- Email: [email protected]
Evaluation of angiogenesis in diabetic lower limb wound healing using a natural medicine: A quantitative approach
Accepted Manuscript Title: Evaluation of Angiogenesis in Diabetic Lower Limb Wound Healing using a natural medicine: a Quantitative Approach Author: Ananya Barui Provas Banerjee Amrita Chaudhuri Sailesh Conjeti Bikas K. Mondal Susmita Dey Jyotirmoy Chatterjee PII: DOI: Reference:
S2213-9095(14)00045-7 http://dx.doi.org/doi:10.1016/j.wndm.2014.09.003 WNDM 46
To appear in: Received date: Accepted date:
27-5-2014 29-9-2014
Please cite this article as: Barui A, Banerjee P, Chaudhuri A, Conjeti S, Mondal BK, Dey S, Chatterjee J, Evaluation of Angiogenesis in Diabetic Lower Limb Wound Healing using a natural medicine: a Quantitative Approach, Wound Med. (2014), http://dx.doi.org/10.1016/j.wndm.2014.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of Angiogenesis in Diabetic Lower Limb Wound Healing using a natural medicine: a Quantitative Approach
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Ananya Barui1 PhD, Provas Banerjee2 PhD, Amrita Chaudhuri3 MSC, Sailesh Conjeti3 MS, Bikas K Mondal3MSC, Susmita Dey4 MSC, Jyotirmoy Chatterjee3 PhD
Centre for Healthcare Science and Technology, IIEST, Shibpur, Howarh 711103, West Bengal, India Banerjee’s Biomedical Research Foundation, Sainthia, India-700 005
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School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India
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Electrical Engineering, B. P. Poddar Institute of Management and Technology, Kolkata, West Bengal, India
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*To whom correspondence should be addressed: Dr. Ananya Barui Assistant Professor Address: Centre for Healthcare Science and Technology, IIEST, Shibpur, Howarh 711103, West Bengal, India Email: [email protected] Phone: 91-9733388223
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Abstract: Increasing incidents of diabetes (mellitus) induced non healing lower extremity wounds and disease associated amputations have raised significant concerns related to quality of
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life of afflicted patients. High glucose level in diabetic wounds inhibits the
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transactivation of angiogenesis related molecules resulting delayed healing progression.
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Present study investigates the impact of a natural medicine like honey in angiogenesis of
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non healing diabetic lower limb wounds. Quantitative assessment of different vessel
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parameters was performed on in vitro CAM model for validation of angiogenic potential
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of honey. Further the upregulation of angiogenesis related prime molecular markers like
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HIF-1α, VEGFA, VEGFR2 are under the therapeutic intervention of honey indicated
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improved angiogensis which in turn promote the healing rate. These results may
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facilitate in determining the healing impact of this natural product in treatment of
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diabetic wounds and it may also help in developing alternative cost effective therapeutic
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modality.
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Key Words: Diabetic wound, Angiogenesis, Honey, HIF1α, VEGFA.
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1. Introduction Successful management of diabetic chronic foot ulceration is an unmet clinical
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challenge and often leads to disease-associated amputations. In diabetic patients,
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impaired healing process has been attributed to micro and macro vascular alterations
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causes peripheral neuropathy and tissue hypoxia while abnormalities in inflammatory
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pathways lead to development of infectious non healing foot wounds [1]. In such
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situations, besides anti-bacterial activity, stimulation of angiogenesis becomes necessary
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pre-requisite to promote adequate healing. Though hypoxic chronic wound ambience
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induces hypoxic-inducible factors-α (HIFα) that stimulates new blood vessels formation,
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high glucose level in diabetic wound prevents transactivation of HIF-1α and impairs
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angiogenesis by blocking the transcription of cascade of angiogenic activators like
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vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF),
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stromal-derived factor-1(SDF-1) etc. [2-3]. Dysfunction of HIF-1α is majorly caused by
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reactive oxygen species which modify its co-activator p300 [4].
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In wound beds angiogenesis is mainly controlled by the balance between
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angiogenic (like VEGF) and anti-angiogenic factors (like endostatin) [5]. VEGF
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signaling is a critical rate limiting step in sustained neo-vascularization. Its isoforms
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especially VEGF-A stimulates the angiogenesis process in paracrine manner after
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cutaneous injury and have been detected on the blood vessels of newly formed
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granulation tissues [6]. VEGF signaling is interceded by activation of transmembrane
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tyrosine kinase receptors - VEGFR-1 and VEGFR-2. During angiogenesis VEGF binds
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to VEGFR-2 leading to formation of new vasculature [7].
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To manage chronic diabetes complications, different therapeutic strategies have
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been employed [8-10]. However, no single therapy have been shown to adequately
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support the multi-dimensional requirements of diabetic wound beds. In this direction,
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there is relentless search for appropriate therapeutic modalities that promote rapid
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healing as well as substantially provide a cost effective holistic support. Different
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alternative approaches for diabetes therapy includes various herbal preparations, dietary
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components and other natural products [11]. Amongst them, honey has been used as
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natural therapeutic agents as wound dressing for over 100 years. Its healing role as a
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topical agent for both infected and non-infected wounds [12] has been reported. In last 3
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few years, increased evidence-based reports on beneficial effects of honey encourage its
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therapeutic applications in diverse disease conditions including diabetes mellitus [13].
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Amongst other therapeutic activities of honey, broad spectrum antimicrobial effects of
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honey are variably ascribed to acidic pH (3.2-4.5), H2O2 content and osmotic properties
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in addition to role of other components in low doses [14-15]. Different in vitro as well as
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in vivo studies have revealed interesting results that administration of honey along with
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other anti-diabetic drugs (like glibenclamide or metformin) can more efficiently reduce
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the serum glucose level in diabetic rats [16]. Moreover, honey is known to substantially
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decrease the serum fructosamine concentration which is not observed with standard anti-
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diabetic drugs. Combination of honey and anti-diabetic drugs are shown to improve the
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antioxidant defenses and reduce oxidative damage [17] which has impact in
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angiogenesis. Hydrogen peroxide present in honey plays stimulatory role in angiogenic
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which further facilitates granulation tissue formation. Cumulative effect of low pH and
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angiogenesis help to release oxygen in wound bed that stimulates tissue regeneration
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process [14, 18]. Clinical evidences confirm rapid healing impact of honey with
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substantial angiogenesis role in diabetic wound repair process. However lack of
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scientific validations limits its application in contemporary medicine. Though some
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recent in vitro and in vivo studies have been scientifically interpreted the healing
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mechanism of honey but as the molecular basis mainly examined in animal models, they
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have a limited translational potential. Moreover, as origin of honey is a vital issue for
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quality control and to ensure consistent healing outcome, hence quantitative validation is
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necessary for developing a standard assessment process. This study intends to bridge the
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gaps between experimental outcome and clinical observations through quantitatively
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analyzing the different aspects of angiogenesis and expressions of related molecular
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markers under honey in lower limb diabetic wounds.
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In this current study we sought to evaluate the angiogenic potential of physico-
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chemically characterized honey on treatment of non-healing diabetic foot wounds. In
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order to examine the vascular adaptation of this therapeutic agent, in vitro CAM assay
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has been performed and quantitative evaluation of vessel structure may predict the
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stimulatory role of honey in neo-vascularization. Further the modulation in the
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expressions of angiogenesis related prime molecules (like HIF1α, VEGF-A, VEGFR-2) 4
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during healing of diabetic wounds under therapeutic intervention of honey may provide
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scientific validation of its well known healing potential. However, further studies are
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required to elucidate in depth molecular mechanism that may answer the angiogenesis
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paradox of diabetic wounds and to establish therapeutic impact of honey in diabetic
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wound healing.
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2. Materials and Methods
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2.1 Collection of honey
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Honey- (ripe and dark amber colour honey), collected from bee keepers of greater
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Kolkata, India [19].
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2.2 Physico-chemical characterization
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pH, electrical conductivity and water content assessment
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Electrical conductance and pH of honey was measured at 37 ºC temperature with
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pH-conductivity meter (420A, Orion, UK). For pH measurement ROSS ultra glass
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combination glass electrode was used (Orion 8102BNUWP). Electrical conductivity was
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determined through Orion DuraProbe 4-Electrode Conductivity Cells (013605MD) with
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0.55 cm-1 cell constant.
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To measure the free water content, frozen honey was vacuum dried by lyophilizer
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(Laboratory freeze dryer, IIC Indus. Corp.). Water loss of the sample was measured at
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different time intervals (i.e. 30, 60, 90, 120 min etc.) up to the constant weight reached.
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Estimation of total phenolic content:
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Total phenolic content in honey was determined by the method using Follin-
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Ciocalteu’s phenol reagent (Fluka analytical, Germany). Aqueous solution of raw honey
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(i.e. 5 gm of honey dissolved in 50 ml of distilled water) was initially filtered (millipore
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filter -22 μm) and 500 µl of diluted honey was treated with 2.5 ml of Follin-Ciocalteu’s
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phenol reagent (0.2 N) for 5 min. 2 ml sodium carbonate solution (75 g/l) was added to
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the above mixture and incubated for 2 h at room temperature in dark [20]. Absorbance at
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760 nm was measured through UV-Vis spectrophotometer (V-1601, UV-Visible
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Spectrophotometer, Shimadzu, Japan) against the blank methanol without honey. The
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concentration of phenolic compound was determined from the standard curve of gallic
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acid.
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Estimation of Catalase Enzyme Activity (H2O2 Activity) and DPPH Radical Scavenging Activity
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For determination of catalase activity, 2 ml of 0.02 M hydrogen peroxide (H2O2)
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was added in 0.5 ml of honey dilutions (v/v in 2.5 ml PBS). 1 ml of this mixture was
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further mixed with 2 ml 5 % potassium dichromate/ acetic acid solution at time intervals
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of 0, 60 and 120 seconds. On addition of potassium dichromate, the enzyme begins to
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react with the honey forming a deep blue color and also bubbling of the liquid was seen
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due to the breakdown of hydrogen peroxide into hydrogen and oxygen. The solution was
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incubated in a boiling water bath for 10 minute and OD was taken at an absorbance of
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620 nm.
