Honey Extracted Polyphenolics Reduce Experimental Hypoxia in

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Honey Extracted Polyphenolics Reduces Experimental Hypoxia in Human Keartinocytes Culture Amrita Chaudhary, Swarnendu Bag, Provas Banerjee, and Jyotirmoy Chatterjee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00366 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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Journal of Agricultural and Food Chemistry

Mode of action and molecular targets associated with the therapeutic activity of physico-chemically characterized Jamun honey extracted polyphenolics (HEP) on HaCaT cells under hypoxia. 301x196mm (96 x 96 DPI)

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HEP Reduce Hypoxic Assault in vitro 1

Honey Extracted Polyphenolics Reduces Experimental Hypoxia in Human Keartinocytes

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Culture

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Amrita Chaudhary1*, Swarnendu Bag2,, Provas Banerjee3, Jyotirmoy Chatterjee1*

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1

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Kharagpur- 721302, West Bengal, India

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2

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Ravangla Sub-Division South Sikkim - 737 139

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3

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*To whom correspondence should be addressed: Jyotirmoy Chatterjee & Amrita Chaudhary

School of Medical Science and Technology, Indian Institute of Technology Kharagpur,

Depatment of Biotechnology, National Institute of Technology Sikkim, Barfung Block

Banerjees’ Biomedical Research Foundation, Sainthia-731234, Birbhum, West Bengal, India

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Address: School of Medical Science and Technology,

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Indian Institute of Technology, Kharagpur-721302.

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Email : [email protected]/[email protected]

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Mobile No.: +91-9434830474/+91-9475355875

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Phone No. +91-3222-282302

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Fax: +91 32222 82221

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1 ACS Paragon Plus Environment

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Abstract

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Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy

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cells / molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic

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cytotoxicity in this study. Different honey samples were physico-chemically characterized to

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identify preferred (Jamun) honey [pH=3.55±0.04, conductivity (µs/cm)=6.66±0.14, water

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content % (w/w) =14.70±0.35, total solid content % (w/w) =85.30±0.35, phenol content (mg

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GAE/100 g) = 403.55±0.35, flavonoid content (mg QE/100 g) = 276.76±4.10, radical scavenging

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activity (% 500 µl) = 147.75±3.13, catalase activity (absorbance at 620 nm.) = 0.226±0.01]. HEP

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was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and

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antioxidant assays. The cardinal molecular expressions like cadherin-catenin-cytoskeleton

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complex (viz. E-cadherin, β-catenin and F-actin), hypoxia marker (Hif 1 α), proliferation marker

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(Ki67) and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and

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qRT-PCR. The 0.063 mg/ml HEP demonstrated better vitality and functionality of HaCaT cells

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as per viability assay (*P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated

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restoration of cellular survival and homeostasis under 0.063 mg/ml HEP after hypoxic assault.

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Further, major spectral changes for nucleic acids and membrane phospholipids reorganizations

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by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/ml HEP on

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hypoxic cells considering proliferation and cellular integrity. It was concluded that specific dose

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of Jamun HEP reduces the hypoxic cytotoxicity.

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Keywords: Antioxidant; Honey; HaCaT; Hypoxia; Proliferation

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INTRODUCTION

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Exploring the utility of natural materials in bio-medical applications is an expanding field of

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research 1. Reactive oxygen species are the byproduct of biological reactions which reacts with

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healthy cells / molecules and create detrimental oxidative effects 2. These ROS are quenched by

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antioxidants 3. In this regard, role of honey as antioxidant bears immense significance especially

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in preventing ageing and facilitating wound healing 4. Medicinal value of honey has been

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recognized since ancient time in Ayurveda 5. It contains variety of phyto-chemicals including

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anti-oxidants. Besides high sugar contents, enzymes like glucose oxidase, catalase and methyl

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syringate are important constituents of honey 6. It also contains proline which alters intracellular

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redox environment and reduces ROS 7. The flavonoids and phenolics are well recognized

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components of honey but these varies as per floral origin 8. Recent reports indicated that honey

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in higher dilutions protect cells from detrimental oxidative stress due to presence of anti-oxidants

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9

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disorders 10.

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A number of earlier work showed the antioxidant activity of honey containing polyphenolics and

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their possible role in apoptosis, cellular differentiation and cell cycle regulation of certain

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specific cells

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radical scavenging activity of its polyphenolic contents.

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dimensions of

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(protein/gene) level mechanism especially on hypoxia induced cell are lacking. Only a few

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studies have reported the effect of different combination of polyphenolics on in vitro system 13.

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The present study not only explored the free-radical scavenging activity of HEP (Honey

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Extracted Polyphenolics) but also deciphered the expressions of prime epithelial genes viz. E-

. Dzialo et al., 2016 also reported the antioxidant activity of plant derived phenolics on skin

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. Although many studies depicted antioxidant potential of honey due to the free 12

However, studies on temporal

anti-oxidative effect of honey extracted poly-phenolics and its molecular


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cadherin, β-catenin, F-actin, Hif-1α, p63, Ki67 by immuno-cytochemistry (ICC), western blot

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and qRT-PCR (quantitative real time polymerase chain reaction) analysis to elucidate the

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antioxidant impact of HEP on HaCaT cell line under hypoxic assaults.

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The migration of cells is known to be influenced by extra-cellular matrix and is linked with

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modulation in membranous expression of adherent protein E-cadherin and it’s intracellular

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partner protein β-catenin

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cytoskeleton (cytoskeletal proteins) re-organization. Collectively all these modifications lead to

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cell migration 14. Further, hypoxic exposure to cells persuades stabilization and up-regulation of

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Hif 1α expression

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essentially involved and Ki67 is known as cell proliferation marker

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master regulatory role in epithelial cell proliferation and differentiation 17.

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Existing techniques for identifying cardinal molecular expression and viability are based on

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spectro-photometric and microscopic techniques [viz. ICC, flow cytometry, MTT {3-(4, 5-

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dimethylthiazol-2-y)-2, 5-diphenyltetrazolium bromide}, NBT (Nitro Blue Tetrazolium) and

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Live Dead cell assay etc.] having their own inadequacy in evaluating multimodal cellular

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behavior. In this context, it may be noted that Fourier transform infrared spectroscopy (FTIR)

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observation of cell samples provides valuable informations about functional groups18. Thus,

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concurrent FTIR experiment and expression of E-cadherin, β-catenin, F-actin, Hif 1 α, p63 and

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Ki67 under honey extracted phenolics (HEP) in normoxia and hypoxia provide integrated clues

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regarding impacts of such interventions on cellular vitality and functionality.

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14

. The extracellular matrix persuades the motility of cells via actin

. In wound healing, migration and proliferation of basal keratinocytes are 16

whereas p63, plays a

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MATERIALS AND METHODS

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Chemicals: All chemicals and reagents used were of analytical grade obtained from Sigma

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Aldrich like Phosphate Buffer Saline (PBS) 52% Perchloric acid, Anthrone and 95% Sulfuric

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acid (H2SO4) Methanol, DPPH (2, 2-diphenyl-1-picryylhydrazyl), Ascorbic acid Hydrogen

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Peroxide (H2O2), Potassium Di-Cromate

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Carbonate (Na2CO3), Gallic acid, Aluminium tri-Chloride (AlCl3) and Quercetin. Delbecco’s

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Modified Eagle Media (DMEM-F12), Antimycotic antibiotic, Fetal bovine serum (FBS), L-

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glutamine and 0.05% trypsin-EDTA solution, Phosphate buffered saline (PBS), Bovine serum

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albumin (BSA) and MTT reagent (TC191-1G), were purchased from Himedia (India), DAPI (4′,

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6 diamidino-2 phenylindole, dihydrochloride), TCA (Trichloroacetic acid), TBA (2-

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Thiobarbituric acid) (Sigma-Aldrich, St. Louis, MO). Primary antibodies: Anti-p63 antibody

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[EPR5701] ab124762, Anti-Ki67 antibody [PP-67] ab6526, Anti-beta Catenin antibody [15B8]

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ab6301, Anti-E-Cadherin, clone EP700Y, Cat. No. ab40772, Anti-HIF-1-alpha antibody

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[EP1215Y] ab51608 were purchased from Abcam (Cambridge, United Kingdom). Secondary

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Antibodies: For fluorescence detection fluorophore-conjugated secondary anti-bodies were used;

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Goat anti-Mouse IgG (H+L) Alexa Fluor® 594 conjugate Cat No A-11005 & Alexa Fluor® 488

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conjugate Cat No A-11008 both obtained from Thermo Fisher (Waltham, MA USA).

