Continental J.

Pharmaceutical Sciences 4: 28 - 34, 2010

© Wilolud Journals, 2010

ANTIOXIDANT ACTIVITY IN BARK AND ROOTS OF NEEM (Azadirachta indica) AND MAHANEEM
(Melia azedarach)

Gayatri Nahak and R. K. Sahu

Department of Botany, B.J.B. (A) College, Bhubaneswar, Orissa

ABSTRACT
The aim of this study was to investigate the antioxidant activity of the multi-solvent
extracts (aqueous, methanolic and ethanolic) of Root and Bark of two Medicinal plant i.e.
Azadirachta indica A. Juss and Melia azedarach of Meliaceae family using 2, 2-diphenyl-
1-picrylhydrazyl (DPPH) - scavenging assay. The dry powder of Root and Bark of the two
neem tree were extracted using soxhlet extraction followed by vacuum rotary evaporator
methods. The extracts were tested for antioxidant activity using DPPH-scavenging assay.
Total phenol contents of extracts were also determined by folin cicalteu reagent method.
Experimental results revealed the highest fraction of crude extract, phenol content as well
as antioxidant activity in Barks and Roots of Mahaneem (Melia azedaarach) in
comparison to Neem (Azadirachta indica). The total phenolic concentration of Bark, Root
of Melia showed a positive correlation with antioxidant capacity. Similarly IC50 values in
Bark and Root of Mahaneem was as low as Ascorbic acid. The plant Mahaneem and its
plant parts may be exploited for clinical medicine as potent factor because of its high
antioxidant activity. (CJBiolSci/2010/018)

KEYWORDS: Neem, Mahaneem, Antioxidant activity, Phenol content, Bark and Root.

INTRODUCTION
It is an established fact that polyphenolic compounds possess remarkable antioxidant activities which are
present quite commonly in the plant family Meliaceae (Siddiqui, et al., 1992; Sultana, et al., 2007), It has also
been reported that phenolic contents of neem can be influenced by geographical locations and other abiotic
factors (Ermel, et al., 1986; Kaura, et al., 1998; Kaushik, et al., 2007). A. indica is well known in India and its
neighboring countries for more than 2000 years as one of the most versatile medicinal plants having a wide
spectrum of biological activity. A. indica and M. azedarach are two closely related species of Meliaceae family.
The former is popularly known as Indian Neem (Margosa tree) or India lilac, and the latter as Mahaneem or
Persian lilac. All parts of the plant have been used for medicinal purposes including fruits, seeds, leaves, roots
and barks (Anon, 1985). Neem has been extensively used in Ayurveda, Unani and homoepathic medicine and
has become a Synonym of modern medicine. The Neem tree contains more than 100 bioactive ingredients. The
most important bioactive compound is azadirichtin. Melia azedarach, the Persian Lilac is popularly known as
Mahaneem tree and cultivated in all stations. It is a large evergreen tree found throughout India and very similar
to Neem. It is native to upper Burmah region. It’s Flowering time is May-June and Fruiting time is Nov-Dec.
The inner bark contains a resinous alkaloid substance and is used as an anthelmintic. Various scientific studies
reported the analgesic, anticancer, antiviral, antimalarial, antibacterial, and antifungal, antifeedent and
antifertility activity of this plant (Vishnukanta & Rana, 2008)

Leaf and bark extract of A. indica has been studied for its anti-oxidant activity (Ghimeray, et al., 2009; Sultana,
et al., 2007). However anti-oxidant activity of Mahaneem (M. azedarach) another very important medicine
plant has not been investigated. In present work bark and root of two trees, A. indica & M. azedarach belonging
to family Meliaceae extracted in water, ethanol & methanol were investigated for the presence of phenol content
and antioxidant activity in a comparative way.

MATERIALS AND METHODS

Chemicals and Reagents
Folin-Ciocalteu reagent (Merck Pvt. Ltd, India), Sodium chloride (S.D. Fine Chem, India), Sodium carbonet
(Merck Pvt. Ltd, India), Catechol (Himedia Lab., India), 2, 2-Diphenyle-2-picryl hydrazyl (DPPH) and Ascorbic
acid are obtained from (Himedia Lab., India). Stock solutions of the test extracts were prepared in ethanol.
Appropriate blanks were used for individual assays.

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 Gayatri Nahak and R. K. Sahu: Continental J. Pharmaceutical Sciences 4: 28 - 34, 2010

Plant Materials
The Roots (root with root bark) and Barks of the two species i.e. A. indica & Melia azedarach of Meliaceae
family were collected from the Medicinal Garden of B.J.B (A) College, Bhubaneswar, Orissa. The Barks and
Roots of two plants were rinsed severally with clean tap water to make it dust and debris free. Then the Barks
and Roots were spread evenly and dried. Then the dried samples were ground in electric chopper to get fine
powder form for further use.

