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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

Mohd Sadiq S. Zarger*1, Fehmeeda Khatoon, Nida Akhtar2

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,


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.
Plants contain a wide variety of compounds called phytochemicals, mainly described as those
compounds having medicinal properties. Scientists have identified thousands of

Vol 3, Issue 10, 2014.


com Vol 3. although only a small fraction has been studied closely. Salix alba (White willow) of the family Salicaceae. back pain. Stearic acid. The most famous chemical constituents of Salix alba leaves are Salicyl alcohol. antimicrobial efficacies from plants have to be explored. toothache and menstrual cramps. they also give beneficial healthful effects in humans. Since Salix alba is a commonly known medicinal plant. but because of their antioxidant or hormone like actions. Sumerian. can cause diseases. 2014. Many species are harmless commensals or endosymbionts of hosts including humans. 4. Essentially all areas of the human gastrointestinal tract can harbour Candida [4. Salix alba is a species of willow native to Europe and Western and central Asia. Candida tropicalis and Candida glabrata [8. Linolenic acid. ancient people recorded the use of white willow to cure pain and inflammation. Since that time. including the Assyrian. Willow bark has been used to treat different kinds of pain. 4-Acetoxy-3-methoxycinnamic acid. It is also used to relieve sore throat. or harmless species in the wrong location. fever and headache associated with upper respiratory tract infections and influenza [11]. 6-O-nonylidene. especially in immunocompromised patients. World Journal of Pharmacy and Pharmaceutical Sciences phytochemicals. white willow has continued to be used to ease pain and inflammation [10] . China. including rheumatic pain. Subsequently.wjpps. Egyptian. Some of the better known phytochemicals include beta carotene and other carotenoids. It is very interesting to look anticandidal activity of Salix alba leaves. Plants containing phytochemicals not only have effect to some particular diseases.Records suggest that. The increase of fungal infection. inflammation and pain. toxicity of some antifungal agents. but other species. Its name is derived from the white tone to the underside of the leaves. ascorbic acid (vitamin c). Issue 10. 7] . which is found in western and central Asia. Candida albicans can cause diseases (candidiasis or thrush) in humans and other animals. 3] . Galactose. white willow was used in Mesopotamia. 1321 .] Candida albicans is a causal agent of opportunistic oral and genital infections in humans. as far back as 6000 years ago. The most commonly isolated species from the human gastrointestinal tract is Candida albicans followed by Candida parapsilosis.Zarger et al. Caribbean. Candida is a genus of yeast. It is now cultivated in India. genus Salix is well known for making Cricket bats [9] . folic acid and vitamin E. Greek and Roman civilizations. Stearyl www. Hence. They also prescribed a brew of willow leaves to ease the excruciating pains of childbirth. interaction with different kind of drugs and development of resistance of some species of fungi have led to search for new antifungal agents [1. Chinese. Hippocrates recommended chewing willow bark to patients suffering from fever. Babylonian.

but this study is an attempt to show anticandidal role of Salix alba leaves extract against some recently obtained Candida isolates. It has been given in dyspepsia.Zarger et al. Phytochemical and anticandidal evaluation of leaves of this medicinal plant. 2014. a glucosid previously obtained (Pirin 1851) by acting upon populin with nitric acid (Compare Salicinum) which has great medicinal importance. The extract was used for the phytochemical and antimicrobial investigation after clearance of biosafety and ethical committee of the institute. alkaloids (Mayer’s/ Wagner’s test) and saponins [14. Very less study has been done on Salix alba leaves and the mode of action is not given yet. 13] World Journal of Pharmacy and Pharmaceutical Sciences . at this time the leaves are completely grown and modified. Issue 10. The leaves were washed. Phytochemical Analysis The methanolic extract was subjected to various phytochemical tests to find out the major Vol 3. NaOH/H2SO4). Johanson (1875) showed the presence of benzohelicin. Our Study involved the Collection. This nursery is grown by the department of forestry district Baramulla Jammu and Kashmir. 1322 . Extraction. glycoside (Keller-Killiani Test). chronic mucous discharges and it has also been given chronic diarrhoea and dysentery. steroids and terpenoids (Salkowski test). then it was filtered with the Whattman filter paper and filtrate was then evaporated to dryness. www. this nursery is well known for different species of Salix. Some Scientific studies have shown that the bark of willow contains main constituent glucosid Salicin according to Pelletier and Caventou.wjpps. This study has shown great effect on Candida cells and this ability of the extract was tested and identified by the minimum inhibitory concentration. The samples were stored in the laboratory for the further reference. Samples were collected in September-October 2012. 17]. aldehyde [12. tannins (Braemer’s test). The tests were performed for Phenols/flavanoids (NaOH. filter disc assay and growth curve studies. dried and grinded to make the powder and extracted with methanol in the Soxhlet apparatus for the period of 72h. MATERIALS AND METHODS Collection and Preparation of plant extract The leaves of Salix alba were collected from Duroo Sopore plant nursery.

