See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/304142010
Phytochemical Screening, Free Radical Scavenging and Antioxidant Activity of
Azadirachta indica Resin
Article · March 2016
CITATIONS
READS
0
90
1 author:
P.Muthukumaran -.
DMI-St. Eugene University Chibombo, Zambia
31 PUBLICATIONS 44 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
research article View project
All content following this page was uploaded by P.Muthukumaran -. on 20 June 2016.
The user has requested enhancement of the downloaded file.
450 in Biosciences 9(7), Print : ISSN 0974-8431,
Trends
Trends
450-455,
in Biosciences
2016
9 (7), 2016
Phytochemical Screening, Free Radical Scavenging and Antioxidant
Activity of Azadirachta indica Resin
P. MUTHUKUMARAN AND S.KUMARAVEL
Indian Institute of Crop Processing Technology, Thanjavur,
Tamilnadu 613005
ABSTRACT
The aim of this study was to investigate the
Phytochemical and antioxidant effect of Azadirachta
Indica Resin. The antioxidant activity was evaluated
by various antioxidant assays, including 1, 1-diphenyl2- picrylhydrazyl (DPPH), 2, 2’-azino-bis (3ethylbenzthiazoline-6-sulfonic acid) (ABTS), and
Hydrogen peroxide scavenging method. The
antioxidant activities were compared to standard
antioxidant ascorbic acid. Azadirachta Indica Resin
extract showed a significant antioxidant activity in
DPPH, ABTS and H2O2 scavenging methods. The
findings of the present study suggest that
Azadirachta Indica Resin could be a potential source
of natural antioxidant that could have greater
importance as therapeutic agent in preventing or
slowing oxidative stress related degenerative diseases.
Key words
DPPH, ABTS ,H 2O 2, Azadirachta
Indica,Resin
Medicinal plants show antioxidant property
is used to prevent oxidative damage caused by free
radicals (Maxwell, 1995). Reactive oxygen species
(ROS) are consisting of free radicals (O2, HO) and
non free radicals (H2O2). Free radicals produced
from oxidation reaction start the chain reaction that
damage the cell get involved in immune suppression,
cell membrane disintegration, membrane protein
damage and DNA mutation, which can further
initiate the development of many diseases like
cancer, liver injury, cardiovascular diseases,
inflammation, diabetes, atherosclerosis etc
(Devasagayam et al .,2004). The most reactive free
radical is the hydroxyl radical which is known to
initiate lipid peroxidation and cause fragmentation
of DNA leading to mutations(Haillwell,
1997).Although the body possesses such defense
mechanisms as enzymes and antioxidant nutrients,
which arrest the damaging properties by removing,
free radical intermediates and inhibit other oxidation
reactions.
Many antioxidant compounds, naturally
occurring from plant sources, have been identified
oxygen scavengers (Duh, 1998). Recently, interest
has increased considerably in finding naturally
occurring antioxidant for use in foods or medicinal
materials to replace synthetic antioxidants, which
are being restricted due to their side effects such
as carcinogenicity. Natural antioxidants can protect
the human body from free radicals and retard the
progress of many chronic diseases as well as retard
lipid oxidative rancidity in foods (Lai et al .,2001).
Azadirachtaindica Commonly known as
Neem is widely distributed in Indian subcontinent
and its neighbouring countries for more than a
thousand years as one of the most versatile medicinal
plant having wide spectrum biological activity. It
has been an inherent part of folklore and the
traditional medicinal system since one can recall.
Every part of the tree is used as medicine for
household remedies against various human ailments
and even as bio-pesticides for agricultural purpose.
Tender shoots and leaves are taken as food in India;
in Assam the tiny leaves were oil fried to eat with
the rice in early summer like in the month of MarchApril, which function as an appetizer. The twigs
are often used to clean the teeth.Neem oil, leaves,
bark, stem products have been therapeutically used
for the treatment of respiratory disorder,
inflammation, constipation, skin infection (Sithisarn
et al., 2006 ) arthritic disorders, fever and diabetes
etc (Van Der Nat et al., 1992) . The present study
was thus undertaken to access the antioxidant
activity of Azadirachtaindica Resin using DPPH
radical scavenging ABTS and H 2O2 Scavenging
assay.
