Evaluation
of Antioxidant, Antino Potentialities of Methanolic
Extract of Memecylon umbellatum
Nowshin N. Rumzhum*, Md.
Mostafizur Rahman, Most. Nazma Parvin, Sadia A. Chowdhury
Laboratory
of Pharmacognosy and Pharmacology, Department of Pharmacy, Stamford University
Bangladesh, Dhaka-1217, Bangladesh
ABSTRACT:
The methanolic extract of leaves of
Memecylon umbellatum
(Melastomtaceae) was evaluated for in vitro
antioxidant activity by determination
of hydrogen peroxide scavenging activity, total antioxidant capacity,
assay of nitric oxide scavenging activity and reducing power test and in vivo antinociceptive effect in
acetic acid induced writhing model in Swiss albino mice. The plant extract was
also subjected for brine shrimp lethality bio-assay to evaluate its cytotoxic property. The results revealed the presence of
pronounced antioxidant property as compared with ascorbic acid used as standard
and a dose-dependent (250 and 500 mg/kg) analgesic effect. Investigation also
showed that it has strong lelthality (LC50
1.178 µg/ml) against brine shrimp nauplii, Vincristine Sulphate
was used as positive control. The antioxidant, antinociceptive
and cytotoxic properties observed seem to be in good
accordance with the traditional uses of Memecylon
umbellatum.
KEY WORDS: Memecylon umbellatum, Melastomataceae, Antioxidant, Antinociceptive,
Swiss albino mice, Cytotoxicity, Vincristine
Sulphate.
INTRODUCTION:
The importance of plant is well known. Life and its growth cannot
be imagined without plants. They not only produce food for survival but also
create healthy environment and eco-friendly atmosphere to live. Historically
plants have provided a good source of medicine which is highly effective
instruments in the fight against various diseases. Natural products, either as
pure compounds or as standardized plant extracts, provide unlimited
opportunities for new drug leads because of the unmatched availability of
chemical diversity. Therefore, researchers are increasingly turning their
attention to folk medicine and looking for new leads day by day to develop
better drugs against different diseases.
Large scale evaluation of the local flora exploited in traditional medicine for
various biological activities is a necessary first step in the isolation and
characterization of the active principle and further leading to drug development1.
A large number of plants have been screened as a viable source of natural
antioxidants including tocopherols, vitamin C, carotenoids and phenolic
compounds which are responsible for maintenance of health, to help the human
body reduce oxidative damage and protection from coronary heart diseases and
cancer2. In view of these Memecylon
umbellatum was studied exhaustively for its
potential antioxidant, analgesic and cytotoxic
effects.
Memecylon umbellatum
(Common name: Iron-wood tree, Bengali name: Anjan;
Family- Melastomataceae) is a large, fast growing,
evergreen tree which grows to a height of 3 meters, with spreading branches and
many aerial roots. It is found mostly in the coastal regions of the Deccan
peninsula, the eastern and southern parts of India and in the Andaman Islands3.
Previous phytochemical studies revealed the
presence of umbelactone
(4-hydroxymethyl-3-methyl-but-2-ane-4,7-olide), amyrin,
sitosterol, tartaric acid, malic
acid,oleanolic acid, urseolic
acid4, 5, tannins6, fatty acids like octocosonoic acid, cerotic acid, ethyl palmitate,
palmitic acid and butyric acid7. Few records are found in the literature with
respect to the isolation of anthocyanins from Memecylon species8. All the parts of
the plant is used as medicine, the fruits are edible, are eaten in time of
famine and they are quite safe9.
Its leaves are used gonorrhea, leucorrhoea, herpes, diabetes
and for wound healing activities10-14, the seeds are used to cure
cough and sedative15, the roots are used in menorrhagia
and the bark is used for the treatment of bruises16. In this paper,
the antioxidant, antinociceptive and the cytotoxic activities of the methanolic extract of Memecylon umbellatum
are being reported to validate the traditional use of the crude drug through in
vivo and in vitro evaluation.
MATERIAL AND METHODS:
Plant materials
Memecylon umbellatum was collected from Gagipur in May 2008 and was identified by Bangladesh
National Herbarium, Mirpur, Dhaka, where a voucher
specimen (DCAB accession no: 31101) has been deposited. The leaves of the plant
were first sun dried and then ground into coarse powder.
