Academia Journal of Medicinal Plants 2(2): 026-031, February 2014

DOI: http://dx.doi.org/10.15413/ajmp.2013.0138
ISSN: 2315-7720
2014 Academia Publishing

Research Paper

Phytochemical screening and antioxidant activity of extracts of the leaf and bark of
Albizzia lebbeck (Benth)

Accepted 20th January, 2014

Abstract

Albizzia lebbeck (L) Benth is an important medicinal tree found in India. The
present study was aimed to evaluate the phytochemical constituents and
antioxidant activity of leaf and bark extracts of Albizzia lebbeck. Antioxidant
activity was carried out using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and
Nitric oxide. The phytochemical screening of leaves and barks of A. lebbeck
revealed the presence of phenols, steroids, tannins, saponins and alkaloids in the
hydroalcohol extract. The percentage yield of hydroalcohol extract of the leaf of A.
lebbeck was higher (13.55) than that of the bark. Also quantitative analysis
Padamanabhan Vasanthi1, Manimekalai showed that percentage of phenols was higher (17.47) in the leaf extract. The
Ganapathy2 , Vasthi kennedy results of DPPH scavenging activity for leaf hydroalcohol showed (14.87%) and
Evanjelene3, Nirmala Ayyavuv4 and
Jagajothi Angamuthu5.
bark hydroalcohol showed (12.95%). When compared to standard the leaf
hydroalcohol showed better activity (14.84%). The percentage antioxidant activity
1.Govt Arts college (Autonomous), Salem- is high in leaf extract than bark extract and in Nitric oxide assay the percentage
7. inhibition was also higher than the bark extract when compared to the Ascorbic
2.K.K.C, Paramathy velur, Namakkal Dt.
3.Alpha Omega Hi- Tech Bio Research
acid (standard). It indicates that hydroalcohol leaves extract the plant has the
Centre, Salem- 8. potency of scavenging free radicals and it may provide leads in the ongoing search
4.Sri Sarada College for Women for natural antioxidants from various medicinal plants to be used in treating
(Autonomous), Salem- 16. diseases related to free radical reactions.
5.Govt Arts college (Autonomous), Salem-

Corresponding author email: Key words: Phytochemicals, antioxidant, free radicals, Albizzia lebbeck, DPPH,
[email protected] nitric oxide assay.

INTRODUCTION

Plants have been associated with the human health from flavonoids, which are potential source of drugs. Nearly one
time immemorial and they are the important sources of third of the pharmaceuticals are plant origin. As all the
medicines since the down of human civilization. In spite of plants are able to synthesize a multitude of organic
tremendous development in the field of allopathic molecules/phytochemicals, they are referred to as
medicines during 20th century, plants still remain one of the "secondary metabolites (Harborne, 1982).
major sources of drugs in modern as well as in traditional Plants derived compounds are playing an important role
systems of medicine. Herbal medicines are universally in the development of several clinically useful medicines
accepted because of the fact that Medicinal plants continue (Madhuri and Pandey, 2009). Scientist first started
to play important role in healthcare system of a large extracting and isolating chemicals from plants in the 18th
number of world's population. In fact there are several century and since the time we have grown a custom of
medicinal plants all over the world which are being used looking at herbs and their effects in terms of the active
traditionally in the prevention and treatment of several constituents they contain. It is generally accepted that
diseases. The medicinal plants are rich source of secondary plants based medicines are better than synthetic drugs as
metabolites like alkaloids, glycosides, steroids and these are much safer for human beings. The ascendance of
Academia Journal of Medicinal Plants; Vasanthi et al. 027

much touted synthetic drugs over phytomedicine thrived antioxidant activity.