Collection and Preparation of Extracts
Azadirachtaindica resin collected from
Thanjavur, Tamilnadu, India. The resins were then
dried under shade 15 days. The dried resins was
coarsely powdered and used for following
estimations.
MUTHUKUMARAN and KUMARAVEL, Phytochemical Screening, Free Radical Scavenging and Antioxidant Activity 451
Fig. 1. Phytochemical quantification
25 g of air dried powder of resins were taken
separately with 150 ml of Ethanol and were soaked
for 24 hrs till the colour of the plant material
disappeared. After which the extracts were
collected and stored at 4oC in airtight bottles and
were qualitatively tested for the presence of various
phytocompounds.
Total polyphenolic content (TPC)
Total polyphenol content (soluble and bound)
was determined by the method described by
Singleton and Rossi (1965) .Known volume of
extract was taken and made up to 250µl with
distilled water and the sample was mixed with 250
µl of folinciocalteau reagent and 500 µl of 20%
Na 2Co3. The final reaction mixture volume was
adjusted to 5ml using distilled water. The reaction
mixture was incubated in dark for 30 minutes and
samples were then centrifuged at 2000 rpm for 5
minutes after which the supernatant absorbance
Fig. 2. DPPH S cavenging Activity
452
Trends in Biosciences 9 (7), 2016
Fig. 3. Hydrogen Peroxide Scavenging Activity
was measured at 760 nm. A calibration curve was
constructed with different concentrations of gallic
acid as standard. The results were expressed as
mg of gallic acid equivalent/100g of sample.
Total flavonoids content
This was assayed as described by Jia et
al.,(1999) with slight modification. A known
amount of above mentioned extract was taken and
made up to 500µl with distilled water. To this
mixture 75µl of 5% sodium nitrite was added and
allowed to stand for 5 minutes after which 150 µl
of 10% AlCl3 was added and after 6 minutes 0.5
ml of 1M NaOH were added and sample mixture
was incubated at dark for 15 minutes. It was then
centrifuged at 2500 rpm for 5 minutes and
absorbance was recorded at 512nm. The flavanoid
content was expressed as mg of catechin
equivalent/100g of sample.
Fig. 4. Reducing Power
MUTHUKUMARAN and KUMARAVEL, Phytochemical Screening, Free Radical Scavenging and Antioxidant Activity 453
Total Tannins content
scavenging activity was calculated by the formula:
The quantitative tannin content in samples was
estimated by the method of Price and Butler (1977),
with some modifications. Known concentration of
methanolic extract were taken and made up to 0.5ml
using distilled water. To this reaction mixture, 1 ml
1% K3Fe(CN)6 and 1ml 1% FeCl3 were added,
and the volume was made up to 10 ml with distilled
water. The reading of the resultant solution was
measured spectrophotometrically at 720nm after 5
min using tannic acid as a standard. The tannin
content was expressed as mg of tannic acid
equivalent/100g of sample.
DPPH Radical Scavenging assay
Accurately weighed 4.3 mg of DPPH was
dissolved in 3.3 ml of methanol in a test tube
(Ionita., 2005). Solution was protected from light
by covering with aluminum foil. 150 ìl of above
solution was taken and diluted up to 3 ml with
methanol, the absorbance of this solution was taken
immediately at 516 nm on UV spectrophotometer
using methanol as blank. This reading was served
as control reading. For the test and standard, the
aliquots of different concentration ranging were
prepared. For the assay 150 ìl of the test or std
solution was added to 150 ìl of DPPH solution and
diluted up to 3 ml with methanol, the absorbance
of this solution was taken after 15 min at 516 nm
on UV spectrophotometer using methanol as blank.
