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International Journal of Materials and Biomaterials Applications

Universal Research Publications. All rights reserved

ISSN 22499679
Original Article
Adenium obesum flower Extractaction and Application to synthesis of Silver
Nanoparticles and Antioxidant
R.M.Kershi1*, Naji Ebrahim2
1. Physics Department, Faculty of science, Ibb University, Ibb, Yemen.
2.
Department of Plant Production, Faculty of Agriculture and Veterinary Science, Ibb University, Ibb,Yemen.
*Email:-rkershi1@gmail.com
Received 19 February 2014; accepted 26 February 2014
Abstract
In this study environmentally friendly biosynthesis of sliver nanoparticles (AgNPs) using leaves extract of Adenium
obesum have been achieved. AgNPs can be prepared with lower amounts of leaf extract in short time. Stable AgNPs were
formed by treating solution using the plant leaf extracts as reducing agents. Biosynthesized nanoparticles were
characterized by UVV spectrophotometer. Also, this study aims to investigate and compare Adenium obesum L. extracts
obtained in Ultrasonic condition with different water/methanol and water/ethanol extraction mixture acidified with 0.1%
HCl. The extracts were analyzed for monomeric anthocyanins contents and antioxidant activities. The highest anthocy-
anins content (18340.9 mg/L) and the best free radical scavenging activity were obtained for the Adenium obesum extract
with 100% methanol. Also, there is a good correlations between antioxidant activity (R2 = 0.9368) for water/ethanol series
extracts.
2013 Universal Research Publications. All rights reserved
Keywords: Adenium obesum , anthocyanins, antioxidant activity, sliver nanoparticles.
Introduction [10] and tamarind [11]. Anthocyanins are representative of
Nanotechnology has a wide variety of applications in plant pigments widely distribut-ed in colored fruits and
various fields like optics, electronics, catalysis, bio- flowers. Because anthocyanins are widely consumed,
medicine, magnetics, mechanics, energy science, etc. finding out additional biological activities related to these
Nanobiotechnology is a multidisciplinary field involving compounds would be of great interest [12]. Anthocyanins
research and development of technology in different fields are normally obtained by extraction from plants and the
of science like biotechnology, nanotechnology, physics, extraction methods currently employed are with the use
chemistry, and materials science [1-2]. Metal nanoparticles metha-nol, ethanol, acetone, water or mixtures as solvents.
are usually in sizes smaller than 100 nm and possess unique In fact, the color stability of anthocyanins depends on a
physical and chemical properties. Especially, silver combination of factors, such as the structure and
nanoparticles have been attributed to their small size, more concentration of the anthocyanin, pH, temperature and
surface bulk atoms and high surface area permits them to presence of complex agents such as phenols and metals
highly interact with microbial membranes. Besides, silver [13]. The most common solvents used for anthocyanins
is used in the medical field and water purification. Many extraction are aqueous mixtures of ethanol, methanol or
physical or chemical methods that are currently available acetone [14]. The adverse effects of oxidative stress on
for silver nanoparticle production but many of them have human health have become a serious issue [15]. Under
several disadvantages such as high cost, hazard. Recently, stress, our bodies produce more reactive oxygen spe-cies
biosynthesis of silver nanoparticles has received a special (ROS) such as; superoxide anion radicals, hydroxyl radicals
attention due to environmentally friendly green synthesis and hydrogen peroxide than enzymatic antioxidants such
and easy to scale-up. In recent years, several plant extracts as; superox-ide dismutase [16], glutathione peroxidase, and
have been successfully used and reported for fast and catalase and non-enzymatic antioxidants such as; ascorbic
efficient extracellular synthesis of metal nanoparticles such acids, glutathi-one, carotenoids, and flavonoids. This
as silver, copper and gold nanoparticles by using broth imbalance leads to damage of biological structures such as
extracts of neem [3], Aloe vera [4], tamarind [5], Avena proteins, lipids and DNA and induce a variety of human
sativa [6], wheat [7], alfalfa [8], geranium [9], lemongrass diseases [17- 22]. Antioxidants from fruits and vegetables,

International Journal of Materials and Biomaterials Applications 2014; 4(1): 14-18