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Radical scavenging activity was determined by 2, 2-diphenyl-1-picryylhydrazyl
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(DPPH). 0.5 ml of different honey dilutions, 1 ml of methanol and 100 μl of DPPH was
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added to test tubes. Blank was prepared by adding 0.5 ml PBS, 1 ml of methanol and
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100 μl of DPPH. The tubes were kept in the dark and absorbance was takes after 60
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minutes at 517 nm in UV visible spectrophotometer. Ascorbic acid was used as positive
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control. Changing of color indicates the reducing activity of DPPH to DPPHH. Yellow
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color indicated the scavenging efficiency of samples.
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Scavenging activity in % = A- B/A × 100
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A – is the absorbance of DPPH
B – is the absorbance of DPPH and honey combination
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2.3. Biological Characterization
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Anti-microbial activity
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Bacterial strains like Staphylococcus aureus, Staphylococcus epidermidis,
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Pseudomonas aeruginosa, Klebsiella pneumonae and Escherichia coli were collected
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from B S Medical College, West Bengal, India. Disc diffusion test and minimum
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inhibitory concentration (MIC) assays were performed to determine the anti-bacterial
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activity of selected honey against these bacterial strains.
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In disc diffusion technique, 100 μl of the standard bacterial nutrient broths of
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above mentioned isolates (cultures of the isolates at the density of 1.5 x 108 CFU /ml)
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were spread on Muller Hinton agar (HiMedia) plates. Honey impregnated filter paper
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disc (8 mm in diameter) was placed on each types of bacteria inoculated plates.
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Commercially available tetracycline disc (30 mcg) and sterilized distilled water were set
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as positive and negative control respectively. Plates were incubated at 37 ºC for 24 and
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48 h under aerobic condition. After incubation period, the inhibition zones around discs
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were measured in millimeters (mm). Tests were performed in triplicate with fresh
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subcultures.
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In MIC assay, honey was serially diluted with de-ionized sterilized distilled
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water [concentration of 70%, 60%, 50%, 40%, 30%, 15%, 10% and 5 % (v/v) honey in
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de-ionized sterilized distilled water]. Diluted solutions were initially screened against
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the bacterial strains by disc diffusion technique and the lowest concentration of honey
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that exhibited bacterial resistance was selected for MIC. This study was performed in
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triplicate.
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Chorioallantoic membrane (CAM) analysis
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A batch of fertilized chick eggs (n=60) were washed with potassium
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permanganate and wiped with 70% alcohol and incubated at 37 ºC with optimum
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humidity for 48 h. Afterwards, 2 ml of egg albumin was removed without disturbing the
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yolk and again incubated for 7 d at 37 ºC. Eggs were divided into 4 equal groups viz. (a)
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positive control (treated with angiogenin factor - Angiogenin Human recombinant no.
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A6955, Sigma Aldrich), (b) negative control (treated with water), (c) raw honey. Discs
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(1 mm) containing the therapeutic agents were inserted inside the egg followed by 2 d of
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incubation.
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Assessment of blood vessel formation under different study groups in CAM
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assay was performed using stereo zoom microscopy (Olympus MVX10, Japan) under
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40x (NA 0.5). Images were grabbed digitally by DP-72 camera (Olympus, Japan) at
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1360x1024 pixels.
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2.4 Application of honey on non-healing diabetec foot wounds:
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Clinical study: Patients (n=6) of either gender (age = 45-65 years) with non-healing lower
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extremity wounds (traumatic origin) having exudation of pus, foul smell, and necrotic
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tissues and non-responding to conventional topical antibiotics were included under
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informed written consent. Ethical clearance was obtained from institutional ethical
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committee according to Helsinki declaration.
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Subsequently, honey based occlusive dressing (i.e., honey-soaked gauge
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followed by a layer of dry cotton tied with crepe elastic bandage) was applied on LLW.
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Redressing was performed with an interval of 24 h for initial 7-8 d having foul odor,
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exudation and necrotic tissues in the wounds and with progression of healing interval
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increased to 48–72 h. Clinically, pain, malodor, oedema, debridement, granulation tissue
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formation, and epithelialisation were recorded.
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Inclusion and Exclusion Criteria:
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Six patients with type 2 diabetes of age 45-65 years; body weight- 80.2 ± 3.5 kg; BMI-
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29.5 ± 1.9 kg/m2; FPG, 170 ± 10 mg/dL; and diabetes duration, 2.5 ± 0.5 years were
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studied. Patients were suffered from non-healing lower extremity wounds and non-
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responding to conventional topical antibiotics (for soframycin) were included under
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informed written consent [21].
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Collection of biopsies:
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Incisional biopsies from wound edge were collected from patients (n = 6) under
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local anaesthesia (Xylocaine) before and after (i.e., 15th and 22nd d) honey dressing.
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Normal skin samples collected from superfluous tissues of surgical interventions.
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Tissue processing:
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Biopsies fixed with 10% phosphate buffered formalin and processed for 4 μm
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thick paraffin sections on poly-L-lysine (Cat.No. P 8920 Sigma-Aldrich, St. Louis, MO,
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USA) coated slides.
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Immunohistochemistry:
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Tissue sections baked and deparaffinized then hydrated for antigen retrieval in
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10mM citrate buffer (pH 6.0) using EZ-Retriever System V.2 (Bio-Genex, San Ramon,
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California, USA) and immunostained with kit (i.e., Super Sensitive Polymer-HRP IHC 8
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Detection System Cat. no: QD400-60K BioGenex). Sections incubated with primary
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antibodies (Anti-HIF1 alpha antibody [EP1215Y]-(ab51608); abcam; Anti-VEGF
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antibody [14-124]- (ab16883); Abcam; Anti-VEGF Receptor 2 antibody [SP123]-
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(ab115805); Abcam) in 1 : 500 dilution. Primary antibody binding visualized using a
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horseradish peroxidise conjugated secondary antibody using the chromogen 3, 3’-
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diaminobenzidine (DAB) and counterstained with hematoxylin. Appropriate controls
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were put up to validate the experiments.
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Microscopic studies
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Immunohistochemical assessments were performed using Zeiss Observer.Z1
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Microscope (Carl Zeiss, Germany) under 20x (NA 0.8; pixel resolution 0.31) and 40x oil
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(NA 1.3; pixel resolution 0.16). Images were taken digitally by CCD camera (AxioCam
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MRC, Zeiss) at 1388 × 1040 pixels.
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2.5 Image processing
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Image Analysis of CAM Assay
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Angiogenic features on CAM model were quantitatively evaluated for all three
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classes of interventions. Parameters like – 1. vascular area, length of vessel skeleton, 2.
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first order features like vessel branch point density and end point density and 3. vessel
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density (minor and major) were extracted through via appropriate image analytics
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techniques schematized in Figure 1. Maximal-contrast green color channel was digitally
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extracted from the input CAM image and contrast was enhanced using contrast limited
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adaptive histogram equalization method [22]. Multi-scale second order Hessian based
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Frangi Vesselness filter was employed to selectively enhance the vessel like tubular
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structures including the minor vessels and suppresses avascular structures [23]. To
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preserve the inherent vessel structure, an edge-sensitive locally adaptive thresholding
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approach was used which also aided in calibration and generation of the corresponding
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blood vessel binary image [24]. Finally, the blood vessel skeleton was extracted using
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Medial Axis Transform and spurious end points, however the non-vascular structures
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were iteratively pruned subsequently. Table 1 summerized the formula used in
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extraction of features quantifying global angiogenesis along with derived first order
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features from the vessel skeleton [25-28].
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For analysis of IHC images (n=14) for HIF1 alpha expression, the target features
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(image intensity, lengthwise distribution) of different groups (pre, post-intervention and
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normal) were segmented by K-mean clustering method. Each image was subsequently
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analyzed by their intensity values. And the length of spread of distribution from
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basement membrane to point of interest then measured for each group. Paired‘t’ test was
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performed to observe the significance level (p<0.05) at different time points of healing.
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3. Results and Discussion
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pH, electrical conductivity, water and phenolic content of honey and their impact
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in altering diabetic wound ambience
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Indian honey under investigation exhibited acidic pH and conductivity ~18.39
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μS/cm. This acidic pH of raw honey has potential impact on repair of non-
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healing/chronic wounds which have tendency to acquire alkaline ambience. Available
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literature reported that lowering the pH in chronic wound has strong influence on
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cellular, molecular cascades in wound bed consequently aiding progressive healing [29].
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Further, this honey contains optimum amount of free water (~13%) and phenolic
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compound (~118 μg/ml) which stimulate its anti-bacterial activities. The viscous and
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hygroscopic nature of raw honey provides a protective barrier from various infections.
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Large amount of sugars along with minimum amount of free water in honey may also
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facilitate optimal moisture retention in wound bed.
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Peroxide content and free radical scavenging activity of honey and their association
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with diaetic wound healing
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H2O2 production in honey is due to the action of its inherent component glucose
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oxidase which is known to oxidize glucose to gluconic acid and produces peroxide as
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byproduct. Much of the literature indicated that H2O2 production is greatly increased
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with dilution of honey [30] and our current data (Fig. 2a) also indicated that catalase
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activity is highest at 20% (v/v) honey dilution. Generally dilutaion rate of honey
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depends on the amount of wound exudates. So it is important to know the concentration
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of H2O2 at different honey concentration. It has been found that with dilution,
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approximately 1-2 mmol/L H2O2 is produced and with this concentration the possibility
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of cyto-toxic damage in healing bed is very low [30]. 10
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During healing process of any wound types, cells generally enter from an
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quiescent state to highly proliferative and migratory state. Besides stimulatory role of
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different growth factors/cytokines, H2O2 in low dose is found to replicate the growth
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factor signaling especially related to cellular proliferation and migration. In addition to
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its well known antibacterial activity by stimulating body’s inflammatory cells, H2O2 is
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also known to promote the angiogenesis by triggering important angiogenic factor like
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VEGF through oxidant induction meditated by activated nutrophils [31].