The absorbance was taken in triplicate manner. The
% Scavenging activity was found by using
following formula:
O.D of control – O.D of test
% scavenging activity = ——————— × 100
O.D of control
Hydroxyl Radical Scavenging Activity
The Hydroxyl radical scavenging activity was
measured by studying the competition between
deoxyribose and the extract for hydroxyl radicals
generated from the Fe3+/ascorbate/ EDTA/ H2O2
system (Jayaprakasha et al .,2004). The reaction
mixture contained deoxy ribose (2-8mM), Fecl
(0.1mM), EDTA (0.1mM), H2O2 (lmM), ascorbate
(0.1mM), KH PO - KOH buffer (20mM, 2 4 pH
7.4) and various concentrations (25- 400 ìm of
extracts and std 10 to 80 ìm /ml) of standard drug
in a final volume of 1 ml. The reaction mixture
was incubated for 1hr at 37°C; deoxyribose
degradation was measured with spectrophotometer
at 532 nm. The percentage of hydroxyl radical
O.D of control – O.D of test
% scavenging activity = ——————— × 100
O.D of control
Reducing power
The reducing power of a compound serves
as significant indicator of its potential antioxidant
activity. Increased absorbance of the reaction
mixture indicates increased reducing power.
Various conc. of the extracts in 1ml of water were
mixed with phosphate buffer (2.5 ml, 0.2 M pH
6.6) and 1% potassium ferricyanide (2.5 ml). The
mixture was incubated at 50ºC for 20 min. Aliquots
of trichloroacetic acid (2.5 ml, 10%) were added
to the mixture, which was then centrifuged al
3000g for 10 min. upper layer of solution (2.5 ml)
was mixed with distilled water (2.5 ml) and freshly
prepared FeCl 3 solution (0.5ml, 0.1%). The
absorbance was measured at 700nm (Oyaizu
,1986).
Control – Test
Reducing power formation (%) = ———— X100
Test
Statistical analysis
All experiments were performed thrice and
the results averaged Data were expressed as
Mean ± SD
RESULTS AND DISCUSSION
The phytochemicals are the most prevalent
secondary metabolites in the plant kingdom. They
are naturally occurring antioxidant components and
are capable of mitigating the harmful effects of
diseases related to oxidative stress (Li et al., 2008).
Quantification
constituents
of
phytochemical
The Quantified Phenolic contents of
Azadirachta Indica Resin extracts were 19.40 (mg/
100g GAE), The Flavonoid content 19.14 (mg/100g
GAE), Tannic acid 30.8 (mg/100g). Results of
quantified Phenol, Flavonoids and Tannic acid
contents were showed in Figure 1. Chemical
compounds from plants are known to be useful in
the treatment of many diseases(Cheenpracha
et al., 2010).
Phenolic compounds used as nutraceuticals,
and found in apples, green-tea, and red-wine and
454
Trends in Biosciences 9 (7), 2016
in many medicinal plants as phytochemical or
secondary metabolites. Phenolic compound has
enormous ability to combat cancer and are also
thought to prevent heart ailments to an appreciable
degree and sometimes are anti-inflammatory agents.
They are potent vasodilator (Padilla et al., 2005)
and for the presence of hydroxyl group they
possessing potent scavenging activity (Rajan and
Muthukrishnana.,2013). The Resin showed
considerably high amount of phenolic content
(75.39 ± 3.19 mg/g). The detection and
quantification of total phenolic compound present
in the Resin of Azadirachta Indica Resin may in
future contribute in field of herbal remedy as potent
antioxidant.
DPPH activity. In this study, at 50ìg /ml, the resin
extract showed highest inhibition of DPPH activity
shown in (Figure 2). The experimental data of the
extracts revealed that the extracts are likely to have
the effects of scavenging free radicals. From the
result we observe that a dose dependent relationship
in the DPPH radical scavenging activity. The
involvement of free radicals, especially their
increased production, appears to be a feature of
most of the human diseases including
cardiovascular
diseases
and
cancer
(Deighton et al., 2000). The results of DPPH-free
radical scavenging assay suggest that the
Azadirachta Indica Resin is more capable of
scavenging free radicals.