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HaCaT cells were purchased from NCCS Pune, India.

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Collection of honey samples: Sixteen Indian honey samples were used to determine the physio-

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chemical and antioxidant properties. Within these sixteen samples, four samples were unifloral

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and twelve were multifloral. Supplementary Table (ST) 1 showed all the details of selected

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honey samples.

(K2Cr2O7), Folin-Ciocalteau reagent, Sodium


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Honey Characterization: pH and conductivity measured by using Thermo Scientific, USA pH

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and conductivity meter19 The absorbance of 50 % honey solution (w/v) at 635 nm. were recorded

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to determine color at pfund scale19 using following formula

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Where, mm Pfund= -38.70+371.39×absorbance

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Water content and solid content19 were identified by drying method (105ºC for 3 hours) and

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calculated using following formula:

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Moisture (%) = (w1-w2)/w1 ×100; where w1= honey sample before drying, w2= honey sample

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before drying

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Total solid (%) =100- moisture content

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FTIR- The FTIR honey samples were prepared by using 0.05mg honey on KBr palates and

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absorbance were recorded in the range of 500-4000 cm-1 wavelength in Thermo Nicolet

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spectrometer (Model - NEXUS-870, Thermo Nicolet Corporation, Madison, WI, USA). The

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spectral peaks were identified by OMNIC (Nicolet, Madison, WI, USA) software19.

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Total phenolics and flavonoids in honey samples were examined by Folin-Ciocalteau (gallic acid

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as standard) and Dowd (Quertcetin as standard) method repectively20, 21.

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DPPH radical scavenging activity (RSA)- The protocol for DPPH RSA was adopted by

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Hussein et al. (2011) with some modification22. Briefly, the stock solutions for honey samples

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(10mg/ml. honey in methanol) were prepared. 0.5 ml of honey solution was added to 1 ml. of 0.1

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mM DPPH solution in methanol and was incubated for 30 minutes in dark at room temperature.

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Ascorbic acid 10mg/ml. were treated as control. Further, the absorbances were taken at 515 nm



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using Multiskan GO UV/Visible spectrophotometer. The percentage RSA calculated by

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following formula:

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Scavenging activity in % = A- B/A × 100

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(A = the absorbance of DPPH and B = the absorbance of DPPH and honey combination)

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Proline content: The proline content of honey estimated by ninhydrin biochemical test using

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(0.032 mg/ml) proline as standard20. Briefly, 500 µl of honey solution (0.5g/ml.) and 1 ml. of

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80% formic acid added with 1 ml of ninhydrin reagent solution in 3% in ethylene glycol

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monomethylether. Then mixed intensely for 15 min to boil and incubated for 10 min at 700C.

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Further, 5 ml of 50% 2-propanol (Merck Millipore) was added and cooled to room temperature.

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The absorbance was taken at 510 nm. The total proline content in honey was calculated using the

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following formula:

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(mg/kg) = (Es/Ea) × (E1/E2) × 80 Where, Es = Absorbance of sample solution;

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Ea= Absorbance of standard solution;

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E1 = Weight of proline (mg) in standard solution;

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E2 = Weight of honey (kg);

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80 is the dilution

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Catalase enzyme activity: The catalase activity protocol was adopted from Asru K. Sinha

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(1972) with some modification23. Briefly, 0.5 ml. of raw honey (undiluted) dissolved in 2.5 ml of

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0.01 M Phosphate buffer (PBS) and 2 ml of 0.2 M Hydrogen peroxide (H2O2). Further, 0.5 ml of

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this reaction mixture incubated with 1 ml. of 5% potassium dichromate solution in glacial acetic


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acid and then heated for 10 minutes. The blue precipitate was transformed into green, indicating

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reduction of hydrogen peroxide into hydrogen and oxygen. The absorbance was taken at 620nm.

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Column chromatography (CC) of honey: The total polyphenolics of honey were extracted by

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solid phase extraction technique 24. Briefly, honey samples (100g) mixed with 50 ml of acidified

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water and filtered by sterile cotton to remove solid particles. This solution was passed through

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amberlite XAD-2 column to remove all the saccharides and polar compounds. Further, the

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adsorbed polyphenols eluted with methanol followed by column wash with water. The volume of

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extracted polyphenolics reduced by rotary evaporator to dryness and the stock solution of HEP

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was prepared by dissolving in methanol and filtering through 0.22µm sterile filter.

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Cell culture: HaCaT cells (immortal keratinocytes) were cultured in Delbecco’s Modified Eagle

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Media (DMEM/F 12) complete media at 37 °C temperature with 5 % carbon dioxide CO2, 20 %

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oxygen O2, balance nitrogen and humidity for normoxia. However, hypoxic condition achieved

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by incubating cells under hypoxic incubator (5 % carbon dioxide CO2, 3 % oxygen O2, balance

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nitrogen ). Briefly, cells were grown at 50 % confluency under normoxia; then treated with HEP

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dilutions (mg/ml.) in DMEM/F 12 complete media (the dry weight of HEP calculated by

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reducing the methanolic extracted poly-phenolics volume using rotary evaporator which was

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found 200mg/100ml of honey) and transferred into hypoxic incubator for 24 hrs. After that cells

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were harvested for subsequent assays.

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Phase contrast microscopy: Phase contrast microscopy was performed to observe the cellular

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morphological alterations under HEP interventions. For this cells were seeded on 1 % poly L

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lysine coated cover slip and grown under interventions for 24 hrs. Further, the images were

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grabbed at 10x objective by Ziess, microscope. 8 ACS Paragon Plus Environment


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MTT assay: In MTT assay 3000 cells per well were seeded in 96 well microtiter plate and

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grown in DMEM F-12 media for 12 hrs. Further, cells were treated with HEP dilutions (0.25,

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0.125, 0.063 and 0.031) mg/ml in triplicate and incubated for 24 hrs. MTT reagent was applied

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for 2 hrs. The cellular contents dissolved in DMSO and absorbance was recorded at 595 nm.

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Appropriate control (media without honey dilution) and blank (DMSO) were put up. Survival

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rate % calculated by following formula:

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Survival Rate (%) = (Asample– Ab) / (Ac – Ab) x 100

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Asample = Absorbance of test sample, Ab = Absorbance of blank & Ac = Absorbance of control

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In addition, cell were treated with 50 µM ascorbic acid25( positive control) and 200 µM H2O2

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(negative control)26 for 12 hrs followed by 12 hrs incubation of above mentioned HEP dilutions.

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NBT assay: In NBT assay presence of intracellular superoxide anions were evaluated. In short,

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cells (both treated and untreated) were incubated with NBT reagent (0.1 mg/ml media) for 3 hrs.

