Antimicrobial and antioxidant potentials of biosynthesized colloidal zinc oxide nanoparticles for a fortified cold cream formulation: A potent nanocosmeceutical application

Accepted Manuscript Antimicrobial and antioxidant potentials of biosynthesized colloidal zinc oxide nanoparticles for a fortified cold cream formulation: A potent nanocosmeceutical application

S. Sonia, H. Linda Jeeva Kumari, K. Ruckmani, M. Sivakumar PII: DOI: Reference:

S0928-4931(16)31138-9 doi: 10.1016/j.msec.2017.05.059 MSC 8041

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

2 September 2016 27 April 2017 10 May 2017

Please cite this article as: S. Sonia, H. Linda Jeeva Kumari, K. Ruckmani, M. Sivakumar , Antimicrobial and antioxidant potentials of biosynthesized colloidal zinc oxide nanoparticles for a fortified cold cream formulation: A potent nanocosmeceutical application, Materials Science & Engineering C (2017), doi: 10.1016/j.msec.2017.05.059

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ACCEPTED MANUSCRIPT

Antimicrobial and antioxidant potentials of biosynthesized colloidal zinc oxide nanoparticles for a fortified cold cream formulation: a

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potent nanocosmeceutical application

Division of Nanoscience and Technology, Bharathidasan Institute of Technology, Anna University,

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a

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S. Sonia a,1, H. Linda Jeeva Kumari b,c,1, K. Ruckmani b,c,*, M. Sivakumar a,**

b

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Tiruchirappalli 620024, Tamil Nadu, India

National Facility for Drug Development (NFDD) for Academia, Pharmaceutical and Allied Industries,

c

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Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil Nadu, India Department of Pharmaceutical Technology, Centre for Excellence in Nanobio Translational Research

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(CENTRE), Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil

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Nadu, India

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* Corresponding author at: Department of Pharmaceutical Technology, Centre for Excellence in Nanobio Translational Research (CENTRE), Bharathidasan Institute of Technology, Anna University,

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Tiruchirappalli 620024, Tamil Nadu, India. Tel.: +91-431-2407978; fax: +91-431-2407910.

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** Corresponding author at: Division of Nanoscience and Technology, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620024, Tamil Nadu, India. Tel.: +91-431-2407959. E-mail addresses: [email protected] (K. Ruckmani), [email protected] (M. Sivakumar). 1

Contributed equally

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ACCEPTED MANUSCRIPT ABSTRACT Nanocosmeceuticals are promising applications of nanotechnology in personal care industries. Zinc oxide is an inorganic material that is non-toxic and skin compatible with selfcleansing and microbicidal properties. Herein, the exploitation of colloidal zinc oxide

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nanoparticles (ZnONps) as potent biomaterial for a topical formulation of cosmetic and

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dermatological significance is employed. ZnONps were green synthesized using environmentally benign Adhatoda vasica leaf extract and characterized by UV–Vis absorption spectroscopy,

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Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), high-resolution transmission electron microscopy (HR-TEM) and energy-

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dispersive X-ray spectroscopy (EDX). The results reveal that the biosynthesized ZnONps exhibit

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an absorption peak at 352 nm. XRD and HR-TEM analyses confirm the hexagonal wurtzite structure of ZnONps with particle size of about 10 nm to 12 nm. Elemental analysis by EDX

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confirms the presence of zinc and oxygen. Zeta potential of −24.6 mV affirms the stability of

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nanoparticles. The antibacterial and antifungal activities of biosynthesized ZnONps exhibit mean zone of inhibition from 08.667 ± 0.282 to 21.666 ± 0.447 (mm) and 09.000 ± 0.177 to 19.000 ±

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0.307 (mm) respectively, in a dose-dependent manner. The IC50 value exerted from the

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antioxidant activity of ZnONps is found to be 139.27 µg mL-1. ZnONps infused cold cream formulation of microbicidal and antioxidant properties was further tested against clinical skin pathogens. The nano-based cold cream exhibited significant inhibitory action against Candida sp., which showed resistance against a commercial antifungal cream (2 %). Therefore, this study demonstrates the exploitation of ZnONps as promising colloidal drug carriers in cosmeceuticals that can significantly alleviate human skin infections and oxidative stress induced cellular damage. 2

ACCEPTED MANUSCRIPT Keywords: Zinc oxide nanoparticles, antibacterial, antifungal, antioxidant, nanocosmeceutical, cold cream 1. Introduction

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Cutaneous infections are one of the major concerns in pharmaceutical and cosmetic

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sectors. The global market involving technologies for the treatment of skin diseases reached

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$17.1 billion in 2015, and is expected to reach $20.4 billion by 2020 [1]. Among the major pharmaceutical companies, innovative therapies for certain skin diseases have renewed interest

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in the market. Novel dermatological and cosmetic formulations are employed with antibacterial

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and antifungal potentials to combat various skin conditions and infections [2]. These formulations comprise of fragrance, oils and fats that can undergo auto-oxidation and chemical

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degradation when exposed to air, thereby becoming odorless. Addition of antioxidants can

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preserve them, increase their shelf life and also protect the skin from cellular damages [3].