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. Sample (0. Hi Chrome Agar. carbon and nitrogen assimilation test and ascospore production on the malt-extract agar was done. Cells were resuspended in a 0. All chemicals and solvents were of analytical grade and obtained from Merck (India).wjpps. New Delhi.9% normal saline solution to give an optical density at 600nm (OD6oo) of 0. The working strains were maintained on YEPD slants containing 2% (w/v) glucose. Vardhaman Mahavir Medical College. Determination of Minimum Inhibitory Concentration MICs of the strains were determined using a broth microdilution method. Stock solutions of the test extract were prepared in 1% DMSO. All media constituents were obtained from Hi-Media (India). 1323 . using a modified Kirby-Bauer disc diffusion method. Helium was used as carrier gas. India. 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). Issue 10.LIB and NISTO5s Strains and growth media Clinical Isolates of Candida guilliermondii. 2% peptone and 1% yeast extract at -20°c and were subcultured twice prior to testing to ensure viability and purity. Broth cultures swabbed onto agar to achieve a lawn of confluent yeast growth. All the organisms were stored at -20°C until use. Candida glabrata and Candida parapsilosis used in this study were collected from Department of Microbiology. 2014.1. World Journal of Pharmacy and Pharmaceutical Sciences GC-MS analysis GC-MS analysis of the extract was carried out using a Shimadzu 2010 gas chromatograph fitted with an AB-Wax Vol 3. Cells were grown at 30°C in YEPD broth (approximately 105 cells/ml) and were passaged at least twice on solid agar.1ml) was injected in the splitless mode. 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] .Zarger et al. microscopic morphology on Corn Meal Agar. Paper discs impregnated with www. For all experimental studies the yeast cells were maintained on the yeast extract-peptone-dextrose (YEPD) medium at 30°c. For confirmation of the isolates germ tube test. A drug free control was also included.

++. Stearic acid 12. World Journal of Pharmacy and Pharmaceutical Sciences different extract concentrations were placed on each plate. phenols. 2 Cyclohexanediol 12.wjpps. molecular weight.17% and Galactose 4. Table 1.Faint Bluish Saponins Frothing Colourless Note: .com Name of test Wagner Mayer Vol 3. after 48 hours. The growth rates of cells alone and with inhibitor were performed. Deep yellow.+ +. tannins. Optical density was recorded for each concentration against time (hrs). glycosides and steroids as positive while as saponins were absent in the test extract.+ ++. Required concentrations of test extract were added to culture. The diameter of the zone of inhibition was recorded in millimetres. Phenols/ Flavonoids NaOH. Growth was followed turbidometrically at 595nm using spectrophotometer. Phytochemical analysis of Salix alba Leaves extract.19%.66%. ++ moderately present www. The result showed the alkaloids. 2014. The test was based on colour intensity.absent. Issue 10.Iodine Dark Blue. Colour intensity +. The major compounds of the extract were 1. Growth curve studies This experiment carried 106 cells (optical density A595=0.49%. The growth rate is equivalent to slope log (optical density) versus time duration the exponential phase [19] . ALCl3 Colour less yellow Steriods / Terpenoids Salkowaski Red colour Glycosides H2SO4 Dark Brown Keller Killiani Brown Ring Tannins Braemer.+ ++ +.89%. + slightly present. retention time and percentage of presence.+ - 1324 .6-O-nonylidene 11.1) of strains which were grown aerobically in automated shaker set at 30°C until stationary phase. The plates were incubated at 30°C for 48 hrs. The identification of phytochemicals is based on the molecular formula. Phytochemicals Alkaloids Colour observed Pale yellow Brown ppt. Salicyl Alcohol 7. In the GC-MS 40 phytochemical compounds were identified in the methanolic extract of Salix alba leaves. One disc impregnated with 1% DMSO was placed in the centre of the plate that served as solvent control. RESULTS AND DISCUSSION (Table 1) shows the result of preliminary phytochemical analysis.Zarger et al. Linolenic acid 16.NaOH/ H2SO4. As indicated in the (Table 2) out of which seven were found in abundance and rest thirty three were in traces.