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Further, NBT-formazan crystals were dissolved by KOH (2M/DMSO solution) followed by

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methanol wash. KOH 2M/DMSO solution were put up as blank and absorbance was carried out

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at 630 nm27. Ascorbic acid (50µM) and H2O2 (200 µM) were used as positive and negative

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control respectively.

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Native Page for SOD activity: Crude protein from the cells were isolated and quantified by

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Bradford assay.10 % Native Page gel electrophoresis performed for NBT assay where the

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protocol mentioned by Datkhile et al. (2008) was followed

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scanner to observe an achromatic band of SOD protein activity. Further, the relative percentage

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area of bands was quantified by ImageJ software.

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Lipid per-oxidation test: Lipid per-oxidation of cells was evaluated by measuring the

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malondialdehyde formation29. Cells were grown on petridish (60*15 mm) and treated with HEP

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and gel was visualized by gel


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doses for 24 hrs. Further, cells were lysed with 2 ml of 20% TCA and 1 ml. of 0.67 % TBA

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followed by three times wash with PBS. The reaction mixture was heated on boiling water bath

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for 15 min. and centrifuged at 15000 rpm for 15 min. The pellet discarded and absorbance of

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supernatant carried out at 532 and 600 nm. 1.56 x 105 M-1cm-1 was considered as molar

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extinction coefficient for malondialdehyde and concentration was calculated by the following

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formula

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Concentration of MDA (µM) = (A532 - A600)/ 1.56 x 105

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Ascorbic acid (50µM) and H2O2 (200 µM) were used as positive and negative control

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respectively.

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Live cell time lapse imaging: The cells were grown at 50 % confluency and phase contrast

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imaging were performed at 0 hrs, 4 hrs, 8 hrs and 16 hrs of the same area of interest to observe

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the cell population growing rate under interventions.

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Immuno-cytochemistry: Immuno-cytochemical assay was performed by fixing cells with 4%

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paraformaldehyde in PBS, serum blocking (10% goat serum in PBST) and incubating with

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diluted primary antibodies (Ki67, p63, β-catenin & Hif 1α). Horseradish peroxidase conjugated

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secondary antibody used for chromogenic staining and fluorophore-conjugated secondary anti-

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bodies were used for fluorescence staining. Further, the cells were counter stained by Meyers

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Hematoxylin (for chromogenic detection) and DAPI (for fluorescence detection).

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Western Blot: Western blot analysis was performed by comparing the expression of endogenous

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control β-tubulin molecule. A semi-dry transfer blotting system (Bio-Rad, United States) was

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used. Briefly, transferred protein (from sodium dodecyl sulfate polyacrylamide gel to a

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polyvinylidene fluoride membrane) was kept into blocking solution [10 ml 5% BSA (Bovine

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Serum Albumin) in TBST (Tris-Buffered Saline and Tween 20)] for 2 hours with mild agitation



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at room temperature. After that, the diluted primary antibody was added on the membrane at 40C

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for overnight incubation. Further, secondary antibody was added on the membrane followed by

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washing and incubated for 2 hours at room temperature. Development of X-ray film was

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performed by Western ECL Substrate (Catalogue No- #170-5061, Bio-Rad, USA). The

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percentage area of the band was calculated by Image j software.

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RNA extraction, cDNA synthesis and qRT PCR: For gene expression study m-RNA was

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pulled out by TRIzol reagent and c-DNA was obtained by High-Capacity cDNA Reverse

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Transcription Kit (Applied Biosystems) in a Arktik™ Thermal Cycler according to the

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manufacturer's protocol. The real time-PCR reactions were accomplished in triplicates on Light

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Cycler® 480 II (Roche Diagnostics GmbH, Mannheim, Germany), using Light Cycler® 480

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SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany). GAPDH

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(Glyceraldehyde 3-phosphate dehydrogenase) were used as endogenous control. The primer

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sequences of selected gene and cycling conditions were mentioned in table 1.

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Sample preparation for cell FTIR spectroscopy: Briefly, cells were trypsinized with trypsin

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EDTA solution from Himedia, India followed by washing with PBS solution in order to remove

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growth medium and centrifuged for 2000 rpm for 2 minutes to pellet down. Further, the cells

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were suspended in 300 µl PBS and were fixed with 70 % alcohol. The fixed cells were stored at

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40C for 2 days for FTIR study. FTIR analysis was performed by Thermo Nicolet spectrometer

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(Model - NEXUS-870, Thermo Nicolet Corporation, Madison, WI, USA) in order to identify

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important chemical signatures.

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Statistical analysis: Results were depicted as mean ± standard deviation (SD) in the tables of

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supporting document. Correlation matrix was developed using IBM SPSS statistic 20 software

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and 2D Score plot was formed by XLSTAT software.


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Semi-quantification of nuclear expression of p63 and Ki67 expression: The nuclear

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expression of p63 and Ki67 was semi-quantified using grey scale intensity values by image J

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software and notch box plots were constructed by Matlab 2012b software to depict changes in

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the expression levels. In correlation analysis first we arranged all the data set (Ki67 and p63

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expression intensity) into 0-10 scale and subsequently used a scatter plot to find linear

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relationship. Further, the pearson’s correlation coefficient was calculated by Microsoft Office

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Excel.

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RESULTS

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Physico-chemical characterization of honey samples

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Sixteen honey samples (ST 1) were physico-chemically analyzed and the findings were

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illustrated in table ST 2 depicting acidic pH (1.9-3.9), electrical conductivity (2.7-32.6 µs/cm),

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water content (12.52-16.23 w/w %), solid content (83.77-87.48 w/w %) and proline content

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45.93-1265.69 mg/kg.

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Principal component analysis (PCA) of FTIR spectra

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Supplementary Figure (SF) 1 demonstrated comparable absorption spectra with some dissimilar

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regional intensity. The unsupervised principal component analysis (PCA) of FTIR spectra was

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effective to obtain overall distinguishable features. The Eigen-analysis in PCA of honey samples

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for correlation matrix was performed by using XLSTAT software and three primary principal

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components i.e. F1 (62.66%), F2 (29.48%) and F3 (3.33%) were noted. The clusters of honey

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samples based on chemical composition were illustrated in the schematic representation (Figure

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1A). The score plot depicted total variance of 92.14% for first two components (F1 and F2) and

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95.47% for other three (F1, F2 & F3). All the studied honey samples distribution in bi-plot varies

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±1 for F1 (62.66%) and F2 (29.48%) except samples O and H (Figure 1A). The FTIR spectra 12 ACS Paragon Plus Environment


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were effective for carbohydrate analysis of the selected honey samples by 2D Scree plot. In this

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context, melanoidins formation as a result of Millard reaction noted to be important due to it’s

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significant anti-radical activity30. Therefore, proper detection of sugar spectrum was relevant in

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this study. The different intensities were distinguished from the spectra for sugar region from

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750-1500 cm-1. The spectral peaks for sugar region showed comparatively less disparity

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(approximately between ±2) in the first principle component (F1), but the difference was

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observed for second principle component (F2) as well as for the third principle component (F2)

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(Figure 1A).

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Anti-oxidant potential

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The color of honey samples varied from water white to amber on pfund scale, average phenolic

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content ranged from 209.09 to 403 mg GAE/100 g, flavonoid ranged from 274.44 to 519.87 mg

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QE/100 g, DPPH RSA ranged from 50.93 to 147.75 % in 500 µl and catalase activity ranged

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from 0.063 to 0.226 absorbance at 620 nm (ST 3). In order to identify the adulteration in honey,

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proline determination is very important because it has major contributory role for amino acid

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content and anti-oxidant activity 20. The total proline content was more than 180 mg/kg in most

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of the tested sample (except sample A, B, D and E) ST 3. These differences in proline content

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were due to different floral origin 20. Particularly in jamun honey (sample N) the proline content

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was highest among all the selected samples.