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Formulations of biological origin are advantageous over synthetic formulations due to better patient tolerance, cost effectiveness and minimal adverse effects [4]. Several plants have

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been investigated for the treatment of various skin diseases ranging from itching to skin cancer

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[5]. The active phytoconstituents like alkaloids, flavonoids, tannins, saponins, glycosides, coumarins and lignins possess antibacterial, antifungal and antioxidant properties which are requisites for the treatment of skin diseases [6]. Therefore, cream, gel and soap formulations that contain these secondary metabolites of plants are effective in the treatment of various skin disorders caused by microbial infections [7]. Chemical preservatives are used in cosmetics to increase their shelf life by avoiding microbial spoilage [8]. Compared to organic materials, inorganic powders like zinc oxide is 3

ACCEPTED MANUSCRIPT stable and capable of withstanding harsh processes represent a promising alternative to chemical preservatives with microbicidal properties [9]. ZnO is also considered as one of the generally recognized as safe (GRAS) materials for humans [10]. It inhibits bacterial enzymes such as thiol peroxidases, glutathione reductases and dehydrogenases, owing to its pronounced antibacterial

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property [11]. The antifungal activity of ZnO occurs by the deformation of fungal hyphae and

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preventing the development of conidiophores leading to the death of fungal hyphae causing

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cellular destruction [12]. ZnO is used as a promising antioxidant in cosmeceuticals that is capable of penetrating the stratum corneum of skin and provides protection against reactive

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oxygen species (ROS) by inactivating the production of free radicals that damage the skin, and

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also help in promoting cellular repair and healing [13]. The efficacy majorly depends not only on

and the degree of polydispersity [14].

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the concentration of the active substance, but also on the size of the particles, their modification,

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Incorporation of nano-sized particles in cosmetic formulations can improve the stability

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of active constituents like vitamins, unsaturated fatty acids and antioxidants, thereby enhancing their therapeutic value [15]. Nanotechnology is a burgeoning field that has greater significance to

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revolutionize cosmetics and pharmaceuticals. Colloidal drug carriers like nanoparticles have

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tremendous scope in cosmetic and dermatological sectors due to their nanometric size and potential applications [16]. Modern cosmetic-oriented products consist of nano-sized particles, which are envisaged to replace conventional preservatives used in cosmetics [17]. They offer better skin penetration and UV protection, increased color and finish quality, long-lasting effects [18], and also eradicate or control the activity of various microorganisms [19]. Highly ionic nanoparticulate metal oxides such as zinc oxide nanoparticles (ZnONPs) are promising biomaterials in nanomedicine that can be produced with larger surface areas and 4

ACCEPTED MANUSCRIPT longer shelf life, and has synergistic effect with organic antimicrobial agents [17]. They are biocompatible to human cells [20], whereas, toxic towards a wide range of micro-organisms like bacteria [21] and fungi [22]. The antimicrobial activity of colloidal ZnONps has been assessed against human [23] and plant [24] pathogens. Antimicrobial properties of nanoscale ZnO

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particles have been the focus of various industrial applications as biocides coating in water

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treatment, paints and cosmetics [25]. The risk for humans from the usage of ZnONps in various

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sunscreens or cosmetics is considered negligible when used at concentrations upto 25 % and in vitro genotoxic and photogenotoxic profiles of ZnONps results in no consequence to human

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health [20].

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Eco-friendly synthesis of metal and metal oxide nanoparticles is preferred over chemical

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methods since it is a simple, non-toxic and cost-effective approach [26]. Colloidal ZnONps have been green synthesized using extracts of various parts of plants or trees like Ruta graveolens

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[27], Camellia sinensis [28], Cassia fistula [29], Pithecellobium dulce [19], Lagenaria siceraria

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[19], Calotropis procera [21], Parthenium hysterophorus [24] and Rosa canina [30].

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Adhatoda vasica Nees (Acanthaceae), also known as Malabar nut tree is a native of South Asia which is being widely used in the Indian traditional medicine for the treatment of various

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respiratory disorders like asthma, chronic bronchitis and tuberculosis [31]. The leaves of A.vasica are also being used in Ayurvedic medicine exclusive for the treatment of various skin related diseases [32]. These medicinal qualities of A. vasica attribute to their active phytoconstituents like alkaloids, polysaccharides, vitamin C, polyphenols, proteins, glycosides, quinines, flavones, coumarins, triterpenes and essential oils. The principal constituents are quinazoline alkaloids and the chief alkaloid is vasicine [33]. Alkaloids have excellent antibacterial [34], antifungal [35] and antioxidant [36] properties. Such bioactive compounds act 5

ACCEPTED MANUSCRIPT as reducing, capping and stabilizing agents in the synthesis of nanoparticles and also render biocompatible functionalities to the nanoparticles for various biomedical applications [26]. A.vasica is used in the synthesis of silver nanoparticles and assessed for antibacterial and antifungal [37], larvicidal [38] and electrochemical [39] activities. Herein, we report an eco-

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friendly synthesis of colloidal ZnONps by the biological reduction of Zn2+ ion using A. vasica

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aqueous leaf extract. Their physicochemical characterizations and evaluation of antibacterial,

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antifungal and antioxidant properties have been performed and exploited for a potent nano-based

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cosmeceutical application. 2. Materials and methods

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2.1. Materials

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Zinc acetate dihydrate [Zn(CH3COO)2.2H2O] and DPPH (2,2-diphenyl-1-picrylhydrazyl)

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were purchased from Hi-media laboratories, Mumbai, India and was used as received. All other reagents were of analytical grade. Deionized (Milli-Q) water was used throughout the

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experiments. Nutrient broth (NB), Mueller Hinton agar (MHA), Sabouraud dextrose broth (SDB)

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and sabouraud dextrose agar (SDA) used for the cultivation of bacteria and fungi were obtained from Hi-media laboratories, Mumbai, India. Standard bacterial and fungal strains of clinical

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significance such as Staphylococcus epidermidis MTCC 435 (equivalent ATCC 155), Escherichia coli MTCC 443 (equivalent ATCC 25922), Pseudomonas aeruginosa MTCC 741 (equivalent ATCC 25668), Aspergillus fumigatus MTCC 6594, Trichophyton rubrum MTCC 296 and Microsporum audouinii MTCC 8197 were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. Clinical isolates Staphylococcus sp., Streptococcus sp., Candida sp. and Fusarium sp. were obtained from a local hospital. 6

ACCEPTED MANUSCRIPT 2.2.