25 1.endo-2.6 11.36 12.1 7.4-Trimethyl-2-hexene Palmitic acid 1. monomethyl ester Octahydro-1-nitroso-1H-azonine 8-Nonenoic acid Glycerol 5-Methoxy-2-methylaniline Endo. 4-hexen-1-yl ester 2.19 18.37 0.17 1325 .22 14. Retention % age time of Presence 5.82 0.5 10.02 1.Zarger et al.2 1.5. World Journal of Pharmacy and Pharmaceutical Sciences Table 2: Chemical composition of Salix alba leaves extract S.44 0.3-Benzenetriol.41 12.2-Benzenediol Benzyl isopentyl ether 3α-Cholesterol methyl ether 2-Propyl-tetrahydropyran-3-ol Salicyl Alcohol Galactose.03 31. Issue 10.23 6.25 0.46 11.43 0.84 0.3 3.06 7.5. 2014.69 0. 4.15 12.72 0.93 14.22 11.13 0. 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 2.6-Dimethylheptadecane Succinic acid.56 17.67 16.47 0.3-bornanediol Isovanillic acid 1-Amino-4-methylpiperazine Geranyl acetone 5-Methyl-5-octen-1-ol Isomenthol 2.43 7.45 5.25 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 Vol 3.2-Dibromocholestanone Palmitic acid.75 28.06 0.07 0.57 0.07 6.35 10.17 4.09 6.42 0.02 0. No.95 0.64 0.2 Cyclohexanediol Linolenic acid www.66 3.7 12.14 8.44 1.wjpps.49 14.92 0.69 2.92 4.12-Pentacosadiynoic acid 4-Acetoxy-3-methoxycinnamic acid Picolinohydroxamic acid Ethylene glycol m-cresyl ether Tetradecanoic acid Acetyl monoglyceride 1.58 10.54 3.14 9.59 0.beta.22 0. methyl ester Stearyl aldehyde 3.39 1.58 0.67 3. Hexanoic acid.29 3.42 16.79 0.5-Trimethyl-3-hexyn-2-ol 10.44 10.89 16.6-O-nonylidene Stearic acid 1.88 11.07 0.4.12 6.04 0.57 7.28 11.8-Trimethyltetralin Limonene oxide.38 5.66 0. triacetate 3-Hydroxy-.93 6.2.35 9.07 0.23 12.-damascone 2.27 3.

com C.57 10.57 12. Hence in the current study the broth dilution method was used in determining the activities measured as MIC. The results obtained provide us clear evidence that the test extract used in the study has substantial level of antifungal activity. World Journal of Pharmacy and Pharmaceutical Sciences Biological Activity Investigation.57 - 1326 .66±0.68 16. parapsilosis (SN1980) 6.33±0. When treated with same concentration of the test extract.90 16. In comparison ketoconazole at 100 μg/ml showed 15mm.33±0. Issue 10. Filter disc assay In Vitro Antifungal activity of the test extract was studied against three isolates at different three concentrations 400mg/ml. Table 3. as solvent is having no effect on the tested organisms. glabrata (SN2266) 7.66±0.57 9.58 11.33±0. Hence we can effectively conclude that whole of the antifungal effect is because of different concentration of the test extract used in the study.59 10. respectively. C.33±0. The results summarised in (Table 3) Highest zone of inhibition i. In using this kind of method.66±0.Zarger et al.57 - C. The results evaluated that the test extract showed more killing in case of Candida guilliermondii and Candida glabrata. 16mm and 16mm zone of inhibition in Candida guilliermondii. higher degrees of difference in susceptibility among Candida species were noted. It would reveal that Candida parapsilosis is the less sensitive yeast to the test extract.81 15. Candida glabrata and Candida parapsilosis respectively. 800μg/ml and 1600μg/ml against Candida guilliermondii. The crude methanol extract was found to have minimum inhibitory concentration Of 800μg/ml.66±0. Antifungal activity screening data for different test extract concentrations and ketoconazole Concentration of extract 400 mg/ml 800 mg/ml 1200 mg/ml ketoconazoleª (100 μg/ml) Control www. The results showed that in case of control disc no zone of inhibition was seen so far as our study is concerned 1%DMSO.57 - Vol 3.33±0. 2014. In case of Candida guilliermondii at highest concentration of the test extract the zone of inhibition was only 10mm.e 12mm was measured in Candida glabrata when treated against 1200mg/ml of the test extract followed by 11mm measured in Candida parapsilosis. guilliermondii (SN2006) 6.58 10±0. 800mg/ml and 1200mg/ml respectively. Candida glabrata and Candida parapsilosis.66±0. Minimum inhibitory concentration An accurate method of assessment is the broth dilution technique.66±0.wjpps. Comparison between antifungal activities of the different extract concentrations and standard drug is shown in Fig 1.

