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ZnO superstructures as an antifungal for effective control of Malassezia furfur, dermatologically prevalent yeast: prepared by aloe vera assisted combustion method Kavya Shree, Shilpa C J, H. Nagabhushana, Daruka Prasad B, Sreelatha G L, Sharma S C, Ashoka Siddaramanna, Ananda Kumari J, and H B Premkumar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/sc500784p • Publication Date (Web): 04 May 2015 Downloaded from http://pubs.acs.org on May 10, 2015

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ACS Sustainable Chemistry & Engineering

1 2 3 4 5 6

ZnO superstructures as an antifungal for effective 7 8 9 10 1

control of Malassezia furfur, dermatologically 12 13 14 15

prevalent yeast: prepared by aloe vera assisted 16 17 18 19

combustion method 20 21 2 23 24

D. Kavyashree1, C. J. Shilpa2, H. Nagabhushana2,*, B. Daruka Prasad3, G. L. Sreelatha4, 25 26 27

S.C. Sharma5, S. Ashoka6, J. Anandakumari7, H. B. Premkumar8 28 29 30

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31 2

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3

Department of Physics, Channabasaveshwara Institute of Technology, Gubbi–572216, India

Prof. C.N.R Rao Centre for Advanced Materials, Tumkur University, Tumkur-572 103, India. 3

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Department of Physics, B.M. S. Institute of Technology, Bangalore - 560 064, India. 4

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Department of Microbiology & Biotechnology, Jnana Bharathi Campus, Bangalore University, Bangalore - 560056, India.

38 39

5

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Dayananda Sagar University, Shavige Malleshwara hills, Kumaraswamy layout,

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Bangalore 560 078, India 42

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Department of Chemistry, Post graduate center, Vijaya College,

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R.V. Road, Bangalore – 560004, India. 45 7

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Department of Physics, Sree Siddaganga College for women, Tumkur-572 103, India

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Department of Physics, Acharya Institute of Technology, Bangalore - 560090, India.

*Corresponding author: [email protected], +91-9663177440 (Mobile Phone) 50 51 52 54

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KEYWORDS: Zinc Oxide superstructures, Green Synthesis, Photoluminescence, Antidandruff, 5 56

Antimalassezial activity. 58

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ACS Paragon Plus Environment


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ABSTRACT: In this paper, a robust and simple biogenic route has been developed to synthesize 5

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self-assembled ZnO superstructures in short interval of time using naturally available aloe vera 6 7

plant gel and zinc nitrate as starting materials. The stabilization of zinc ions with 10

9

8

polysaccharides wrapped chains along with the support of proteins, lipids and physterols of aloe 12

1

vera gel followed by combustion derives the ZnO superstructures. 13

The obtained ZnO

14

superstructures shows hexagonal crystal phase and exhibit semiconducting behaviour with the 17

16

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energy band gap varies from 2.92 – 3.08 eV. The aloe vera gel derived ZnO superstructures 19

18

exhibit unique and strong orange-red emission centered at 600 nm. 20

The better structural,

2

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morphological and photoluminescence results are obtained for ZnO prepared with 16.6 % W/V 24

23

of zinc nitrate with aloe vera content compared to other concentrations of aloe vera. The 25 26

prepared compounds are tested for antimalassezial activity against Malassezia furfur 27 29

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dermatologically prevalent yeast and were found to have Minimum Inhibitory Concentration 31

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(MIC) values ranging from 8 µg/ml to 125 µg/ml. Fluorescence microscopic analysis revealed 32 34

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that yeast cells treated with ZnO superstructures have the chromatin as orange instead of green 36

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show casing the cell aggregation suggests that ZnO superstructures have an immense potential as 38

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antifungal agent. 39

Hence, the explored method of preparation shows high efficient ZnO

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superstructures derived from the aloe vera plant gel have potential applications in medicine, 43

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biomedical and cosmetic industries. 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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INTRODUCTION 4 5

Dandruff is a common embarrassing scalp disorder affecting a large portion of 6 7

population. It is caused due to excessive flaking of dead skin cells from the scalp. 10

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finding 12

1

confirmed that

Malassezia

furfur

Recent

(M. furfur), lipophilic, saprophytic and

basidiomycetous yeast belongs to resident flora of human skin [1] have been responsible for the 13 14

pathogenesis of several chronic diseases such as pityriasisversicolor [2], folliculitis [3], 17

16

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seborrhoeic dermatitis [4] and some forms of atopic dermatiti [5], psoriasis [6] and confluent and 19

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reticulate papillomatosis [7]. It has been isolated from sebum-rich areas of the body such as the 20 2

21

upper trunk, head and face. 23 24 25 26

M. furfur has special requirements for exogenous lipids due to its inability to synthesize 27 29

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long-chain fatty acids [8]. The topical and systemic treatments available today, typically do not 31

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eradicate but only helps in reinstating the yeast‟s population to the commensally condition. The 32 3

available active ingredients included in treatment options are zinc pyrithione, salicyclic acid, 36

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corticosteroids, imidazole derivatives, glycolic acid, steroids, selenium suplhide, sulphur coal tar 38

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derivatives. These drugs are toxic and expensive and require prolonged treatment [9]. Therefore, 39 41

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it is imperative to develop alternative antifungal agents with versatile features such as effective 43

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combination of antimicrobial potency, wide availability, less toxicity and good compatibility. 4 45 46 48

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In this view, nanostructured metal oxides have gained intensive interest, due to their 50

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electrostatic attraction between negatively charged microbial cells and positively charged 51 52

nanoparticles for the activity of nanoparticles as antifungal agents [10, 11]. These materials 53 5

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have the potential to revolutionize medicine because of their ability to interact at molecular and 57

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cellular levels of organs and tissues of a human body [12]. Their unique size dependent 58 60

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properties make these materials superior and indispensable in many more areas of research like



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solar cells [13] gas sensors [14] photo sensors [15,16], piezoelectric devices [17], waste water 5

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treatment [18], and so on. 6

The nanostructures were already tested for their antimicrobial

7

properties as antibacterial, antifungal and anti-yeast [19 - 22]. 10

9

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Thus, innovative and scalable

synthesis routes need to be developed in order to control the production and morphology of the 12

1

various metal oxides at nano and microscale. 13 14 15 17

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Several synthesis approaches have been successfully explored for the controlled 19

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synthesis of a variety of ZnO superstructures (ZnO – SS) viz. spray-pyrolysis, precipitation, 20 2

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hydrothermal, chemical vapor deposition, sol–gel, thermal evaporation solution combustion etc. 24

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[23 -30]. Chemical procedures involved in these synthesis methods produce the main products 25 26

and also releases the hazardous waste products to the environment. Thus, there is a need for 27 29