Preparation of extract and phytochemical analyses The fresh leaves of Adhatoda vasica were harvested in the month of September from

Tiruchirappalli, Tamil Nadu, India, and were authenticated at the Department of Botany, St. Joseph’s College of Arts and Science, Tiruchirappalli. The leaves were washed thoroughly twice

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and dried. 10 g of finely cut leaves were weighed and boiled with 100 mL of deionized water at

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100°C for 10 min. The residues were removed by Whatman no.1 filter paper. The filtrate was cooled down and qualitatively analyzed for various phytochemical constituents by standard

Biosynthesis of colloidal ZnONps

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2.3.

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procedure [40]. The extract was further used for the synthesis of ZnONps.

ZnONps were synthesized by adding 1 mL of A. vasica aqueous leaf extract to

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Zn(CH3COO)2.2H2O solution (0.1 M, 50 mL) with rapid stirring for 10 min at room temperature.

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The pH was adjusted to 10 with NaOH and the mixture was continued to stir at 400 rpm for 1 h. The formation of ZnONps was observed visually by color change. The synthesized colloidal

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ZnONps were also subjected to phytochemical analyses as mentioned above to confirm the

mechanism.

UV-Vis absorption spectroscopy

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2.4.

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phytoconstituents that act as stabilizing and capping agents responsible for the reduction

The colloidal ZnONps were characterized by UV-Vis absorption spectroscopy (Shimadzu/UV-2000, Japan) with wavelength range between 200 and 800 nm to confirm the reduction of Zn2+ ions. Then, the obtained nanoparticles were centrifuged thrice at 7,500 rpm for 10 min at 4 0C by re-dispersing in milliQ water repeatedly to remove any uncoordinated

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ACCEPTED MANUSCRIPT biomoieties. This process of purification was performed to ensure better separation of ZnONPs. The obtained pellet was air dried and used for further characterizations. 2.5.

Fourier transform infrared spectroscopy (FTIR)

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FTIR spectral studies were performed to identify the possible biomolecules responsible for

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the reduction and stability of the metal oxide nanoparticles. FTIR spectra of A. vasica leaf extract

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and the biosynthesized ZnONps were recorded by FTIR spectrophotometer (JASCO/FTIR-6300, Japan). The samples were prepared as KBr pellets in the ratio 1:100 using hydraulic pellet press

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at a pressure of 10 tons for 5 min. The prepared pellet was fitted in the sample holder and

X-ray diffraction (XRD)

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2.6.

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scanned from 4000 cm-1 to 400 cm-1 with a scan speed of 1 cm s-1.

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XRD is a versatile, non-destructive analytical method for the detection and quantitative determination of crystalline phases. Powder XRD data were collected via Philips PW 1710

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diffractometer with CuKα radiation (λ = 1.5406 A⁰) and graphite monochromator, operated at 45

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kV; 30mA and 25 ⁰C. The crystallite size was calculated from the width of XRD peaks using

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Debye- Scherrer’s formula,

……………………………………………………………………….(1)

where, D is the average crystallite domain size, λ is the X-ray wavelength, β is the full width at half maximum, and θ is the diffraction angle. 2.7.

Dynamic light scattering (DLS)

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ACCEPTED MANUSCRIPT Particle size and zeta potential analyses were carried out by means of DLS using Zeta sizer Nano ZS series (Malvern, UK). The purified ZnONps were dispersed in deionized water and the measurements were recorded. The zeta potential analysis was also performed to confirm the stability and homogeneity of the synthesized ZnONps.

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2.8. Morphological studies

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Size and structural conformations of the biosynthesized ZnONps were characterized by Phillips TECHNAI FE 12 high-resolution transmission electron microscopy (HR-TEM) operated

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at 190 V of 200 kV and the phase analysis was performed by selected area electron diffraction

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(SAED) pattern.

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2.9. Elemental analysis

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The elemental analysis of ZnONps was performed by energy-dispersive X-ray

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spectroscopy (EDX) using HITACHI-S3400N.

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2.10. Evaluation of antimicrobial activities The antimicrobial action of ZnONPs was studied using standard and clinical microbial

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strains. Well diffusion technique was followed to perform antimicrobial susceptibility test as described in our previous study [41]. MHA and SDA were used for the cultivation of bacteria and fungi respectively. Briefly, freshly grown overnight bacterial cultures were used, whereas, fungal cultures were grown in SDB at 25 °C for 72 h. The turbidity of suspensions was adjusted to 0.5 McFarland standards (1×108 CFU mL−1 and 1×106 CFU mL−1 for bacterial and fungal cultures respectively). 100 µL of them were spread plated uniformly on 20 mL of solidified and dried agar plates. Wells were punctured at equidistance using sterile cork borer of diameter 8 9

ACCEPTED MANUSCRIPT mm. ZnONps at a stock concentration 100 µg mL−1 was prepared in sterile Milli-Q water, dispersed and ultrasonicated to obtain a homogeneous solution. Streptomycin and fluconazole (10 µg) were used as positive controls for bacterial and fungal strains respectively. ZnONps were dispensed into the wells to obtain 5, 10, 15 and 20 µg per well. Sterile Milli-Q water was used as

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negative control. The plates were incubated at 37 ⁰C for 24 h and upto a week for bacterial and

performed in triplicate.

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2.11. Statistical analysis and graphical representation

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fungal cultures respectively. The zone of inhibition was measured in mm. The assays were

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All the results were expressed as mean ± standard deviation (SD). The data of antibacterial and antifungal activities of ZnONps were analyzed for statistical significance using

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student’s unpaired t-test. The analysis was carried out using statistical package of social sciences

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(SPSS software, Version 14.0, Chicago, ILA, USA). The percentages of antibacterial and antifungal efficacies of the biosynthesized ZnONps on standard and clinical bacterial and fungal

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strains were graphically described by a pie-chart representation to depict the highly susceptible

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organisms.