Extraction
of plant materials
About
60 gm of powered material was taken in a clean, flat-bottomed glass container
and soaked in 800 ml of methanol. The container with its contents was sealed
and kept for a period of 20 days accompanying occasional shaking and stirring.
The whole mixture then underwent a coarse filtration by a piece of clean, white
cotton material. The filtrate (methanol extract) obtained was evaporated under
ceiling fan and in a water- bath until dried. It rendered a gummy concentrate
of blackish color. The gummy concentrate was designated as crude extract of
methanol (3.5 gm).
To get
preliminary idea about the active constituents present in the plant leaves
extract different chemical tests were performed and showed the presence of
steroids, alkaloid, flavonoids, tannins and gums17.
Antioxidant property
i) Qualitative assay: A
suitably diluted stock solutions ( sample solutions) were spotted on pre-coated Silica gel
TLC (Thin layer chromatography) plates and the plates were developed in solvent
systems of different polarities (polar, medium polar and non-polar) to resolve
polar and non-polar components of the extract and to choose the solvent system
in which stock solutions run well. The plates were dried at room temperature
and were sprayed with 0.02% DPPH (2,2-diphenyl-1-picrylhydrazine) in ethanol. Bleaching of DPPH by the resolved
bands was observed for 10 minutes and the color changes (yellow on purple
background) were noted18.
ii) Quantitative assay: Free radical scavenging activity of the methanol extract was evaluated
by determination of hydrogen peroxide scavenging activity, total
antioxidant capacity, assay of nitric oxide scavenging activity and reducing
power test. In all methods ascorbic acid is used as standard.
a. Scavenging
of hydrogen peroxide
The ability of the methanolic
extract of Memecylon umbellatum
to scavenge hydrogen peroxide was determined according to the method
of Ruch et al.19.
A solution of hydrogen peroxide (2 mmol/l)
was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration
was determined spectrophotometrically from absorption at 230 nm with
molar absorbtivity 81 (mol/l)-1 cm-1
in a spectrophotometer (Hach, DR-4000U). Extracts (50–250mg/ml) in distilled
water were added to a hydrogen peroxide solution (0.6 ml, 40 mM). Absorbance of hydrogen peroxide at 230 nm was
determined after ten minutes against a blank solution containing in
phosphate buffer without hydrogen peroxide. For each concentration, a
separate blank sample was used for background subtraction. The percentage of
scavenging of hydrogen peroxide of methanolic
extract of Memecylon umbellatum
and standard compounds was calculated using the following
equation:
Percent scavenged [H2O2] = (A0
− A1)/ A0 × 100
Where, A0 was the absorbance of the control, and A1
was the absorbance in the presence of methanolic
extract of Memecylon umbellatum
and standards20.
b. Determination of
total antioxidant capacity
The antioxidant
activity of the extract was evaluated by the phosphomolybdenum
method according to the procedure of Prieto et al.21. The assay is based on the reduction of
Mo (VI) – Mo (V) by the extract and subsequent formation of a green phosphate /
Mo (V) complex at acid pH. 0.3 ml extract was
combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The tubes containing the
reaction solution were incubated at 95οC for 90 min. Then the
absorbance of the solution was measured at 695 nm using a spectrophotometer (Hach, DR-4000U) against blank after cooling to room
temperature. Methanol (0.3 ml) in the place of extract is used as the blank.
The antioxidant activity is expressed as the number of equivalents of ascorbic
acid.
c. Assay of Nitric
oxide scavenging activity
The procedure is
based on the method, where sodium nitroprusside in
aqueous solution at physiological pH spontaneously generates nitric oxide,
which interacts with oxygen to produce nitrite ions that can be estimated using
Greiss reagent. Scavengers of nitric oxide compete
with oxygen leading to reduced production of nitrite ions. For the experiment,
sodium nitroprusside (10 mM)
in phosphate buffered saline was mixed with different concentrations of
methanolic extract of Memecylon umbellatum dissolved in methanol and incubated at room
temperature for 150 min. The same reaction mixture without the ethanol extract
but the equivalent amount of methanol served as the control. After the
incubation period, 0.5 ml of Griess reagent (1%
sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride
was added. The absorbance of the chromophore formed
was read at 546 nm22.
d. Reducing power
test
The reducing power
of methanolic extract was determined according to the method of Oyaizu23.