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“green chemistry”, which includes a clean, nontoxic and environmentally benign routes. 31

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Additionally, the large-scale synthesis of pure ZnO – SS with unique properties at relatively low 32 3

temperatures is still remains a challenge. 35

34 36 38

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As a result, investigation of simple and fast synthesis route that can control the shape and 39 41

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size of ZnO - SS with high yield under ambient conditions received significant attention. The 43

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use of naturally available plant based materials such as leaf; root, latex, seed, and stem appear to 45

4

be the best for the large scale synthesis [31-35]. The aloe vera plant gel which is having many 46 48

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health benefits have been attributed to the polysaccharides contained in the gel of the leaves. 50

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These biological entities help in wound healing, antifungal activity, hypoglycaemic, anticancer 51 52

and immunomodulatory properties. 53

This plant gel has been previously employed for the

5

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preparation of size and shape controlled metallic superstructures. However, the potential use of 57

56

an aloe vera plant gel as a bio template for the synthesis of different ZnO –SS is not yet explored 58 60

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in detail.


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Therefore, in this communication, we have employed modified solution combustion method 5

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for the production of self-assembled ZnO – SS using low cost and naturally available aloe vera 6 7

plant gel. Aloe vera plant gel acts as a fuel, surfactant and as a sacrificial bio template. The 10

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overall synthesis time required for the preparation of self- assembled ZnO – SS is less than 20 12

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min. The typical green synthesis involves the simple heat treatment of thoroughly mixed 13 14

aqueous solution of the naturally available aloe vera plant and zinc nitrate at 300 °C. The 17

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prepared samples were further tested for the management of superficial infections caused by M. 19

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furfur yeast. 20 21 2 24

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EXPERIMENTAL METHOD 25 26

All the chemicals used for the synthesis of ZnO - SS are of analytical grade and used 27 29

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without further purification. Zinc nitrate hexahydrate (Zn(NO3)2.6H2O; 99% pure) is procured 31

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from Sigma Aldrich (99 % pure). Aloe vera plant gel is collected according to the previous 32 3

literature reported by Patel et al [35]. The 20 % V/V of pure aloe vera gel is mixed thoroughly 36

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with double distilled water and prepared the mother solution using a magnetic stirrer. The 38

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resulting aloe vera gel solution of different volume is used along with zinc nitrate hexahydrate 39 41

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for synthesis of ZnO - SS. In a typical synthesis, four numbers of concentrations are selected, 43

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one without the use of aloe vera to prepare ZnO where oxyalyldihyrazide is used as a fuel. The 45

4

other three samples are prepared with the concentrations of 50% W/V, 25 % W/V and 16.6 % 46 48

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W/V of zinc nitrate hexahydrate along with above-mentioned mother solution contains aloe vera 50

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gel. The resulting solutions are stirred constantly by using magnetic stirrer for ~10 min. Then 51 52

the obtained solutions are kept in a pre-heated muffle furnace maintained at 300 ± 10 C. 5

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Within few minutes, the solution catches fire and undergoes smoldering type of burning and 57

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ends up with foamy and bulged white powder. The powders are collected, characterized and 58 60

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tested for antifungal activity.



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Microorganism, media and chemicals 5

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Pure culture M. furfur (Strain no: 1374) was obtained from Microbial Culture Collection 6 7

Centre and Genebank (MTCC). The culture was maintained in modified Emmon‟s agar medium 10

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and Saborauds Dextrose Agar (SDA) supplemented with milk. Leeming - Notman agar medium 12

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was used for carrying out antimicrobial studies. The plates were incubated at 32 ± 2C for four 13 15

14

days [36]. 16 17 18 19

In vitro antimalassezial susceptibility assay 2

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Inoculum preparation was done by direct colony suspension method recommended by 24

23

National Committee for Clinical and Laboratory Standards (NCCLS). The disc diffusion assay 25 26

was used to screen the antifungal activity of all the extracts. Inoculum suspension previously 29

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adjusted to 0.5 McFarland standard was swabbed evenly over the sterile LNA agar medium 31

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plates set for the disc diffusion assay. This procedure yielded a yeast stock suspension of 3 x 106 32 34

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cells per mL and produced semi-confluent growth with M. furfur. Sterile 6mm diameter discs 36

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were placed equidistantly round the margin of petridish. 10µl of test samples (1 mgml -1 and 0.5 38

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mgml-1) was dispensed on the discs. Ketoconazole was used as positive reference standard. 39 41

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Negative control was prepared using the same solvent (Ethanol) used to dissolve test sample. 43

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The inoculated plates were incubated at 32 ± 2C for 48 h. Each test was performed in triplicate. 4 46

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The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48

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48 h of incubation. 49 50 51 53

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Evaluation of Minimum Inhibitory Concentration (MIC) by Microbroth Dilution Assay 5

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MICs were determined in 96-well microlitre well plates recommended by the National 56 57

Committee for Clinical Laboratory Standards (NCCLS). The ZnO – SS/ZnO nanoparticles 60

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(without aloe vera) were diluted by two fold serial dilution ranging from


1 mgml-1 - 0.004

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1 2

mgml-1. 5

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100 µl of ZnO – SS solution was transferred into each well. 100 µl of standardized

inoculum suspension (3 x 106 cells/mL) of yeast cells was added to the wells. The plates were 6 7

then incubated at 32 ± 2C for 48 hours. After 48 h of incubation, 15 µl of Iodonitrotetrazolium 10

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(INT) chloride was added in each well and further incubated for 4 h to obtain colour change 12

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from colourless to purple. MICs were determined as the lowest concentration of the drug that 13 15

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prevented the colour change. The standard reference used in these studies was ketoconazole at 17

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the concentration of 0.156 mgml-1. A well was reserved in each plate as growth control and 18 19

blank. The experiment was performed in triplicate under meticulous aseptic conditions [37]. 21

20 2 24

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Fluorescence Microscopy analysis 25 26

100 µL of the inoculums suspension was incubated with 50 µL of minimal dosage of 29

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ZnO – SS which favoured inhibition of yeast cells. The cells were centrifuged at 6000 rpm for 31

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10 min and washed twice with 1x phosphate buffered saline (PBS) and stained with Ethidium 32 34

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bromide and Acridine orange (EB/AO). EB/AO stock solution (100 µg/mL) was prepared in 36

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Milli Q water and stored in dark at -20 C. 37 38 39 40 41 43

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CHARACTERIZATION 4 46

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The surface morphology of ZnO – Superstructures is characterized by scanning electron 48

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microscope (SEM) (Hitachi-3000). Transmission electron microscopy (TEM), High resolution 50

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transmission electron microscopy (HRTEM) and selected-area electron diffraction (SAED) 51 53