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Antioxidant correlation analysis

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Among sixteen honey samples (ST 1), I and N showed amber colors which were very indicative

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in relation to high phenolic content, DPPH RSA and catalase activity. Therefore, a correlation

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matrix for total phenolics, flavonoids and antioxidant properties (i.e. DPPH RSA and catalase

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activity) were drawn using SPSS software. The correlation matrix (Table 2) showed a 13 ACS Paragon Plus Environment

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significantly positive and strong co-relation [at the 0.01 level (2-tailed)] between total phenol to

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DPPH RSA and catalase. Further, sample I showed highest level of total flavonoid content

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(519.87 mg QE/100 g) with analogous catalase activity (0.199 absorbance at 620 nm.) and DPPH

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RSA (149.08 in 500 µl).

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Moreover, sample N also exhibited highest level of total phenolic content (403.55mg GAE/100

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g), catalase activity (0.226 absorbance at 620 nm.) and corresponding DPPH RSA (147.75 in 500

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µl) with highest proline content (1265.69 mg/kg) indicating it’s maximum natural antioxidant

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potential as reported by Islam et al., (2012) 31.

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Phenolic acids in jamun honey and its anti-oxidant activity

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The antioxidant activity of phenolics varies according to their different molecular structure (i.e

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hydroxylation and methylation). Therefore, the selected honey (sample N; based on optimum

304

physico-chemical properties and antioxidant co-relation analysis) polyphenolics were analyzed

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by HPLC & MALDI TOF MS studies. Figure 1B represented the characteristic HPLC spectra

306

(Protocol was given in supplementary document). From this spectrum four major retention time

307

(Rt) fractions were collected to observe the antioxidant behavior through DPPH RSA. Rt 12.11

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min. depicted highest free radical scavenging activity in comparison to other fractions [where

309

same volume (5ml) of each fraction taken for DPPH RSA] (Figure 1C). Further, the HPLC

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collected four major Rt fractions were analyzed by MALDI TOF MS (Protocol was given in

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supplementary document). This chromatographic technique coupled with mass spectroscopy

312

effectively identified different active phytochemicals and plant secondary metabolites viz.

313

flavonoids, phenolic acids, anthocyanins and tannins (ST 4) having well known anti-oxidant

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properties 32, 33. This Rt (12.11min.) also exhibited multiple peaks in MALDI-MS.

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Cell morphology, viability, anti-oxidant and temporal behavior under HEP dilution

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For in vitro evaluation, sample N (i.e. Jamun honey) was selected based on better antioxidant

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activity. Figure 2A depicted the phase contrast images of HaCaT population under interventions.

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The control cell population under hypoxia showed loss of cell-cell adhesion i.e. cells were more

320

segregated and cellular morphology was also altered from flattened densely packed to round and

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oval. However, under hypoxia 0.063 % dilution most of the cells flattened and dividing like

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normoxia 0.063 % dilution.

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The cellular vitality of HaCaT cells was evaluated by MTT assay, which was based on

324

mitochondrial dehydrogenase enzyme activity. In this test metabolically active cell absorbed the

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MTT reagent and changes the color of reagent from pale yellow to purple by reduction. Further,

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the formazan crystals (purple color) were dissolved in DMSO solvent. The cell survival assay

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provides valuable information concerning the effect of HEP dilution on HaCaT cells under

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normoxia and hypoxia. For this work different HEP dilutions in DMEM F12 complete media

329

were prepared. The HaCaT cell survival rate under 0.25, 0.125, 0.063 and 0.031 mg/ml HEP was

330

calculated both for normoxic and hypoxic conditions. The percentage of cell survival illustrated

331

the extent of cellular vitality and functionality under different HEP dilution (Figure 2B).Figure

332

2B depicted significant (P < 0.01) increase in cell survival % under 0.063 mg/ml HEP dilution

333

for both normoxic and hypoxic conditions.

334

Generation of intracellular reactive oxygen species increases under hypoxic condition5.

335

Therefore, evaluation of anti-oxidant property of HEP dilution is imperative in this regard.

336

Nitroblue tetrazolium (NBT) reduction assay was performed to observe the superoxide

337

scavenging activity of HEP dilutions under normoxic and hypoxic conditions. Figure 2C

338

depicted the decreased anti-oxidant behavior of cells under hypoxia control in comparison to


Page 17 of 46


339

normoxia as the obtained absorbance at 630 nm. was highest in control than other dilutions, this

340

indicated more ROS. The lowest absorbance was observed in 0.063 mg/ml (P≤ 0.05) so this

341

dilution was found to be efficient in reducing ROS. In addition, native PAGE gel electrophoresis

342

also performed to examine superoxide dismutase (SOD) activity at protein level. Figure 2F

343

depicted the specific band for SOD activity, the observed percentage of band were significantly

344

highest (*P≤0.005) under 0.063 mg/ml HEP for both normoxia and hypoxia (Figure 2E).

345

Further, the cellular oxidative stress were quantified in terms of lipid peroxidation test where μΜ

346

concentration of MDA (by product of lipid peroxidation) formation was measured. Figure 2D

347

showed significant (P≤ 0.01) reduction in lipid peroxidation for 0.063 mg/ml both under

348

normoxia and hypoxia as compared to control and other dilutions. Therefore, this particular

349

dilution’s (0.063 mg/ml) effect was less on cellular lipid peroxidation.

350

The temporal changes in cell population under interventions investigated by real time imaging.

351

Figure 3A represented phase contrast images (100x) of HaCaT cells under interventions at 0 hrs,

352

4hrs, 8 hrs, 16 hrs. The total cell count at different temporal points were normalized into 0-10

353

scale, figure 3B showed the growing rate of cell population which reduced for hypoxic control

354

cells and increased for normoxia control, normoxia (0.63mg/ml HEP) and hypoxia (0.63mg/ml

355

HEP).

356

Cell-cell interaction, cytoskeletal changes and Hif 1 α expression

357

To clarify the disparity between normoxic and hypoxic cell-cell interaction immunostaining of

358

cell-cell adhesion protein E-cadherin was performed. Figure 4 depicted the altered expression

359

pattern of

360

illustrated the predominant expression of E-cadherin in cell-cell junctions. The other HEP

361

interventions in normoxia group (Figure 4 b1, c1, d1) showed comparatively less membranous

E-cadherin under interventions. Figure 4 a1 showed normoxic control which



Page 18 of 46

362

expression of E-cadherin indicating cell migration. In hypoxia group, control figure 4 a2 the

363

membranous localization of E-cadherin was disturbed (Figure 4 a3), however under 0.063 and

364

0.031 mg/ml of HEP, the membranous integrity moved towards restoration as represented by

365

enlarge view of figure 4 c2 and 4 d2 (Figure 4 c3, d3). Further, western blot analysis of

366

extracted proteins of 0.063 mg/ml treated cells (both for normoxia and hypoxia) was performed.

367

Significant (**P≤0.05) alterations was found in normoxic and hypoxic treated cells for E-

368

cadherin expression (Figure 8A & B). In addition, the m-RNA expression of E-cadherin for

369

0.063 mg/ml of HEP under normoxia and hypoxia conditions was significantly (*P ≤ 0.1) up-

370

regulated than control counterpart as per qRT-PCR study (Figure 8C).