Different amounts of methanolic extracts (100 – 1000 µg) in 1 ml of methanol
were mixed with phosphate buffer (2.5 ml, 0.2 mol/l, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml,
1%). The mixture was incubated at 50οC for 20 min. A portion
(2.5 ml) of trichloroacetic acid (10%) was added to
the mixture, which was then centrifuged (650 x g at room temperature) for 10
min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5
ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700
nm. Increased absorbance of the reaction mixture indicated increased reducing
power.
Antinociceptive property
Evaluation of antinociceptive property was performed by acetic acid
induced writhing model in mice24. The acetic acid induced writhing
method is an analgesic behavioral observation assessment method that
demonstrates a noxious stimulation in mice. The test consists of injecting the
0.7% acetic acid solution intraperitoneally and then
observing the animal for specific contraction of body referred as ‘writhing’. A
comparison of writhing was made between positive control
(Diclofenac-Na). Control and test sample are given orally 30 minutes prior to
acetic acid injection. If the sample possesses analgesic activity, the animal
that received the sample will give lower number of writhing than the control,
i.e. the sample having analgesic activity will inhibit writhing.
Cytotoxicity study
Brine shrimp
lethality bioassay25, 26 technique was
applied for the determination of cytotoxic property
of the crude extract.
Preparation of
positive control group
Vincristine sulphate
was used as the positive control. Measured amount of the vincristine
sulphate was dissolved in DMSO to get an initial
concentration of 20 μg/ml from which serial
dilutions were made using DMSO to get 10 μg/ml,
5 μg/ml, 2.5 μg/ml,
1.25 μg/ml,
0.625 μg/ml,
0.3125 μg/ml,
0.15625 μg/ml, 0.078125 μg/ml,
0.0390 μg/ml.
Then then positive control solutions were added to
the premarked vials containing ten living brine
shrimp nauplii in 5 ml simulated sea water to get the
positive control groups.
Preparation of
negative control group
100 μl of DMSO was added to each of three premarked glass vials containing 5 ml of simulated sea
water and 10 shrimp nauplii to use as control groups.
If the brine shrimps in these vials show a rapid mortality rate, then the test
is considered as invalid as the nauplii died due to
some reason other than the cytotoxicity of the
compounds.
Counting of nauplii
After 24 h, the vials
were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From this data, the
percent (%) of lethality of the brine shrimp nauplii
was calculated for each concentration.
RESULTS AND
DISCUSSION:
Antioxidant property
i) Qualitative assay: The color changes
(yellow on purple background) on the TLC plates were observed due to the
bleaching of DPPH by the resolved bands.
ii) Quantitative
assay
a. Scavenging
of hydrogen peroxide
Scavenging of H2O2 by extracts may be
attributed to their phenolic constituents, which can
donate electrons to H2O2, thus neutralizing it to water.
The methanolic extracts of Memecylon umbellatum
were capable of scavenging hydrogen peroxide in a concentration-dependent
manner
(Figure 1).
b. Total antioxidant
capacity
Total antioxidant
capacity exerted by the extract was concentration dependent. It was observed
that the extract was likely to have the capacity of reduction of Mo (VI) to Mo
(V) by the antioxidant principle and the formation of a green phosphate / Mo
(V) complex with a maximal absorption at 695 nm. The antioxidant activity is
expressed as the number of equivalents of ascorbic acid (Table 1).
Table 1: Total antioxidant capacity of
methanolic extract of Memecylon umbellatum
|
Materials |
Concentration (μg/mL) |
Equivalent to ascorbic acid |
|
Methanol extract of Memecylon umbellatum |
10 25 50 125 250 500 |
0.066±0.11 0.121±0.09 0.238±0.13 0.526±0.12 1.074±0.10 1.925±0.08 |
c. Nitric oxide
scavenging activity
From Figure 2,
it is observed that the extract is likely to have concentration dependent
nitric oxide scavenging activity. The leaves may have the property to
counteract the effect of NO formation and in turn may be of considerable
interest in preventing the ill effects of excessive NO generation in the human
body. Further, the scavenging activity may also help to arrest the chain of
reactions initiated by excess generation of NO that are detrimental to the
human health. Nitric oxide is also implicated for inflammation, cancer and
other pathological conditions27.