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pattern are done using JEOL 2100 HRTEM. The crystal structure and composition of ZnO 5

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superstructures are determined by Shimadzu powder X-ray diffractometer (PXRD) using Cu Kα 56 57

radiation in the 2θ range of 20−80°. The X-ray photoelectron spectroscopy (XPS) of ZnO is 60

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examined using ultra-high vacuum set-up equipped with a high resolution Gamma data-Scienta



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SES 2002 analyzer. Fourier transform infrared (FTIR) spectrum is recorded in absorption mode 5

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with Perkin Elmer spectrometer (Spectrum 1000) along with KBr pellets. The presence of 6 7

organic and inorganic traces and their type of bonding are analyzed using FTIR. UV-Vis 10

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spectrum of the sample is recorded by dispersing the powder in liquid paraffin with the Specord 12

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S600 - 212C205 spectrometer. 13

The optical properties are investigated from Horiba Flurolog

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spectrofluorimeter recorded at room temperature using a xenon lamp as the excitation source. 17

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The excitation wavelength of 325 nm is used. 18 19 20 2

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Each ZnO – SS sample was mixed with stock solution of fluorescent dye just prior to 24

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microscopy and the samples were incubated in the dark for 30 min. Morphology of exposed cells 25 26

was determined under fluorescent microscope after labeling with EB/AO. 27

10 μl of cell

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suspension was placed onto a microscopic slide; covered with a glass cover slip, and was 31

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examined using a Carll Zeiss Fluorescence Microscope (excitation filter BP 490; barrier filter O 32 3

515), at 40x magnification. Observe the fluorescent image under a Fluorescence Microscope [38 36

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– 40]. 37 38 39 41

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RESULTS AND DISCUSSION 43

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SEM and TEM together provide detailed characterization to confirm the nano size and to 45

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analyze the surface morphology / particles size of the prepared ZnO compounds. The size and 46 48

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shape of the superstructures obtained represents the selected high and low magnified SEM 50

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images of the ZnO – SS with and without aloe vera gel (Fig.1 – high magnification and Fig.2 – 51 52

low magnification). Simple decomposition of zinc nitrate in water has been carried out to 53 5

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compare the effect of aloe vera on ZnO nano/micro structure formation. The SEM image of 57

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ZnO prepared without aloe vera gel consists of strongly agglomerated flakes type structure 58 60

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(Fig.1a). Fig. 1b – 1d shows the SEM images for ZnO prepared with varying concentrations of


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aqueous Zn (NO3)3 and aloe vera gel of 50% W/V, 25 % W/V and 16.6 % W/V respectively. 5

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The pictures observed in horizontal line of Fig.1 shows the digitized form of the selected portion 6 7

of the image, selected portion of the image for digitization, image shown with 10 m scale, 10

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image shown with 50/30 m scale and the equivalent naturally existing picture. 1 12 13 15

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The images clearly indicate that the micrographs are composed of several micro/nano 17

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entities namely bowl and platelets. Fig. 1(b) shows the SEM images of the ZnO prepared using 18 20

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50% W/V zinc nitrate with aloe vera gel; shows the presence of well-ordered microstructures 2

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consists of pyramids organized one above the other resembles the group of layered nests. The 24

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other SEM images of the ZnO prepared using different concentrations of aloe vera gel consists 25 27

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of acoron caps and group of lily flowers respectively for 25% W/V and 16.6 % W/V 29

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concentrations of zinc nitrate with aloe vera gel . The SEM image of the ZnO prepared using 30 31

16.6 % W/V concentrated reaction mixture consists of clusters of symmetrically arranged small 32 34

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plates with superstructure resemblance to lily flowers (Fig. 1d). This confirms that the aloe vera 36

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gel acts as a sacrificial bio-template in the formation of ZnO – SS during combustion. To 37 38

support for the formation of ZnO-SS, many number of similar type of superstructures in the 41

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larger area are shown in Fig. 2 (a-d). Fig. 3 (A – D) shows the TEM images of the four samples, 43

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the ZnO without aloe vera shows the loosely bounded particles of irregular shape, while the 4 46

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TEM images with different concentrations of aloe vera shows the increased bounding and flakes 48

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type ordered particles. Further, the nanosize of the particles is supported with HRTEM and 50

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indexed SAED pattern for the sample with highest aloe vera concentration (Fig. 4(A) and (B)) 51 53

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shows well patterned d-spacing of 0.25 nm. 54 5 56 57

Aloe vera gel consists of several multifunctional organic compounds which include 60

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water/moisture, soluble polysaccharides, monosaccharides, proteins, lipids, physterols, amino



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acids, glycoproteins, enzymes, vitamins such as ascorbic acid, complex B: thiamin, riboflavin, 5

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niacin, folic acid carotenoids, tocopherols [41]. Further, it also contains minerals and trace 6 7

elements with some kind of gum materials. The active ingredient in aloe vera extraction is the 10

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polysaccharides. This shows stability in harsh environments associated with high temperature 12

1

and pH. It shows affinity towards water and incorporates strongly in biological structure. When 13 14

zinc nitrate mixed with aloe vera plant gel, the Zn2+ ions will distribute uniformly thereby 17

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forming a three-dimensional polymeric network structure. The resulting polymeric networks 19

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undergo slow decomposition when subjected to heat treatment. Similar work has been reported 20 2

21

by Maliyekkal et al [42], where cellulose is used as a template during the decomposition of 24

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magnesium nitrate in the mixture of urea and glycine to prevent the agglomeration and escape of 25 26

combustion products from the reaction vessel. In addition, the formation of polymer network 27 29

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controls the combustion rate, as a result a well-ordered uniform ZnO – SS. The pictorial form of 31

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mechanism of formation of superstructures is shown in Fig. 5a. Polysaccharides exhibit surface 32 3

/interfacial activity; it has been attributed to the presence of protein impurities. 36

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molecules combine polysaccharide and protein elements. 38

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Macro-

The detailed study is needed to

understand the formation of different ZnO – SS. The complexity and variety of polysaccharides 39 41

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can be explained by two ways, firstly monosaccharides can be linked together in different ways 43

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(1 to 2, 1 to 3, 1 to 4, 1 to 5 and 1 to 6, in an  and  configuration) and secondly due to the 4 45

presence of branched side chains [43, 44]. Many investigators have identified acemannan as the 48

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primary polysaccharide of the aloe vera gel [45 - 47], while few reports mentioned as pectic 50

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substance as the primary polysaccharide [48]. 51

Acemannan with the configuration -(1,4)-

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glycosidic bond is important for its active reaction and pectic substance with amorphous 5

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carbohydrate present in aloe vera with chain of 1,4 linked R-D-galacturonic acid units 56 57

interrupted by 1,2 linked L-rhamnopyranosyl residues in alternative positions acts as a 60

