WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Zarger et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue10, 1320-1330.

Research Article

ISSN 2278 4357

PHYTOCHEMICAL INVESTIGATION AND GROWTH INHIBITING

EFFECTS OF SALIX ALBA LEAVES AGAINST SOME PATHOGENIC
FUNGAL ISOLATES
Mohd Sadiq S. Zarger*1, Fehmeeda Khatoon, Nida Akhtar2
1

Department of Applied Science & Humanities, Jamia Millia Islamia (Central University),
New Delhi- 110025, India.

Department of Chemistry, Faculty of Science, Jamia Hamdard, New Delhi-110062, India.

Article Received on
25 July 2014,

ABSTRACT

Revised on 20 August 2014,

Accepted on 15 Sept 2014

inherent part of life in world. Plant based medicines are the basis of

Since ancient times, use of plants as a source of medicines has been the

many modern pharmaceuticals used in day today life to cure different

kind of illnesses. Salix alba has been used from a long time as
*Correspondence for Author
Mohd Sadiq S. Zarger

medicine. In the Current study an attempt was made to determine

Department of Applied Science

Phytochemical Composition and in vitro effects of Methanolic extract

& Humanities, Jamia Millia

of Salix alba leaves against Candida guilliermondii, Candida glabrata

Islamia (Central University),

and Candida parapsilosis. Preliminary Phytochemical analysis

New Delhi- 110025, India.

revealed the presence of steroids, alkaloids, Phenols, glycosides and

tannins in different concentration. In GC-Mass analysis, the major

chemical compounds were Salicyl Alcohol, Linolenic acid, Galactose, 4, 6-O-nonylidene, 4Acetoxy-3-methoxycinnamic acid, Stearic acid, Stearyl aldehyde. Biological activity was
carried out in terms of Minimum Inhibitory Concentration, Filter disc assay and growth curve
study. From the data we conclude, Salix alba leaves extract as an option can be used further
for safe and efficacious drug for Candidiasis.
KEY WORDS: Salix, Candida, Phytochemicals, Biological Activity, Gas ChromatographyMass Spectrometry.
INTRODUCTION
Plants contain a wide variety of compounds called phytochemicals, mainly described as those
compounds having medicinal properties. Scientists have identified thousands of

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phytochemicals, although only a small fraction has been studied closely. Some of the better
known phytochemicals include beta carotene and other carotenoids, ascorbic acid (vitamin c),
folic acid and vitamin E. Plants containing phytochemicals not only have effect to some
particular diseases, but because of their antioxidant or hormone like actions, they also give
beneficial healthful effects in humans. The increase of fungal infection, toxicity of some
antifungal agents, interaction with different kind of drugs and development of resistance of
some species of fungi have led to search for new antifungal agents

[1, 3]

. Hence, antimicrobial

efficacies from plants have to be explored. Since Salix alba is a commonly known medicinal
plant, which is found in western and central Asia. Candida is a genus of yeast. Many species
are harmless commensals or endosymbionts of hosts including humans, but other species, or
harmless species in the wrong location, can cause diseases. Candida albicans can cause
diseases

(candidiasis

or

thrush)

in

humans

and

other

animals,

especially

in

immunocompromised patients. Essentially all areas of the human gastrointestinal tract can
harbour Candida

[4, 7]

. The most commonly isolated species from the human gastrointestinal

tract is Candida albicans followed by Candida parapsilosis, Candida tropicalis and Candida
glabrata [8.] Candida albicans is a causal agent of opportunistic oral and genital infections in
humans. It is very interesting to look anticandidal activity of Salix alba leaves.
Salix alba is a species of willow native to Europe and Western and central Asia. Its name is
derived from the white tone to the underside of the leaves. It is now cultivated in India,
China, Caribbean. Salix alba (White willow) of the family Salicaceae, genus Salix is well
known for making Cricket bats

[9]

.Records suggest that, as far back as 6000 years ago, white

willow was used in Mesopotamia. Subsequently, ancient people recorded the use of white
willow to cure pain and inflammation, including the Assyrian, Babylonian, Sumerian,
Egyptian, Chinese, Greek and Roman civilizations. Hippocrates recommended chewing
willow bark to patients suffering from fever, inflammation and pain. They also prescribed a
brew of willow leaves to ease the excruciating pains of childbirth. Since that time, white
willow has continued to be used to ease pain and inflammation