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2.12. Determination of MIC, MBC and MFC The minimum inhibitory concentration (MIC) of ZnONps was further determined by broth macro-dilution method based on Clinical and Laboratory Standards Institute (CLSI) guidelines as reported [42]. ZnONps were prepared at a stock concentration of 1024 µg mL-1. 500 µL of the stock solution was incorporated with 500 µL of NB medium for bacterial cultures or SDB medium for fungal cultures to obtain a concentration of 512 µg mL-1. Two-fold serial dilutions were performed to obtain 256, 128, 64, 32, 16 and 8 µg mL-1. 50 µL of 1 x 106 CFU mL-1 10

ACCEPTED MANUSCRIPT microbial suspensions were transferred to each tube of varying concentrations of ZnONps. Inoculums and media were used to compare the turbidity visually. All the tubes containing bacterial culture were incubated at 37 ⁰C, 250 rpm shaking for 24 h. The tubes containing fungal spores were incubated at 25 ⁰C for a week without shaking. The lowest concentration that

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inhibited the growth of bacteria and fungi determines the MIC. A loopful of culture from each

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tube was inoculated on sterile MHA plates for bacterial cultures incubated at 37 ⁰C for 24 h,

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whereas, for fungal cultures sterile SDA plates were used incubated at 25 ⁰C for a week. The

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lowest concentrations at which 99.9 % bacteria and fungi are killed determine minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) respectively.

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All assays were performed in triplicate.

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2.13. Free radical scavenging activity

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Free radical scavenging is a primary mechanism by which antioxidants inhibit oxidative processes. DPPH radical scavenging assay is a widely used method for evaluating the ability to

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scavenge free radicals generated from DPPH reagent. DPPH, a stable free radical, can be

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reduced by a proton-donating substrate such as an antioxidant causing the decolorization of purple-colored DPPH. The assay was performed using a modified microtitre plate method [43].

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Briefly, 100 µL of the synthesized ZnONps at varying concentrations (10-300 µg in methanol) was added with 100 µL of 0.2 mM DPPH in methanol solution. The mixture was incubated at room temperature in dark for 30 min. Quercetin was used as a standard control. The absorbance was measured at 517 nm in a multimode microplate reader (EnSpire, PerkinElmer, Singapore). The assays were performed in triplicate. The percentage of inhibition at various concentrations was calculated using,

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ACCEPTED MANUSCRIPT % inhibition = (Ac – At)/Ac X100 ...…………………………………………………………….(2) where, Ac is the absorbance of the control and At is the absorbance of the test sample A graph was plotted between the concentration of the sample and percentage of inhibition,

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thereby determining the IC50 value.

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2.14. Nano cream formulation and antimicrobial activity

The biosynthesized colloidal ZnONps were further employed in a cosmetic and

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dermatological formulation because of its antioxidant potential and antimicrobial efficacy against

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bacterial and fungal skin pathogens. The nano-based cold cream formulation was performed as described previously [44]. Briefly, 2 g bees wax and 6 mL liquid paraffin were taken in a beaker

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and heated in a water bath at 70 °C to prepare the oil phase. The aqueous phase was prepared by

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heating 0.1 g of borax in 2 mL of Milli-Q water in another beaker at 50°C. The aqueous phase was transferred to the mixture of bees wax and liquid paraffin. This preparation was performed

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in duplicates for testing two concentrations. At room temperature, the synthesized colloidal

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ZnONPs were added and continuously stirred until the solution becomes completely homogeneous. These oil-in-water emulsions resulted as 1% and 2% nano-based cold cream

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formulations. These formulations were stored in airtight containers for further studies. The antimicrobial activity of the formulated cold cream was assessed similarly as mentioned in section 2.9 with slight modifications. As the creams were semi-solid in nature, we adopted to check the efficacy of formulations by streaking onto the cultured plates with a sterile inoculation loop instead of well puncture technique. The zone of inhibition was visually observed around the streak and compared with the standard controls. Commercial antibacterial and antifungal creams

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ACCEPTED MANUSCRIPT (2 %) were used as standard controls for bacterial and fungal strains respectively. Cream base without ZnONps served as a negative control. All experiments were performed in triplicate. 3. Results and discussion Phytochemical analyses and mechanism of ZnONps formation

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3.1.

A. vasica aqueous leaf extract shows the presence of alkaloids, glycosides, phenols,

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reducing sugars, lignins, proteins, carbohydrates, steroids and tannins. The biosynthesized

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ZnONps also revealed the presence of alkaloids, glycosides, phenols, reducing sugars and proteins. These secondary metabolites possess antimicrobial and antioxidant properties, and also

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act as reducing, capping and stabilizing agents for the efficient reduction of zinc ion to metallic

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ZnONps. Vasicine, the chief alkaloid present in A. vasica leaf extract might play a vital role in the reduction of metal oxide precursor Zn(CH3COO)2.2H2O forming ZnONps. The plausible

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mechanism of reaction (Fig. 1A) is illustrated as follows: ZnONPs typically have neutral

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hydroxyl groups attached to their surface that play a key role in their surface change behavior. Generally, the synthesis of ZnONps from aqueous solutions starts with a reaction between Zn2+

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and OH− ions followed by an aggregation process. In aqueous medium and basic pH, the

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chemisorbed protons (H+) are released from the surface of the particles resulting in a negatively charged surface with partially bonded oxygen atoms (ZnO-) (equations 3-5). The colloidal solution is maintained at pH 10 as basic conditions are highly conducive to the direct preparation of ZnO crystals [45]. Therefore, ZnONPs will have strong negative surface charge at basic pH, owing to highly stable nanoparticles, for which, the aqueous leaf extract of A. vasica rich in phytoconstituents act as a reducing and surface stabilizing agents in the formation of ZnONPs. Zn(CH3COO)2.2H2O  Zn2+ + 2CH3COO- + 2H2O…………………………...(3) 13

ACCEPTED MANUSCRIPT Zn2+ +2 OH−  Zn(OH)2………………………………………………………...(4) Zn(OH)2  ZnO− + H2O………………………………………………………...(5) 3.2.