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Now the question remains whether excessive ROS which is produced during
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healing process caused poor healing? Different studies on the expression of biomarkers
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related to oxidative damage indicated that ROS may cause damage if they are not
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properly alleviated by anti-oxidants. However, even with the incomplete knowledge
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regarding the impact of oxidative damage in healing process several studies reported the
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beneficial effect of anti-oxidants in diabetic wound healing [32]. Anti-oxidants that
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quench free radicals are generated in chronic inflammatory loop in diabetic wounds.
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Honey is a unique therapeutic agent which has anti-oxidant activity along with
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capability of producing low dose peroxide [33]. Anti-oxidant activity of honey was
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measured through its DPPH free radical scavenging activity (Fig. 2b). The % inhibition
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of free radical increases with honey dilutions from 60% to 5%. But in standard (ascorbic
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acid) the % inhibition of free radical decreases with dilutions. So in comparison to
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standard the % inhibition of free radical is inversely related to the honey dilutions.
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It is believed that high level of ROS especially H2O2 (~ 166 mM) may caused
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prolonged inflammation and neutrofil associated proteolytic environment which leads to
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delayed healing process. However very low concentration of H2O2 may promote the
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healing process in diabetic wound through increasing cellular migration and
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angiogenesis [34]. Here the unique compositional balance of honey with low dose H2O2
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production capability along with radical scavenging activity for excess ROS generated
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especially in non-healing wound may establish this natural healing agent as an important
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candidate for diabetic wound healing. However, in this regard more studies are required
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to unveil the exact molecular mechanism under honey intervention.
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Anti-microbical role of honey: Diabetis induced foot infections are serious problem often developed due to
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resistance to antibiotics. Moreover the bacterial proteases digests extra cellular matrix
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important for tissue regeneration. Outer coat of bacteria stimulates sever inflammatory
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response that often causes ulcerations by activation of the proteases. Most commonly
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occurring pathogenic strains in diabetic foot ulcer are Staphylococcus aureus,
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Pseudomonas aeruginosa, and Proteus [35]. Different studies revealed the broad
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spectrum antibacterial activity of honey against the most commonly encountered
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infections (like MRSA12, 21-24 and VRE12) that have been encountered during
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diabetes induced foot infections [36]. In present study, disc diffusion test revealed
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antibacterial properties of the Indian honey in terms of zone of inhibition against five
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different human pathogenic strains like Staphylococcus aureus, Escherichia coli,
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Pseudomonas aeruginosa, Klebsiella pneumonae and Staphylococcus epidermidis.
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Honey although exhibited significant inhibition zone against the Staphylococcus aureus,
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Escherichia coli, and Pseudomonas aeruginosa but no inhibition on Klebsiella
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pneumonae, Staphylococcus epidermidis.
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Further Minimum inhibitory concentration (MIC) study indicates that at 70%,
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60% , 50% and 40% (v/v) concentrations honey shows inhibitory effects against the
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pathogens like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
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(Table 2). MIC assay revealed 5% concentration of the honey produced no antibacterial
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effect where at concentration of 10% a small inhibition zone against Staphylococcus
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aureus and Escherichia coli were found and for Pseudomonas aeruginosa, 40% of
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honey produced a small inhibition zone.
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Quatitative analysis of angiogenesis potential of honey on in vitro CAM model
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CAM assay is an established reliable model to evaluate the pro- or anti-
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angiogenic potentials of various drugs/molecules. In the present study, vascularization
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was remarkably more (Fig. 3) under treatment of honey in comparison to negative
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control. Analysis of different parameters of angiogenesis in study gropus depicted that
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under intervention, the mean vascular area under honey was increased by 75.54% with
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respect to negative control and by 15.05% with respect to positive control (Fig 4). Total
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length of the vessel skeleton also increased two-fold under intervention when compared 12
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to negative control. These global features are indicative of the increased degree of
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angiogenesis under the intervention and overall enhanced vasoproliferative potential of
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honey. In CAM study, the pattern of vasculature depends on the combined action of
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both deterministic and random processes. During development, initially an abundant
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capillary meshwork is formed with an increasing number of branches and bifurcations
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that connect with various capillary beds to their stem vessels. Vascular network
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formation proceeds along three main stages: (i) migration and early network formation;
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(ii) network remodeling and (iii) differentiation in tubular structures as well as the
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development of capillary networks. These capillary networks are further characterized
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by typical intercapillary distances ranging from 50 to 300 mm which is important for
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optimal metabolic exchanges. In general the complexity of CAM vasculature refers to
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the density of the blood vessels. However, under different molecular interventions,
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vascular complexity may change depending on the altered growth and distribution of
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vessels. Several studies have been performed to understand the logic of vascular
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network growth. Previously feature extraction from CAM images were tedious and have
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significant inter-observer variability due to the use of low magnification bright field
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images [28]. To overcome these shortcomings, digital image analysis packages for
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extraction of blood vessels and their quantification have been employed. In this study,
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robust and locally adaptive edge-sensitive vessel extraction algorithm has been proposed
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which handles variations in illumination [37].
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Figure 4 indicated that the factors related to vascular network development
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significantly increased under honey. Vessel branch point density increased by 81.20%
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and 22.64% with respect to negative control positive control respectively, while the
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corresponding vessel end point density exhibited 46.79% and 17.17% with respect to
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negative and positive control. Major vessels density also increased 2.6 times in
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comparison to the negative control. Corresponding minor vessels observed 77 % in
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increase in their density with respect to the negative control and 20.3 % increase with
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respect to the positive control. All together the results indicate remarkable angiogenic
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potential of honey that may stimulate the rapid healing progression of non-healing
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diabetic wounds. 13
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Honey modulates the expressions of angiogenesis related markers during lower
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extremity wound healing: HIF-1α plays a crucial role in stimulating multiple angiogenic growth factors
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[14] in chronic wound ambience. Different studies have shown hyperglycemia in
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diabetic wound impairs the function and stability of this molecule [2]. Accordingly in
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present study, expression of HIF-1α was found to be significantly (p<0.001) low in pre
401
intervention biopsies (5c) in comparison to 15 days post intervention (5b). Expression of
402
this molecule was substantially increased both in terms of intensity as well as length of
403
spread of distribution from basement membrane in post intervention samples (data not
404
shown). Whereas, in normal tissue expression level was comparably low than post
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intervention biopsies indicated the absence of chronic ambience (Fig. 5a). Several
406
studies have reported the down regulation of HIF-1α in diabetic wounds resulted due to
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inhibition of transactivation of this molecule under hyperglycemic ambience that
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specifically impair binding of HIF-1alpha to the coactivator p300 [39]. Although detail
409
molecular mechanisms underlying impairment of HIF-1α in diabetes is poorly
410
understood but some recent studies predict that high glucose ambience augments
411
oxidative stress and stimulates excess ROS generation that in turn affects HIF-1α
412
regulation [39]. Application of different hydroxylase inhibitors, (DMOG, DFX etc.) with
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anti-oxidant activity has been studied to stabilize and activate HIF-1 α in diabetic
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wounds [2]. Here, anti-oxidant components of honey and their radical scavenging
415
activity possibly help in structural and functional stability of HIF-1α in diabetic wound
416
ambience.
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Inhibition of transactivation of HIF-1α also inhibits another prime angiogenesis
418
factor - VEGFA which is an essential mediator of neovascularization during healing
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process [38] . In pre-intervention tissues (Fig. 5b), expression of VEGFA is substantially
420
low and mostly confined in the basal region whereas in 15 days post intervention (Fig.
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5c), expression level is remarkably increased. Expression of VEGFR2 (Fig. 5 g-i) is also
422
improved in post-intervention biopsies (Fig. 5i) which is almost not detectable in pre-
423
intervention sample (Fig. 5h).These observations show a positive corroboration with
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HIF-1α expressions and it may indicate that with application of honey the
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hyperglycemic environment in diabetic wounds is getting modulated. Recent report by 14
Page 14 of 26
Erejuwa O. hypothesized an interesting fact that besides anti-oxidant role, other
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components of this sugar-rich product especially fructose and oligosaccharides might
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have some hypoglycemic effect [40]. In addition to its inhibitory role on oxidative stress
429
and hyperglycemia, honey ameliorates other metabolic disorders like hepatic trans-
430
aminases, triglycerides, HDL cholesterol etc. [41].
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Though application of sugar-rich material like honey (almost 70% of its
432
composition) in diabetic wounds is a bit starling however, beside high content of sugars,
433
anti-microbial and radical scavenging anti-oxidant activities of honey have a promising
434
role in quenching of excessive ROS produced in non healing diabetic wounds which stall
435
the healing progression. Interestingly, different oligosaccharides and fructose components
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of this natural healing agent have been reported to contribute in reversing the
437
hyperglycemic condition [40]. These budding evidences hypothesize that honey possible
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produce a conducive healing ambience and promote the pro-healing molecular cascades
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for successful repair of diabetic wounds. Present investigation tries to enlight the pro-
440
angiogenic potential of honey and its impact on diabetic lower limb wound healing
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through modulating the angiogenesis related molecular events. However, more studies
442
are needed to identify the component specific contribution of honey and related
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cellular/molecular signaling pathway that trigger the angiogenesis process.
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4. Conclusion
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Healing impact of honey in diabetic wounds have been reported. This study
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quantitatively depicted pro-angiogenic potential of honey on in vitro CAM model. This
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quanitative assessment may provide a standard for angiogenesis pattern of honey that
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can possibly differ with origin of honey samples and helps in quality control prior to
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clinical application. Further clinical study suggest that wound healing in subjects with
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type 2 diabetes was accelerated under honey dressing which modulate the expressions of
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prime angiogenic factors like HIF-1α, VEGFA, VEGFR2 which actually inhibited in
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hyperglycemic ambience in diabetic wounds. Antioxidant content and radical
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scavenging activities of honey possibly play a crucial role in controlling over production
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ROS in diabetic wounds. Assessment of these molecular events evidenced the healing
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impact of honey in diabetic lower limb wounds by facilitating the angiogenesis events
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which otherwise stalled in hyperglycemic wound ambience.
