Natural Honeycomb Flavone Chrysin (5,7-dihydroxyflavone)-Reduced

Oct 27, 2017 - National Centre for Nanosciences and Nanotechnology, University of Madras, Guindy Campus, Chennai-600025, Tamil Nadu, India...
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Natural Honeycomb Flavone Chrysin (5 7-dihydroxyflavone) Reduced Graphene Oxide Nanosheets Fabrication for Improved Bactericidal and Skin Regeneration Sathishkumar G, Premkumar P, Pradeep K Jha, Jeyaraj M, Manikandan K, Asif Mohammed Khan M, and Sivaramakrishnan S ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02603 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Natural Honeycomb Flavone Chrysin (5 7-

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dihydroxyflavone) Reduced Graphene Oxide Nanosheets

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Fabrication for Improved Bactericidal and Skin

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Regeneration

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Sathishkumar G†, Premkumar P§, Pradeep K Jha‡, Jeyaraj M⊥, Manikandan K∥, Asif Mohammed

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Khan M†, Sivaramakrishnan S†*

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Department of Biotechnology, Bharathidasan University, Tiruchirappalli-620024, Tamilnadu, India. § Department of Biochemistry, Bharathidasan University, Tiruchirappalli-620024, Tamilnadu, India. ‡ School of Medical Science and Technology, Indian Institute of Technology, Kharagpur-721302, West Bengal, India. ⊥National Centre for Nanosciences and Nanotechnology, University of Madras, Guindy campus, Chennai 600025, India. ∥Nonlinear Optical Materials Laboratory, School of Physics, Bharathidasan University, Tiruchirappalli-620024, Tamilnadu, India.

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Corresponding author*

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Tel: 0431-2407086: Fax: 0431-2407045

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Email: [email protected] (S.Sivaramakrishnan)

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ABSTRACT

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Functionalization of the biocompounds on nanomaterials surface will decrease their

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detrimental side effects and escapes them from immunological rejection. In this study, we

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developed a sustainable green chemistry route to fabricate natural honeycomb flavone chrysin (5,

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7-dihydroxyflavone ChR) reduced graphene oxide nanosheets (ChR-rGONSs) using a simple

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experimental set-up. The hydroxyl (O-H) functional group of ChR wires the reduction and

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generation of functionalized ChR-rGONSs, and it was characterized through Raman, Fourier

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transform infrared (FT-IR) and X-ray photo electron (XPS) spectroscopic analysis. Effective

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reduction of graphene oxide (GO) into ChR-rGONSs was further revealed with X-ray Diffraction

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(XRD), atomic force microscope (AFM), field emission scanning electron microscopic (FE-

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SEM) measurements. Using the high resolution transmission electron microscopic (HR-TEM)

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images, Zeta potential, and Energy dispersive X-ray spectroscopic (EDAX) analysis we have

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shown the fine morphological features, surface charge and stableness of fabricated ChR-

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rGONSs. Thermo-stable properties of ChR-rGONSs were much greater than ChR and GO. In

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disc diffusion study, the ChR-rGONSs showed an excellent inhibitory action against 11 bacterial

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pathogens comparatively with raw graphite (G), free ChR and GO which clearly depicts their

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enhanced antimicrobial value. Moreover, in vivo studies were proved that the ChR-rGONSs

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promotes rapid skin regeneration and wound closure action relatively with other treatments such

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as raw-G, GO, and free ChR. The ChR-rGONSs were exhibited no signs of toxicity against

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treated animal model, also causes less RBCs lysis which represents their biocompatibility for

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direct wound dressing and other regenerative medicine applications.

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KEYWORDS: Green chemistry, Chrysin (5, 7-dihydroxyflavone ChR), Reduced graphene

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oxide, Wound healing, Bactericidal, X-ray Photoelectron Spectroscopy

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INTRODUCTION

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Multifaceted two-dimensional (2D) graphene sheet bonded with sp2 carbon atoms was

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considered as an important nanomaterial because of its outstanding optical, electronic,

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mechanical, and catalytic properties1. Especially, the Graphene oxide (GO) possess unique

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operative physical, chemical and biological properties, as it possess oxygenated functional

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groups2. Also, the GO was receiving huge importance on account of its easy availability,

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dispersity, flexibility for chemical reaction, tunable surface chemistry and a high-level

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biocompatibility. This prominent material was employed for wide spectrum of biomedical

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applications such as biosensing3, drug/gene delivery4, phototherapy5, imaging6, tissue

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engineering7, regenerative medicine8 and theranostics9. Reduced GO (rGO) nanosheets

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hybridized with oxygen-containing chemical groups have gained colossal interests because of

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their exclusive surface properties. The active functional groups such as epoxy, hydroxyl, and

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carboxyl makes GO an important nanomaterial to carry the active ‘payloads’ such as drugs and

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molecules10.