371

The intracellular cell-cell adherence protein β-catenin was notably expressed in membrane under

372

normoxic control (Figure 5Aa). However, under hypoxia group this molecule showed much

373

disintegrated expression in control (Figure 5Ae2) and 0.125 mg/ml of HEP (Figure 5Af2). The

374

0.063 (Figure 5Ag2) and 0.031 (Figure 5Ah2) mg/ml of HEP under hypoxia showed

375

membranous restitution of this molecule (Figure 5). The membranous distribution of β-catenin

376

were illustrated by yellow arrow in figure g2a and h2a. The β-catenin expression in western blot

377

analysis of 0.063 mg/ml. HEP treated normoxic and hypoxic cells depicted non significant

378

(*P>0.05) alterations (Figure 8B). The m-RNA expression for β-catenin also found to be up-

379

regulated (*P ≤ 0.1) for 0.063 mg/ml of HEP under normoxia and hypoxia conditions (Figure

380

8C).

381

In order to access cytoskeletal changes the F-actin filament organization of HaCaT cells under

382

interventions was observed. The F-actin fibres mainly confined to the periphery of cells under

383

normoxia control (cells without HEP intervention) (Figure 6a1), while loss of peripheral/cortical

384

fibers were observed under hypoxia control cells (Figure 6a2) and actin molecules scattered all


Page 19 of 46


385

over the cytoplasm. Cells under 0.063 mg/ml of HEP hypoxia, these actin molecules present at

386

both scattered as well as peripheral/cortical form (Figure 6c2, marked by yellow arrows).

387

The Hif 1α expression was not clear in normoxia condition. Therefore, the Hif 1α expression

388

under hypoxia condition was studied. Most of cells expressed this protein under hypoxia control

389

(Figure 5Ae3) and 0.125 mg/ml HEP dilution (Figure 5Af3). However, only few cells were

390

found to express this protein in 0.063 (Figure 5Ag3) and 0.031 (Figure 5Ah3) dilution under

391

hypoxia. Further, the separated Hif 1α images grey scale intensity was quantified to create notch

392

box plot. Figure 5B depicted that the Hif 1α expression was significantly reduced under HEP

393

dilutions (0.125, 0.063 and 0.031 mg/ml) in comparison to control. The western blot analysis of

394

Hif 1α expression for 0.063 mg/ml. HEP treated normoxic and hypoxic cells represented a

395

significant (**P≤0.05) change (Figure 8B). The gene expression for Hif 1α, 0.063 mg/ml dilution

396

observed to be reduced (**P > 0.1) under normoxia and hypoxia conditions (Figure 8C).

397

Proliferation of HaCaT cells under interventions

398

Findings of chromogenic ICC images for expressions of Ki67 which is known as non-histone

399

protein (performs a crucial role in cellular proliferation) depicted the differences in nuclear

400

expression of Ki67 (Figure 7A a-d). The grey scale intensity value for each study group images

401

of the nucleus was quantified to create notch box plot. The notch box plot (Figure 7B) analysis

402

revealed that under 0.063 mg/ml of HEP the expression of Ki67 was significantly up-regulated

403

for normoxia (p=5.243 E-05) and hypoxia (p= 9.736 E-08) as compared to other HEP dilutions

404

(0.125 and 0.031). The western blot analysis of Ki67 expression showed a non-significant

405

(*P>0.05) change in expression of 0.063 mg/ml normoxic and hypoxic cells (Figure 8B). Here,

406

the relative quantification of total m-RNA level i.e. qRT-PCR (Figure 8C) of 0.063 mg/ml of

407

HEP for Ki67 was found to be up-regulated (**P>0.1) under both nomoxic and hypoxic



Page 20 of 46

408

conditions.

409

Not only Ki67 but also the expressional alteration of another crucial nuclear protein, p63

410

(epithelial master regulator) having involvement on cellular proliferation and differentiation were

411

very indicative.

412

whereas under hypoxia some cells showed intense p63 expression (dark brown nucleus) and

413

other showed very faint p63 expression (purple nucleus). Figure 7D represented notch box plot

414

analysis of the grey scale intensities of p63 expression in control vs. study groups; in which

415

0.063 mg/ml of HEP showed significantly highest (p=1.61773E-23) under normoxia and hypoxia

416

(p=3.72928E-11). The proteomic expression of p63 as per western blot analysis, no significant

417

(*P>0.05) changes between normoxic and hypoxic cells under 0.063 mg/ml HEP were found.

418

The result of qRT-PCR (Figure 8C) also indicated the comparative up-regulated expression of

419

p63 under 0.063 mg/ml of HEP in normoxia and hypoxia conditions.

420

Figure 9 represented the FTIR spectral signature (500-4000 cm-1) of HaCaT cells under

421

interventions. For this, the cell sample preparation protocol as mentioned by Pereira et al. (2012)

422

was adopted18. According, to this report cell fixation by 70% alcohol produced less alteration in

423

bio-chemical parameter as compared to formalin and methacarn fixation. Further, these selected

424

spectra were analyzed by OMNIC software to find the specific functional group peaks. From this

425

spectrum, the changes in nucleic acid region (DNA/RNA) (1800–900 cm-1), lipid (2800-2950

426

cm-1) and protein region (2800-3700 cm-1) 35 were clearly visible (Figure 9). It was observed that

427

normoxic control and 0.063 mg/ml represented similar spectra but at different intensity. The

428

major changes in nucleic acids like symmetric PO2- stretching in RNA and DNA; PO2-

429

symmetric (phosphate II) of the phosphodiester group of nucleic acids and membrane

430

phospholipids, and partially protein (amide III) were identified (ST 5). The bands originating

34

Figure 7C (a-d) showed all the cells expressed p63 but at different extent


Page 21 of 46


431

from sugar chains (C-OH band) were overlapped, mostly phospholipids phosphate and partly

432

from oligosaccharide C-OH bonds phosphate esters. Symmetric PO2- stretching in RNA and

433

DNA (1043.80 cm-1) was more intense in 0.063 mg/ml than control under hypoxia (ST 5).

434

DISCUSSION

435

In the context of quality control of honey, besides compositional analysis, evaluation of its anti-

436

oxidant potential, under both normoxic / hypoxic conditions is important

437

screened the compositional variability of sixteen different honey samples and have selected JH

438

as better one considering composition as well as anti-oxidant contents (ST3) and anti-oxidative

439

roles (Table 2). The observations of table 2 indicated a strong positive co-relation between total

440

phenolics and anti-oxidant potential of JH. Further, it was corroborative with the reports of

441

Alsanad et al. (2014) regarding principal roles of phenolic compounds in antioxidant activity. 36

442

However, we didn’t find any significant correlation of flavonoids with total phenolic content,

443

catalase and DPPH RSA activity. This was supported the findings of Pontis et al. (2014) in

444

respect to lack of any correlation between the sub-group of flavonoids like flavonones and

445

dihydroflavonols with total phenol and antioxidant properties 37.

446

In precise analysis of anti-oxidant constituents of JH the CC, HPLC and MALDI-TOF MS were

447

successfully employed. These analysis were effective to identify phytochemicals and plant

448

secondary metabolites viz. flavonoids (hesperetin, meciadanol, formononetin, isosakuranetin,

449

quercetin, etc.); phenolic acids (gastrodin, oleocanthal, 4-O-methylhonokiol, ellagic acid,

450

hyperoside, etc.), anthocyanins (6-hydroxycyanidin, malvidin, myrtilin, peonidin 3-O-glucoside,

451

etc.) and tannins (castalin, digalloyl-beta-D-glucose, tetraphlorethol C ) (ST 4) in HEP having

452

well known anti-oxidant properties

453

pertinent which described a range of secondary metabolites in Jamun

32, 33, 38

27

. Present work thus

. In this context, report of Chagas et al., 2015 is (Syzygium cumini)



Page 22 of 46

454

possessing anti-hyperglycemic, anti-inflammatory, antibacterial and antioxidant properties is

455

pertinent

456

efficacy for RSA (Figure 1C).