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biopolymer chain having a high anionic nature. During the process, pectin and acemannan


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serves as bio templates to restrict the growth of ZnO structures, but also serves as an assembling 5

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agent to get superstructures. 6

Similarly cellulose, hemicelluloses, chitosan, xylan etc. also

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contributes in stabilization of zinc ions to obtain various morphologies. The nature prefers the 10

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least utilization of energy in any of the self-assemblies. Hence in this method of preparation the 12

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supramolecular structures of ZnO are obtained by natures least energy utilization mechanisms. 13 14 15 17

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Different morphologies are obtained with different aloe vera content. The reason behind 19

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this is the polymeric network formed during the stirring process decomposed in ordered manner 20 2

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when subjected to heat treatment during the combustion synthesis. The effective heat liberated 24

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during combustion process is in the layered manner and it may be interlocked with quantity of 25 26

aloe vera content. At low temperature, the rate of crystal growth is higher than the nucleation. 27 29

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Whereas, with increase in temperature, the nucleation rate gradually increases and overtake the 31

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rate of crystalline growth. Hence, smaller sized and smoother surface structures would be 3

32

produced at higher temperatures. In all the processes, the Zn2+ ions gets stabilized with various 36

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polysaccharide chains by interacting with O(-ve) ions. The overall mechanism involves some 38

37

phenolic compounds, proteins that are bound to the surface of ZnO – SS during stirring process. 39 41

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The stability of ZnO – SS may be due to the free amino and carboxylic groups that have 43

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interacted with the zinc surface. The bonds of functional groups are derived from heterocyclic 45

4

compounds of the protein chains. 46

These functional groups act as capping ligands of the

48

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superstructures. Further, the proteins present in the medium prevent agglomeration and aids in 50

49

the stabilization by forming a coat, covering the ZnO – SS. 51 52 53 5

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The aloe vera gel mainly composed of sugar molecules such as glucose, galactose and 57

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acetylmanose, which are linked into chains of various lengths. The mechanism of incorporation 58 60

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of ZnO particles into the polysaccharide chains can be described by a model called “egg box”



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(Fig.5b). This model consists of polysaccharide molecules that interact with trivalent / divalent 5

4

cations, forming bridges between two carboxyl groups of acetyl groups from two different 6 7

chains comes in close contact [49, 50]. The divalent ions keep the molecules together and form 10

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superstructures along with supportive binding of two or more chains of polysaccharide. This 12

1

mechanism forms the polymeric nature of bindings and their agglomeration further binds the 13 15

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ZnO+ more strongly. This polymeric binding is responsible for the conjunction of all these 17

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families of compounds present in the gel and exhibit a synergistic effect to get the complex 18 19

structures. Further, the interaction of proteins and polysaccharides as attractive or repulsive 2

21

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interaction, the synergistic effect fine tunes. 23 24 25 26

It is well known that ZnO belongs to the p63mc space group and (0001) and (000-1) 29

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planes are rich in Zn (positive polar plane) and O (negative polar plane) respectively. These 31

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planes have different surface energy and thereby the different growth rate [51].The existence of 32 34

3

some water soluble organic compounds (phenols, terpenoids or proteins) present in aloe vera gel 36

35

is adhered to ZnO surface thereby promoting the growth in a specific direction [41, 52, 53]. Fig. 37 38

6 shows the powder X-ray diffraction (PXRD) patterns of ZnO – SS synthesized using various 39 41

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concentration of aloe vera gel. All the diffraction peaks can be indexed to hexagonal wurtzite 43

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structure [JCPDS 89-7102] and space group - p63mc (186) with slight variation in the lattice 4 45

constants from the standard values (Table.1) confirms the formation of crystallite size. Though 48

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the aloe vera gel contains trace elements such as Fe, Cu, Zn, Mn, Al, Se and Cr, the PXRD 50

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pattern does not show any of the impurity peaks corresponding to the trace elements and their 51 52

compounds. Generally, (101) plane is dominant over the other planes. This result well matches 5

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with the previously reported by Sangeetha et al., for the preparation of ZnO nano ZnO – SS 57

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using aloe vera gel as a surfactant via precipitation method [34]. But in the present method of 58 60

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preparation, with increase in the aloe vera content, the (002) plane gets dominate over the other


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planes, shows the change in growth mechanism with concentration of bio template. 5

4

Further it

is observed that the preferred orientation of (002) plane of the ZnO – SS obtained in the presence 6 7

of aloe vera extract is slightly shifted to the lower angle side when compared to without aloe 10

9

8

vera used ZnO product (Fig.6(B)). In a more general way, the intensity depends of the number 12

1

of atom layers correctly ordered perpendicular to the plane considered. It depends on the 13 14

material and its preparation environment. In our case due to the aloe vera gel as a surfactant, the 17

16

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reflections in the plane have almost constant intensities while the reflections due to the stacking 19

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in the c-axis may be very intense if a lot of layers are correctly ordered. As an intermediate 20 2

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situation, the intensity increases while ordering the stacking of the layers is observed with the 24

23

increase in the aloe vera content. From the full widths at half-maximum (FWHMs) of major 25 26

reflection peaks, the sizes of the coherently scattering domains in the „a‟ and „c‟ directions, Da 27 29

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and Dc, are calculated using Scherrer formula. Further, the aspect ratio (AR) = (D a / Dc) is used 31

30

to describe the anisotropy of the crystalline subunits of the agglomerations [51]. AR value 32 3

decreased slightly with increase in aloe vera content and the average crystallite size is found to 36

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be ~11 nm for all the compositions as listed in Table.1. The powder diffraction pattern is 38

37

simulated with the help of the Rietveld‟s refinement software, the FULLPROF suite 2.05 39 41

40

program, by providing necessary structural information (Fig. 7) [54]. The background is 43

42

successfully fitted with a Chebyshev function (2) with a variable number of coefficients 4 45

depending on its complexity and found good match. The detailed outputs of the refinement are 48

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tabulated in Table.1. The X-ray density is observed highest for ZnO prepared with 16.6 % W/V 50

49

zinc nitrate with aloe vera gel content. This may be attributed to the highest electron density for 51 52

this composition of aloe vera. 54

53 5 57

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The XPS of the ZnO prepared using 16.6 % W/V zinc nitrate with aloe vera gel is as 58 60

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shown in Fig. 8. Fig. 8(a) shows the wide range of binding energy from 200 – 1500 eV and Fig.