UV Visible spectral analysis

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The synthesized ZnONps are visually observed as pale white colloidal solution due to the excitation of surface plasmon resonance (Fig.1B inset). The UV-Visible absorption spectrum

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(Fig. 1B) exhibits a characteristic absorption peak at 352 nm typical for the formation of

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ZnONps, which can be assigned to the intrinsic band-gap absorption of ZnO due to the electron transitions from valence band to conduction band [29]. The reduction of zinc is due to the

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protons supplied by bioreducing agents like alkaloids and phenols present in the aqueous

FTIR spectral analysis

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3.3.

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environment of A. vasica leaf extract.

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The FTIR spectra of A. vasica aqueous leaf extract and the biosynthesized ZnONps (Fig. 2A) were analyzed for the possible functional groups responsible for the formation of ZnONps.

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The characteristic bands of A. vasica observed at 3430 cm-1 corresponds to stretching of -OH or phenolic groups, band at 2090 cm-1 corresponds to C≡C stretching, band at 1573 cm-1

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corresponds to C-O stretching, band at 1400 cm-1 responsible for acid group present in C=O group, band at 1124 cm-1 corresponds to C-N stretching, and the peak at 689 cm-1 is responsible for C-Cl stretching vibrations. The bio-synthesized ZnONps show all the corresponding bands of plant extract. Additionally, a peak is observed at 514 cm-1 which confirms the existence of metal oxide, this peak in the region between 400 and 600 cm-1 is considered as ZnO [24, 46]. The biosynthesized ZnONps are reduced by A. vasica leaf extract, and are mainly surrounded by

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ACCEPTED MANUSCRIPT phytochemicals with functional groups alcohols, amides, ketones, aldehyde and carboxylic acids. From the FTIR analysis, it can be stated that the hydroxyl and carbonyl groups present in carbohydrates, alkaloids, flavonoids, terpenoids and phenolic compounds are powerful reducing agents and they may be accountable for the bioreduction of Zn2+ ion to metallic ZnONps. The

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proteins and other secondary metabolites are strongly bound to the metal oxide nanoparticles

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capping ZnONps and protecting them from agglomeration, thereby stabilizing the nanoparticles.

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Therefore, the functional biomoieties play dual role in the formation and stabilization of ZnONps

XRD spectral analysis

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3.4.

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in aqueous medium.

The structural properties and purity of ZnONps were analyzed by XRD spectrum. The

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diffraction pattern of ZnONps (Fig. 2B) is consistent with the wurtzite structure of ZnO as

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reported earlier [47]. The diffraction peaks were recorded at 2θ values of 31.75⁰, 34.44⁰, 36.25⁰, 47.54⁰, 56.55⁰, 62.87⁰ and 67.91⁰ corresponding to the crystal phases [100], [002], [101], [102],

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[110], [103] and [112] planes, respectively. The diffraction peaks matched with standard JCPDS

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No. 00-005-0664 confirming the formation of crystalline monoclinic ZnO with face-centered cubic (fcc) geometry. No other impurity phase is observed in the XRD pattern of ZnONps

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indicating the purity of biosynthesized nanoparticles. The crystallite size is found to be about 12 nm as determined by Debye-Scherrer’s formula. 3.5.

Particle size and zeta potential analyses The particle size distribution and zeta potential were measured by DLS method to

determine the hydrodynamic diameters of ZnONps. The mean hydrodynamic diameter of ZnONps is found to be 74.48 ± 1.9 nm (Fig. 3A), confirming the nanometric size of the particles. 15

ACCEPTED MANUSCRIPT Monodispersity is a significant attribute to improve biomedical applications. A low polydispersity index (PDI) indicates the homogeneity of particles [48]. The PDI of synthesized ZnONps is found to be 0.175 signifying that the particles formed are of monodispersive in nature. Zeta potential is a vital parameter to study the surface charge and colloidal stability of

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nanoparticles. The zeta potential of ZnONps is found to be −24.6 mV (Fig. 3B) suggesting the

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formation of highly stable nanoparticles at basic pH as discussed in section 3.1. The negative

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value could be attributed to the capping and stabilizing properties of phytoconstituents present in A. vasica extract. The repulsive forces between the negatively charged particles prevent them

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from agglomeration, leading to a long term stability of ZnONps.

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3.6. Morphological studies

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The HR-TEM micrograph shows that the surface morphology of ZnONps is spherical and

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hexagonal (Fig. 4A). The average particle size is calculated from clearly visible particles with perfect boundaries and is found to be about 10 nm to 12 nm, which is also in agreement with the

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particle size calculated from XRD spectra. A. vasica leaf extract which acts as a reducing agent

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in the synthesis of ZnONps may contribute to the nanometric size of the particles. The SAED pattern of ZnONps (Fig. 4A inset) is recorded by directing the electron beam perpendicular to

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one of the individual nanoparticle. The characteristic bright circular fringes of corresponding (hkl) values is indexed to [101], [100], [002], [110], [103] and [112] of fcc lattice structure similar to that of the XRD pattern, implying the nano-crystalline nature of ZnONps [47]. 3.7. Elemental analysis

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ACCEPTED MANUSCRIPT EDX analysis was performed to determine the elemental composition of ZnONps. The EDX spectrum of ZnONps (Fig. 4B) exhibits strong signals for the presence of Zn (69.07 weight %) and O (30.93 weight %).