457

The protective integrity of human skin is affected in many ways under environmental assaults 40.

458

The skin is slightly hypoxic in nature which exacerbates further in wound due to vascular

459

damage and poor blood supply

460

oxidation of membrane lipids and MDA formation 42. Although human body has defense system

461

to mitigate the level of O2•− radicals, under chronic hypoxia this ability is impaired 43. Therefore,

462

we intended to observe the anti-oxidant activity of HEP on human keratinocytes (HaCaT) under

463

hypoxia.

464

The limited life span and stringent culture conditions of primary keratinocytes motivated to use

465

HaCaT (spontaneously immortalized human keratinocyte) cell line as keratinocytes model in this

466

study 44.

467

In our previous study we reported effect of honey dilutions on hypoxic HaCaT cell wound

468

model47. Hypoxic situation has connotation

469

inflammatory conditions embedding higher oxidative stress

470

hypoxia by augmenting endogenous defense system of skin 46. Our present finding on the ability

471

of HEP to reduce hypoxia induced oxidative stress in HaCaT cells is meaningful in the context of

472

intensifying cutaneous anti-oxidative defense. Furthermore, present study illustrated differential

473

efficacy of HEP dilutions (viz. 0.25, 0.125, 0.063 and 0.031 mg/ml) in combating the hypoxic

474

assault on HaCaT cells using survival assay, NBT assay and lipid per-oxidation test (Figure 2).

475

The

476

attachments and contact inhibition (Figure 2A) of epithelial cells

39

. However, for the first time we are reporting their presence in Jamun honey and its

41

. Hypoxia generates superoxide (O2•−) radicals causing per-

with different delayed wound healing and 45

. Natural antioxidants reduce

hypoxia causes alteration in regular shape (cobblestone), compactness, cell-cell 47

and these are again


Page 23 of 46


477

associated with damaging impacts of ROS 48. Our study showed that HaCaT cells under hypoxia

478

started to divide with 0.063 mg/ml HEP, justifying the anti-oxidative/protective role of this

479

dilution (Figure 2 A g). The efficacy of 0.063 mg/ml HEP to combat cellular hypoxic assault has

480

been further justified with the findings on O2•− radicals scavenging activity (Figure 2C, E, F),

481

MDA formation study (Figure 2D) and time lapse imaging (Figure 3). The counter-balancing

482

impact of HEP in hypoxic assaults maintenance of cell-cell integrity, proliferation, membrane

483

phospholipids reorganizations, nucleic acids, PO2- stretching were illustrated for HaCaT cells

484

(Figure 9). The relinquishing impact of HEP under hypoxic stress was also appraised by

485

transcriptomic and proteomic findings on prime epithelial genes in HaCaT. The expressional

486

modulations in E-cadherin-β-catenin-F-actin complex, Hif 1α (induced by hypoxia), Ki67 and

487

p63 under different HEP dilutions were remarkable (Figure 4-7). E-cadherin, the

488

molecule of cadherin-catenin-cytoskeleton complex and imperative in scheming cell migration as

489

well as proliferation 49. It has been opined that besides controlling ROS generation, anti-oxidants

490

have vital role in protecting membranous E-cadherin protein 50. Thus, our findings on restoration

491

of epithelial cell-cell adhesion and membranous E-cadherin with 0.063 and 0.031 HEP dilutions

492

(Figure 4c2, 4d2; Figure 8C) even under hypoxia were corroborative to the said notion. The

493

results were again supported by NBT assay on hypoxic cells under 0.063 and 0.031 HEP

494

dilutions showing significant (p < 0.05) increase in superoxide scavenging activity (Figure 2C, E

495

& F) along with decrease in lipid per-oxidation (p ≤ 0. 01) (Figure 2D).

496

HEP impacts on β-catenin could not be neglected as expression of this gene significantly

497

contributes in terminal differentiation and migration of keratinocytes

498

intercellular adhesion process52. It is reported that the expression of β-catenin becomes

499

cytoplasmic under low oxygen/ hypoxic condition 53 and our study demonstrated reversal of that

51

crucial

and plays a vital role in



Page 24 of 46

500

in cells under hypoxia with 0.063mg/ml HEP (Figure 5). Thus, it validated the efficacy of HEP to

501

renovate cellular integrity at molecular levels.

502

The cytoskeletal protein like F-actin has very important role in maintaining cellular mechanical

503

support and elasticity

504

filamentous web like actin into parallel stress fibres

505

alteration i.e. round and scattered HaCaT cells under hypoxia (Figure 2A e) along with F-actin

506

rearrangement but under 0.063 mg/ml HEP the cellular compactness was increased (Figure 2A g)

507

and F-actin arrangement was modified with reappearance of some interconnecting peripheral

508

fibers (Figure 6 c2). It indicated cells were recovering from hypoxia induced stress.

509

The hypoxic condition commonly occurs in chronic wound which shows up-regulation of Hif 1α

510

15

511

(0.063mg/ml) has shown positive impacts on hypoxic epithelial cells through modulating Hif 1α

512

expression (Figure 5). Interestingly, this study again brought forward the possible dose–effect

513

relation of anti-oxidants by establishing superior result under 0.063mg/ml HEP in comparison to

514

other dilutions (Figure 2).

515

The Ki67 is a crucial marker (nuclear protein) known to be involved in cell cycle which gets

516

attenuated after termination of mitotic phase

517

contributory for validating cellular proliferation under a given set of intervention on HaCaT

518

population 57. Ki67 expression in 0.063 mg/ml HEP under normoxia and hypoxia was more than

519

other HEP dilutions exhibiting cellular proliferation even under hypoxia (Figure 7B).

520

Present findings on the epithelial master regulator p6334 was informative in the context of

521

protective effects of HEP dilutions on proliferation and differentiation of keratinocytes57 under

522

hypoxia. The highest expression of p63 in HaCaT cells (Figure 7C, 8B & 8C) under 0.063 mg/ml

54

but hypoxia provokes changes in actin cytoskeleton i.e. conversion of 55

. Our work exhibited morphological

and there are reports regarding potential of honey in healing of such wounds

56

45

. HEP

. Therefore, present observation on Ki67 was


Page 25 of 46


523

HEP (**P ≤ 0.1) at normoxic and hypoxic conditions depicted strong positive correlation (r =

524

0.87-0.99) with Ki67 expressions demonstrating efficacy of honey polyphenolics to counter

525

damaging impact of hypoxia on cellular proliferation and differentiation (SF 2).

526

Towards understanding possible mode of HEP’s anti-oxidant activity and the targets it was

527

found that HEP inhibited membrane lipid peroxidation, intracellular ROS production and Hif 1α

528

expression under hypoxia. Sustenance of cell membrane integrity was also illustrated by

529

membranous expression of E-cadherin and β-catenin which again persuade peripheral F-actin re-

530

organizations. Other intracellular targets of HEP included p63 and Ki67, which were found to be

531

up-regulated. As result of less ROS production, DNA damage was reduced (Figure 10).

532

In conclusion, this study demonstrated for the first time the characteristic HEP in JH and its

533

potent anti-oxidant effects to counter hypoxic assaults on HaCaT cells in vitro. The findings

534

indicated topical applicability of JH HEP (0.063 mg/ml) as an effective antioxidant in

535

minimizing cutaneous oxidative stress. However, in depth study is required to elucidate exact

536

doses of such polyphenolics and their time dependent impact on cells. Present observation thus

537

motivated us to record cellular dynamics under specific HEP dose via real time live cell imaging

538

and cell tracking. In addition it paved the path to endorse HEP in dietary intake to reduce

539

oxidative stress and in devising novel topical therapy.

