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In vitro antioxidant activity of saraca asoca roxb. De wilde stem bark extracts from various extraction processes

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Sunita Panchawat at Mohan Lal Sukhadia University
Sunita Panchawat
Siddhraj S Sisodia at Bhupal Nobles' College of Pharmacy
Siddhraj S Sisodia
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Abstract

Antioxidant potential of various extracts i.e. ethanolic, hydroalcoholic and acetone prepared by different extraction methods (Soxhlet extraction, Ultrasonication extraction and Microwave assisted extraction) of stem bark of Saraca asoca Roxb. De Wilde was studied by using DPPH (1,1, diphenyl-2 picryl hydrazyl) in-vitro model. The highest IC50 value 193.88 μg/ml were showed by hydroalcoholic (ethanol 60%) extract prepared by soxhlet extraction method. The acetone extract prepared by ultrasonication extraction method exhibited the lowest IC50 value 97.82 μg/ml. The results indicate that the antioxidant property of the extract may be due to high content of phenolic compounds.
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231
Research Article
IN VITRO ANTIOXIDANT ACTIVITY OF SARACA ASOCA ROXB. DE WILDE STEM BARK
EXTRACTS FROM VARIOUS EXTRACTION PROCESSES
PANCHAWAT S*, SISODIA SS
Bhupal Noble’s College of Pharmacy, Udaipur, Rajasthan, India. E-mail: sunita_pharma2008@rediffmail.com
ABSTRACT
Antioxidant potential of various extracts i.e. ethanolic, hydroalcoholic and acetone prepared by different extraction methods (Soxhlet extraction,
Ultrasonication extraction and Microwave assisted extraction) of stem bark of Saraca asoca Roxb. De Wilde was studied by using DPPH (1,1,
diphenyl-2 picryl hydrazyl) in-vitro model. The highest IC50 value 193.88 µg/ml were showed by hydroalcoholic (ethanol 60%) extract prepared by
soxhlet extraction method. The acetone extract prepared by ultrasonication extraction method exhibited the lowest IC50 value 97.82 µg/ml. The
results indicate that the antioxidant property of the extract may be due to high content of phenolic compounds.
Key words: Antioxidant, Free radicals, Saraca asoca, DPPH radical scavenging.
INTRODUCTION
There is no doubt that plants are a good source of biologically active
natural products. In the investigation of bioactive natural
compounds, it is essential to have access to simple biological tests to
locate required activities1. Free radicals, powerful oxidants are
species that contain unpaired electrons. They are capable of
randomly damaging all components of the body (lipid, proteins, DNA
and saccharides) and are involved in mutations. Radical reactions
are also important in the development of chronic diseases that are
life limiting like Cancers, hypertensions and cardiac infraction,
atherosclerosis, rheumatism and also in cataract2. Reactive oxygen
species (ROS) are generated continuously in the body by both
endogenous and exogenous factors like normal aerobic respiration,
by stimulated polymorpho-nuclear leukocytes, macrophages and
exposure to various pollutants like tobacco smoke, ionizing
radiation, organic solvents and pesticides3, 4.
Many degenerative human diseases have been recognized as being a
consequence of free radical damage. There have been many studies
undertaken on how to delay or prevent the onset of these diseases5.
The most likely and practical way to fight against degenerative
diseases is to improve body antioxidant status which could be
achieved by higher consumption of vegetables and fruits. Foods
from plant origin usually contain natural antioxidants that can
scavenge free radical6. The antioxidant may mediate their effect by
directly reacting with ROS, quenching them and/or chelating the
catalytic metal ions7.
Phenolic compounds are naturally occurring substances in fruit,
vegetables, nuts, seeds, flowers and some herb beverages and are an
integral part of the human diet. Several studies have indicated that
the antioxidant activities of some fruits and vegetables were highly
correlated with their total phenolic contents. Antioxidants are
compounds that can delay or inhibit the oxidation of lipids or other
molecules by inhibiting the initiation or propagation of oxidative
chain reactions8, 9. The antioxidant activity of phenolic compounds is
mainly due to their redox properties, which can play an important
role in absorbing and neutralizing free radicals, quenching singlet
and triplet oxygen, or decomposing peroxides10. Flavonoids,
phenolic acids and phenolic diterpenes, lignans are the examples of
phenolic compotents with antioxidant properties11, 12.