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1. Tahergorabi Z, Khazaei M. Imbalance of angiogenesis in diabetic complications: the mechanisms. Int J Prev Med 2012; 3:827-38. 2. Botusan IR, Sunkari VG, Savu O, Catrina AI, Grunler J, Lindberg S, et al. Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci U S A 2008;105:19426-31. 3. Tandara AA, Mustoe TA. Oxygen in wound healing--more than a nutrient. World J Surg 2004;28:294-300. 4. Covello KL, Simon MC. HIFs, hypoxia, and vascular development. Curr Top Dev Biol 2004;62:37-54. 5. Baraka AM, Guemei A, Gawad HA. Role of modulation of vascular endothelial growth factor and tumor necrosis factor-alpha in gastric ulcer healing in diabetic rats. Biochem Pharmacol 2010;79:1634-9. 6. Eming SA, Krieg T. Molecular Mechanisms of VEGF-A Action during Tissue Repair. J Investig Dermatol Symp P 2006;11:79-86. 7. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359-71. 8. Allen Jr RJ, Soares MA, Haberman ID, Szpalski C, Schachar J, Lin CD, et al. Combination Therapy Accelerates Diabetic Wound Closure. PLoS ONE 2014;9:e92667. 9. Lee P-yR. Cell/Gene Therapy For Diabetic Wound Healing, 2005. 10. Greer N, Foman NA, MacDonald R, Dorrian J, Fitzgerald P, Rutks I, et al. Advanced wound care therapies for nonhealing diabetic, venous, and arterial ulcers: a systematic review. Ann Intern Med 2013;159:532-42. 11. Chawla R, Thakur P, Chowdhry A, Jaiswal S, Sharma A, Goel R, et al. Evidence based herbal drug standardization approach in coping with challenges of holistic management of diabetes: a dreadful lifestyle disorder of 21st century. J Diabetes Metab Disord 2013;12:35. 12. McInerney RJ. Honey--a remedy rediscovered. J R Soc Med 1990;83:127. 13. Molan PC. Re-introducing honey in the management of wounds and ulcers theory and practice. Ostomy Wound Manage 2002;48:28-40. 14. Sell SA, Wolfe PS, Spence AJ, Rodriguez IA, McCool JM, Petrella RL, et al. A preliminary study on the potential of manuka honey and platelet-rich plasma in wound healing. Int J Biomater 2012;2012:313781. 15. Yaghoobi R, Kazerouni A, Kazerouni O. Evidence for Clinical Use of Honey in Wound Healing as an Anti-bacterial, Anti-inflammatory Anti-oxidant and Anti-viral Agent: A Review. Jundishapur J Nat Pharm Prod 2013;8:100-4. 16. Erejuwa OO, Sulaiman SA, Wahab MS, Sirajudeen KN, Salleh MS, Gurtu S. Glibenclamide or metformin combined with honey improves glycemic control in streptozotocin-induced diabetic rats. Int J Biol Sci 2011;7:244-52. 17. Erejuwa OO, Sulaiman SA, Wahab MS, Salam SK, Salleh MS, Gurtu S. Antioxidant protective effect of glibenclamide and metformin in combination with honey in pancreas of streptozotocin-induced diabetic rats. Int J Mol Sci 2010;11(5):2056-66.
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18. Mandal MD, Mandal S. Honey: its medicinal property and antibacterial activity. Asian Pac J Trop Biomed 2011;:154-60. 19. Barui A, Banerjee P, Das RK, Basu SK, Dhara S, Chatterjee J. Immunohistochemical Evaluation of p63, E-Cadherin, Collagen I and III Expression in Lower Limb Wound Healing under Honey. Evid Based Complement Alternat Med 2011;2011:239864. 20. Moreira L, Dias LG, Pereira JA, Estevinho L. Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal. Food Chem Toxicol 2008;46:3482-5. 21. Powell RJ, Goodney P, Mendelsohn FO, Moen EK, Annex BH. Safety and efficacy of patient specific intramuscular injection of HGF plasmid gene therapy on limb perfusion and wound healing in patients with ischemic lower extremity ulceration: results of the HGF-0205 trial. J Vasc Surg 2010;52:1525-30. 22. Karel Z. Contrast limited adaptive histogram equalization. In: Paul SH, editor. Graphics gems IV: Academic Press Professional, Inc., 1994:474-85. 23. Wells W, Colchester A, Delp S, Frangi A, Niessen W, Vincken K, et al. Multiscale vessel enhancement filtering. Medical Image Computing and ComputerAssisted Interventation — MICCAI’98: Springer Berlin Heidelberg, Inc; 1998, p. 130-7. 24. Ray N, Saha BN. Edge Sensitive Variational Image Thresholding. Proceedings of the Image Processing, 2007 ICIP 2007 IEEE International Conference on; 2007 Sept. 16 2007-Oct. 19 2007; VI - 37-VI - 40. 25. Nowak-Sliwinska P, Ballini JP, Wagnieres G, van den Bergh H. Processing of fluorescence angiograms for the quantification of vascular effects induced by antiangiogenic agents in the CAM model. Microvasc Res 2009;79:21-8. 26. Cemil K, Francis Q. A review of vessel extraction techniques and algorithms. ACM Comput Surv 2004;36:81-121. 27. Kirchner LM, Schmidt SP, Gruber BS. Quantitation of angiogenesis in the chick chorioallantoic membrane model using fractal analysis. Microvasc Res 1996;51(1):2-14. 28. Blacher S, Devy L, Hlushchuk R, Larger E, Lamandé N, Burri P, et al. QUANTIFICATION OF ANGIOGENESIS IN THE CHICKEN CHORIOALLANTOIC MEMBRANE (CAM). 2011;24. 29. Shukla VK, Shukla D, Tiwary SK, Agrawal S, Rastogi A. Evaluation of pH measurement as a method of wound assessment. J Wound Care 2007;16:291-4. 30. Bang LM, Buntting C, Molan P. The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing. J Altern Complement Med 2003;9:267-73. 31. Cho M, Hunt TK, Hussain MZ. Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release. Am J Physiol Heart Circ Physiol 2001;280:H2357-63. 32. Hallberg CK, Trocme SD, Ansari NH. Acceleration of corneal wound healing in diabetic rats by the antioxidant trolox. Res Commun Mol Pathol Pharmacol 1996;93:312. 33. van den Berg AJ, van den Worm E, van Ufford HC, Halkes SB, Hoekstra MJ, Beukelman CJ. An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey. J Wound Care 2008;17(4):172-4, 6-8.
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34. Kassim M, Achoui M, Mustafa MR, Mohd MA, Yusoff KM. Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro antiinflammatory activity. Nutr Res 2010;30(9):650-9. 35. Sharma VK, Khadka PB, Joshi A, Sharma R. Common pathogens isolated in diabetic foot infection in Bir Hospital. Kathmandu Univ Med J (KUMJ) 2006;4(3):295301. 36. Molan PC, Betts JA. Using honey to heal diabetic foot ulcers. Adv Skin Wound Care 2008;21(7):313-6. 37. Costa PZ, Soares R. Neovascularization in diabetes and its complications. Unraveling the angiogenic paradox. Life Sci;92(22):1037-45. 38. Thangarajah H, Yao D, Chang EI, Shi Y, Jazayeri L, Vial IN, et al. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Proceedings of the National Academy of Sciences 2009. 39. Teixeira AS, Andrade SP. Glucose-induced inhibition of angiogenesis in the rat sponge granuloma is prevented by aminoguanidine. Life Sci 1999;64(8):655-62. 40. Erejuwa OO, Sulaiman SA, Wahab MS. Fructose might contribute to the hypoglycemic effect of honey. Molecules 2012;17(2):1900-15. 41. Busserolles J, Gueux E, Rock E, Mazur A, Rayssiguier Y. Substituting honey for refined carbohydrates protects rats from hypertriglyceridemic and prooxidative effects of fructose. J Nutr 2002;132(11):3379-82.
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569 570 Table 1 Features Evaluating Angiogenic Potential along with their mathematical formulation and physiological importance Mathematical Formulation
Physiological Importance
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Name of Angiogenic Feature
It measures the percent contribution of vascular and avascular tissues in the given field of view. It is a global measure quantifying the degree of vasoproliferative response of the angiogenic drug being administered. It quantifies the total length of the vascular tree in the field of view.
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% Vascular Area
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Length of Vessel Skeleton
Vessel Branch Point Density Vessel End Point Density Major Vessel Density Minor Vessel Density
It is a first order derived feature from the vessel skeleton. It quantifies the degree of branching of the vascular network. It is a first order derived feature from the vessel skeleton. It quantifies the degree of transition of the vessels from vessels to the capillary plexus. It quantifies the major micro-vessel density in the field of view (Vessels > 200 µm). It quantifies the minor micro-vessel density in the field of view (Vessels ≤ 200 µm).
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Table 2 Minimum inhibitory concentration of honey
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Zone of Inhibition (in mm) E. coli P. aeruginosa 23.1±0.3 16.6±0.5 22.4±0.4 13.3±0.5 22.0±0.1 8±0.1 17.3±0.3 5.1±0.1 14.9±0.1 ND 14.2±0.4 ND 8.1±0.2 ND ND ND ND ND
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70% 60% 50% 40% 30% 15% 10% 5% H20
S. aureus 22.9±0.1 20.9±0.8 20.2±0.4 19.0±0.1 8.9±0.1 7.0±0.1 5±0.1 ND ND
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Honey sample
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570 571 572 573
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574 575 576 577 578 579 580 581 582 583 584
Figure 1. Schema for Image Analytics and Quantitative Feature Extraction from CAM Images
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depicting their trends under different interventional agents. These are: (a) % Vascular
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Area, (b) Length of Vessel Skeleton, (c) Vessel Branch Point Density, (d) Vessel End
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Point Density, (e) Major Vessel Density and (f) Minor Vessel Density. The increasing
588
trend of these features indicates increasing pro-angiogenic potential and increased
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complexity and extent of the underlying vascular structures.