due to their fast action.
Phytochemicals are bioactive compounds found in plants
that work with nutrients and dietary fiber to protect MATERIALS AND METHODS
against diseases. They are non-nutritive compounds. These
phytochemicals are the secondary metabolities present in Collection of plant material
smaller quantities in higher plants and they include the
alkaloids, Steroids, flavonoids, terpenoids, tannins and The leaves and barks of A. lebbeck were collected and the
many others (Peteros, 2010). Many phytochemicals have specimen deposited in the Alpha Omega Hi- Tech Bio
antioxidant activity and reduce the risk many diseases. It is research centre. The fresh leaves and barks of A. lebbeck
crucial to know the type of phytochemical constituent, thus were authenticated by ABS Botanical conservation,
knowing the type of biological activity which might be Research & Training Centre, Salem (Dt).
exhibited by the plant (Agbafor and Nwachukwu, 2011).
The importance of medicinal plants and the contribution of
phytomedicine to the well-being of a significant member of Extraction of the plant material
the world's population have attracted interest from diverse
disciplines. The fresh plant materials were washed with running tap
Antioxidants are substances that may protect our body water and shade dried. The leaves and barks were crushed
cells against the effects of free radicals. Free radicals are to coarse powder by a grinder. The coarse powder (25g)
molecules produced when our body breaks down food. was then subjected to successive extraction in 250 ml of
They can also be produced by environmental exposures like each solvent hexane, ethyl acetate and hydroalcohol by
tobacco smoke and radiation. Free radicals can damage using Soxhlet apparatus. The collected extracts were stored
cells, and may play a role in heart disease, cancer and other and then taken up for further investigations. Dimethyl
diseases. Oxidative damage can lead to a break down or sufloxide (DMSO) is used as solvent for dissolving the
even hardening of lipids, which is the major composition of extracts.
all cell walls. This breakdown or hardening is due to lipid
peroxidation and leads to death of cell or loss of normal cell
function. In addition, other biological molecules including Quantitative phytochemical analysis
RNA, DNA and protein enzymes are also susceptible to
oxidative damage. Environmental agents initiate free Estimation of flavonoids
radical generation, which leads to different complications in
body. The toxicity of lead, pesticides, cadmium, ionizing The total flavonoid content in the sample was estimated by
radiation, alcohol, cigarette smoke, UV light and pollution the method of Chang et al. (2002). The extract prepared for
may all be due to their free radical initiating capability the estimation of total phenolics was used as sample for
(Langseth, 1996; Davies 1991; Halliwell, 1993). this assay. A volume of 0.25 ml of the sample was diluted to
Antioxidants cause protective effect by neutralizing free 1.25 ml with distilled water. A volume of 75 l of 5%
radicals which are toxic by products of natural cell sodium nitrite was added and after six minutes 0.15 ml of
metabolism. The human body naturally produces aluminium chloride solution was added. A volume of 0.5 ml
antioxidants but the process is not 100% effective in case of of 0.1M NaOH was added after 5 min and made up to 2.5 ml
over whelming production of free radicals and that with distilled water. The solution was mixed well and the
effectiveness also declines with age (Sies, 1999). Increasing absorbance was read at 510 nm in comparison with
the anti-oxidant intake can prevent diseases and lower standard quercetin at 5-25 g concentration. The results
health problems. Research is increasingly showing that are expressed as mg of flavonoids as quercetin equivalent/
antioxidant rich foods and herbs have health benefits. gm of dried sample.
Medicinal herbs are the richest sources of antioxidants
compounds (Sies et al., 1992).
The need of the hour is to study the medicinal plant for Estimation of alkaloids
promising biological antioxidant activity. Considering the
aforesaid, the medicinal plant Albizzia lebbeck (L) Benth Alkaloid was determined using Harborne (1973) method.
belongs to the family Mimosaceae. The plant in common is Five grams of the sample was weighed into a 250 ml beaker
called as Siris tree and in tamil it is called as vaagei. It is a and 200 ml of 10% acetic acid in ethanol was added and
deciduous tree with compound leaves, flat oblong fruits, covered and allowed to stand for 4 h. This was filtered and
round cream colored seeds and it grows wild. The Plant is the extract was concentrated on a water bath to one
found throughout India, Bangladesh, tropical and quarter of the original volume. Concentrated ammonium
subtropical Asia and Africa (Kirtikar and Basu, 1980). The hydroxide was added drop wise to the extract until the
present investigation is on Quantitative phytochemicals and precipitation was complete. The whole solution was
Academia Journal of Medicinal Plants; Vasanthi et al. 028

allowed to settle and the precipitated was collected and occasional shaking and diluted to the mark with distilled
washed with dilute ammonium hydroxide and then filtered. water. The absorbance was measured at 780 nm against the
The residue is the alkaloid, which was dried and weighed. reagent blank.