Flavonoids are important group of polyphenol
widely distributed among the plant flora and
containing a benzopyrone which use as antioxidants
or free radical scavengers and also have cardio
protective roll (Kar, 2007). By inhibiting the estrogen
producing enzyme Flavonoid suppress the
progression of cancer. For example, Flavonoid
inhibited the estrogen synthesis which binds
estrogen to its receptor. The percentage of total
Flavonoid in Azadirachta Indica Resin was (19.14
mg/100g GAE). This moderately the level of
Flavonoid present in the Resin could be attributed
to its antioxidant capacity.
Hydrogen peroxide
Tannin is Phenolic compounds of high
molecular weight used as antiseptic and this activity
is due to presence of the Phenolic group. This is
also associated with antiviral activity. In presence
of very high quantity of tannin, there are many
viruses like polio virus, herpes simplex viruses have
been found to get inactivated (Bajaj, 1998). The
Resin of Azadirachta Indica contains very high level
of tannin (30.8 mg/100g). The presence of very
high quantity of tannin supports the Resin of this
plant has antimicrobial activity.
DPPH radical Scavenging assay
In the present study several free radical
scavenging activities of ethanolic extract of
Azadirachta Indica Resin were evaluated by DPPH
scavenging assay. Ethanolic extracts of Resin have
got profound antioxidant activity. DPPH antioxidant
assay is based on the ability of DPPH, a stable free
radical, which gets decolorized in the presence of
antioxidants (Burits and Bucar ,2000). 19) . The
ethanolic extracts of Azadirachta Indica Resin
exhibited a significant dose dependent inhibition of
Hydrogen peroxide itself is not very reactive,
but sometimes is toxic to cell because it may give
rise to hydroxyl radical in the cells (Halliwell, 1991).
Therefore, removing of hydrogen peroxide is very
important for antioxidant defense in cell system.
In this study, at 50ìg /ml, the resin extract showed
highest inhibition of H2O2 activity shown in (Figure
3). The composition of hydrogen peroxide into
water may occur according to the antioxidant
compounds as the antioxidant components present
in the extract are good electron donors, they may
accelerate the conversion of H 2 O 2 to H 2 O.
Polyphenols have also been shown to protect
mammalian cells from damage induced by hydrogen
peroxide, especially compounds with the
orthohydroxy Phenolic compounds like Quercetin,
Gallic acid, Caffeic acid and catechin (Nakayama
,1994). Therefore, the Phenolic compounds of the
Phenol of Azadirachta Indica Resin may probably
be involved in scavenging hydrogen peroxide.
The reducing capacity
The reducing capacity of a biological
compound acts as a significant indicator of its
antioxidant potential (Moein et al., 2008) .The
reducing properties are generally associated with
the presence of reductones. Reductones are also
reported to react with certain precursors of
peroxide, thus preventing peroxide formation.
Figure 4 shows the reductive capabilities of the
Azadirachta indica resin compared to ascorbic acid.
Higher ascorbic acid equivalent value indicates
higher reducing capacity of samples, thus greater
antioxidant potential. The Azadirachta indica resin
with potent reducing capacity. The reducing power
MUTHUKUMARAN and KUMARAVEL, Phytochemical Screening, Free Radical Scavenging and Antioxidant Activity 455
of ethanolic extract of Azadirachta indica resin was
found remarkable and the reducing power of the
extract was observed to rise as the concentration
of the extract gradually increased.
The result of scavenging activity assay in this
study indicates that the resin was potently active.
This suggests that the resin contain compounds
that are capable of donating hydrogen to a free
radical in order to remove odd electron which is
responsible for radical’s reactivity. The extracts
were capable of scavenging DPPH, hydroxyl and
Reducing Power activity in a concentration
dependent manner. Further investigation of the
compounds responsible for the antioxidant activity,
its isolation and in vivo studies are needed.
LITERATURE CITED
Bajaj YPS. 1988 .Medicinal and aromatic plants
(Biotechnology in agriculture and forestry). B er lin :
Springer-Verlag.
Burits, M., Bucar, F.2000. Antioxidant activity of Nigella
sativa essential oil. Phytotherapy Research, 14:323–
328.
Cheenpracha S, Park EJ, Yoshida WY, Barit C, Wall M,
Pezzuto JM .2010. Potential anti-inflammatory phenolic
glycosides from the medicinal plant Moringa oleifera
fruits. Bioorg Med Chem , 18(17): 6598-6602.