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3.8. Antimicrobial activities and determination of MIC, MBC, MFC

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The antibacterial and antifungal effects of ZnONps were evaluated against standard and

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clinical bacterial and fungal skin pathogens. Fig. 5A shows the antibacterial effect of ZnONps at varying concentrations against S. epidermidis MTCC 435, the highest susceptible bacterial strain.

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Fig. 5B shows the antifungal effect of ZnONps at varying concentrations against A. fumigatus

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MTCC 6594, the highest susceptible fungal strain. Fig. 6A shows the antimicrobial effect of ZnONps on various bacterial and fungal skin pathogens. For bacterial strains, the positive control

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streptomycin produced mean zone of inhibition from 08.333 ± 0.279 to 20.333 ± 0.177 (mm),

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and the zone of inhibition for ZnONps ranged between 08.667 ± 0.282 and 21.666 ± 0.447 (mm). For fungal strains, the positive control fluconazole produced mean zone of inhibition from

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09.333 ± 0.237 to 17.000 ± 0.222 (mm), and the zone of inhibition for ZnONps ranged between

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09.000 ± 0.177 and 19.000 ± 0.307 (mm). The antimicrobial activity of ZnONps increased in a dose-dependent manner against all the test organisms. There was no activity observed for the

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negative controls.

The MIC and MBC values of ZnONps ranged from 16 to 128 µg mL-1 and 16 to 256 µg mL-1 against bacterial pathogens respectively (Table 1). For fungal pathogens, MIC and MFC values were from 32 to 256 µg mL-1 and 32 to 512 µg mL-1 respectively. For antibacterial activity, ZnONps showed the highest mean zone of inhibition 21.666 ± 0.447 (mm) and lowest MIC (16 µg mL-1) and MBC (16 µg mL-1) against S. epidermidis. For antifungal activity,

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ACCEPTED MANUSCRIPT ZnONps showed the highest mean zone of inhibition 19.000 ± 0.307 (mm) and lowest MIC (32 µg mL-1) and MFC (32 µg mL-1) against A. fumigatus. The MBC/ MFC ranges were two-fold higher than MIC values indicating the bacteriostatic/ fungistatic effects of ZnONps respectively due to the bound phytochemicals responsible for capping and stabilizing effects [49].

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Exceptionally, bacteria (S. epidermidis, Staphylococcus sp.) and fungi (A. fumigatus, Candida

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sp.) exhibit bactericidal and fungicidal effects respectively. However, the results show that the

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synthesized ZnONps are effective bacteriostatic, fungistatic, bactericidal and fungicidal agents claiming its broad spectrum of antimicrobial action [23]. This is due to the effective antibacterial

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[34] and antifungal [35] activities of alkaloids present in A. vasica that shows activity against a

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wide range of microbes, particularly skin pathogens [32].

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3.9. Mechanism of action

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The synthesized ZnONps strongly inhibit the growth of all test organisms even at very low concentrations (5, 10, 15, 20 µg). Gram-positive Staphylococcus sp. is highly sensitive to

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ZnONps than Gram-negative E. coli, which is in accordance with previous reports [23, 27, 28].

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This is due to the fact that Gram-positive bacteria lack outer membrane and have a thick peptidoglycan layer rich in teichoic acids which are sensitive to ZnONps unlike Gram-negative

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bacteria that have an outer membrane with a thin peptidoglycan layer rich in lipopolysaccharides, lipids and lipoproteins which are quite resistant to ZnONps. However, significant antimicrobial effect is observed against a broad spectrum of micro-organisms including Gram-positive and Gram-negative bacteria, and pathogenic fungi. The mechanism of antibacterial action may be due to their nanometric size and larger surface area that owe greater adsorption onto the surface of bacterial cells and generation of

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ACCEPTED MANUSCRIPT superoxide radical and peroxide anions to inhibit cell growth [23]. Oxygen present in the metal oxide reacts with the sulfhydryl (-S-H) groups on cell wall to eliminate hydrogen atoms as water molecules. This blocks respiration by the formation of R-S-S-R bond with sulfur atoms, thereby inactivates bacterial protein synthesis and DNA replication. ZnONps penetrate through the

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bacterial cell wall, block transport channels and alter membrane permeability, resulting in

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intracellular leakage and disruption of nuclear functions, causing cell death [12]. The secondary

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metabolites of A. vasica like alkaloids and polyphenols that act as reducing and capping agents in the formation of ZnONps also are effective microbicidals [32]. The inhibitory effect of ZnONps

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is pronounced for A. fumigatus, which is in agreement with previous reports [28,46]. The

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antifungal effect of ZnONps may be due to the increased nucleic acid contents resulting from stress response of fungal hyphae. The defense mechanism of fungi against ZnONps also leads to

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increased carbohydrates [12]. Therefore, excessive accumulation of nucleic acids and

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carbohydrates may result in the deformed structures of fungal hyphae, causing fungal cell death.