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Conventional methodologies to fabricate rGO nanosheets were involved with the

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utilization of toxic chemical reductants like hydrazines and sodium borohydride11. Most of these

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reducing agents and their resultant by-products were unsafe to environment as well as life forms,

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which makes them not suitable for scaled-up production and further bio-related applications12.

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Therefore, there was a growing demand to find an alternative facile green chemistry route for

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reduced GONSs fabrication. Diverse new and eco-friendlier reducing agents such as, vitamin

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C13,14, amino acids15, alkyl amines16, sugar17, glucose18, bovine serum albumin19, hydrogen-rich

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water20, metal nanoparticles21 and powders22, supercritical alcohols23, polyphenols of green

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tea24,25, ginseng26, bacteria27,28, baker’s yeast29, wild carrot root30, and phytoextract31 were

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reported as the potential replacement of toxic reductants. Apart from this, few other sustainable

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methods to fabricate reduced GO nanosheets were flash photo reduction32, laser reduction33,

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catalytic34 and photocatalytic35-41. However, understanding the mechanism of reduction and

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toxicological insights of bio-based rGONSs were very much important for their applications. As

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for as our knowledge this might be the maiden report where a single dietary flavone Chrysin (5,

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7- dihydroxyflavone ChR) has been utilized to convert GO into sole rGO nanosheets, also it

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represents a comprehensive view on the mechanistic aspects of green chemistry synthesis.

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Natural bioactive dietary flavone Chrysin (5, 7- dihydroxyflavone ChR) was commonly

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found in different bio entities including honeycomb and propolis owns remarkable antimicrobial,

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anti-inflammatory, antioxidant, anticancer, hepatoprotective and anti-diabetic effects42. It was

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widely consumed as a steroid supplement by male bodybuilders for large muscle development,

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because it hinders the participation of aromatase in estrogens biosynthesis. An interesting

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advantage of ChR was it actively scavenges the free-radicals and act against various factors of

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aging related disorders43,44. The anti-inflammatory activity of ChR was noteworthy, as it holds an

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ability to suppress the Cyclooxygenase-2 enzyme level45.

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Nevertheless, ChR possesses diverse biological activities it always suffers with certain

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limitations such as poor solubility, bioavailability and dose-limiting detrimental side effects46. In

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general, flavones were well known for their extraordinary antioxidant potentials due to the active

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hydroxyl and ketone functional groups. They also holds an ability to combat against oxidative

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stress associated diseases like cancer, diabetes, cardiovascular and neurological disorders47.

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Surface functionalization or capping of these active flavonoids with nano-graphene oxide sheets

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will eventually improve their solubility, cellular permeability, bioavailability and compatibility

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for value added biomedical applications.

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Wound healing and remodeling comprises with a number of intrinsic cell as well as

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molecular cascades which ensues in four major stages such as hemostatic, inflammatory, re-

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epithelialization or proliferative and restoration48. The pathological process of skin regeneration

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begins with epidermis and keratinocytes reproduction followed by the contraction of

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extracellular matrix (ECM). Presence of valuable flavonoids such as chrysin, kaempferol,

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apigenin, pinocembrin, galangin, hesperetin, and quercetin in the honey and its by-products

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significantly upsurges the ECM components at initial stages of wound repair. Also, it triggers the

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production of a major cytokine i.e. transforming growth factor beta (TGF-β) which partakes an

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important role in hemostasis and inflammation49. Generally, Oxygen was considered as the major

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requirement to disinfect wounds, fuel hauling, redox signals, and actively participates in repair

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cascade of physical trauma50. Hence, in this work we present a facile green chemistry method to

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fabricate ChR-rGONSs using the honeycomb flavone Chrysin (5 7-dihydroxyflavone) for

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biomedical applications.

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EXPERIMENTAL DETAILS

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Materials

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Graphite flakes