Figure 2: (a) Catalase activity of different honey dilutions at different intervals, (b) DPPH radical scavenging activity of different honey dilutions
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Figure 3: Stereo-zoom microscopic images (40x) of angiogenesis in CAM developmet under (a) positive control (b) negative control (c) honey. Arrow indicated healing agent impregnated disc
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Figure 4: Notch box plots of quantitative features for assessment of CAM assay
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Figure 5: Immunohistochemical photomicrographs (20x) of skin biopsies (normal and
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leg wound periphery): (a)–(c) depicted expression of HIF1α in normal (a), before (b),
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15th days after (c) topical intervention of honey: (d)–(f) demonstrated VEGFA
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expression in in normal (d) skin and in peripheral biopsies of before (e) and after said
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days of interventions (f); (g)–(i) depicted expression of VEGFR2 in normal skin (g), pre-
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intervention (h) and postintervention (i). Expressions of HIF1α, VEGFA and VEGFR2
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substantially low in pre intervention sample (a,d,g) in comparison to post intervention
598
and normal biopsies.
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599
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b
0sec
60 sec
DPPH Radical Scavanging Activity 120
60 % v/v honey dilution in PBS 40 % v/v honey dilution in PBS 20 % v/v honey dilution in PBS 10 % v/v honey dilution in PBS 5 % v/v honey dilution PBS
100 80 60 40 20 0 60%
120 sec
40%
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% of Inhibition
control (Raw honey)
20%
cr
Catalase Activity
Absorbance at 620 nm.
599 600 601 602a 603 0.7 0.6 604 0.5 605 0.4 606 0.3 607 0.2 608 0.1 609 0 610 611 612 613
10%
standard honey dil.
5%
Honey dilutions in PBS
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Time (sec.)
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613
b
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a
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613
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613 15 days Post
Normal
a
b
d
e
g
h
Pre
c
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HIF1-α
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VEGFR2
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VEGF A
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S2213-9095(14)00045-7 http://dx.doi.org/doi:10.1016/j.wndm.2014.09.003 WNDM 46
To appear in: Received date: Accepted date:
27-5-2014 29-9-2014
Please cite this article as: Barui A, Banerjee P, Chaudhuri A, Conjeti S, Mondal BK, Dey S, Chatterjee J, Evaluation of Angiogenesis in Diabetic Lower Limb Wound Healing using a natural medicine: a Quantitative Approach, Wound Med. (2014), http://dx.doi.org/10.1016/j.wndm.2014.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of Angiogenesis in Diabetic Lower Limb Wound Healing using a natural medicine: a Quantitative Approach
1 2
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Ananya Barui1 PhD, Provas Banerjee2 PhD, Amrita Chaudhuri3 MSC, Sailesh Conjeti3 MS, Bikas K Mondal3MSC, Susmita Dey4 MSC, Jyotirmoy Chatterjee3 PhD
Centre for Healthcare Science and Technology, IIEST, Shibpur, Howarh 711103, West Bengal, India Banerjee’s Biomedical Research Foundation, Sainthia, India-700 005
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School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India
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Electrical Engineering, B. P. Poddar Institute of Management and Technology, Kolkata, West Bengal, India
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*To whom correspondence should be addressed: Dr. Ananya Barui Assistant Professor Address: Centre for Healthcare Science and Technology, IIEST, Shibpur, Howarh 711103, West Bengal, India Email: [email protected] Phone: 91-9733388223
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Abstract: Increasing incidents of diabetes (mellitus) induced non healing lower extremity wounds and disease associated amputations have raised significant concerns related to quality of
35
life of afflicted patients. High glucose level in diabetic wounds inhibits the
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transactivation of angiogenesis related molecules resulting delayed healing progression.
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Present study investigates the impact of a natural medicine like honey in angiogenesis of
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non healing diabetic lower limb wounds. Quantitative assessment of different vessel
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parameters was performed on in vitro CAM model for validation of angiogenic potential
40
of honey. Further the upregulation of angiogenesis related prime molecular markers like
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HIF-1α, VEGFA, VEGFR2 are under the therapeutic intervention of honey indicated
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improved angiogensis which in turn promote the healing rate. These results may
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facilitate in determining the healing impact of this natural product in treatment of
44
diabetic wounds and it may also help in developing alternative cost effective therapeutic
45
modality.
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Key Words: Diabetic wound, Angiogenesis, Honey, HIF1α, VEGFA.
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1. Introduction Successful management of diabetic chronic foot ulceration is an unmet clinical
57
challenge and often leads to disease-associated amputations. In diabetic patients,
58
impaired healing process has been attributed to micro and macro vascular alterations
59
causes peripheral neuropathy and tissue hypoxia while abnormalities in inflammatory
60
pathways lead to development of infectious non healing foot wounds [1]. In such
61
situations, besides anti-bacterial activity, stimulation of angiogenesis becomes necessary
62
pre-requisite to promote adequate healing. Though hypoxic chronic wound ambience
63
induces hypoxic-inducible factors-α (HIFα) that stimulates new blood vessels formation,
64
high glucose level in diabetic wound prevents transactivation of HIF-1α and impairs
65
angiogenesis by blocking the transcription of cascade of angiogenic activators like
66
vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF),
67
stromal-derived factor-1(SDF-1) etc. [2-3]. Dysfunction of HIF-1α is majorly caused by
68
reactive oxygen species which modify its co-activator p300 [4].
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In wound beds angiogenesis is mainly controlled by the balance between
70
angiogenic (like VEGF) and anti-angiogenic factors (like endostatin) [5]. VEGF
71
signaling is a critical rate limiting step in sustained neo-vascularization. Its isoforms
72
especially VEGF-A stimulates the angiogenesis process in paracrine manner after
73
cutaneous injury and have been detected on the blood vessels of newly formed
74
granulation tissues [6]. VEGF signaling is interceded by activation of transmembrane
75
tyrosine kinase receptors - VEGFR-1 and VEGFR-2. During angiogenesis VEGF binds
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to VEGFR-2 leading to formation of new vasculature [7].
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To manage chronic diabetes complications, different therapeutic strategies have
78
been employed [8-10]. However, no single therapy have been shown to adequately
79
support the multi-dimensional requirements of diabetic wound beds. In this direction,
80
there is relentless search for appropriate therapeutic modalities that promote rapid
81
healing as well as substantially provide a cost effective holistic support. Different
82
alternative approaches for diabetes therapy includes various herbal preparations, dietary
83
components and other natural products [11]. Amongst them, honey has been used as
84
natural therapeutic agents as wound dressing for over 100 years. Its healing role as a
85
topical agent for both infected and non-infected wounds [12] has been reported. In last 3
Page 3 of 26
few years, increased evidence-based reports on beneficial effects of honey encourage its
87
therapeutic applications in diverse disease conditions including diabetes mellitus [13].
88
Amongst other therapeutic activities of honey, broad spectrum antimicrobial effects of
89
honey are variably ascribed to acidic pH (3.2-4.5), H2O2 content and osmotic properties
90
in addition to role of other components in low doses [14-15]. Different in vitro as well as
91
in vivo studies have revealed interesting results that administration of honey along with
92
other anti-diabetic drugs (like glibenclamide or metformin) can more efficiently reduce
93
the serum glucose level in diabetic rats [16]. Moreover, honey is known to substantially
94
decrease the serum fructosamine concentration which is not observed with standard anti-
95
diabetic drugs. Combination of honey and anti-diabetic drugs are shown to improve the
96
antioxidant defenses and reduce oxidative damage [17] which has impact in
97
angiogenesis. Hydrogen peroxide present in honey plays stimulatory role in angiogenic
98
which further facilitates granulation tissue formation. Cumulative effect of low pH and
99
angiogenesis help to release oxygen in wound bed that stimulates tissue regeneration
100
process [14, 18]. Clinical evidences confirm rapid healing impact of honey with
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substantial angiogenesis role in diabetic wound repair process. However lack of
102
scientific validations limits its application in contemporary medicine. Though some
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recent in vitro and in vivo studies have been scientifically interpreted the healing
104
mechanism of honey but as the molecular basis mainly examined in animal models, they
105
have a limited translational potential. Moreover, as origin of honey is a vital issue for
106
quality control and to ensure consistent healing outcome, hence quantitative validation is
107
necessary for developing a standard assessment process. This study intends to bridge the
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gaps between experimental outcome and clinical observations through quantitatively
109
analyzing the different aspects of angiogenesis and expressions of related molecular
110
markers under honey in lower limb diabetic wounds.
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In this current study we sought to evaluate the angiogenic potential of physico-
112
chemically characterized honey on treatment of non-healing diabetic foot wounds. In
113
order to examine the vascular adaptation of this therapeutic agent, in vitro CAM assay
114
has been performed and quantitative evaluation of vessel structure may predict the
115
stimulatory role of honey in neo-vascularization. Further the modulation in the
116
expressions of angiogenesis related prime molecules (like HIF1α, VEGF-A, VEGFR-2) 4
Page 4 of 26
during healing of diabetic wounds under therapeutic intervention of honey may provide
118
scientific validation of its well known healing potential. However, further studies are
119
required to elucidate in depth molecular mechanism that may answer the angiogenesis
120
paradox of diabetic wounds and to establish therapeutic impact of honey in diabetic
121
wound healing.
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122
2. Materials and Methods
124
2.1 Collection of honey
125
Honey- (ripe and dark amber colour honey), collected from bee keepers of greater
126
Kolkata, India [19].
127 128
2.2 Physico-chemical characterization
129
pH, electrical conductivity and water content assessment
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Electrical conductance and pH of honey was measured at 37 ºC temperature with
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pH-conductivity meter (420A, Orion, UK). For pH measurement ROSS ultra glass
132
combination glass electrode was used (Orion 8102BNUWP). Electrical conductivity was
133
determined through Orion DuraProbe 4-Electrode Conductivity Cells (013605MD) with
134
0.55 cm-1 cell constant.