Figure 2: Nitric oxide scavenging activity of methanolic
extract of Memecylon umbellatum.
d. Reducing power
activity
Reduction ability of
the extract has been investigated from the Fe+++ - Fe++
transformation using the method followed by Oyaizu23. Earlier
authors28, 29 have observed a direct correlation between antioxidant
activity and reducing power of certain plant extracts. The reducing properties
are generally associated with the presence of reductones28 which
have been shown to exert antioxidant action by breaking the free radical chain
by donating a hydrogen atom30. Reductones
are also reported to react with certain precursors of peroxide, thus preventing
peroxide formation. Figure 3 shows the reduction ability of Memecylon umbellatum.
Figure 3: The reducing power of methanolic extract of Memecylon umbellatum
Antinociceptive property
The methanolic
extract of plant leaves produced 85.19% and 64.82% writhing inhibition at the
doses of 500 mg/kg and 250 mg/kg body weight respectively, in acetic acid
induced writhing model in mice which are comparable to Diclofenac sodium
(45.59% at the dose of 25 mg/kg). Table-2 shows the antinociceptive
activity of Memecylon umbellatum.
Table 2: Antinociceptive
property of methanolic extract of Memecylon
umbellatum in acetic acid induced writhing in
mice
|
Animal
group/Treatment |
Number of Writhing (% writhing) |
Inhibition (%) |
|
Control 1% Tween-80
solution in water, 10 ml/kg, p.o. n=4 Positive control Diclofenac
sodium 25 mg/kg, p.o. n=4 Test group I Methanolic
extract 250 mg/kg, p.o. n=4 Test group II Methanolic
Extract 500 mg/kg, p.o. n=4 |
27±0.11a (100) 15.5±0.03a (57.41) 9.5±0.08a (35.18) 4±0.14a (14.81) |
-- 42.59 64.82 85.19 |
Cytotoxic effect
Following the
procedure of Mayer31 the
lethality of the
crude extract to
brine shrimp was determined on Artemia
salina. Table 3 shows the results of the brine
shrimp lethality testing after 24 hours of exposure to the methanolic extract
and the positive control, vincristine sulphate. The LC50 obtained from the best-fit
line slope was found to be 1.178 µg/ml in comparison with Vincristine
Sulphate (LC50 0.323 µg/ml).
Table 3: LC50
data of the test sample of Memecylon umbellatum in
brine shrimp lethality bioassay.
|
Sample |
LC50 ( µg/ml) |
|
Vincristin Sulphate |
0.323 |
|
Methanolic
extract of Memecylon umbellatum |
1.178 |
Antioxidant has been
suggested to possess both anti-inflammatory and analgesic activity
in humans32. A
combination of antioxidants with analgesics normalized the oxidative stress
which, suggest that the administration of antioxidants in pain treatment may be
employed to decrease the doses of analgesics. Antioxidant-based pain killers may one day become a viable alternative to
addictive medications such as morphine. Therefore, medicinal plants like Memecylon umbellatum which
possesses both antioxidant and antinociceptive
properties can be used in the treatment of pain stimulated oxidative stress
condition through their pharmacological validation. Further, several reports
suggest that most of the cytotoxic compounds isolated
from natural resources are good candidates for anticancer drugs33.
CONCLUSION:
Our present study,
demonstrates the antioxidant, antinociceptive and cytotoxic properties of Memecylon
umbellatum, which validate its use in traditional
medicine.
ACKNOWLEDGMENTS:
Authors are grateful
to the authority of Stamford University Bangladesh and the Chairman, Department
of Pharmacy of the same for extending their cordial supports to perform these
investigations.
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Received on 29.10.2011
Modified on 10.11.2011
Accepted on 22.11.2011
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
4(2): March-April 2012,
84-88