[10]

. Willow bark has been

used to treat different kinds of pain, including rheumatic pain, back pain, toothache and
menstrual cramps. It is also used to relieve sore throat, fever and headache associated with
upper respiratory tract infections and influenza [11].
The most famous chemical constituents of Salix alba leaves are Salicyl alcohol, Linolenic
acid, Galactose, 4, 6-O-nonylidene, 4-Acetoxy-3-methoxycinnamic acid, Stearic acid, Stearyl

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aldehyde

[12, 13]

World Journal of Pharmacy and Pharmaceutical Sciences

. Some Scientific studies have shown that the bark of willow contains main

constituent glucosid Salicin according to Pelletier and Caventou. Johanson (1875) showed the
presence of benzohelicin, a glucosid previously obtained (Pirin 1851) by acting upon populin
with nitric acid (Compare Salicinum) which has great medicinal importance. It has been
given in dyspepsia, chronic mucous discharges and it has also been given chronic diarrhoea
and dysentery. Very less study has been done on Salix alba leaves and the mode of action is
not given yet, but this study is an attempt to show anticandidal role of Salix alba leaves
extract against some recently obtained Candida isolates. This study has shown great effect on
Candida cells and this ability of the extract was tested and identified by the minimum
inhibitory concentration, filter disc assay and growth curve studies. Our Study involved the
Collection, Extraction, Phytochemical and anticandidal evaluation of leaves of this medicinal
plant.
MATERIALS AND METHODS
Collection and Preparation of plant extract
The leaves of Salix alba were collected from Duroo Sopore plant nursery. This nursery is
grown by the department of forestry district Baramulla Jammu and Kashmir, this nursery is
well known for different species of Salix.
Samples were collected in September-October 2012, at this time the leaves are completely
grown and modified. The samples were stored in the laboratory for the further reference. The
leaves were washed, dried and grinded to make the powder and extracted with methanol in
the Soxhlet apparatus for the period of 72h, then it was filtered with the Whattman filter
paper and filtrate was then evaporated to dryness. The extract was used for the phytochemical
and antimicrobial investigation after clearance of biosafety and ethical committee of the
institute.
Phytochemical Analysis
The methanolic extract was subjected to various phytochemical tests to find out the major
constituents. The tests were performed for Phenols/flavanoids (NaOH, NaOH/H2SO4),
glycoside (Keller-Killiani Test), tannins (Braemers test), steroids and terpenoids (Salkowski
test), alkaloids (Mayers/ Wagners test) and saponins [14, 17].

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GC-MS analysis
GC-MS analysis of the extract was carried out using a Shimadzu 2010 gas chromatograph
fitted with an AB-Wax column. Helium was used as carrier gas. Sample (0.1ml) was injected
in the splitless mode. The chemical component from the extract was identified by comparing
the retention time of the chromatographic peaks with those of authentic compound using the
WILEY8.LIB and NISTO5s
Strains and growth media
Clinical Isolates of Candida guilliermondii, Candida glabrata and Candida parapsilosis used
in this study were collected from Department of Microbiology, Vardhaman Mahavir Medical
College, New Delhi, India. For confirmation of the isolates germ tube test, microscopic
morphology on Corn Meal Agar, Hi Chrome Agar, carbon and nitrogen assimilation test and
ascospore production on the malt-extract agar was done. The working strains were
maintained on YEPD slants containing 2% (w/v) glucose, 2% peptone and 1% yeast extract
at -20c and were subcultured twice prior to testing to ensure viability and purity. All media
constituents were obtained from Hi-Media (India). All chemicals and solvents were of
analytical grade and obtained from Merck (India). For all experimental studies the yeast cells
were maintained on the yeast extract-peptone-dextrose (YEPD) medium at 30c.
Determination of Minimum Inhibitory Concentration
MICs of the strains were determined using a broth microdilution method. Cells were
resuspended in a 0.9% normal saline solution to give an optical density at 600nm (OD6oo) of
0.1. The diluted cell suspensions were added to the wells of round-bottomed 96 well
microtiter plates containing equal volumes of medium and different concentrations of test
extract

[18]