540 541

Conflict of interest: None

542

List of symbols and abbreviations

543

AlCl3, aluminium tri-chloride; BSA, bovine serum albumin; CC, column chromatography; HEP,

544

honey extracted polyphenolics; DAPI, 4', 6-diamidino-2-phenylindole; DMEM-F12, delbecco’s

545

modified eagle media-nutrient mixture F12 Ham; DPPH, 2, 2-diphenyl-1-picryylhydrazyl; FBS,



Page 26 of 46

546

fetal bovine serum; GAE, gallic acid equivalence; GAPDH, glyceraldehyde 3-phosphate

547

dehydrogenase; H2O2, hydrogen peroxide; HaCaT cells, human immortal keratinocytes; ICC,

548

immuno-cytochemistry;

549

malondialdehyde;

550

diphenyltetrazolium bromide; Na2CO3, sodium carbonate; PBS, phosphate buffered saline; QE,

551

quercetin equivalence; ROS, reactive oxygen species; RSA, radical scavenging activity; RT-

552

PCR, real time polymerase chain reaction; TBA, 2-thiobarbituric acid ;TCA, trichloroacetic acid

553

Acknowledgements

554

Source of Funding: This work was financially supported by Ministry of Human Resource

555

Development, Government of India, New Delhi, India (IIT/SRIC/SMST/NIC/2013-14/228,

556

dated: 16-04-2014) and University Grant Commission, New Delhi, India (Ref: F. No. 19-

557

6/2011(i) EU-IV, dated, 30.11.2011).

JH,

mRNA,

jamun

honey;

messenger

K2Cr2O7,

RNA;

MTT,

potassium

di-cromate;

MDA,

3-(4,5-dimethylthiazol-2-yl)-2,5-

558 559

Supporting Information description

560

Supporting Table 1. Source of different honey samples; ST 2. Physico-chemical properties of

561

honey; ST 3. Anti-oxidant properties of honey; ST 4. Phenolics / Flavonoids identification in

562

selected major fraction; ST 5. Peak assignment for FTIR spectra of HaCaT cells;

563

Supplementary Figure (SF) 1 FTIR spectrum of different honey samples from spectral range

564

from 500-4000 cm-1 and an enlarged view of sugar region from 750-1500 cm-1; SF 2. Pearson’s

565

correlation between Ki 67 and p63 expression in different HEP concentration: A. Control

566

(Normoxia) r = 0.99; B. 0.125 (Normoxia) r = 0.87; C. 0.063 (Normoxia) r =0.97; D. 0.013

567

(Normoxia) r = 0.9; E. Control (Hypoxia) r = 0.91; F. 0.125 (Hypoxia) r = 0.98; G. 0.063

568

(Hypoxia) r = 0.98; H. 0.013 (Hypoxia) r = 0.9 (P < 0.005 for all the selected groups). 25 ACS Paragon Plus Environment

Page 27 of 46


569

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32. Zhang, H.; Tsao, R., Dietary polyphenols, oxidative stress and antioxidant and antiinflammatory effects. Curr. Opin. Food Sci. 2016, 8, 33-42. 33. Zheng, W.; Wang, S. Y., Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 2001, 49, 5165-5170. 34. Carroll, D. K.; Carroll, J. S.; Leong, C.-O.; Cheng, F.; Brown, M.; Mills, A. A.; Brugge, J. S.; Ellisen, L. W., p63 regulates an adhesion programme and cell survival in epithelial cells. Nature Cell Biol. 2006, 8, 551-561. 35. Colagar, A. H.; Chaichi, M. J.; Khadjvand, T., Fourier transform infrared microspectroscopy as a diagnostic tool for distinguishing between normal and malignant human gastric tissue. J. Biosci. 2011, 36, 669-677. 36. Alsanad, S. M.; Williamson, E. M.; Howard, R. L., Cancer patients at risk of herb/food supplement–drug interactions: a systematic review. Phytother. Res. 2014, 28, 1749-1755. 37. Pontis, J. A.; Costa, L. A. M. A. d.; Silva, S. J. R. d.; Flach, A., Color, phenolic and flavonoid content, and antioxidant activity of honey from Roraima, Brazil. Food Sci. Technol. (Campinas)2014, 34, 69-73. 38. Mouhoubi-Tafinine, Z.; Ouchemoukh, S.; Tamendjari, A., Antioxydant activity of some algerian honey and propolis. Ind. Crops Prod. 2016, 88, 85-90. 39. Chagas, V. T.; França, L. M.; Malik, S.; de Andrade Paes, A. M., Syzygium cumini (L.) skeels: aprominent source of bioactive molecules against cardiometabolic diseases. Front. Pharmacol. 2015, 6. 40. Li, M.; Lin, X.-f.; Lu, J.; Zhou, B.-r.; Luo, D., Hesperidin ameliorates UV radiation-induced skin damage by abrogation of oxidative stress and inflammatory in HaCaT cells. J. Photochem. Photobiol. B. 2016, 165, 240-245. 41. Nauta, T. D.; van Hinsbergh, V. W.; Koolwijk, P., Hypoxic signaling during tissue repair and regenerative medicine. Int. J. Mol. Sci. 2014, 15, 19791-19815. 42. Apak, R. a.; Özyürek, M.; Güçlü, K.; Çapanoğlu, E., Antioxidant Activity/Capacity Measurement. 3. Reactive Oxygen and Nitrogen Species (ROS/RNS) Scavenging Assays, Oxidative Stress Biomarkers, and Chromatographic/Chemometric Assays. J. Agric. Food Chem. 2016, 64, 1046-1070. 43. Sureda, A.; Batle, J. M.; Martorell, M.; Capó, X.; Tejada, S.; Tur, J. A.; Pons, A., Antioxidant Response of Chronic Wounds to Hyperbaric Oxygen Therapy. PloS one 2016, 11, e0163371. 44. Spörl, F.; Schellenberg, K.; Blatt, T.; Wenck, H.; Wittern, K.-P.; Schrader, A.; Kramer, A., A circadian clock in HaCaT keratinocytes. J. Invest. Dermatol. 2011, 131, 338-348. 45. Barui, A.; Banerjee, P.; Chaudhary, A.; Conjeti, S.; Mondal, B.; Dey, S.; Chatterjee, J., Evaluation of angiogenesis in diabetic lower limb wound healing using a natural medicine: A quantitative approach. Wound Med. 2014, 6, 26-33. 46. Ryter, S. W.; Kim, H. P.; Hoetzel, A.; Park, J. W.; Nakahira, K.; Wang, X.; Choi, A. M., Mechanisms of cell death in oxidative stress. Antioxid. Redox Signal. 2007, 9, 49-89. 47. Coleman, P. R.; Chang, G.; Hutas, G.; Grimshaw, M.; Vadas, M. A.; Gamble, J. R., Ageassociated stresses induce an anti-inflammatory senescent phenotype in endothelial cells. Aging (Albany NY) 2013, 5, 913-924. 48. Facino, R. M., Antioxidant and radical scavenging activity of honey in endothelial cell cultures (EA. hy926). Planta Med. 2007, 73, 1182-1189.



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721 722 723 724 725 726 727 728 729 730 731


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732

Figure Legends

733

Figure 1(A). Score of percent alteration in overall biochemical composition of different honey

734

samples represented by blue dots and absorption peaks for sugar region (750-1500 cm-1) by red

735

dots; a. F2 vs F1; b. F2 vs F1 (B). HPLC spectra of HEP at 254 nm (C) DPPH RSA activity of

736

HEP retention time fractions.