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8 (b and c) shows the narrow range of O (1S) and Zn (2p). The Zn(2p) XPS spectrum consists 5

4

of Zn 2p1/2 and 2p3/2 doublet peak. Lower binding energy of 1022.2 eV corresponds to the Zn 7

6

2

10

9

8

p3/2 and the highest binding energy of 1045.6 eV corresponds to Zn 2p1/2 [55]. The O (1s)

spectrum of the ZnO contains one peak due to the presence of oxide, hydroxides and adsorbed 12

1

water. The intense peak is at 953.4 eV can be attributed to O2- type of species associated with 13 14

the oxide of Zn, which is in the form of ZnO. The intensity of the O (1s) peak shows that the 17

16

15

amount of oxide associated with Zn is more than the hydroxide confirms that the formed 19

18

compound is ZnO. 20 21 2 24

23

Fig. 9 shows the FTIR analysis of ZnO – SS synthesized by bio template method and 25 26

ZnO prepared by combustion method without using aloe vera content. The FTIR of pure ZnO 27 29

28

is not showing any traces of organic contents except the metal-oxygen bonding at ~ 455 cm-1 but 31

30

the FTIR of ZnO prepared using aloe vera content revealed strong band at 1396 cm -1, however, 32 3

peaks at 455 is related to Zn-O bond vibrational frequencies that support the presence of 36

35

34

hexagonal phases. The bands at 3447 cm-1 show the presence of –OH stretching bonded to the 38

37

Zn-OH. 1732 and 1636 cm-1 is due to traces of amide bonds of proteins/enzymes. Further, the 39 40

bands observed at 1396 and 1040 cm-1 have been assigned to alcohols and phenolic groups, C-N 43

42

41

stretching vibrations of aliphatic and aromatic amines respectively [34]. The overall observation 45

4

proves the existence of traces of some phenolic compounds, terpendoids or proteins that are 46 48

47

bound to the surface of ZnO – SS that remained. 49 50 51 52

The optical properties of ZnO crystallites synthesized in presence of aloe vera are 53 5

54

observed in Fig. 10A. In all the spectra, a strong decrease of reflectance can be noticed in the 57

56

range of 360 - 385 nm corresponds to transitions in ZnO. Based on this reflectance data, the 58 60

59

estimated band gap according to kubela-Munk representation is shown in Fig. 10B. The band


Page 15 of 45


1 3

2

gap values are varying between 2.92 – 3.08 eV. The highest band gap of 3.08 eV is observed for 5

4

ZnO prepared using 16.6 % W/V zinc nitrate with aloe vera gel [49]. 6 7 8 10

9

Photoluminescence (PL) study is an effective technique to assess both defects as well as 12

1

optical property for possible applications in device fabrication. Fig.11 presents the 13 14

photoluminescence spectra of the ZnO – SS synthesized using different concentrations of aloe 17

16

15

vera gel with an excitation wavelength of 325 nm. PL spectra display two emission bands, the 19

18

ultraviolet (UV) emission and the broad visible band. The UV peak attributed to the near-band20 2

21

edge (NBE) emission, and the broad visible band is related to the deep-level defects formed by 24

23

vacancies and interstitials of zinc or oxygen in ZnO [56, 57]. Most of the ZnO nano/micro 25 26

structures synthesized by wet chemical methods including hydrothermal, precipitation, sol gel, 27 29

28

and solution combustion and so forth exhibit broad blue, yellow or green emission [58]. 31

30

However, ZnO – SS synthesized using aloe vera plant gel exhibit unique and strong orange 32 3

emission centered at 600 nm. It is widely accepted that the visible luminescence mainly 36

35

34

originates from defect states such as Zn interstitial and oxygen vacancies. Thus, the peak at 600 38

37

nm is assigned to the electron transition from interstitial zinc to an interstitial oxygen (Oi) level 39 41

40

[59]. It is important to mention that the ZnO – SS prepared by precipitation method in the 43

42

presence of aloe vera plant gel exhibit green emission centered at 520 nm. Whereas the present 45

4

study clearly suggests that the method of synthesis and type of nanostructure exhibit different 46 48

47

optical properties. Further it is observed that the emission intensity of ZnO – SS increases with 50

49

increase in volume of aloe vera. As shown in Fig. 11, the intensity ratio of the UV emission to 51 52

the defect- related emission increases with respect to the rise in the aloe vera content except for 53 5

54

16.6 % W/V zinc nitrate with aloe vera gel. This confirms the higher aspect ratio and better 57

56

crystallinity for ZnO prepared using 16.6 % W/V zinc nitrate with aloe vera gel. ZnO prepared 58 60

59

using 16.6 % W/V zinc nitrate with aloe vera gel concentration shows the highest PL intensity



Page 16 of 45

1 3

2

and for the same compound we observed the optimization in its SEM morphology, X-ray 5

4

density, wide energy band gap and pure form of compound. 6 7 8 10

9

Qualitative and quantitative results determined using disc diffusion and broth 12

1

microdilution methods are presented as average values in Tables 2. The preliminary screening 13 14

results of ZnO without aloe vera by disc diffusion showed the zone of inhibition of (10.2 ± 0.33) 17

16

15

mm at 1 mg.ml-1. M. furfur showed different degrees of sensitivity to different tested ZnO – SS 19

18

and ZnO without Aloe vera (Fig. 12). Fig.12 A for 50 % W/V zinc nitrate with aloe vera gel 20 2

21

concentration, Fig. 12B for 25% W/V zinc nitrate with aloe vera gel concentration and Fig.12C 24

23

shows the ZnO – SS obtained from 16.6 % W/V zinc nitrate with aloe vera gel concentration. 25 26

Among them Fig.12C showed significantly high inhibitory effect against the growth of M. furfur 27 29

28

with MIC 0.028 mg/ml. The mean inhibition zone of all the tested ZnO – SS ranged from 11 to 31

30

23 mm indicating a remarkable antimalassezial effect when compared with that of ketoconazole 32 3

with 24 mm tested at 0.125 mg/ml. To determine the MIC, concentrations of the ZnO – SS 36

35

34

varying between 1 mgml-1 and 0.04 mgml-1 were tested. On 96-well microdilution followed by 38

37

conformational streaking, it was found that ZnO without Aloe vera exhibited higher MIC of 39 41

40

0.500 mg/ml which was relatively high compared with ZnO-SS which showed MIC range of 43

42

0.008 to 0.063 mg/ml when tested at different concentrations of zinc nitrate with aloe vera gel. 45

4

The pronounced antifungal activity of ZnO-SS can be due to its relatively small size and high 46 48

47

surface to volume ratios compared with Zinc oxide without aloe vera. The present study clearly 50

49

signifies the potentiality of ZnO-SS as antifungal agents over ZnO without Aloe vera gel. Table 51 52

2 and Fig. 13 show the different zone of inhibition with MIC and MFC of the extracts against the 53 5