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3.10. Statistical analysis and graphical presentation

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Statistical analysis is a tool to correlate the antimicrobial efficacy of ZnONps. Student’s t-test analysis was carried out to evaluate the significance of ZnONps (5 µg). Differences at p < is

considered

statistically

significant

when

compared

with

standard

drugs

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0.05

streptomycin/fluconazole at 10 µg (n=3). The highest percentage of antimicrobial effect of ZnONps is recorded against a Gram-positive bacterium S. epidermidis MTCC 435 as 13%, followed by Staphylococcus sp. and fungus A. fumigatus MTCC 6594 as 11 %. Gram-negative bacterium E. coli MTCC 443, fungi T. rubrum MTCC 296 and Fusarium sp. hold 10 %. The percentages of P. aeruginosa MTCC 741, Streptococcus sp. and Candida sp. are 9 % and M. audouinii MTCC 8197 is 8 % (Fig. 7A). 19

ACCEPTED MANUSCRIPT 3.11. Free radical scavenging activity Free radical scavenging activity of ZnONps was assessed by DPPH assay. The freshly prepared DPPH solution exhibited a deep purple color with a maximum absorbance at 517 nm. The color of DPPH solution gradually changed to pale yellow in the presence of ZnONps by

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transferring electron density present at oxygen atom to the odd electron present at nitrogen atom

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in DPPH. The decrease in absorbance with increase in concentration of ZnONps is taken as a measure for radical scavenging activity. Free radical scavenging activity or the antioxidant

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potential of ZnONPs on DPPH radical is found to increase with increase in concentration dosedependently with a maximum inhibition of 78 % at 300 µg mL-1 (Fig. 6B). This is similar to

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standard quercetin that exhibits 80% inhibition at this concentration. The IC50 value for

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biosynthesized ZnONps is found to be 139.27µg mL-1 proving their efficacy in quenching the

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free radicals.

The plausible mechanism of antioxidant activity of ZnONps is as follows: free radicals

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are generated by normal metabolic processes or by external factors that can damage the skin.

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DPPH is a stable lipophilic, nitrogen-centered free radical that acts as hydrogen ion acceptor to oxidize the free radicals. The antioxidant activity of A. vasica leaf extract is reported to be 68 µg

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mL-1 [50]. The significant antioxidant potential of ZnONps is due to the hydrogen donating ability of the active phytochemicals of A. vasica like alkaloids, proteins and phenols that are capped over ZnONps. The hydroxyl group of the principal constituent, vasicine, could donate hydrogen ion to neutralize free radicals and prevent oxidation of lipids, proteins and nucleic acids that could reduce cellular damages caused by oxidative stress. Therefore, A. vasica rich in such antioxidants act as potential reducing agents to obtain functionally superior ZnONps which are further exploited for a cosmetic and dermatological application. 20

ACCEPTED MANUSCRIPT 3.12. Nano cream formulation and antimicrobial evaluation ZnO is widely being used in ointments, creams and lotions as a potential antimicrobial agent in treating various skin infections, and also to protect skin against sunburn and cellular damages caused by UV radiations. As an ingredient in sunscreen, ZnO blocks both UVA (320–

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400 nm) and UVB (280–320 nm) rays of UV light. ZnO is considered to be non-allergenic, non-

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irritating and non-comedogenic. Many sunscreens use nanoparticles of ZnO as they appear transparent by not reflecting light, therefore they are cosmetically appealing unlike the

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incorporation of larger particles of ZnO in traditional sunscreens that give white chalky appearance when applied to the skin. These nanoparticles do not penetrate beyond the stratum

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corneum of skin, hence are well tolerated on the skin overcoming toxicity concerns [51]. Such

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nano creams also increase the sun protection factor (SPF) with better UV filtration and

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absorption properties.

The cosmeceutical formulation enriched with ZnONps of microbicidal, antioxidant and

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moisturizing properties is shown in Fig. 7B (inset). The antibacterial and antifungal activities of

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the formulation were compared visually with commercial antibacterial and antifungal creams respectively. The efficacy of the formulation was similar to that of commercial antimicrobial

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creams. Moreover, the formulation was stable due to the presence of highly stable ZnONps that showed similar effects even after ninety days, retaining its moisturizing properties without any skin irritations. The antifungal effect of the formulation was excellent against a clinical skin pathogen, Candida sp. and surprisingly, the effect outweighed the commercial antifungal cream at 2 %. Candidiasis is considered to be the second most prevalent fungal skin infection in humans caused by Candida species, which are observed in the dermatomycoses of nails, hands and feet. The clinical isolate under study showed resistance against the commercial cream, 21

ACCEPTED MANUSCRIPT whereas, the formulated ZnONps cold cream was susceptible at 2% (Fig. 7B). The cream base that served as negative control did not show any activity. This shows that the antioxidantenriched nano-based cold creams can significantly alleviate human skin infections and oxidative stress induced cellular damage.

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4. Conclusions

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The present study deals with the facile synthesis of multifunctional colloidal ZnONps using aqueous Adhatoda vasica leaf extract and its physicochemical characterizations by several

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techniques. Characteristic absorption peak at 352 nm due to surface plasmon resonance

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confirmed the formation of ZnONps. The possible functional groups responsible for the reduction mechanism were identified by FTIR analysis. Phase purity and grain size were

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determined by XRD analysis. HR-TEM micrographs exhibited particle size about 10 nm to 12

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nm. EDX analysis confirmed the presence of zinc and oxygen. The zeta potential of ZnONps was found to be -24.6 mV confirming the stability of nanoparticles. The antibacterial and

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antifungal activities of biosynthesized ZnONps exhibited mean zone of inhibition from 08.667 ±

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0.282 to 21.666 ± 0.447 (mm) and 09.000 ± 0.177 to 19.000 ± 0.307 (mm) respectively, in a concentration-dependent manner similar to earlier studies [23, 27, 28]. The highest antibacterial

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and antifungal activities were observed against S. epidermidis (MIC and MBC 16 µg mL-1) and A. fumigatus (MIC and MFC 32 µg mL-1) respectively. The antioxidant activity of ZnONps exerted an IC50 value of 139.27 µg mL-1. Colloidal ZnONps of biological origin exhibited pronounced antimicrobial and antioxidant properties due to their nanometric size, which were further incorporated in a nanocosmeceutical formulation and tested against clinical skin pathogens. It is interesting to note that, significant inhibitory action was observed against Candida sp., the causative agent of the most prevalent fungal skin infection candidiasis, which 22

ACCEPTED MANUSCRIPT showed resistance against a commercial antifungal cream (2 %). This study demonstrates the exploitation of biogenic ZnONps as promising biomaterials in nanomedicine that can significantly alleviate human skin infections and oxidative stress induced cellular damages. Further studies are envisaged in the applications of ZnONps as colloidal drug carriers in

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cosmetic formulations to improve the bioavailability of active constituents.