. A drug free control was also included. The MIC test end was evaluated visually

and is defined as the lowest extract concentration that showed significant inhibition of growth
compared to the controls.
Filter Disc Assay
Filter disc assay was performed according to the standard guidelines (M2-A7) of the national
committee for clinical laboratory standards (NCCLS), using a modified Kirby-Bauer disc
diffusion method. All the organisms were stored at -20C until use. Cells were grown at 30C
in YEPD broth (approximately 105 cells/ml) and were passaged at least twice on solid agar.
Broth cultures swabbed onto agar to achieve a lawn of confluent yeast growth. Stock
solutions of the test extract were prepared in 1% DMSO. Paper discs impregnated with

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different extract concentrations were placed on each plate. One disc impregnated with 1%
DMSO was placed in the centre of the plate that served as solvent control. The plates were
incubated at 30C for 48 hrs. The diameter of the zone of inhibition was recorded in
millimetres, after 48 hours.
Growth curve studies
This experiment carried 106 cells (optical density A595=0.1) of strains which were grown
aerobically in automated shaker set at 30C until stationary phase. Growth was followed
turbidometrically at 595nm using spectrophotometer. Required concentrations of test extract
were added to culture. The growth rates of cells alone and with inhibitor were performed.
Optical density was recorded for each concentration against time (hrs). The growth rate is
equivalent to slope log (optical density) versus time duration the exponential phase

[19]

RESULTS AND DISCUSSION

(Table 1) shows the result of preliminary phytochemical analysis. The result showed the
alkaloids, phenols, tannins, glycosides and steroids as positive while as saponins were absent
in the test extract. In the GC-MS 40 phytochemical compounds were identified in the
methanolic extract of Salix alba leaves. As indicated in the (Table 2) out of which seven were
found in abundance and rest thirty three were in traces. The identification of phytochemicals
is based on the molecular formula, molecular weight, retention time and percentage of
presence. The major compounds of the extract were 1, 2 Cyclohexanediol 12.89%, Salicyl
Alcohol 7.19%, Stearic acid 12.66%, Linolenic acid 16.17% and Galactose 4,6-O-nonylidene
11.49%.
Table 1. Phytochemical analysis of Salix alba Leaves extract. The test was based on
colour intensity.
Phytochemicals
Alkaloids

Colour observed
Pale yellow
Brown ppt.
Phenols/ Flavonoids
NaOH,NaOH/ H2SO4, Deep yellow,
ALCl3
Colour less yellow
Steriods / Terpenoids
Salkowaski
Red colour
Glycosides
H2SO4
Dark Brown
Keller Killiani
Brown Ring
Tannins
Braemer,Iodine
Dark Blue,Faint Bluish
Saponins
Frothing
Colourless
Note: - absent, + slightly present, ++ moderately present

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Name of test
Wagner Mayer

Vol 3, Issue 10, 2014.

Colour intensity
+,+
++,++,+
++
+,+
+,+
-

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Table 2: Chemical composition of Salix alba leaves extract

S. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

Name of Compound
2-Butynyl p-toluenesulfonate
3-Allyl-2-methoxyphenol
2,5,8-Trimethyltetralin
Limonene oxide,
Hexanoic acid, 4-hexen-1-yl ester
2,5,5-Trimethyl-3-hexyn-2-ol
10,12-Pentacosadiynoic acid
4-Acetoxy-3-methoxycinnamic acid
Picolinohydroxamic acid
Ethylene glycol m-cresyl ether
Tetradecanoic acid
Acetyl monoglyceride
1,2,3-Benzenetriol, triacetate
3-Hydroxy-.beta.-damascone
2,6-Dimethylheptadecane
Succinic acid, monomethyl ester
Octahydro-1-nitroso-1H-azonine
8-Nonenoic acid
Glycerol
5-Methoxy-2-methylaniline
Endo,endo-2,3-bornanediol
Isovanillic acid
1-Amino-4-methylpiperazine
Geranyl acetone
5-Methyl-5-octen-1-ol
Isomenthol
2,2-Dibromocholestanone
Palmitic acid, methyl ester
Stearyl aldehyde
3,4,4-Trimethyl-2-hexene
Palmitic acid
1,2-Benzenediol
Benzyl isopentyl ether
3-Cholesterol methyl ether
2-Propyl-tetrahydropyran-3-ol
Salicyl Alcohol
Galactose, 4,6-O-nonylidene
Stearic acid
1,2 Cyclohexanediol
Linolenic acid