54

organism. 56 57 58 59 60


Page 17 of 45


1 3

2

To know the morphological changes that could be attributed to antimalassezial activity. 5

4

This is done by the inclusion-exclusion using fluorochromes viz Acridine Orange 6 7

(AO)/Ethidium bromide (EB) dyes. Live and Dead cells were identified by nuclear condensation 10

9

8

of chromatin stained by AO (green colour) or EB (orange colour), respectively; thereby 12

1

indicating cell membrane damage. Dead cells were identified by uniform labelling of the cell 13 14

with EB. Live cells were dispersed and alive with normal nuclei staining which present 17

16

15

yellowish to green chromatin (Fig. 15D) with organized structures. The yeast cells treated with 19

18

ZnO - SS have similar normal nuclei staining as live cells except the chromatin is orange instead 20 2

21

of green showcasing the cell aggregation (Fig. 15A, 15B and 15C) suggesting that ZnO - SS 24

23

have an immense potential as antifungal agent . 25 26 27 29

28

CONCLUSIONS 31

30

Aloe vera gel assisted solution combustion method is fast, simple, convenient, 32 3

economical, and environmentally benign method compared to the previously reported 36

35

34

conventional solution combustion syntheses. The ZnO without aloe vera is very reluctant to the 38

37

antifungal activity, whereas ZnO –SS with different aloe vera concentration showed the better 39 41

40

antifungal activity for very least concentration of about 0.5 mg/ml. The synthesized ZnO - SS 43

42

show highest band gap and purest form when synthesized with 16.6 % W/V zinc nitrate with 45

4

aloe vera gel concentration. 46

The synthesized ZnO - SS exhibits unique orange emission at 600

48

47

nm. The yeast cells treated with ZnO - SS have similar normal nuclei staining as live cells 50

49

except the chromatin is orange instead of green showcasing the cell aggregation suggesting that 51 52

ZnO - SS have an immense potential as antifungal agent. 53

Hence, the explored method of

5

54

preparation shows high efficient ZnO -SS derived from the aloe vera plant gel is expected to 57

56

have extensive applications in medicine, biomedical and in cosmetic industries. 58 59 60



Page 18 of 45

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2

ACKNOWLEDGEMENT 5

4

The author Dr. H Nagabhushana thanks to DST Nano Mission (Project No. SR/NM/NS7

6

48 /2010) New Delhi for the sanction of this Project. 8 9 10 1 12 13

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Page 26 of 45

1 3

2 TABLE CAPTIONS

5

4

Table.1. Crystal Data of ZnO for different aloe vera gel: JCPDS No. 89-7102.

7

6

Table.2.Antifungal activity of ZnO -SS as determined by the agar diffusion method (diameter in mm) and their Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) analyzed by Microbroth dilution method

1

10

9

8

FIGURE CAPTIONS Figure. 1 SEM images of the ZnO - SS synthesized using aloe vera gel of different concentration. { (a) without aloe vera gel (b) 50 % W/V zinc nitrate with aloe vera gel. (c) 25 % W/V zinc nitrate with aloe vera gel and (d) 16.6 % W/V zinc nitrate with aloe vera gel. Figure. 2 Large area SEM images of ZnO - SS obtained using various concentration of aloe vera gel. (a) without aloe vera gel (b) 50 % W/V zinc nitrate with aloe vera gel (c) 25 % W/V zinc nitrate

17

16

15

14

13

12

with aloe vera gel and (d) 16.6 % W/V zinc nitrate with aloe vera gel.

19

18

Figure. 3 TEM images of ZnO prepared (A) without aloe vera gel (B) 50 % W/V (C) 25 % W/V and (D) 16.6 % W/V zinc nitrate with aloe vera gel.

20 21

Figure. 4 (A) HRTEM and (B) SAED pattern of ZnO prepared with 16.6 % W/V zinc nitrate with aloe vera gel.

23

2

Figure.5 (A) Pictorial representation of contents of aloe vera gel and ZnO formation due to the trapping of Zn 2+ with polysaccharide chains (B) pictorial representation of ZnO - SS formation according to the egg box model in the environment of aloe vera extract.

26

25

24

27 Figure.6 PXRD pattern of ZnO products with various concentrations of aloe vera gel content (A) the 2 range from 20 – 80  and (B) The 2 specific range of 34 – 37 .

29

28 30

Figure.7 Rietveld refined profiles for ZnO with different aloe vera content.

31 32

Figure.8 XPS spectra (A)Wide range of binding energy spectrum (B) O (1s) and (C) Zn (2p) elements of prepared ZnO using 16.6 % W/V zinc nitrate with aloe vera gel..

34

3 35

Figure. 9 FTIR of synthesized ZnO - SS with various concentrations of aloe vera gel.

36 37

Figure.10 UV Visible spectra and energy band gap of ZnO - SS.

38 39

Figure. 11 PL intensity of ZnO with different aloe vera concentration.

41

40

Figure. 12 Antimalassezial activity of ZnO - SS on dermatologically prevalent yeast M. furfur (A) 50 % W/V Zinc nitrate and aloe vera gel, (B) 25 % W/V Zinc nitrate and aloe vera gel and (C) 16.6 % W/V Zinc nitrate and aloe vera gel (D) ZnO without aloe vera gel[ 1- positive control, 2-negative control, 3 – 0.5 mg/ml of ZnO and 4 – 1 ml /mg of ZnO –SS.

46

45

4

43

42

Figure. 13 Determination of Minimum Inhibitory Concentration (MIC) of ZnO – SS on M. furfur by 96-well plate method (A) - ZnO obtained using 50 % W/V zinc nitrate with aloe vera gel; (B) – ZnO obtained 25 % W/V zinc nitrate with aloe vera gel and (C) - ZnO obtained using 16.6 % W/V zinc nitrate with aloe vera gel [B– Blank; GC– Growth Control; PC– Positive control; 1- 1; 2– 0.5 ; 3– 0.25 ; 4– 0.125; 5-0.063; 6-0.032; 7-0.016; 8-0.008; 9-0.004(mg/ml)]

54

53

52

51

50

49

48

47

Figure. 14 Determination of MIC and MYC of ZnO- SS on M. furfur by steak plate method. [B– Blank; GC– Growth Control; PC– Positive control; 1- 1; 2– 0.5 ; 3– 0.25 ; 4– 0.125; 5-0.063; 60.032; 7-0.016; 8-0.008; 9-0.004(mg/ml)]

60

59

58

57

56

5

Figure. 15 Fluorescence imaging of M. furfur treated with (A) ZnO (@ 50 % W/V zinc nitrate with aloe vera gel; (B) ZnO (@ 25 % W/V zinc nitrate with aloe vera gel and (C) ZnO (@ 16.6 % W/V zinc nitrate with aloe vera gel and (D) cells without treatment.