Deighton N, Brennan R, Finn C, Davies HV.2000. Antioxidant
properties of domesticated and wild Rubus species. J.
Sci. Food Agric, 80; 1307-1313.
Devasagayam T.P.A, Tilak J.C., Boloor K.K., Sane K.S.,
Ghaskadbi S.S and.Lele R.D .2004. Review: Free radicals
and antioxidants in human health: Current status and
future prospects, J. Assoc. Phys. India, ,52, 794-804.
Duh P D .1998.Antioxidant activity of burdock (Arctium
lappa Linne): Its scavenging effect on free radical and
active oxygen, Journal of the American Oil Chemist’s
Society, 75: 455– 465
Haillwell.B.1997. Oxygen Radicals and Disease Process
(Hardwood Academic Publisher, The Netherlands, , 114.
Halliwell B .1991. Reactive oxygen species in living systems:
Source biochemistry and role in human disease. Am J
Med; 91 Suppl., 14–22.
Ionita P .2005. Is DPPH Stable free radical a good scavenger
for oxygen active species? Chem., 59, 11-6.
Jayaprakasha GK, Jaganmohan RL, Sakariah KK (2004).
“Antioxidant activities of flavidin in different in vitro
model systems.” Bioorg. Med. Chem. 12(19):5141-5146.
Jia, Z.S., Tang, M.C and Wu J.M (1999).The determination
of Flavonoid contents in mulberry and their scavenging
effects on superoxide radicals,”Food Chem, 64 (4): 555559.
Kar, A (2007). Pharmaocgnosy and Pharmacobiotechnology
(Revised-Expanded Second Edition). New Delhi: New
Age International LimtedPublishres, 332-600.
Lai L.S., Chou S.T., Chao W.W(2001).Studies on the
antioxidativeactivities of hsian-tsao (Mesona
procumbens Hemsl) leaf gum, Journal of Agricultural
and Food Chemistry, 49: 963–968.
Li HB, Wong CC, Cheng KW, Chen F (2008). Antioxidant
properties in vitro and total phenolic contents in
methanol extracts from medicinal plants. LWTF ood Sci
Technol 41: 385-390.
Maxwell SRJ (1995). Prospects for the use of antioxidant
therapies. Drugs, 45: 345-361.
Moein MR, Moein S, Ahmadizadeh S (2008). Radical
scavenging and reducing power of Salvia mirzayanii
Subfractions. Molecules 13:2804-2813.
Nakayama T (1994).Suppression of hydroperoxide-induced
cytotoxicity by polyphenols. Cancer Res, 54; 19911993.
Oyaizu, M. 1986 Studies on product of browning reaction
prepared from glucose amine. Japanese Journal of
Nutrition 44: 307-315,
Padilla E, Ruiz E, Redondo S, Gordillo-Moscoso A, Slowing
K, Tejerina T(2005) Relationship between vasodilation
capacity and phenolic content of Spanish wines. Eur J
Pharmacol. 517(1-2):84–91.
Price, M.L and Butler, L.G (1977) Rapid visual estimation
and spectrophotometric determination of tannin content
of sorghum grain,”J Agricul Food Chem,vol: 25 (6), 12681273.
Rajan T, Muthukrishnana S (2013). Characterization of
phenolic compounds in pseudarthria viscida root extract
by hplc and ft-ir analysis. Asian J Pharm Clin Res ,
6(2): 274-276.
Singleton, V.L and Rossi, J.A (1965).Colorimetry of total
phenolics with phosphomolybdicphosphotungstic acid
reagent,” Am J Enol Viticult, 16 (3), 144-158.
Sithisarn P., Supabphol R., andGritsanapan W
(2006).Internat. Jr. of the Kuwait University Health
Sciences Centre, 15, 3.
Van Der Nat, M. G., Van Der Sluis, K. and Labadie, R. P
(1991).Biological activities and medicinal properties of
neem (Azadirachta indica). J Ethnopharmacol., 35,1–
24.
Received on 17-03-2016
View publication stats
Accepted on 20-03-2016