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To measure the free water content, frozen honey was vacuum dried by lyophilizer
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(Laboratory freeze dryer, IIC Indus. Corp.). Water loss of the sample was measured at
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different time intervals (i.e. 30, 60, 90, 120 min etc.) up to the constant weight reached.
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Estimation of total phenolic content:
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Total phenolic content in honey was determined by the method using Follin-
140
Ciocalteu’s phenol reagent (Fluka analytical, Germany). Aqueous solution of raw honey
141
(i.e. 5 gm of honey dissolved in 50 ml of distilled water) was initially filtered (millipore
142
filter -22 μm) and 500 µl of diluted honey was treated with 2.5 ml of Follin-Ciocalteu’s
143
phenol reagent (0.2 N) for 5 min. 2 ml sodium carbonate solution (75 g/l) was added to
144
the above mixture and incubated for 2 h at room temperature in dark [20]. Absorbance at
145
760 nm was measured through UV-Vis spectrophotometer (V-1601, UV-Visible
146
Spectrophotometer, Shimadzu, Japan) against the blank methanol without honey. The
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Page 5 of 26
concentration of phenolic compound was determined from the standard curve of gallic
148
acid.
149 150
Estimation of Catalase Enzyme Activity (H2O2 Activity) and DPPH Radical Scavenging Activity
151
For determination of catalase activity, 2 ml of 0.02 M hydrogen peroxide (H2O2)
152
was added in 0.5 ml of honey dilutions (v/v in 2.5 ml PBS). 1 ml of this mixture was
153
further mixed with 2 ml 5 % potassium dichromate/ acetic acid solution at time intervals
154
of 0, 60 and 120 seconds. On addition of potassium dichromate, the enzyme begins to
155
react with the honey forming a deep blue color and also bubbling of the liquid was seen
156
due to the breakdown of hydrogen peroxide into hydrogen and oxygen. The solution was
157
incubated in a boiling water bath for 10 minute and OD was taken at an absorbance of
158
620 nm.
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Radical scavenging activity was determined by 2, 2-diphenyl-1-picryylhydrazyl
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(DPPH). 0.5 ml of different honey dilutions, 1 ml of methanol and 100 μl of DPPH was
161
added to test tubes. Blank was prepared by adding 0.5 ml PBS, 1 ml of methanol and
162
100 μl of DPPH. The tubes were kept in the dark and absorbance was takes after 60
163
minutes at 517 nm in UV visible spectrophotometer. Ascorbic acid was used as positive
164
control. Changing of color indicates the reducing activity of DPPH to DPPHH. Yellow
165
color indicated the scavenging efficiency of samples.
167 168 169
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Scavenging activity in % = A- B/A × 100
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A – is the absorbance of DPPH
B – is the absorbance of DPPH and honey combination
170
2.3. Biological Characterization
171
Anti-microbial activity
172
Bacterial strains like Staphylococcus aureus, Staphylococcus epidermidis,
173
Pseudomonas aeruginosa, Klebsiella pneumonae and Escherichia coli were collected
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from B S Medical College, West Bengal, India. Disc diffusion test and minimum
175
inhibitory concentration (MIC) assays were performed to determine the anti-bacterial
176
activity of selected honey against these bacterial strains.
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In disc diffusion technique, 100 μl of the standard bacterial nutrient broths of
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above mentioned isolates (cultures of the isolates at the density of 1.5 x 108 CFU /ml)
179
were spread on Muller Hinton agar (HiMedia) plates. Honey impregnated filter paper
180
disc (8 mm in diameter) was placed on each types of bacteria inoculated plates.
181
Commercially available tetracycline disc (30 mcg) and sterilized distilled water were set
182
as positive and negative control respectively. Plates were incubated at 37 ºC for 24 and
183
48 h under aerobic condition. After incubation period, the inhibition zones around discs
184
were measured in millimeters (mm). Tests were performed in triplicate with fresh
185
subcultures.
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In MIC assay, honey was serially diluted with de-ionized sterilized distilled
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water [concentration of 70%, 60%, 50%, 40%, 30%, 15%, 10% and 5 % (v/v) honey in
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de-ionized sterilized distilled water]. Diluted solutions were initially screened against
189
the bacterial strains by disc diffusion technique and the lowest concentration of honey
190
that exhibited bacterial resistance was selected for MIC. This study was performed in
191
triplicate.
192 193
Chorioallantoic membrane (CAM) analysis
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A batch of fertilized chick eggs (n=60) were washed with potassium
195
permanganate and wiped with 70% alcohol and incubated at 37 ºC with optimum
196
humidity for 48 h. Afterwards, 2 ml of egg albumin was removed without disturbing the
197
yolk and again incubated for 7 d at 37 ºC. Eggs were divided into 4 equal groups viz. (a)
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positive control (treated with angiogenin factor - Angiogenin Human recombinant no.
199
A6955, Sigma Aldrich), (b) negative control (treated with water), (c) raw honey. Discs
200
(1 mm) containing the therapeutic agents were inserted inside the egg followed by 2 d of
201
incubation.
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Assessment of blood vessel formation under different study groups in CAM
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assay was performed using stereo zoom microscopy (Olympus MVX10, Japan) under
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40x (NA 0.5). Images were grabbed digitally by DP-72 camera (Olympus, Japan) at
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1360x1024 pixels.
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2.4 Application of honey on non-healing diabetec foot wounds:
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Clinical study: Patients (n=6) of either gender (age = 45-65 years) with non-healing lower
211
extremity wounds (traumatic origin) having exudation of pus, foul smell, and necrotic
212
tissues and non-responding to conventional topical antibiotics were included under
213
informed written consent. Ethical clearance was obtained from institutional ethical
214
committee according to Helsinki declaration.
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Subsequently, honey based occlusive dressing (i.e., honey-soaked gauge
216
followed by a layer of dry cotton tied with crepe elastic bandage) was applied on LLW.
217
Redressing was performed with an interval of 24 h for initial 7-8 d having foul odor,
218
exudation and necrotic tissues in the wounds and with progression of healing interval
219
increased to 48–72 h. Clinically, pain, malodor, oedema, debridement, granulation tissue
220
formation, and epithelialisation were recorded.
221
Inclusion and Exclusion Criteria:
222
Six patients with type 2 diabetes of age 45-65 years; body weight- 80.2 ± 3.5 kg; BMI-
223
29.5 ± 1.9 kg/m2; FPG, 170 ± 10 mg/dL; and diabetes duration, 2.5 ± 0.5 years were
224
studied. Patients were suffered from non-healing lower extremity wounds and non-
225
responding to conventional topical antibiotics (for soframycin) were included under
226
informed written consent [21].
227
Collection of biopsies:
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Incisional biopsies from wound edge were collected from patients (n = 6) under
229
local anaesthesia (Xylocaine) before and after (i.e., 15th and 22nd d) honey dressing.
230
Normal skin samples collected from superfluous tissues of surgical interventions.
231
Tissue processing:
232
Biopsies fixed with 10% phosphate buffered formalin and processed for 4 μm
233
thick paraffin sections on poly-L-lysine (Cat.No. P 8920 Sigma-Aldrich, St. Louis, MO,
234
USA) coated slides.
235
Immunohistochemistry:
236
Tissue sections baked and deparaffinized then hydrated for antigen retrieval in
237
10mM citrate buffer (pH 6.0) using EZ-Retriever System V.2 (Bio-Genex, San Ramon,
238
California, USA) and immunostained with kit (i.e., Super Sensitive Polymer-HRP IHC 8
Page 8 of 26
Detection System Cat. no: QD400-60K BioGenex). Sections incubated with primary
240
antibodies (Anti-HIF1 alpha antibody [EP1215Y]-(ab51608); abcam; Anti-VEGF
241
antibody [14-124]- (ab16883); Abcam; Anti-VEGF Receptor 2 antibody [SP123]-
242
(ab115805); Abcam) in 1 : 500 dilution. Primary antibody binding visualized using a
243
horseradish peroxidise conjugated secondary antibody using the chromogen 3, 3’-
244
diaminobenzidine (DAB) and counterstained with hematoxylin. Appropriate controls
245
were put up to validate the experiments.
246
Microscopic studies
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Immunohistochemical assessments were performed using Zeiss Observer.Z1
248
Microscope (Carl Zeiss, Germany) under 20x (NA 0.8; pixel resolution 0.31) and 40x oil
249
(NA 1.3; pixel resolution 0.16). Images were taken digitally by CCD camera (AxioCam
250
MRC, Zeiss) at 1388 × 1040 pixels.
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2.5 Image processing
253
Image Analysis of CAM Assay
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Angiogenic features on CAM model were quantitatively evaluated for all three
255
classes of interventions. Parameters like – 1. vascular area, length of vessel skeleton, 2.
256
first order features like vessel branch point density and end point density and 3. vessel
257
density (minor and major) were extracted through via appropriate image analytics
258
techniques schematized in Figure 1. Maximal-contrast green color channel was digitally
259
extracted from the input CAM image and contrast was enhanced using contrast limited
260
adaptive histogram equalization method [22]. Multi-scale second order Hessian based
261
Frangi Vesselness filter was employed to selectively enhance the vessel like tubular
262
structures including the minor vessels and suppresses avascular structures [23]. To
263
preserve the inherent vessel structure, an edge-sensitive locally adaptive thresholding
264
approach was used which also aided in calibration and generation of the corresponding
265
blood vessel binary image [24]. Finally, the blood vessel skeleton was extracted using
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Medial Axis Transform and spurious end points, however the non-vascular structures
267
were iteratively pruned subsequently. Table 1 summerized the formula used in
268
extraction of features quantifying global angiogenesis along with derived first order
269
features from the vessel skeleton [25-28].
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For analysis of IHC images (n=14) for HIF1 alpha expression, the target features
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(image intensity, lengthwise distribution) of different groups (pre, post-intervention and
272
normal) were segmented by K-mean clustering method. Each image was subsequently
273
analyzed by their intensity values. And the length of spread of distribution from
274
basement membrane to point of interest then measured for each group. Paired‘t’ test was
275
performed to observe the significance level (p<0.05) at different time points of healing.