Instrumentations
Collection of multi-solvent extract was done by Soxhlet apparatus (J.S.G.W) with varying temperatures
according to the B.P. of the solvents. The samples were evaporated through the Rotary vacuum evaporator at
60-1000C according to the B.P. of supplied solvents. Absorbance spectrophotometery was carried out using a
UV-vis spectrophotometer (EI, model-1371).Wavelength scans and absorbance measurements were in 1ml
quartz cells of 1cm path length.

Preparation of plant extracts

The dried and powdered Neem and Maha-neem Barks and Roots (each 50g) were extracted successively with
multi-solvent extraction by using double distilled water, ethanol and methanol (each 400ml.) for 10-12 h.
through Soxhlet apparatus. Then collected solutions were filtered through Whatman No-1 filer paper. The
extracts were evaporated to dryness under reduced pressure at 900C by Rotary vacuum evaporator to obtain the
respective extracts and stored in a freeze condition at −180C until used for further analysis.

Phenolic Estimation
The total phenolic content of plant extracts were determined by using Folin-Ciocalteu Spectrophotometric
method according to the method described (Kim, et al., 2007). Reading samples on a UV-vis Spectrophotometer
at 650 nm. Results were expressed as catechol equivalents (µg/mg).

Antioxidative activity
The antioxidant activity of the Neem and Mahaneem (Barks and Roots) on the basis of the scavenging activity
of the stable 2, 2- diphenyl-2-picrylhydrazyl (DPPH) free radical was determined according to the method
described in (Brand-Williams, et al., 1995) with slight modification. The following concentrations of extracts
were prepared 40µg/mL, 80µg/mL, 120µg/mL, 160 µg/mL and 200µg/mL. All the solutions were prepared with
methanol. 5 ml of each prepared concentration was mixed with 0.5mL of 1mM DPPH solution in methanol.
Experiment was done in triplicate. The test tubes were incubated for 30 min. at room temperature and the
absorbance measured at 517nm. Lower the absorbance of the reaction mixture indicates higher free radical
scavenging activity. Ascorbic acid was used as a standard and the same concentrations were prepared as the test
solutions. The different in absorbance between the test and the control (DPPH in ethanol) was calculated and
expressed as % scavenging of DPPH radical. The capability to scavenge the DPPH radical was calculated by
using the following equation.

Scavenging effect (%) = (1-As/Ac) ×100

As is the absorbance of the sample at t =0 min.

Ac is the absorbance of the control at t=30 min.

RESULTS AND DISCUSSION

The Effect of Different Solvents on the Yields of Neem and Mahaneem Leaf Extracts
The significant variation in the yields of Azadirachta and Melia extracts were shown using various fraction
solvents. The yield of extracts using Water, Methanol and Ethanol in case of Azadirachta were 3.90gm, 4.45gm
and 4.35gm (in Bark) and 3.02gm, 3.45gm and 3.56gm (in Root) respectively. Likewise in case of Melia extract
also followed the same order as the Azadirachta extracts, and they were 4.34gm, 5.20gm and 5.85gm (in Bark)
and 3.29gm, 3.56gm and 3.29gm (in Root) respectively. The variation in yield may be due to the polarity of the
solvents used in the extraction process (Table-1).

Free Radical and Antioxidant Activity

Table-2 and Table-3 show the results of the free radical (DPPH) scavenging activity in % inhibition in
Azadirachta and Melia respectively. The result revealed that the ethanol fraction of Bark in case of Melia
exhibited the highest radical scavenging activity with 83.38±0.01 followed by its methanol extract with
72.88±0.04 and aqueous extract with 53.89±0.07. In comparison to Melia the Azadirachta extract shows less

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 Gayatri Nahak and R. K. Sahu: Continental J. Pharmaceutical Sciences 4: 28 - 34, 2010

scavenging activity. The Azadirachta extract of Bark obtained from ethanol shows 66.94±0.02.i.e. highest
scavenging activity followed by its methanol extract with 55.00±0.05 and aqueous extract with 37.50±0.03. In
overall comparison the ethanolic extract of Bark in both Azadirachta and Melia show the highest scavenging
activity followed by the methanol and then aqueous. The results showed that the antioxidant activity of Root
extracts of both the plants exhibited decreasing radical scavenging activity as comparison to Bark extracts. The
Root extracts of Melia plant obtained from all the three solvent showed little bit higher antioxidant activity as
compared to Azedarachta Root extracts. The ethanolic fraction of root in case of Melia plant exhibited highest
antioxidant activity i.e. 63.55±0.04 followed by the methanolic fraction i.e. 47.96±0.02 and aqueous fraction i.e.
36.44±0.04. Similarly in case of Azedarachta plant the ethanolic fraction of root extract showed the highest i.e.
50.84±0.03 followed by the methanolic fraction i.e. 42.71±0.04 and aqueous fraction i.e. 30.50±0.04. The
Methanol and ethanol has been proven as effective solvent to extract phenolic compounds (Siddhuraju &
Becker, 2003). In the present study, the values of ethanolic and methanolic extracts were higher than those of
aqueous ones. Among solvents used in this study ethanol has showed the best effectiveness extracting phenolic
components. Ethanol is preferred for the extraction of antioxidant compounds mainly because its lowers toxicity
(Karadeniz, et al., 2005) Fig.1. Shows the comparative study of radical scavenging activity between Melia and
Azadirachta with respect to Ascorbic acid as standard.