Page 27 of 45


1 2 3 4 5 6 7 Table.1. Crystal Data of ZnO for different aloe vera gel: JCPDS No. 89-7102.

8 9 10

Crystal structure Space group Hall symbol Lattice Parameters (Å)

Hexagonal P63mc (186) P 6c -2c a = 3.2462 c = 5.2007

ZnO (@ 50 % W/V zinc nitrate with aloe vera gel.) Hexagonal P63mc (186) P 6c -2c a = 3.2458 c = 5.1999

2

0.926 3.370 4.320 4.390 4.740 47.463

0.974 3.260 3.410 4.280 4.390 47.44

1.094 3.260 3.570 4.330 3.958 47.47

1.171 5.350 11.30 9.380 8.010 47.00

5.717 11.49

5.720 11.37

5.068 11.34

5.728 11.40

2.33

2.32

2.28

2.26

1

ZnO (Without Aloe vera) 12 13

19

18

17

16

15

14

20

23

2

21

Rp RBragg RWP Rexp Volume of unit cell / formula unit (Å3) X-ray density (g/cm3) Average crystallite size (nm) (From Scherrer formula) Aspect ratio in a and c directions (Da/Dc) 37

36

35

34

3

32

31

30

29

28

27

26

25

24

38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


ZnO (@ 25 % W/V zinc nitrate with aloe vera gel.) Hexagonal P63mc (186) P 6c -2c a = 3.2467 c = 5.2010

ZnO (@ 16.6 % W/V zinc nitrate with aloe vera gel.) Hexagonal P63mc (186) P 6c -2c a = 3.2451 c = 5.1993


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1 2 4

3 Table.2.Antifungal activity of ZnO -SS as determined by the agar diffusion method (diameter in mm) and their Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) analyzed by Microbroth dilution method

7

6

5

Sl.No.: Nanoparticle screened

8 9

Zone of Inhibition (mm) 1 mgml-1

0.5 mgml-1

1 ZnO (@ 50 % W/V zinc nitrate with aloe vera gel

13.625 ± 0.13

11.625 ± 0.13

2 ZnO (@ 25 % W/V zinc nitrate with aloe vera gel

22.5 ± 0.25

14.875 ± 0.13

ZnO (@ 16.6 % W/V zinc nitrate with aloe vera gel 4 ZnO without aloe vera gel *Nd - not detected

23.25  0.25 10.2 ± 0.33

10 1 12

16

15

14

13

Ketoconazole (Positive control) (0.125 mgml 1)

MIC mgml-1

MFC mgml-1

Ethanol (Negative control ) 0.008

0.016

0.063

0.125

17.5  0.25

0.066

0.132

------

0.500

1.000

2

21

Nd

20

19

18

24.00 ± 0.0

17

23 25

24 3

31

30

29

28

27

26

32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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1 2 3

(a) 4 5 6 7 8 9

(b) 10 1 12 13 14 16

15

(c) 17 18 19 20 21 2

(d) 23 24 25 26 27 28 29

Figure. 1 SEM images of the ZnO - SS synthesized using aloe vera gel of different concentration. 31

30

{ (a) without aloe vera gel (b) 50 % W/V zinc nitrate with aloe vera gel. (c) 25 % W/V zinc nitrate with aloe vera gel and (d) 16.6 % W/V zinc nitrate with aloe vera gel.

32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60



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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 3

32

Figure. 2 36

35

34

Large area SEM images of ZnO - SS obtained using various concentration of aloe vera gel. (a) without aloe vera gel (b) 50 % W/V zinc nitrate with aloe vera gel (c) 25 % W/V zinc nitrate with aloe vera gel and (d) 16.6 % W/V zinc nitrate with aloe vera gel.

37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


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1 2 3 (A)

4

(B)

5 6 7 8 9 10 1 12 13 14 15 16 17 18 20

19 ( C)

21

(D)

2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 38

37 Figure. 3 TEM images of ZnO prepared (A) without aloe vera gel (B) 50 % W/V (C) 25 % W/V and

40

39

(D) 16.6 % W/V zinc nitrate with aloe vera gel.

41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 4

3

(A ) 5

(B)

6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 26

25 Figure. 4 (A) HRTEM and (B) SAED pattern of ZnO prepared with 16.6 % W/V zinc nitrate with aloe vera gel.

27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 32

31 Figure.5 (A) Pictorial representation of contents of aloe vera gel and ZnO formation due to the trapping of Zn 2+ with polysaccharide chains (B) pictorial representation of ZnO - SS formation according to the egg box model in the environment of aloe vera extract.

35

34

3

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1 2 3 4 5

(i) ZnO @ 0 % W/V aloe vera gel

JCPDS No.: 89-7102

(A)

19

18

17

16

15

14

13

(202)

(004)

(iv)

(103)

(100)

(iv) ZnO @16.6% W/V aloe vera gel

(200) (112) (201)

12

1

Intensity (a.u.)

10

9

(iii) ZnO @ 25 % W/V aloe vera gel

(110)

8

(102)

7

(002) (101)

(ii) ZnO @ 50 % W/V aloe vera gel

6

(iii)

(ii)

20 21 2 23

(i) 24 25

30 26

40

27

50

2 (Degree)

60

70

80

28 29 30

(B) 31


JCPDS No.: 89-7102

(ii) ZnO @ 50 % W/V aloe vera gel

32 3


4

43

42

41

40

39

38

37

36

Intensity (a.u)

35

34

(iv)


(iii)

(ii)

45 46 47

(i) 48 50

49

34.5 53

52

51

35.0

35.5

2 (Degree)

36.0

36.5

Figure.6 PXRD pattern of ZnO products with various concentrations of aloe vera gel content (A) the 2 range from 20 – 80  and (B) The 2 specific range of 34 – 37 .

5

54 56 57 58 59 60


35


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1 2 3


4

YObserved

4000

5 6

12

1

10

9

Intensity (a.u)

8

YObserved - YCalculated Bragg's positions

3000

1000

13

2000

1000

0

15

YCalculated

Braggs positions

2000

14

YObserved

YObserved - YCalculated

Intensity (a.u.)

3000

7

ZnO @ 50 % W/V aloe vera gel

4000

YCalculated

0

16 -1000

17

30

18

40

50

60

70

-1000

80

30

40

2 (Degree)

19

50

60

70

80

2 (Degree)

20 21

8000


2

YObserved

4000

23

Bragg's positions Intensity (a.u.)

Intensity (a.u.)

30

29

28

27


6000

Bragg's positions

3000

26

YObserved YCalculated


24 25


YCalculated

2000

1000

4000

2000

32

31 0

0

34

3 -1000

35

30

40

50

36

2 (Degree)

60

70

80

-2000 30

40

50

60

70

80

2 (Degree)

37 38 Figure.7 Rietveld refined profiles for ZnO with different aloe vera content.