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Molecular Molecular
Formula
weight
C11H12O3S
224
C10H12O2
164
C13H18
174
C10H16O
152
C12H22O2
198
C9H16O
140
C25H42O2
374
C12H12O5
236
C6H6N2O2
138
C9H12O2
152
C14H28O2
228
C5H10O4
134
C12H12O6
252
C13H20O2
208
C19H40
268
C5H8O4
132
C8H16N2O
156
C9H16O2
156
C3H8O3
92
C8H11NO
137
C10H18O2
170
C8H8O4
168
C5H13N3
115
C13H22O
194
C9H18O
142
C10H20O
156
C27H44Br2O
542
C17H34O2
270
C18H36O
268
C9H18
126
C16H32O2
256
C6H6O2
110
C12H18O
178
C28H48O
400
C8H16O2
144
C7H8O2
124
C15H28O6
304
C18H36O2
284
C6H12O2
116
C18H30O2
278

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Retention % age
time
of Presence
5.79
0.06
7.64
0.07
6.13
0.1
7.69
0.12
6.95
0.14
9.82
0.14
8.06
0.15
12.04
0.17
4.44
0.22
11.42
0.23
12.58
0.23
6.22
0.27
3.59
0.28
11.02
0.29
3.66
0.3
3.84
0.35
9.37
0.35
10.25
0.38
5.07
0.41
12.43
0.44
10.07
0.5
10.72
0.57
7.25
0.58
10.47
0.6
11.57
0.67
16.07
0.88
11.92
0.93
14.25
1.03
31.2
1.09
6.02
1.22
14.39
1.45
5.44
1.56
17.69
2.75
28.67
3.92
4.54
3.93
6.43
7.19
18.46
11.49
14.7
12.66
3.36
12.89
16.42
16.17

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World Journal of Pharmacy and Pharmaceutical Sciences

Biological Activity Investigation, Minimum inhibitory concentration

An accurate method of assessment is the broth dilution technique. Hence in the current study
the broth dilution method was used in determining the activities measured as MIC. In using
this kind of method, higher degrees of difference in susceptibility among Candida species
were noted. The crude methanol extract was found to have minimum inhibitory concentration
Of 800g/ml, 800g/ml and 1600g/ml against Candida guilliermondii, Candida glabrata
and Candida parapsilosis respectively. The results evaluated that the test extract showed
more killing in case of Candida guilliermondii and Candida glabrata. It would reveal that
Candida parapsilosis is the less sensitive yeast to the test extract. The results obtained
provide us clear evidence that the test extract used in the study has substantial level of
antifungal activity.

Filter disc assay

In Vitro Antifungal activity of the test extract was studied against three isolates at different
three concentrations 400mg/ml, 800mg/ml and 1200mg/ml respectively. The results
summarised in (Table 3) Highest zone of inhibition i,e 12mm was measured in Candida
glabrata when treated against 1200mg/ml of the test extract followed by 11mm measured in
Candida parapsilosis. When treated with same concentration of the test extract, respectively.
In case of Candida guilliermondii at highest concentration of the test extract the zone of
inhibition was only 10mm. In comparison ketoconazole at 100 g/ml showed 15mm, 16mm
and 16mm zone of inhibition in Candida guilliermondii, Candida glabrata and Candida
parapsilosis. Comparison between antifungal activities of the different extract concentrations
and standard drug is shown in Fig 1. The results showed that in case of control disc no zone
of inhibition was seen so far as our study is concerned 1%DMSO, as solvent is having no
effect on the tested organisms. Hence we can effectively conclude that whole of the
antifungal effect is because of different concentration of the test extract used in the study.
Table 3. Antifungal activity screening data for different test extract concentrations and
ketoconazole
Concentration of extract
400 mg/ml
800 mg/ml
1200 mg/ml
ketoconazole (100 g/ml)
Control

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C. guilliermondii
(SN2006)
6.330.57
9.330.58
100.81
15.660.57
-