Phenol Content and Antioxidant Activity

It is reported that phenols are responsible for the variation in the antioxidant activity of the plant (Cai, et al.,
2004). They exhibit antioxidant activity by inactivating lipid free radicals or preventing decomposition of
hydroperoxides into free radicals. (Pitchaon, et al., 2007; Pokorny, et al., 2001). Phenolic compounds are
considered to be the most important antioxidative components of herbs and other plant materials, and a good
correlation between the concentrations of plant phenolic and the total antioxidant capacities has been reported
(Madsen, et al., 1996; Pellegrini, et al., 2000). The total phenolic content varied significantly between the two
species of Maliaceae family i.e. Azadirachta indica and Melia azedarach. The contents of total phenolic
compounds in crude ethanolic extracts obtained from these two Neem plants are presented in Table-1. The
results were reported as catechol equivalents (µg/ml). The highest concentration of total phenol was 280µg/ml
present in the ethanolic extract of Bark in Melia plant. The methanolic and aqueous fractions of Bark in case of
Melia showed 220µg/ml and 160µg/ml of phenol contents respectively. Similarly the Azadirachta the ethanolic
extract from Bark exhibited highest phenol contents of i.e.210µg/ml and followed by the methanolic and
aqueous fraction 158µg/ml respectively. In our present investigation we found that in both the Neem plants the
Bark exhibited the higher amount of phenol content as comparison to Root. The total phenol content in case of
Melia Root obtained from ethanol fraction showed highest amount of phenol content i.e. 70µg/ml followed by
methanolic fraction 50µg/ml and aqueous fraction i.e. 20µg/ml.Similaly in case of Azedarachta root obtained
from ethanol fraction showed highest amount of phenol content i.e. 48µg/ml. followed by methanol fraction
30µg/ml and aqueous i.e. 18µg/ml. Much higher antioxidant activity of the alcoholic preparation have given
evident assumption is more useful than the aqueous one in medical approach (Pietta, et al., 1998). Moreover,
such a preparation consists more oxindole alkaloids and a larger spectrum of biologically active constituents.
High percent of yield and high value of phenol content in ethanolic extracts show that phenolic constituents
must be responsible for such properties. It is in agreement with the data of (Goncalves, et al., 2005). It was
reported from our previous paper (Nahak and Sahu, 2010), that the ethanolic fraction of Mahaneem leaves
exhibited highest i.e. 68±0.03% and it showed a low IC50 value of 0.008µg/ml. There was an increasing
concentration of phenol content observed in the root bark of A. indica when compared to the leaf. Also increase
in the stem bark of A. indica was observed when compared to the leaf extract (Nahak and Sahu, 2010; Olabinri,
et al., 2009). Similar case was also observed in case of Melia azedarach (Olabinri, et al., 2009.) The order of
total phenol content for these two neem trees were as follow: stem bark > root bark > leaf. In our present study
we found that there is a positive correlation between total phenolic content and antioxidant activity in both the
neem plants. Some studies have demonstrated a correlation between phenolic content and antioxidant activity
(Yang, et al., 2002).The correlation between total phenolic content and antioxidant capacity in our plant samples
is possible owing to the presence of following factors: the antioxidant activity observed in plant extracts may be
due to the presence of phenolic compounds or polyphenols or flavonoids or tannins.

IC50 Value
IC50 value is defined as the concentration of substrate that causes 50% loss of the DPPH activity and was
calculated by linear regression mentioned of plots of the percentage of antiradical activity against the
concentration of the tested compounds. Results showed in Fig.-3 and Fig.-4 show the reports of IC50 values in
Neem and Mahneem. It shows that there is no IC50 value in water and methanol extraction of Azadirachta

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 Gayatri Nahak and R. K. Sahu: Continental J. Pharmaceutical Sciences 4: 28 - 34, 2010

indica. Only ethanolic extract of Azadirachta root showed an IC50 value where as in both ethanolic and
methanolic extract of Bark fraction showed no IC50 value. In comparison to Azadirachta, all extracts of Melia
showed lower IC50 value in case of Bark extracts, however ethanolic extract of Melia being the lowest (Fig-2).
But in case of Root extracts only the ethanolic extract showed the lowest IC50 value. The ethanolic extract of
Mahaneem exhibited significant activity with low IC50 value in comparison to Azadirachta. A linear relationship
between the reciprocal of IC50 value and the total polyphenol content of Azadirachta and Melia was observed in
this study, indicating that increasing the polyphenol content strengths the antioxidant activity. This finding is
similar to that reported by (Katsube, et al., 2004).