Saraca asoca Roxb. De Wilde – Ashoka is a Sanskrit word which
means”without sorrow” or that gives no grief. Ashoka tree,
universally known by its binomial latin name Saraca asoca (Roxb.),
De wild or Saraca indica belonging family Caesalpinacea13,14. It is a
small evergreen tree 7-10cm high. It occurs up to the altitude 750
m15. Five lignin glycosides, lyoniside, nudiposide, 5-methoxy-9-β-
xylopyranosyl-(-)-isolariciresinol, icariside E3 and schizanriside,
three flavonoids, (-)-epicatechin, epiafzelechin-(4β-8)-epicatechin
and procyanidine B2, together with β-sitosterol glucoside, were
isolated from dried bark16. Saraca asoca exhibited antibacterial
activity17, anticancer activity18, antimenorrhagic activity19,
antioxytocic activity20. It have many uses like to treat skin infections,
CNS function, genitor-urinary functions, uterus pain painful periods,
clots and ammenorhea21,22.
MATERIALS AND METHODS
Collection and identification of plant material
The stem bark of Saraca asoca Roxb.was collected from Bhupal
Nobles Institute Campus, Udaipur (Raj.) India in the month of Sep.-
Oct. 2008. It was dried under shade. Drug sample was identified by
Dr. SS Katewa (Department of Botany), College of Science, MLSU,
Udaipur (Raj.) India.
Materials
Chemicals 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and Gallic acid
were perchased from M/S Sigma Chemical Co. Other chemicals and
reagents used were of analytical grade. UV-visible
spectrophotometer, Shimadzu 1800 was used for recording the
spectra.
Preparation of extracts
Dried stem bark was ground in to moderately coarse powder (≠22)
in a grinder. Extracts were prepared by various extraction methods
(soxhlet, ultrasonication and microwave assisted extraction) by
using three solvents (ethanol 90%, ethanol 60% and acetone). The
parameters used for soxhlet is time-48 hrs., temperature-500C, for
ultrasonication method is time-90 min., temperature-500C and for
microwave assisted extraction method, time- 3 min., temperature
600C, power- 480 htz.
In-vitro antioxidant studies
The prepared extracts were tested for its free radical scavenging
property using DPPH method. All experiments were performed
thrice. In-vitro DPPH (1,1-diphenyl-2-picryl hydrozyl) radical
scavenging activity was carried out by adopting the methopd of
Blois23, Cotelle24, spectrophotometric method25. To a methanolic
extract of DPPH (200µM), 0.05ml of test extracts dissolved in
ethanol were added at different concentrations (100-500 µg/ml). An
equal amount of ethanol was added to the control. After 20 min., the
decrease in absorbance of test mixture (due to quenching of DPPH
free radicals) was read at 517 nm and the percentage inhibition
calculated by using the formula26. The antioxidant activity is
expressed as IC50.
Inhibition (%) = Control-Test \ Control × 100
Statistical Analysis
Linear regression analysis was used to calculate the IC50 values.
RESULTS
Asian Journal of Pharmaceutical and Clinical Research
Vol. 3, Issue 3, 2010 ISSN - 0974-2441
232
Several concentrations ranging from (50-100 µg/ml) of the prepared
extracts by various methods using different parameters were tested
for their antioxidant activity in DPPH- radical scavenging, In-vitro
model. It was observed that free radicals were scavenged by the test
compounds in a concentration dependent manner. From the results
given in Tables 1-3, it was inferred that, with respect to maximum
percentage inhibition, in DPPH model, the extracts prepared by
ultrasonication method showed maximum % inhibition respectively
at 100 µg/ml concentration. However the IC50 value of ethanolic
(90%), ethanolic (60%) and acetone extracts prepared by
ultrasonication method were shown to be 139.78, 126.54, 97.82
µg/ml respectively. The extracts prepared by soxhlet extraction
method showed least inhibition at 100 µg/ml concentration in this
model.
DISCUSSION
Free radicals are chemical entities that can exist separately with one
or more unpaired electrons. The generation of free radicals can
bring about thousands of reactions and thus cause extensive tissue
damage. Lipids, proteins and DNA are all susceptible to attack by
free radicals. Antioxidants may offer resistance against oxidative
stress by scavenging the free radicals. The 1, 1, diphenyl-2-picryl
hydrazyl (DPPH) radical was widely used as the model system to
investigate the scavenging activities of several natural compounds
such as phenolic and anthocyanins or crude mixtures (extracts) of
plants. DPPH radical is scavenged by antioxidants through the
donation of proton forming the reduced DPPH. The proton radical
scavenging action is known to be one of the various mechanisms for
measuring antioxidant activity. DPPH is one of the compounds that
possess a proton free radical and shows a maximum absorption at
517 nm. When DPPH encounter proton radical scavengers, its purple
colour fads rapidly. This assay determines the scavenging of stable
radical species of DPPH by antioxidants. The literature supports that
phytoconstituents such as polyphenolic compounds in drugs are
responsible for the antioxidant potential27, 28, 29. Further, phenolic
compounds are effective hydrogen donors, which make them
antioxidant30.