= Negative (absent). 2a. active exponential phase of 8-10hrs before attaining stationary phase. glabrata 80 0 mg /ml 12 00 mg /ml 100μg/m l 40 0 mg /ml Ketocon azole C. 2c) shows the effect of different concentrations of test extract on growth pattern of Candida guilliermondii. www. Candida glabrata and Candida parapsilosis respectively. Issue 10. active log phase and stationary phase.wjpps. 2b. parapsilosis 80 0 mg /ml 12 00 mg /ml 100μg/ ml Ketocon azole Note: .com Vol 3. Increase in concentration of test extract leads to significant decrease in growth with suppressed and delayed exponential phase with respect to control. 2014. 1% DMS O 40 0 mg /ml World Journal of Pharmacy and Pharmaceutical Sciences 80 0 mg /ml 12 00 mg /ml Contr C. At minimum inhibitory concentration values almost complete stopping of growth was observed which is indicated by flat line. 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. 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. guilliermondii ol 100μg/ ml 40 0 mg /ml ketocon azole C. Control cells showed a normal growth with lag phase 4hrs. ++ = Positive (moderately present) Figure 1: Bar diagram showing comparison between antifungal activities of S. + = Positive (slightly present). The curve depicts a lag phase in the initial phase of growth.Zarger et al. 1327 .

Determination of different concentrations of S. The potential for developing antimicrobials from plants seems rewarding. 1328 . guilliermondii (b) C. glabrata (c) C.wjpps. This is considerable finding Vol 3. Issue 10. World Journal of Pharmacy and Pharmaceutical Sciences (a)Candida guilliermondii (b) Candida glabrata (c) Candida parapsilosis Figure 2.Zarger et al. parapsilosis Natural resources have provided an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. alba leaves extract on growth curve pattern against absorbance at 595nm (hrs) Shows complete inhibition of growth at MIC values against (a) C. as it will lead to the development of phytomedicine to act against different microbes. 2014.

com Vol 3. While as MIC/2 treated cells showed suppressed and delayed exponential phase.Zarger et al. At MIC value S shaped curve declined to flat line showing almost complete death of cells growth (Fig 2). 2014. ACKNOWLEDGMENT The authors are very much thankful to University Grants Commission (UGC) for the financial Support Project: Ref.wjpps. The current study has shown that this test extract considerably inhibit the growth of Candida isolates. Recently obtained clinical isolates were found clearly sensitive to the test extract at varying extents. No. Further investigation and experimentation need to be done. New York. The extract was found highly active against Candida guilliermondii. Candida glabrata and Candida parapsilosis. Chichester John Wile y & Sons. www. We are also thankful to Vardhman Mahavir Medical College (VMMC) for providing us fungal strains. India for providing Laboratory for experimental work. A. REFRENCES 1. Growth kinetic studies as a function of varied concentration also follow the same pattern. Sofowora. which is very essential & important and may help to facilitate its application as future anticandidial agent. On solid media (filter disc assay) effective inhibition of growth of Candida species by test extract was found to increase in concentration dependent manner. The current work is an additional effort to the development of new therapeutic agent which is fungicidal. 1993. Medicinal plants and traditional medicines in Africa. 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. 42-269/2013(SR) and also to Jamia Millia Islamia (Central University) New Delhi. Our findings provide an idea for expanding the utility of plant active principals as antifungal agents. Issue 10. 1329 . less toxic and prevents drug resistance. World Journal of Pharmacy and Pharmaceutical Sciences as Candida is normal resident of oral cavity and genitourinary tract. The test extract displayed considerable MIC against different Candida isolates ranging from 800 to 1600μg/ml. MIC/4 treated cells showed depressed growth curve with clearly differentiated phases. CONCLUSION The extract has shown clear anticandidial activity in both solid and liquid medium. 191-289.

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