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3. Results and Discussion
278
pH, electrical conductivity, water and phenolic content of honey and their impact
279
in altering diabetic wound ambience
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Indian honey under investigation exhibited acidic pH and conductivity ~18.39
281
μS/cm. This acidic pH of raw honey has potential impact on repair of non-
282
healing/chronic wounds which have tendency to acquire alkaline ambience. Available
283
literature reported that lowering the pH in chronic wound has strong influence on
284
cellular, molecular cascades in wound bed consequently aiding progressive healing [29].
285
Further, this honey contains optimum amount of free water (~13%) and phenolic
286
compound (~118 μg/ml) which stimulate its anti-bacterial activities. The viscous and
287
hygroscopic nature of raw honey provides a protective barrier from various infections.
288
Large amount of sugars along with minimum amount of free water in honey may also
289
facilitate optimal moisture retention in wound bed.
290
Peroxide content and free radical scavenging activity of honey and their association
291
with diaetic wound healing
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H2O2 production in honey is due to the action of its inherent component glucose
293
oxidase which is known to oxidize glucose to gluconic acid and produces peroxide as
294
byproduct. Much of the literature indicated that H2O2 production is greatly increased
295
with dilution of honey [30] and our current data (Fig. 2a) also indicated that catalase
296
activity is highest at 20% (v/v) honey dilution. Generally dilutaion rate of honey
297
depends on the amount of wound exudates. So it is important to know the concentration
298
of H2O2 at different honey concentration. It has been found that with dilution,
299
approximately 1-2 mmol/L H2O2 is produced and with this concentration the possibility
300
of cyto-toxic damage in healing bed is very low [30]. 10
Page 10 of 26
During healing process of any wound types, cells generally enter from an
302
quiescent state to highly proliferative and migratory state. Besides stimulatory role of
303
different growth factors/cytokines, H2O2 in low dose is found to replicate the growth
304
factor signaling especially related to cellular proliferation and migration. In addition to
305
its well known antibacterial activity by stimulating body’s inflammatory cells, H2O2 is
306
also known to promote the angiogenesis by triggering important angiogenic factor like
307
VEGF through oxidant induction meditated by activated nutrophils [31].
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Now the question remains whether excessive ROS which is produced during
309
healing process caused poor healing? Different studies on the expression of biomarkers
310
related to oxidative damage indicated that ROS may cause damage if they are not
311
properly alleviated by anti-oxidants. However, even with the incomplete knowledge
312
regarding the impact of oxidative damage in healing process several studies reported the
313
beneficial effect of anti-oxidants in diabetic wound healing [32]. Anti-oxidants that
314
quench free radicals are generated in chronic inflammatory loop in diabetic wounds.
315
Honey is a unique therapeutic agent which has anti-oxidant activity along with
316
capability of producing low dose peroxide [33]. Anti-oxidant activity of honey was
317
measured through its DPPH free radical scavenging activity (Fig. 2b). The % inhibition
318
of free radical increases with honey dilutions from 60% to 5%. But in standard (ascorbic
319
acid) the % inhibition of free radical decreases with dilutions. So in comparison to
320
standard the % inhibition of free radical is inversely related to the honey dilutions.
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It is believed that high level of ROS especially H2O2 (~ 166 mM) may caused
322
prolonged inflammation and neutrofil associated proteolytic environment which leads to
323
delayed healing process. However very low concentration of H2O2 may promote the
324
healing process in diabetic wound through increasing cellular migration and
325
angiogenesis [34]. Here the unique compositional balance of honey with low dose H2O2
326
production capability along with radical scavenging activity for excess ROS generated
327
especially in non-healing wound may establish this natural healing agent as an important
328
candidate for diabetic wound healing. However, in this regard more studies are required
329
to unveil the exact molecular mechanism under honey intervention.
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Page 11 of 26
333
Anti-microbical role of honey: Diabetis induced foot infections are serious problem often developed due to
335
resistance to antibiotics. Moreover the bacterial proteases digests extra cellular matrix
336
important for tissue regeneration. Outer coat of bacteria stimulates sever inflammatory
337
response that often causes ulcerations by activation of the proteases. Most commonly
338
occurring pathogenic strains in diabetic foot ulcer are Staphylococcus aureus,
339
Pseudomonas aeruginosa, and Proteus [35]. Different studies revealed the broad
340
spectrum antibacterial activity of honey against the most commonly encountered
341
infections (like MRSA12, 21-24 and VRE12) that have been encountered during
342
diabetes induced foot infections [36]. In present study, disc diffusion test revealed
343
antibacterial properties of the Indian honey in terms of zone of inhibition against five
344
different human pathogenic strains like Staphylococcus aureus, Escherichia coli,
345
Pseudomonas aeruginosa, Klebsiella pneumonae and Staphylococcus epidermidis.
346
Honey although exhibited significant inhibition zone against the Staphylococcus aureus,
347
Escherichia coli, and Pseudomonas aeruginosa but no inhibition on Klebsiella
348
pneumonae, Staphylococcus epidermidis.
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Further Minimum inhibitory concentration (MIC) study indicates that at 70%,
350
60% , 50% and 40% (v/v) concentrations honey shows inhibitory effects against the
351
pathogens like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
352
(Table 2). MIC assay revealed 5% concentration of the honey produced no antibacterial
353
effect where at concentration of 10% a small inhibition zone against Staphylococcus
354
aureus and Escherichia coli were found and for Pseudomonas aeruginosa, 40% of
355
honey produced a small inhibition zone.
356
Quatitative analysis of angiogenesis potential of honey on in vitro CAM model
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CAM assay is an established reliable model to evaluate the pro- or anti-
358
angiogenic potentials of various drugs/molecules. In the present study, vascularization
359
was remarkably more (Fig. 3) under treatment of honey in comparison to negative
360
control. Analysis of different parameters of angiogenesis in study gropus depicted that
361
under intervention, the mean vascular area under honey was increased by 75.54% with
362
respect to negative control and by 15.05% with respect to positive control (Fig 4). Total
363
length of the vessel skeleton also increased two-fold under intervention when compared 12
Page 12 of 26
364
to negative control. These global features are indicative of the increased degree of
365
angiogenesis under the intervention and overall enhanced vasoproliferative potential of
366
honey. In CAM study, the pattern of vasculature depends on the combined action of
368
both deterministic and random processes. During development, initially an abundant
369
capillary meshwork is formed with an increasing number of branches and bifurcations
370
that connect with various capillary beds to their stem vessels. Vascular network
371
formation proceeds along three main stages: (i) migration and early network formation;
372
(ii) network remodeling and (iii) differentiation in tubular structures as well as the
373
development of capillary networks. These capillary networks are further characterized
374
by typical intercapillary distances ranging from 50 to 300 mm which is important for
375
optimal metabolic exchanges. In general the complexity of CAM vasculature refers to
376
the density of the blood vessels. However, under different molecular interventions,
377
vascular complexity may change depending on the altered growth and distribution of
378
vessels. Several studies have been performed to understand the logic of vascular
379
network growth. Previously feature extraction from CAM images were tedious and have
380
significant inter-observer variability due to the use of low magnification bright field
381
images [28]. To overcome these shortcomings, digital image analysis packages for
382
extraction of blood vessels and their quantification have been employed. In this study,
383
robust and locally adaptive edge-sensitive vessel extraction algorithm has been proposed
384
which handles variations in illumination [37].
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Figure 4 indicated that the factors related to vascular network development
386
significantly increased under honey. Vessel branch point density increased by 81.20%
387
and 22.64% with respect to negative control positive control respectively, while the
388
corresponding vessel end point density exhibited 46.79% and 17.17% with respect to
389
negative and positive control. Major vessels density also increased 2.6 times in
390
comparison to the negative control. Corresponding minor vessels observed 77 % in
391
increase in their density with respect to the negative control and 20.3 % increase with
392
respect to the positive control. All together the results indicate remarkable angiogenic
393
potential of honey that may stimulate the rapid healing progression of non-healing
394
diabetic wounds. 13
Page 13 of 26
395
Honey modulates the expressions of angiogenesis related markers during lower
396
extremity wound healing: HIF-1α plays a crucial role in stimulating multiple angiogenic growth factors
398
[14] in chronic wound ambience. Different studies have shown hyperglycemia in
399
diabetic wound impairs the function and stability of this molecule [2]. Accordingly in
400
present study, expression of HIF-1α was found to be significantly (p<0.001) low in pre
401
intervention biopsies (5c) in comparison to 15 days post intervention (5b). Expression of
402
this molecule was substantially increased both in terms of intensity as well as length of
403
spread of distribution from basement membrane in post intervention samples (data not
404
shown). Whereas, in normal tissue expression level was comparably low than post
405
intervention biopsies indicated the absence of chronic ambience (Fig. 5a). Several
406
studies have reported the down regulation of HIF-1α in diabetic wounds resulted due to
407
inhibition of transactivation of this molecule under hyperglycemic ambience that
408
specifically impair binding of HIF-1alpha to the coactivator p300 [39]. Although detail
409
molecular mechanisms underlying impairment of HIF-1α in diabetes is poorly
410
understood but some recent studies predict that high glucose ambience augments
411
oxidative stress and stimulates excess ROS generation that in turn affects HIF-1α
412
regulation [39]. Application of different hydroxylase inhibitors, (DMOG, DFX etc.) with
413
anti-oxidant activity has been studied to stabilize and activate HIF-1 α in diabetic
414
wounds [2]. Here, anti-oxidant components of honey and their radical scavenging
415
activity possibly help in structural and functional stability of HIF-1α in diabetic wound
416
ambience.
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Inhibition of transactivation of HIF-1α also inhibits another prime angiogenesis
418
factor - VEGFA which is an essential mediator of neovascularization during healing
419
process [38] . In pre-intervention tissues (Fig. 5b), expression of VEGFA is substantially
420
low and mostly confined in the basal region whereas in 15 days post intervention (Fig.