39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


36

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1 2 3

160000

4 5

140000

6 7

ZnO (wide)

(A)

120000

8 9 Intensity (Cps)

100000

10 1 12 13 14

80000 60000 40000

15 16

20000

17 18

0

19 20

-20000 400

21

600

800

1000

1200

1400

Binding Energy (eV)

2 23 24

3000

25

2200

O (1s)

(B)

26

(C) 2000

2500

27

Zn (2p)

1800

Intensity (Cps)

31

30

29

Intensity (Cps)

2000

28

1500

1600

1400

1200

1000

32 1000

3

500

34

800

35

0 948

36

950

952

954

956

958

960

962

430

Binding Energy (eV)

440

450

460

470

Binding Energy (eV)

37 39

38 Figure.8 XPS spectra (A)Wide range of binding energy spectrum (B) O (1s) and (C) Zn (2p) elements of prepared ZnO using 16.6 % W/V zinc nitrate with aloe vera gel..

41

40 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


37


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1 2 3 4

(B) ZnO @ 16.6% W/V Zinc nitrate and aloe vera gel

5

3447

7

1040

6 8

1636

9 10 1

(C) ZnO @ 25% W/V Zinc nitrate and aloe vera gel

12

21

(B) ZnO @ 50% W/V Zinc nitrate and aloe vera gel

1732

20

19

18

17

16

15

14

Transmittance (a.u.)

13

2 23 24 (A) ZnO @ without aloe vera

25 26 28

27 4000

29

3500

3000

2500

2000

1500

1000

500

30

Wavenumber (cm-1)

32

31 Figure. 9 FTIR of synthesized ZnO - SS with various concentrations of aloe vera gel.

3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


38

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1 2 3

100

4

(II)

(I) 5 6

(A) (B) (C) (D)

2000

80

13

12

1500

15 20

17 18

1000

40

14 16

2

60

F(R)

1

10

9

Reflectance (%)

8

7

250

(A) (B) (C) (D) 500

Without aloe vera gel 50 % W/V Zinc nitrate and aloe vera gel 25 % W/V Zinc nitrate and aloe vera gel 16.6 % W/V Zinc nitrate and aloe vera gel 750

1000

1250

500

1500

0 1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Energy (eV)

Wavelength (nm)

19

Without aloe vera gel 50 % W/V Zinc nitrate and aloe vera gel 25 % W/V Zinc nitrate and aloe vera gel 16.6 % W/V Zinc nitrate and aloe vera gel

20 21 Figure.10 UV Visible spectra and energy band gap of ZnO - SS.

2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


39


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1 2 3 7

4

1.0x10

5

excitation = 325 nm

(D)

6

(A) (B) (C) (D)

Without aloe vera gel 50 % W/V Zinc nitrate and aloe vera gel 25 % W/V Zinc nitrate and aloe vera gel 16.6 % W/V Zinc nitrate and aloe vera gel

6

7

8.0x10

16

15

14

13

12

1

10

PL Intensity (a.u.)

9

8 6

6.0x10

(C) 6

4.0x10

(B) 6

2.0x10

17

(A)

18 0.0

19 21

20 450

2

500

550

600

650

700

750

Wavelength (nm)

23 24 26

25 Figure. 11 PL intensity of ZnO with different aloe vera concentration.

27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


40

Page 41 of 45


1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 35

34 Figure. 12 Antimalassezial activity of ZnO - SS on dermatologically prevalent yeast M. furfur (A) 50 % W/V Zinc nitrate and aloe vera gel, (B) 25 % W/V Zinc nitrate and aloe vera gel and (C) 16.6 % W/V Zinc nitrate and aloe vera gel (D) ZnO – SS without aloe vera content[ 1- positive control, 2-negative control, 3 – 0.5 mg/ml of ZnO and 4 – 1 ml /mg of ZnO –SS.

39

38

37

36

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1

A 3

2

B

(A ) 9

8

7

6

5

4

(B )

10 1 12 13 14 15 16 17 18

(C ) 24

23

2

21

20

19

25 26 27 28 29 30 31 32 34

3 Figure. 13 Determination of Minimum Inhibitory Concentration (MIC) of ZnO – SS on M. furfur by 96-well plate method (A) - ZnO obtained using 50 % W/V zinc nitrate with aloe vera gel; (B) – ZnO obtained 25 % W/V zinc nitrate with aloe vera gel and (C) - ZnO obtained using 16.6 % W/V zinc nitrate with aloe vera gel [B– Blank; GC– Growth Control; PC– Positive control; 1- 1; 2– 0.5 ; 3– 0.25 ; 4– 0.125; 5-0.063; 60.032; 7-0.016; 8-0.008; 9-0.004(mg/ml)]

38

37

36

35

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1 2 3

(B)

(A ) 9

8

7

6

5

4

10 1 12 13 14 15 16 17 18 20

19

(C) 23

2

21

24 25 26 27 28 29 30 31 32 3 34 35 36 38

37 Figure. 14 Determination of MIC and MYC of ZnO- SS on M. furfur by steak plate method.

40

39 [B– Blank; GC– Growth Control; PC– Positive control; 1- 1; 2– 0.5 ; 3– 0.25 ; 4– 0.125; 5-0.063; 60.032; 7-0.016; 8-0.008; 9-0.004(mg/ml)]

41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60


43


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1 2

6

5

4

3

(A)

(B)

(C)

(D)

7 8 9 10 1 12 13 14 15 16 17

21

20

19

18

2 23 24 25 26 27 28 29 30 31 32 3 35

34 Figure. 15 Fluorescence imaging of M. furfur treated with (A) ZnO (@ 50 % W/V zinc nitrate with aloe vera gel; (B) ZnO (@ 25 % W/V zinc nitrate with aloe vera gel and (C) ZnO (@ 16.6 % W/V zinc nitrate with aloe vera gel and (D) cells without treatment.

38

37

36

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1 3

2

For Table of Contents Use Only 4 5 6 7 9

8

ZnO superstructures as an antifungal for effective control of Malassezia furfur, dermatologically prevalent yeast: prepared by aloe vera assisted combustion method 1

10

13

12

D. Kavyashree, C. J. Shilpa, H. Nagabhushana, B. Daruka Prasad, G. L. Sreelatha, S.C. Sharm, S. Ashoka, J. Anandakumari, H. B. Premkumar 15

14 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30

Biogenic route to synthesize ZnO superstructures in short interval of time using natural plant gel: optical and dermatological studies 3

32

31

34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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ZnO Superstructures as an Antifungal for Effective ... - ACS Publications

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