C. glabrata
(SN2266)
7.330.59
10.660.57
12.660.90
16.330.57
-

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C. parapsilosis
(SN1980)
6.660.57
10.330.58
11.660.68
16.660.57
-

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1%
DMS
O

40
0
mg
/ml

World Journal of Pharmacy and Pharmaceutical Sciences

80
0
mg
/ml

12
00
mg
/ml

Contr C.
guilliermondii
ol

100g/
ml

40
0
mg
/ml

ketocon
azole

C. glabrata

80
0
mg
/ml

12
00
mg
/ml

100g/m
l

40
0
mg
/ml

Ketocon
azole

C. parapsilosis

80
0
mg
/ml

12
00
mg
/ml

100g/
ml

Ketocon
azole

Note: - = Negative (absent), + = Positive (slightly present), ++ = Positive (moderately present)

Figure 1: Bar diagram showing comparison between antifungal activities of S. alba

leaves methanolic extract at different concentrations and standard antifungal drug
against (a) Candida guilliermondii (b) Candida glabrata (c) Candida parapsilosis
Growth Curve Studies (Turbidometric Measurement)
Growth pattern of the Candida species was investigated at different concentrations of
methanolic extract of Salix alba leaves (Fig.

2a, 2b, 2c) shows the effect of different

concentrations of test extract on growth pattern of Candida guilliermondii, Candida glabrata

and Candida parapsilosis respectively. Control cells showed a normal growth with lag phase
4hrs, active exponential phase of 8-10hrs before attaining stationary phase. The absorbance
obtained for the growth control (only organism) showed that the culture reached the
stationary growth phase after 16h showing a normal growth pattern. The curve depicts a lag
phase in the initial phase of growth, active log phase and stationary phase. Increase in
concentration of test extract leads to significant decrease in growth with suppressed and
delayed exponential phase with respect to control. At minimum inhibitory concentration
values almost complete stopping of growth was observed which is indicated by flat line.

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(a)Candida guilliermondii

(b) Candida glabrata

(c) Candida parapsilosis

Figure 2. Determination of different concentrations of S. alba leaves extract on growth
curve pattern against absorbance at 595nm (hrs) Shows complete inhibition of growth
at MIC values against (a) C. guilliermondii (b) C. glabrata (c) C. parapsilosis
Natural resources have provided an unparalleled source of chemical scaffolds with diverse
biological activities and have profoundly impacted antimicrobial drug discovery. The
potential for developing antimicrobials from plants seems rewarding, as it will lead to the
development of phytomedicine to act against different microbes. This is considerable finding

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as Candida is normal resident of oral cavity and genitourinary tract. Our findings provide an
idea for expanding the utility of plant active principals as antifungal agents. We have found
that methanolic leaves extract of Salix alba exhibits fungicidal activity by filter disc assay
and growth curve study against three different fungal isolates. Recently obtained clinical
isolates were found clearly sensitive to the test extract at varying extents. The current study
has shown that this test extract considerably inhibit the growth of Candida isolates. The test
extract displayed considerable MIC against different Candida isolates ranging from 800 to
1600g/ml. The extract was found highly active against Candida guilliermondii, Candida
glabrata and Candida parapsilosis. Growth kinetic studies as a function of varied
concentration also follow the same pattern. MIC/4 treated cells showed depressed growth
curve with clearly differentiated phases. While as MIC/2 treated cells showed suppressed and
delayed exponential phase. At MIC value S shaped curve declined to flat line showing almost
complete death of cells growth (Fig 2). On solid media (filter disc assay) effective inhibition
of growth of Candida species by test extract was found to increase in concentration dependent
manner.
CONCLUSION
The extract has shown clear anticandidial activity in both solid and liquid medium. The
current work is an additional effort to the development of new therapeutic agent which is
fungicidal, less toxic and prevents drug resistance. Further investigation and experimentation
need to be done, which is very essential & important and may help to facilitate its application
as future anticandidial agent.
ACKNOWLEDGMENT
The authors are very much thankful to University Grants Commission (UGC) for the
financial Support Project: Ref. No. 42-269/2013(SR) and also to Jamia Millia Islamia
(Central University) New Delhi, India for providing Laboratory for experimental work. We
are also thankful to Vardhman Mahavir Medical College (VMMC) for providing us fungal
strains.
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