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Conflicts of Interest

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None

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Acknowledgements

The authors would like to thank the Department of Science and Technology, New Delhi,

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Govt. of India sponsored National Facility for Drug Development (NFDD) for academia,

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pharmaceutical and allied industries (VI-D&P/349/10-11/TDT/1 Dt. 21.10.2010) for its valuable

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support and sophisticated facilities to carry out the present study.

1. Denice

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ACCEPTED MANUSCRIPT List of Figures Figure captions 1.

Fig. 1. A. Schematic diagram for the plausible mechanism of colloidal ZnONps formation

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using aqueous A. vasica leaf extract B. UV-Visible absorption spectra of synthesized ZnONps visualized by color change from clear to white colloidal solution (inset). Fig. 2. A. FTIR spectra of aqueous A. vasica extract and ZnONps B. XRD pattern of

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2.

3.

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ZnONps.

Fig. 3. A. Particle size distribution and B. Zeta potential analysis of biosynthesized

Fig. 4. A. HR-TEM micrograph with SAED pattern (inset) and B. EDX spectrum of

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ZnONps.

ZnONps.

Fig. 5. Antimicrobial susceptibility of biosynthesized ZnONps A. Bacteria S. epidermidis

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5.

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MTCC 435 and B. Fungus A. fumigatus MTCC 6594 - Streptomycin/Fluconazole 10 µg (a), Distilled Water 100 µL (b), ZnONps 5 µg (c), ZnONps 10 µg (d), ZnONps 15 µg (e),

Fig.6. A. Antimicrobial effect of ZnONps against standard and clinical microbial pathogens

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ZnONps 20 µg (f).

(Mean ± SD., n=3) and B. Free radical scavenging activity of biosynthesized ZnONps and standard quercetin by DPPH assay (Mean ± SD., n=3). 7. Fig.7. A. Graphical representation of the percentage significance of antimicrobial activity of

ZnONps against various skin pathogens and B. Antimicrobial activity of the nano-based formulation against a clinical fungal skin pathogen Candida sp. –2 % commercial antifungal

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ACCEPTED MANUSCRIPT cream (a), cream base (b), 1% formulated nano-cream (c), 2% formulated nano-cream (d),

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cold cream formulation with 2 % colloidal ZnONps (inset).

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Fig. 1. A. Schematic diagram for the plausible mechanism of colloidal ZnONps formation

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using aqueous A. vasica leaf extract B. UV-Visible absorption spectra of synthesized

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ZnONps visualized by color change from clear to white colloidal solution (inset).

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ZnONps.

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Fig. 2. A. FTIR spectra of aqueous A. vasica extract and ZnONps B. XRD pattern of

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ZnONps.

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Fig. 3. A. Particle size distribution and B. Zeta potential analysis of biosynthesized

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Fig. 4. A. HR-TEM micrograph with SAED pattern (inset) and B. EDX spectrum of ZnONps.

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Fig. 5. Antimicrobial susceptibility of biosynthesized ZnONps A. Bacteria S. epidermidis MTCC 435 and B. Fungus A. fumigatus MTCC 6594 - Streptomycin/Fluconazole 10 µg (a), Distilled Water 100 µL (b), ZnONps 5 µg (c), ZnONps 10 µg (d), ZnONps 15 µg (e), ZnONps 20 µg (f).

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Fig.6. A. Antimicrobial effect of ZnONps against standard and clinical microbial pathogens (Mean ± SD., n=3) and B. Free radical scavenging activity of biosynthesized ZnONps and standard quercetin by DPPH assay (Mean ± SD., n=3).

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Fig.7. A. Graphical representation of the percentage significance of antimicrobial activity of

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ZnONps against various skin pathogens and B. Antimicrobial activity of the nano-based

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formulation against a clinical fungal skin pathogen Candida sp. –2 % commercial antifungal cream (a), cream base (b), 1% formulated nano-cream (c), 2% formulated nano-cream (d), cold cream formulation with 2 % colloidal ZnONps (inset).

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ACCEPTED MANUSCRIPT List of Tables Table captions

Table 1. MIC and MBC/MFC of biosynthesized ZnONps (µg mL-1) against standard and

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clinical bacterial/fungal skin pathogens.

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Table 1

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MIC and MBC/MFC of biosynthesized ZnONps (µg mL-1) against standard and clinical

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bacterial/fungal skin pathogens.

MBC/MFC

16

16

Escherichia coli MTCC 443

64

128

Pseudomonas aeruginosa MTCC 741

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128

256

Staphylococcus sp.

32

32

Streptococcus sp.

128

256

Aspergillus fumigatus MTCC 6594

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Trichophyton rubrum MTCC 296

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128

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MIC

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Microorganisms

ZnONps (µg mL-1)

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Staphylococcus epidermidis MTCC 435

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512

Fusarium sp.

64

128

Candida sp.

256

256

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Microsporum audouinii MTCC 8197

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GRAPHICAL ABSTRACT

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ACCEPTED MANUSCRIPT HIGHLIGHTS Adhatoda vasica mediated colloidal zinc oxide nanoparticles were synthesized.



Highly stable nanoparticles were formed with size about 12 nm.



Significant increase in antimicrobial activity was observed dose-dependently.



An IC50 value of 139.27 µg mL-1 was determined from the antioxidant assay.



Incorporation of nanoparticles for a cosmeceutical application was employed.

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

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