and flavonoid content
2) IC50 values of A.moschatus extracts on tested radicals
DPPH scavenging activity
Hydrogen peroxide scavenging activity
Superoxide scavenging activity
Hydroxyl radical scavenging activity
Inhibition of Fenton’s reagent-induced strand breaks in plasmid DNA
Inhibition of lipid peroxidation
Antiproliferative activity
MIC –microdilution broth assay
Discussion
According to table.1 the ferric reducing power id higher in AMSI & in AMLIV
than in rest of the samples hence this could be attributed to the presence of
antioxidant phytomolecules.
DPPH radical scavenging abilities was observed in all the extracts and hence
shows that it has high proton donating abiltiy.
The results show that the scavenging of H2O2 by extracts may be attributed to
their phenolics, which can donate electrons to H2O2 and neutralize it to water.
The superoxide radical scavenging property of the extracts is very efficient and
percentage of inhibition increases with increase in concentration.
This can also be used to minimize the adverse effect from
Hydroxyl radicals and also has a good degree of protection
against the oxidative damage.
The cytotoxicity was slightly higher in leaf extracts in both the
cell lines than the seed extract. But the activity is low when
compared to standard drug.
The antimicrobial activity of AMS-I and AML-IV is once
again more efficient which signifies the antibiotic nature of
these extracts.
Conclusion
The present study indicated that the A.moschatus contains
considerable amount of total polyphenols and flavonoids
and exhibit good antioxidant activity by effectively
scavenging the free radicals.
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