Determination of saponin Antioxidant activity

Twenty grams of plant sample was dispersed in 200 ml of DPPH radical scavenging activity
20% ethanol. The suspension was heated over a hot water
bath for 4 h with continuous stirring at about 55C. The The DPPH assay method is based on the reduction of DPPH,
mixture was filtered and the residue re-extracted with a stable free radical. The free radical DPPH with an odd
another 200 ml of 20% ethanol. The combined extracts electron gives a maximum absorption at 517 nm (purple
were reduced to 40 ml over water bath at about 90C. The color). When Antioxidants react with DPPH, which is a
concentrate was transferred into a 250 ml separating stable free radical, it becomes paired off in the presence of a
funnel and 20 ml of diethyl ether was added and shaken hydrogen donor (e.g., a free radical-scavenging antioxidant)
vigorously. The aqueous layer was recovered while the and is reduced to the DPPH and as a consequence, the
ether layer was discarded. The purification process was absorbances decreased from the DPPH.
repeated. A volume of 60 ml of normal butanol extracts
were washed twice with 10 ml of 5% aqueous sodium
chloride. The remaining solution was heated in a water The scavenging reaction between DPPH and
bath. After evaporation the sample were dried in the oven antioxidant
into a constant weight. The saponin content was calculated
in percentage (Nahapetian and Bassiri, 1975). A mass of 4.3 mg of DPPH (1, 1-Diphenyl 2-picrylhydrazyl)
was dissolved in 3.3 ml methanol; it was protected from
light by covering the test tubes with aluminum foil. A
Estimation of saponins volume of 150 l DPPH solutions was added to 3 ml
methanol and absorbance was taken immediately at 517
Plant extract was dissolved in 80% methanol, 2 ml of nm for control reading. A volume of 50 l of various
Vanillin in ethanol was added, mixed well and the 2 ml of concentrations of coumarin compounds as well as standard
72% sulphuric acid solution was added, mixed well and compounds (Ascorbic acid) were taken and the volume was
heated on a water bath at 60C for 10 min, absorbance was made uniformly to 150 l using methanol. Each of the
measured at 544 nm against reagent blank. Diosgeninis was samples was then further diluted with methanol up to 3 ml
used as a standard material and the result was compared and to each 150 l DPPH was added. Absorbance was taken
with Diosgenin equivalents. after 15 min. at 517nm using methanol as blank on UV-
visible spectrometer Shimadzu, UV-1601, Japan. The IC50
values for each compounds as well as standard preparation
Determination of total phenols were calculated. The DPPH free radical scavenging activity
was calculated using the following formula:
The fat free sample was boiled with 50 ml of ether for the
extraction of the phenolic component for 15 min. A volume %scavenging = [Absorbance of control - Absorbance of test
of 5 ml of the extract was pipetted into a 50 ml flask, then sample/Absorbance of control] 100
10ml of distilled water was added. A volume of 2 ml of
ammonium hydroxide solution and 5 ml of concentrated The effective concentration of sample required to scavenge
amyl alcohol were also added. The samples were made up DPPH radical by 50% (IC50
to mark and left to react for 30 min for color development. value) was obtained by linear regression analysis of dose-
This was measured at 505 nm. response curve plotting between %inhibition and
concentrations (Iranshahi et al., 2009).

Estimation of steroids
Nitric oxide free radical scavenging activity
A volume of 1ml of the plant extract of steroid solution was
transferred into 10 ml volumetric flasks. Sulphuric acid (4 The procedure is based on the principle that, sodium nitro-
N, 2 ml) and iron (III) chloride (0.5% w/v, 2 ml), were prusside in hydroalcohol solution at physiological pH
added, followed by potassium hexacyanoferrate (III) spontaneously generates nitric oxide which interacts with
solution (0.5% w/v, 0.5 ml). The mixture was heated in a oxygen to produce nitrite ions that can be estimated using
water-bath maintained at 702C for 30 min with Griess reagent. Scavengers of nitric oxide compete with
Academia Journal of Medicinal Plants; Vasanthi et al. 029

Table 1. Phytochemical quantitative activity of Albizzia lebbeck (Benth).

Name of the Extracts of Albizzia lebbeck Benth

phytochemical constituents (%) Leaf Hydro alcohol Bark hydro alcohol
Alkaloids 0.10 0.16
Flavonoids 10.45 6.73
Steroids 32.18 28.35
Saponin 26.92 21.53
Phenol 40.81 33.34
Tanin 30.69 27.16