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Table 1: Effect of Ethanolic (90%), Ethanolic (60%), Acetone extract of Saraca asoca Roxb.(stem bark) prepared by Soxhlet
method (at 60 °C, 48 hrs.) on DPPH radical scavenging model (Values are mean of three replicates)
Concentration (µg)
Percent Reduction
Ethanol 90%
IC50 (µg/ml)
Percent Reduction
Ethanol 60%
IC50 (µg/ml)
Percent Reduction
Acetone
50
15.48
18.11
16.01
60
18.37
21.25
20.20
70
20.73
187.70
23.35
193.88
23.35
80
21.52
24.40
24.93
90
24.40
26.77
28.34
100
27.55
30.44
29.39
Table 2: Effect of Ethanolic (90%), Ethanolic (60%), Acetone extract of Saraca asoca Roxb. (stem bark) prepared by microwave
method (at 60°C, 480Htz, 3 min.) on DPPH radical scavenging model. (Values are mean of three replicates)
Concentration (µg)
Percent Reduction
Ethanol 90%
IC50 (µg/ml)
Percent Reduction
Ethanol 60%
IC50 (µg/ml)
Percent Reduction
Acetone
IC50 (µg/ml)
50
17.58
18.63
20.73
60
21.25
21.78
23.35
70
22.83
166.80
24.14
165.84
24.67
160.09
80
25.98
27.03
28.08
90
28.34
29.92
30.97
100
29.92
32.02
34.38
Table 3: Effect of Ethanolic (90%), Ethanolic (60%), Acetone extract of Saraca asoca Roxb. (stem bark) prepared by ultrasonication
method (at 50°C, 90 min.) on DPPH radical scavenging model. [Values are mean of three replicates]
Concentration (µg)
Percent Reduction
Ethanol 90%
IC50 (µg/ml)
Percent Reduction
Ethanol 60%
IC50 (µg/ml)
Percent Reduction
Acetone
IC50 (µg/ml)
50
21.25
22.57
26.50
60
23.35
27.03
29.65
70
27.82
139.78
30.44
126.54
35.17
97.82
80
29.65
34.38
40.15
90
34.12
37.27
47.50
100
37.27
40.15
50.91
233
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Drug antioxidant effects. A basis for drug selection?
Article
A free radical is any species capable of independent existence that contains one or more unpaired electrons. Free radical reactions have been implicated in the pathology of more than 50 human diseases. Radicals and other reactive oxygen species are formed constantly in the human body, both by deliberate synthesis (e.g. by activated phagocytes) and by chemical side-reactions. They are removed by enzymic and nonenzymic antioxidant defence systems. Oxidative stress, occurring when antioxidant defences are inadequate, can damage lipids, proteins, carbohydrates and DNA. A few clinical conditions are caused by oxidative stress, but more often the stress results from the disease. Sometimes it then makes a significant contribution to the disease pathology, and sometimes it does not. Several antioxidants are available for therapeutic use. They include molecules naturally present in the body [superoxide dismutase (SOD), alpha-tocopherol, glutathione and its precursors, ascorbic acid, adenosine, lactoferrin and carotenoids] as well as synthetic antioxidants [such as thiols, ebselen (PZ51), xanthine oxidase inhibitors, inhibitors of phagocyte function, iron ion chelators and probucol]. The therapeutic efficacy of SOD, alpha-tocopherol and ascorbic acid in the treatment of human disease is generally unimpressive to date although dietary deficiencies of the last two molecules should certainly be avoided. Xanthine oxidase inhibitors may be of limited relevance as antioxidants for human use. Exciting preliminary results with probucol (antiatherosclerosis), ebselen (anti-inflammatory), and iron ion chelators (in thalassaemia, leukaemia, malaria, stroke, traumatic brain injury and haemorrhagic shock) need to be confirmed by controlled clinical trials. Clinical testing of N-acetylcysteine in HIV-1-positive subjects may also be merited. A few drugs already in clinical use may have some antioxidant properties, but this ability is not widespread and drug-derived radicals may occasionally cause significant damage.