Conclusion
In conclusion, the Bark and Root of Melia azedarach (Mahaneem) proved to be of higher antioxidant potential
in comparison to Neem which is a very important medicinal plant like Neem belonging to family Meliaceae.
Among the plant parts studied Bark proved to be more useful in terms of antioxidant activity which can be
exploited for combating diseases related to oxidative stress.

ACKNOWLEDGMENT
The authors are thankful to University Grants Commission New Delhi, for Financial Assistance in form of
major research project to one of the author (R.K.S) we are also thankful to Head of the Department of Botany
and Principal B.J.B. (A) College for providing necessary facilities for carrying out the experimental work.
Finally we are thankful to Debyani Samantray for helping in computer work without which preparation of the
manuscript would not have been possible.

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Table-1: Crude extracts and phenol contents of Neem and Mahaneem

WATER METHANOL ETHANOL

Plant BARK ROOT BARK ROOT BARK ROOT
name
CE(g) PC CE(g) PC CE(g) PC CE(g) PC CE(g) PC CE(g) PC
(µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml)
NEEM 3.90 120 3.02 18 4.45 158 3.45 30 4.35 210 3.56 48

MAHA 4.34 160 3.29 20 5.20 220 3.56 50 5.85 280 3.29 70
NEEM

Table-2: Antioxidant activities of Azadirachta indica in different solvents

Conc. Antioxidant activity (%)

of extracts Azedarachta indica
(µg/ml)
WATER METHANOL ETHANOL
Bark Root Bark Root Bark Root

40 25.83±0.02 18.64±0.02 40.57±0.01 24.74±0.05 53.33±0.06 29.66±0.06

80 26.66±0.05 24.57±0.11 42.50±0.06 28.98±0.02 55.83±0.05 33.59±0.04
120 29.16±0.06 27.11±0.10 47.50±0.09 31.18±0.03 58.33±0.04 42.37±0.07
160 32.50±0.05 28.81±0.06 50.83±0.07 40.84±0.06 65.33±0.01 47.45±0.02
200 37.50±0.03 30.50±0.04 55.00±0.05 42.71±0.04 66.94±0.02 50.84±0.03

CE- Crude extract, PC-Phenol content

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 Gayatri Nahak and R. K. Sahu: Continental J. Pharmaceutical Sciences 4: 28 - 34, 2010

Table-3: Antioxidant activities of Melia azedarach in different solvents

Conc. Antioxidant activity (%) Melia azedarach

of extracts
WATER METHANOL ETHANOL
(µg/ml)
Bark Root Bark Root Bark Root
40 41.69±0.04 24.57±0.05 60.16±0.05 32.71±0.05 70.16±0.03 46.61±0.03
80 48.64±0.05 25.42±0.12 61.86±0.02 39.83±0.04 72.71±0.11 47.45±0.05
120 49.49±0.03 27.96±0.10 64.40±0.03 44.57±0.03 74.91±0.07 53.38±0.04
160 53.05±0.02 31.35±0.05 67.79±0.05 45.76±0.02 81.69±0.05 57.62±0.06
200 53.89±0.07 36.44±0.04 72.88±0.04 47.96±0.02 83.38±0.01 63.55±0.04

80 90
70 80
60 70
tyi
ivt 50 Ascorbic tyi 60
vi 50
c tc
a 40
t
n acid a 40
t
a
id
x
30
20
n
ad 30
i
x 20
Ascorbi
o
ti o
n
A
10
it
n 10
A
c acid
% 0 %0
0 0 0 0 0 40 80 120 160 200
4 8 12 16 02
Concentration of crude extracts (µg/ml) Concentr ation o f crud extr acts ( µg/ml)

Figure-1: Antioxidant activity of

Figure-2: Antioxidant activity of Neem and Mahaneem (Root)
Neem and Mahaneem (Bark)

Figure-3: IC50 Values of Neem and Figure-4: IC50 Values of Neem and
Mahaneem (Root) Mahaneem (Bark)

Received for Publication: 07/04/2010

Accepted for Publication: 19/04/2010

Corresponding Author:
R. K. Sahu
Department of Botany, B.J.B. (A) College, Bhubaneswar, Orissa
Email: sahurajani@yahoo.co.in

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