oxygen, leading to reduced production of nitrite ions. Large The results of the free radical scavenging activity of the
amounts of NO may lead to tissue damage. A volume of 50 1,1-diphenyl - 2 picryl - hydrazyl (DPPH) assay showed
l of each of the concentrations of coumarin compounds percentage antioxidant activity (%AA) is 14.87% in leaf
previously dissolved in DMSO, as well as Ascorbic acid extract and 12.95% in bark extract and in Nitric oxide
(standard compound) were taken in separate tubes and the assay the percentage inhibition was 13.62% and 11.84% in
volume was uniformly made up to 150 l with methanol. To leaf and bark extract. The Nitric oxide assay of the leaf and
each tube, 2.0 ml of sodium nitroprusside (10 mM) in Bark extract was found to be higher than the Ascorbic acid
phosphate buffer saline was added. The solutions were (standard) value of 11.05% (Table 2).
incubated at room temperature for 150 min. The similar
procedure was repeated with methanol as blank which
served as control. After the incubation, 5 ml of griess DISCUSSION
reagent was added to each tube including control. The
absorbance of chromophore formed was measured at 546 The Medicinal plants are rich in secondary metabolites
nm on UV-visible spectrometer Shimadzu, UV-1601, Japan. which include alkaloids, flavonoids, steroids and related
Ascorbic acid was used as positive control. active metabolites which are of great medicinal value and
The IC50 values for each test compounds as well as have been extensively used in the drug and pharmaceutical
standard preparation were calculated (Bondet et al., 1997; industry. Recently number of studies had been reported on
Hui et al., 2005; Bum-Chun et al., 2007; Jain and Agrawal, the phytochemistry of medicinal plants, particularly on the
2008). vegetative parts like leaves and stems etc (Balakumar et al.,
%scavenging/Reduction = [Absorbance of control - 2011; Paulraj et al., 2011; Premkumar et al., 2011; Anpein
Absorbance of test sample/Absorbance of control] 100. raja et al., 2011; Rajan et al., 2011; kala et al., 2011).
Phytochemicals especially plant phenolics constitute a
major group of compounds that act as primary antioxidants
RESULTS (Hatano et al., 1989). The phenolic compounds have high
redox potential which allow them act as reducing agents,
The results of the phytochemical screening revealed the hydrogen donors and singlet oxygen quenchers (Kahkonen
presence of phenols, steroids, tannins, saponins and et al., 1999). The antioxidant effects of the extract may be
flavonoids in the hydroalcohol leave and bark extracts of A. due to its phenolic content. The delocalization of electrons
lebbeck. over the phenolics and stabilization by the resonance effect
The percentage yields of the constituents of leaf of the aromatic nucleus prevents the continuation of the
hydroalcohol are alkaloids with 0.10%, flavonoids with free radical chain reaction (Tsao and Akhtar, 2005). The
10.45%, steroids with 32.18%, saponin with 26.92%, DPPH assay has been largely used as a quick, reliable and
phenols with 40.81%, tannin with 30.69%. reproducible parameter to search for the in vitro general
The percentage yield of the constituents of bark antioxidant activity of pure compounds as well as plant
hydroalcohol are alkaloids with 0.16%, flavonoids with extracts (Koleva et al., 2002; Goncalves et al., 2005). The
6.73%, steroids with 28.35%, saponin with 21.53%, decrease in absorbance by the DPPH radical with increase
phenols with 33.34%, tannin with 27.16%. Results of in concentration of the extract which manifested in the
quantitative analysis showed that percentage with rapid discolouration of the purple DPPH, suggest that the
reference to phenol in leaf (40.81%) was more than bark methanol extract of A. lebbeck has antioxidant activity due
(33.34%). Also, percentage yield of A. lebbeck bark extract to its proton donating ability. The extract was found to
was 17.45% and ethyl acetate extract was 13.65%. And the highly scavenge free radicals when compared to standard
percentage yield of A. lebbeck leaf extract was 31.1% and antioxidants. The reducing capacity of compounds could
ethyl acetate extract was 19.25% (Table 1 and Figure 1). serve as indicator of potential antioxidant properties (Meir
Academia Journal of Medicinal Plants; Vasanthi et al. 030

Figure 1. Phytochemical quantitative activity of Albizzia lebbeck (Benth).

Table 2. Antioxidant activity of Albizzia lebbeck (Benth).

Percentage of inhibition (%)

Samples
DPPH Nitric oxide
Leaf Hydro alcohol 14.87 13.62
Bark hydro alcohol 12.95 11.84
Standard
Ascorbic acid 14.84 11.05

et al., 1995) and increasing absorbance could indicate an ACKNOWLEDGEMENT

increase in reducing power. Nitric oxide is a free radicals
product and involved in the regulation of various The authors express their thanks to the Managing director
physiological processes, however excess production of of Alpha Omega Hi- Tech Bio Research Centre, Salem for
nitric oxide is associated with several diseases (Lalenti et providing the facility to work in the lab.
al., 1994).
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