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especially with an intense colouration, are considered an
important protection factor against oxidative stress and its
deleterious consequences to human health [23, 24]. The
antioxidative capacity, which is defined as the capacity to
inhibit or delay the oxidation of other molecules,
anthocyanins and their aglycones (anthocyanidins) and
their free radical scavenging activity have been revealed
[25- 27]. The antioxidant activi-ty of berries is directly
proportional to the anthocyanins content [28]. In response
to the increased popularity and greater demand for
medicinal plants, a number of conservation groups are
recommending that wild medicinal plants be brought into
cultivation. Various herbs and spices have been reported to
exhibit antioxidant activity, including Ocimum sanctum,
Piper cubeba, Allium sativum, Terminalia bellerica,
Camellia sinensis and Zingiber officinale [29]. Various
analytical meth-ods have been used to evaluate the
antioxidant properties of phenolic compounds: the 1, 1-
diphenyl- 2-picrylhydrazyl (DPPH) assay proves the
capacity of the antioxidants to quench the PPH radical,
whereas the ORAC method is based on the loss of fluores-
cence of the -phycoerythrin protein or of fluorescein upon
oxida-tion [30, 31]. Reactive oxygen spe-cies (ROS),
including super oxide anion (O2-), hydroxyl radical (OH)
and hydrogen peroxide (H2O2), exist in living organisms
[15]. Adenium obesum belongs to the family Apocy-
anaceae. It is a Succulent shrub or small tree, up to 4(6) m
tall, sometimes with a fleshy taproot; stem swollen at base
up to 1(2) m in diameter; bark pale greyish-green, grey or
brown, smooth, with sticky, clear or white latex; branchlets
glabrescent, pubescent at apex. Leaves arranged spirally,
clustered at the end of branchlets, simple; stipules minute
or absent; petiole up to 4 mm long; blade linear to obviate,
312 (17) cm 0.26 cm, base cuneate, apex acute to A700)pH 4.5, MW is the molecular weight (449.2) and is
rounded or emarginate, entire, slightly glaucous, dull green
or pale green, leathery, pinnately veined with distinct or
indistinct lateral veins [32]. The plant is important in
traditional medicine. In the Sahel a decoction from the
roots, alone or in com-bination with other plants, is used to
treat venereal diseases; a root or bark extract is used as a
bath or lotion to treat skin diseases and to kill lice, while
latex is applied to decaying teeth and septic wounds. In
Somalia a root decoction as nose drops is prescribed for
rhinitis. In northern Kenya latex is rubbed on the head
against lice and powdered stems are applied to kill skin
parasites of camels and cattle. The bark is chewed as an
abortifacient [34]. The red colour of the Adenium obesum
is a consequence of its anthocyanin contents that was not
well investigated in extracts. Therefore in the present study,
blood flower plant was collected from Ibb region in Yemen
to study its anthocyanin contents and antioxidant properties
and its using in synthsis of sliver nanoparticles .
MATERIAL AND METHODS
Fresh petals of the (Adenium obesum) were collected
randomly from the Ibb region during March 2013
Extraction of Anthocyanins
The anthocyanins were extracted according to the
methodology of Lees and Francis (1972). Solvents such as
methanol and ethanol were used at concentrations of 100,
75, 50, 25 and 0.0% in water, acidified with 0.1%
International Journal of Materials and Biomaterials Applications 2014; 4(1): 14-18
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hydrochloric acid (HCl) (Synth 37%) Twenty five g of
fresh petals of Adenium obesum were treated with 100 ml
of different water/alcohol solutions acidified with 0.1%
HCl (Merck, 37%) as extracting material (solid to solvent
ratio 1:4 w/v). Each solution was transferred to a 500 ml
beaker, covered with parafilm and stored overnight at 4 C.
The mixture was then filtered under vacuum using n 1
Whatman paper and a Buchner funnel. Filtrate solution was
taken and then 200 ml of solvent was added to com-plete
the mixture. This mixture was later filtered and the residue
washed with solvent until obtained a total of 500 ml
solution. A 5 ml aliquot was removed from each extract,
placed in a 50 ml volumet-ric flask, the volume completed
with two buffer solutions: potassium chloride buffer 0.025
M (pH 1.0) and sodium acetate buffer 0.4 M (pH 4.5) and
then the absorbance was measured simultaneously at 516
nm and 700 nm after 15 minutes of incubation at room
temper-ature. Absorbance readings were made at room
temperature against distilled water as blank was used for
measurements.
Quantitative Determination of The Anthocyanins
Determination of total monomeric anthocyanins content
was quan-tified using a pH differential method described
by Giusti and Wrolstad (2001). The absorbance was
measured simultaneously at 516 nm and 700 nm after 15
minutes of incubation at room temper-ature. Absorbance
readings were made at room temperature against distilled
water as blank. A Jasco V 530 UV-Vis spectropho-tometer