737

Figure 2 (A). Phase contrast microscopy (100x) images HaCaT cells. a-d represents control,

738

0.125, 0.063 and 0.031 mg/ml HEP dilutions respectively under normoxic condition and e-h

739

represents control, 0.125, 0.063 and 0.031 mg/ml dilutions respectively under hypoxia; (B).

740

Survival % of HaCaT under 0.063 mg/ml. HEP (C). Anti-oxidant activity of HaCaT under 0.063

741

mg/ml. HEP; (D). Lipid peroxidation of HaCaT under 0.063 mg/ml. HEP; (Significant changes

742

in 0.063 mg/ml HEP indicated by black ring in figure B,C,D), (E).Percentage area of the band of

743

superoxide dismutase of HaCaT cells under interventions {P value of Control vs HEP dilutions

744

(*P≤0.005, **P>0.005)}; (F). Native PAGE for NBT Assay.

745

Figure 3 (A). Phase contrast microscopy (100x) images HaCaT cells at different temporal point

746

(i.e. 0 hrs, 4 hrs, 8 hrs, 16 hrs). control normoxia (a1-a4), 0.063 mg/ml. HEP normoxia (b1-b4),

747

control hypoxia (c1-c4), 0.063 mg/ml. HEP hypoxia (d1-d4), HEP dilutions respectively; 3 (B).

748

Graph of cell population growing rate at 0 hrs, 4 hrs, 8 hrs and 16 hrs.

749

Figure 4. Immuno-fluoresence microscopic (200x) images of E-cadherin expression in HaCaT

750

cells. a1-d1 represents control, 0.125, 0.063 and 0.031 mg/ml of HEP respectively under

751

normoxic condition and a2-d2 represents control, 0.125, 0.063 and 0.031 mg/ml of HEP

752

respectively under hypoxia; a3-d3 represents enlarged view of E-cadherin of control, 0.125,

753

0.063 and 0.031 mg/ml of HEP under hypoxia respectively (membranous E-cadherin pointed by



Page 32 of 46

754

red arrows).

755

Figure 5 (A). Microphotographs for immuno-fluoresence expression of multiplexing of β-

756

catenin, Hif 1 α and DAPI (200x) in HaCaT cell population; a-d normoxia control, 0.125, 0.063

757

and 0.031 mg/ml dilutions of HEP respectively; e1, f1, g1 and h1 are merged image of hypoxia

758

group control, 0.125, 0.063 and 0.031 mg/ml of HEP respectively; e2-h2 are separated β-catenin

759

image of hypoxia group control, control, 0.125, 0.063 and 0.031 mg/ml of HEP respectively.

760

Similarly, e3-h3 are separated Hif 1 α image and e4-h4 are separated DAPI image of hypoxia

761

group control, control, 0.125, 0.063 and 0.031 mg/ml dilutions of HEP respectively; g2a and h2a

762

are enlarged view of β-catenin of 0.063 and 0.031 mg/ml of HEP under hypoxia respectively.

763

(membranous restoration of β-catenin indicated by yellow arrows) (B). Notch box plot for grey

764

scale intensity of Hif 1α expression of different study groups.

765

Figure 6. Immuno-fluoresence microscopic (200x) images of F-actin expression in HaCaT cells.

766

a1-d1 represents control, 0.125, 0.063 and 0.031 mg/ml of HEP respectively under normoxic

767

condition and a2-d2 represents control, 0.125, 0.063 and 0.031 mg/ml of HEP respectively under

768

hypoxia.

769

Figure 7 (A). Brightfield microscopic (200x) images Ki67 expression in HaCaT cells. a-d

770

represents control, 0.125, 0.063, 0.031 mg/ml HEP respectively under normoxia and e-h

771

represents control, 0.125, 0.063, 0.031 mg/ml HEP respectively under hypoxia; (B). Notch box

772

plot for grey scale intensity of Ki67 expression among different study groups; (C). Brightfield

773

microscopic (200x) images p63 expression in HaCaT cells. a-d represents control, 0.125, 0.063,

774

0.031 mg/ml HEP respectively under normoxic condition and e-h represents control, 0.125,

775

0.063, 0.031 mg/ml HEP respectively under hypoxia;

776

intensity of p63 expression among different study groups.

(D). Notch box plot for grey scale


Page 33 of 46


777 778

Figure 8 (A). Western blot analysis of the target genes of HaCaT cells under normoxia and

779

hypoxia under 0.063 mg./ml. HEP; (B). Semi-quantitative analysis of target genes using

780

percentage area of band (*P>0.05) (**P≤0.05); (C). Depicting alteration in mRNA expression (in

781

terms of ∆ct value) for target genes of HaCaT population under interventions; (*P ≤ 0.1),

782

(**P>0.01).

783 784

Figure 9. FTIR spectrum of HaCaT cell population under HEP interventions (Nor =Normoxia,

785

Hypo = Hypoxia).

786 787

Figure 10. Mode of action and molecular targets associated with the therapeutic activity of

788

physico-chemically characterized Jamun honey extracted polyphenolics (HEP) on HaCaT cells

789

under hypoxia.

790

791

Table Legends

792

Table 1. Primer sequences and cycling conditions for Real Time PCR

793

Table 2. Correlation matrix for honey anti-oxidant parameters

794



Page 34 of 46

Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Figure 7


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Figure 8



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Figure 9


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Figure 10



Page 44 of 46

Table 1 Primer sequences and cycling conditions for Real Time PCR Genes

Expression Primers

Product size

FP: CGGGAATGCAGTTGAGGATC RP: AGGATGGTGTAAGCGATGGC 201 bp FP: AAGCGGCTGTTAGTCAC 151bp RP: CCCTGTTCCCACTCATAC FP: AGTTTCGACGTGTCCTTCCAG p63 125 bp RP: GTCATCACCTTGATCTGGATG FP: GAACCAAATCCAGAGTCACTGG Hif 1 α 114 bp RP: GGGACTATTAGGCTCAGGTG FP: Ki67 TCCTTTGGTGGGCACCTAAGACCTG 156 bp RP: TGATGGTTGAGGTCGTTCCTTGATG FP:TGCACCACCAACTGCTTAGC GAPDH 150 bp RP:GGCATGGACTGTGGTCATGAG FP for forward primer; RP for reverse primer, bp for base pair. Cycling conditions: 95 °C for 5 min (1 cycle), 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s (40 cycles Ecadherin β-catenin


Page 45 of 46


Table 2 Correlation matrix for honey anti-oxidant parameters Phenol

Flavonoid Catalase

Pearson Correlation 1 .038 Sig. (2-tailed) .888 Sum of Squares and Phenol 35079.780 2033.265 Cross-products Covariance 2338.652 135.551 N 16 16 Pearson Correlation .038 1 Sig. (2-tailed) .888 Sum of Squares and Flavonoid 2033.265 80899.333 Cross-products Covariance 135.551 5393.289 N 16 16 ** Pearson Correlation .713 .262 Sig. (2-tailed) .002 .327 Sum of Squares and Catalase 22.422 12.508 Cross-products Covariance 1.495 .834 N 16 16 ** Pearson Correlation .872 .203 Sig. (2-tailed) .000 .451 Sum of Squares and DPPH RSA 18249.374 6448.538 Cross-products Covariance 1216.625 429.903 N 16 16 ** Correlation is significant at the 0.01 level (2-tailed).

DPPH RSA

.713** .002

.872** .000

22.422

18249.374

1.495 16 .262 .327

1216.625 16 .203 .451

12.508

6448.538

.834 16 1

429.903 16 .735** .001

.028

13.780

.002 16 .735** .001

.919 16 1

13.780

12471.993

.919 16

831.466 16



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TOC


Honey Extracted Polyphenolics Reduce Experimental Hypoxia in

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