was used for measurements. The monomeric anthocyanin
pigment concentration was calculated according to the
following equation:
Monomeric anthocyanin pigment (mg/L) = (A x MW x DF
x 1000)/ (x1) Where A=(A510A700)pH 1.0(A516
the molar absorptivity, (26,900) and DF is the dilution
factor.
Determination of Antioxidant Activity By The DPPH
Method
The 1, 1-diphenyl-2-picrylhydrazine (DPPH) radical
scavenging as-say was first described by Blois in 1958 and
was later modified slightly by numerous researchers. It is
one of the most extensively used antioxidant assays for
plant samples. DPPH is a stable free radical that reacts with
compounds that can donate a hydrogen atom. This method
is based on the scavenging of DPPH through the addition of
a radical species or an antioxidant that decolourizes the
DPPH solution. The antioxidant activity is then measured
by the decrease in absorption at 515 nm. In this method, a
0.1mM solu-tion of DPPH in methanol is prepared (4 mg
DPPH /100 ml meth-anol), and then stored at -20 and 2 ml
of this solution are added to 0.5 ml of the sample solution
in methanol. The mixture was left to stand at room
temperature for 30 min in the dark before Ab-sorbance
measurement at 517 nm to assess the stability of the
coloured reactive action,, A large decrease in the
absorbance of the reaction mixture indicates significant free
radical scavenging activity of the compound. The
antioxidant activity of the extracts was estimated by the
ability to scavenging the DPPH radical. The DPPH
concentration in the reaction medium was calculated from
the calibration curve (Fig.-1) with the following equation
deter-mined by linear regression (R2 = 0.988).
A515=11.368x -0.0437.
Preparation of leaf extract
Plant leaves of Adenium obesum were collected, washed
several times with deionized water to remove the dust. The
plant leaf extract solution was prepared by taking 10 g of
washed and finely cut leaves in 100 ml and then heating the
mixture at 60 oC for 15 min with moving by glass rod.
Preparation of sliver nanoparticles
Silver nitrate was purchased from SigmaAldrich and 1
mM aqueous AgNO3 solution was prepared and added to
Adenium obesum extract with ratio 2:10 respectively.
Characterization of silver nanoparticles (UV-Vis spectral
analysis)
The UV-visible absorption spectrum was carried out on
UV-1601 spectrometer (Germany).The reduction of Ag+ to
Ag0 was monitored by measuring the UV-Vis spectrum of
the mixture of silver nitrate solution and leaf extract) within
the range of 320-550 nm in the UV-Vis spectrophotometer.
RESULTS AND DISCUSSIONS
The paper describes the extraction method of anthocyanins
and antioxidant activity of Adenium obesum selected from
Yemen. The changes in total anthocyanins content
depending on the water/alcohol ratio are presented in
Fig. 1. The monomeric antho-cyanins content increases
with increasing the percentage of meth-anol in the
extraction system. This tendency also observed for
water/ethanol extraction, where high values were obtained
for 100 % ethanol extracting system. The amount of
monomeric an-thocyanins in Adenium obesum extracts
ranged from 18340.9 mg/ L to 946 mg/L for
water/methanol extraction, and from 4870.5 mg/L to 1447
mg/L for water/ethanol extraction. Fig.1 is presented the
percentage of radical scavenging activity after 2 hours of
reaction between the extracts and DPPH radical for the two
studied cases. The higher this value, the higher is anti-
radical efficiency activity of Adenium obesum extracts
increases with increasing the percentage of methanol and
ethanol in the extraction system. Comparing antioxidant
activities of the bilber-ries extracts in the two cases, it is
observed similar antioxidant activities in the ethanol and
methanol series.
between antioxidant activity and anthocyanins content was
obtained, Eq. (1) for ethanol series and Eq. (2) for methanol
series. There is good correlation between antioxidant
activity and anthocyanins content for the Adenium obesum
extracts from methanol series.
Fig. 2. Correlation between anthocyanin contents and
radical scavenging activity
y = 0.005x + 63.203 (R = 0.9158) (1)
y = 0.0009x + 72.216 (R = 0.7101) (2)
Also, notice a good correlation between free radical
scavenging activity and extraction systems content for
methanol series extracts. The values of the determination
coefficients are acceptable, indicat-ing a high correlation by
linear regressions between antioxidant activities and
extraction systems Eq (3)
y = 0.1896x + 69.003 R = 0.9368 (3)
The free radical scavenging by the studied extracts show
similar pattern curves of free radical scavenging activity
versus time. The reaction occurs rapidly in the first minutes
and then slowed. The lower step can be due to the
antioxidant properties of the slow reacting components
originally present in the sample and/or due to the reaction
products formed during rapid phase [34]. The percentage of
free radical scavenging ac-tivity against reaction time is
exemplified in Fig. 3 and 4 for the extracts obtained in
water/methanol system and water/ethanol system
respectively.

Fig. 3. Correlation between alcohol concentrations and

radical scavenging activity

Fig.1. Comparison of radical scavenging activity of

extracts obtained in water/methanol and water/ethanol
systems
The correlations between antioxidant activity and
monomeric anthocyanins content for the two extraction
systems are presented in Fig. 2. The following equations Fig. 4. Free radical scavenging activities for Adenium
determined by linear regres-sion regarding the relationship obesum extracts obtained in water/ethanol system
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Fig. 5.Concentration response curve for DPPHat 515nm
Fig. 5 shows the concentration response curve for DPPH at
515 nm. From the figure one can be noticed that there is a
good correlations between absorbance and DPPH
concentration ( R2 = 0.988).
Results of leaf extract indicated that the reduction of the
Ag+ ion to silver nanoparticles during exposure to the plant
leaf extracts could be detected by the color change. Where
the functional groups played a reducing and controlling role
during the formation of silver nanoparticles in the
solutions, and that the structure of the leaf extract changed
after reaction with silver ions. Formation of the AgNPs
were confirmed by using UVVis spectrophotometer. The
absorption spectrum was measured spectrophotometer in
300550 nm range. Naturally biosynthesized AgNPs of
diameters smaller than 100 nm gave peaks in the visible
region of the electromagnetic spectrum. Fig. 6 shows the
UVvis spectrum recorded from the aqueous solution of 1
mM AgNO3 with leaf broth extract. The maximum
absorbance was observed to occur at 410 nm.
Fig. 6. Absorption peak of silver nanoparticles using UV-
Vis spectrophotometer.
CONCLUSIONS
In this paper we have reported the biosynthesis of silver
nanoparticles (AgNPs) by reduction of silver nitrate
(AgNO3) from leaf extract of Desert Rose (Adenium
obesum).Formation of the AgNPs were confirmed by
surface plasmon spectra using UVVis spectrophotometer
and absorbance peak at 410 nm. Adenium obesum extracts
are a rich source of anthocyanins and possess a significant
antioxidant activity. The best results regarding monomeric
anthocyanins con-tent and antioxidant activity were
obtained at extraction with 100% methanol and ethanol.
International Journal of Materials and Biomaterials Applications 2014; 4(1): 14-18
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However, for food industry, the extractions with ethanolic
solution are more convenient. The correlations be-tween
anthocyanins content, and antioxidant activity depend on
the extraction solvent, the best determination coefficients
was found for Adenium obesum extracts obtained in
water/methanol systems.
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Source of support: Nil; Conflict of interest: None declared

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