421
5c), expression level is remarkably increased. Expression of VEGFR2 (Fig. 5 g-i) is also
422
improved in post-intervention biopsies (Fig. 5i) which is almost not detectable in pre-
423
intervention sample (Fig. 5h).These observations show a positive corroboration with
424
HIF-1α expressions and it may indicate that with application of honey the
425
hyperglycemic environment in diabetic wounds is getting modulated. Recent report by 14
Page 14 of 26
Erejuwa O. hypothesized an interesting fact that besides anti-oxidant role, other
427
components of this sugar-rich product especially fructose and oligosaccharides might
428
have some hypoglycemic effect [40]. In addition to its inhibitory role on oxidative stress
429
and hyperglycemia, honey ameliorates other metabolic disorders like hepatic trans-
430
aminases, triglycerides, HDL cholesterol etc. [41].
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Though application of sugar-rich material like honey (almost 70% of its
432
composition) in diabetic wounds is a bit starling however, beside high content of sugars,
433
anti-microbial and radical scavenging anti-oxidant activities of honey have a promising
434
role in quenching of excessive ROS produced in non healing diabetic wounds which stall
435
the healing progression. Interestingly, different oligosaccharides and fructose components
436
of this natural healing agent have been reported to contribute in reversing the
437
hyperglycemic condition [40]. These budding evidences hypothesize that honey possible
438
produce a conducive healing ambience and promote the pro-healing molecular cascades
439
for successful repair of diabetic wounds. Present investigation tries to enlight the pro-
440
angiogenic potential of honey and its impact on diabetic lower limb wound healing
441
through modulating the angiogenesis related molecular events. However, more studies
442
are needed to identify the component specific contribution of honey and related
443
cellular/molecular signaling pathway that trigger the angiogenesis process.
444
446
4. Conclusion
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Healing impact of honey in diabetic wounds have been reported. This study
447
quantitatively depicted pro-angiogenic potential of honey on in vitro CAM model. This
448
quanitative assessment may provide a standard for angiogenesis pattern of honey that
449
can possibly differ with origin of honey samples and helps in quality control prior to
450
clinical application. Further clinical study suggest that wound healing in subjects with
451
type 2 diabetes was accelerated under honey dressing which modulate the expressions of
452
prime angiogenic factors like HIF-1α, VEGFA, VEGFR2 which actually inhibited in
453
hyperglycemic ambience in diabetic wounds. Antioxidant content and radical
454
scavenging activities of honey possibly play a crucial role in controlling over production
455
ROS in diabetic wounds. Assessment of these molecular events evidenced the healing
15
Page 15 of 26
456
impact of honey in diabetic lower limb wounds by facilitating the angiogenesis events
457
which otherwise stalled in hyperglycemic wound ambience.
458
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1. Tahergorabi Z, Khazaei M. Imbalance of angiogenesis in diabetic complications: the mechanisms. Int J Prev Med 2012; 3:827-38. 2. Botusan IR, Sunkari VG, Savu O, Catrina AI, Grunler J, Lindberg S, et al. Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci U S A 2008;105:19426-31. 3. Tandara AA, Mustoe TA. Oxygen in wound healing--more than a nutrient. World J Surg 2004;28:294-300. 4. Covello KL, Simon MC. HIFs, hypoxia, and vascular development. Curr Top Dev Biol 2004;62:37-54. 5. Baraka AM, Guemei A, Gawad HA. Role of modulation of vascular endothelial growth factor and tumor necrosis factor-alpha in gastric ulcer healing in diabetic rats. Biochem Pharmacol 2010;79:1634-9. 6. Eming SA, Krieg T. Molecular Mechanisms of VEGF-A Action during Tissue Repair. J Investig Dermatol Symp P 2006;11:79-86. 7. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359-71. 8. Allen Jr RJ, Soares MA, Haberman ID, Szpalski C, Schachar J, Lin CD, et al. Combination Therapy Accelerates Diabetic Wound Closure. PLoS ONE 2014;9:e92667. 9. Lee P-yR. Cell/Gene Therapy For Diabetic Wound Healing, 2005. 10. Greer N, Foman NA, MacDonald R, Dorrian J, Fitzgerald P, Rutks I, et al. Advanced wound care therapies for nonhealing diabetic, venous, and arterial ulcers: a systematic review. Ann Intern Med 2013;159:532-42. 11. Chawla R, Thakur P, Chowdhry A, Jaiswal S, Sharma A, Goel R, et al. Evidence based herbal drug standardization approach in coping with challenges of holistic management of diabetes: a dreadful lifestyle disorder of 21st century. J Diabetes Metab Disord 2013;12:35. 12. McInerney RJ. Honey--a remedy rediscovered. J R Soc Med 1990;83:127. 13. Molan PC. Re-introducing honey in the management of wounds and ulcers theory and practice. Ostomy Wound Manage 2002;48:28-40. 14. Sell SA, Wolfe PS, Spence AJ, Rodriguez IA, McCool JM, Petrella RL, et al. A preliminary study on the potential of manuka honey and platelet-rich plasma in wound healing. Int J Biomater 2012;2012:313781. 15. Yaghoobi R, Kazerouni A, Kazerouni O. Evidence for Clinical Use of Honey in Wound Healing as an Anti-bacterial, Anti-inflammatory Anti-oxidant and Anti-viral Agent: A Review. Jundishapur J Nat Pharm Prod 2013;8:100-4. 16. Erejuwa OO, Sulaiman SA, Wahab MS, Sirajudeen KN, Salleh MS, Gurtu S. Glibenclamide or metformin combined with honey improves glycemic control in streptozotocin-induced diabetic rats. Int J Biol Sci 2011;7:244-52. 17. Erejuwa OO, Sulaiman SA, Wahab MS, Salam SK, Salleh MS, Gurtu S. Antioxidant protective effect of glibenclamide and metformin in combination with honey in pancreas of streptozotocin-induced diabetic rats. Int J Mol Sci 2010;11(5):2056-66.
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18. Mandal MD, Mandal S. Honey: its medicinal property and antibacterial activity. Asian Pac J Trop Biomed 2011;:154-60. 19. Barui A, Banerjee P, Das RK, Basu SK, Dhara S, Chatterjee J. Immunohistochemical Evaluation of p63, E-Cadherin, Collagen I and III Expression in Lower Limb Wound Healing under Honey. Evid Based Complement Alternat Med 2011;2011:239864. 20. Moreira L, Dias LG, Pereira JA, Estevinho L. Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal. Food Chem Toxicol 2008;46:3482-5. 21. Powell RJ, Goodney P, Mendelsohn FO, Moen EK, Annex BH. Safety and efficacy of patient specific intramuscular injection of HGF plasmid gene therapy on limb perfusion and wound healing in patients with ischemic lower extremity ulceration: results of the HGF-0205 trial. J Vasc Surg 2010;52:1525-30. 22. Karel Z. Contrast limited adaptive histogram equalization. In: Paul SH, editor. Graphics gems IV: Academic Press Professional, Inc., 1994:474-85. 23. Wells W, Colchester A, Delp S, Frangi A, Niessen W, Vincken K, et al. Multiscale vessel enhancement filtering. Medical Image Computing and ComputerAssisted Interventation — MICCAI’98: Springer Berlin Heidelberg, Inc; 1998, p. 130-7. 24. Ray N, Saha BN. Edge Sensitive Variational Image Thresholding. Proceedings of the Image Processing, 2007 ICIP 2007 IEEE International Conference on; 2007 Sept. 16 2007-Oct. 19 2007; VI - 37-VI - 40. 25. Nowak-Sliwinska P, Ballini JP, Wagnieres G, van den Bergh H. Processing of fluorescence angiograms for the quantification of vascular effects induced by antiangiogenic agents in the CAM model. Microvasc Res 2009;79:21-8. 26. Cemil K, Francis Q. A review of vessel extraction techniques and algorithms. ACM Comput Surv 2004;36:81-121. 27. Kirchner LM, Schmidt SP, Gruber BS. Quantitation of angiogenesis in the chick chorioallantoic membrane model using fractal analysis. Microvasc Res 1996;51(1):2-14. 28. Blacher S, Devy L, Hlushchuk R, Larger E, Lamandé N, Burri P, et al. QUANTIFICATION OF ANGIOGENESIS IN THE CHICKEN CHORIOALLANTOIC MEMBRANE (CAM). 2011;24. 29. Shukla VK, Shukla D, Tiwary SK, Agrawal S, Rastogi A. Evaluation of pH measurement as a method of wound assessment. J Wound Care 2007;16:291-4. 30. Bang LM, Buntting C, Molan P. The effect of dilution on the rate of hydrogen peroxide production in honey and its implications for wound healing. J Altern Complement Med 2003;9:267-73. 31. Cho M, Hunt TK, Hussain MZ. Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release. Am J Physiol Heart Circ Physiol 2001;280:H2357-63. 32. Hallberg CK, Trocme SD, Ansari NH. Acceleration of corneal wound healing in diabetic rats by the antioxidant trolox. Res Commun Mol Pathol Pharmacol 1996;93:312. 33. van den Berg AJ, van den Worm E, van Ufford HC, Halkes SB, Hoekstra MJ, Beukelman CJ. An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey. J Wound Care 2008;17(4):172-4, 6-8.
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34. Kassim M, Achoui M, Mustafa MR, Mohd MA, Yusoff KM. Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro antiinflammatory activity. Nutr Res 2010;30(9):650-9. 35. Sharma VK, Khadka PB, Joshi A, Sharma R. Common pathogens isolated in diabetic foot infection in Bir Hospital. Kathmandu Univ Med J (KUMJ) 2006;4(3):295301. 36. Molan PC, Betts JA. Using honey to heal diabetic foot ulcers. Adv Skin Wound Care 2008;21(7):313-6. 37. Costa PZ, Soares R. Neovascularization in diabetes and its complications. Unraveling the angiogenic paradox. Life Sci;92(22):1037-45. 38. Thangarajah H, Yao D, Chang EI, Shi Y, Jazayeri L, Vial IN, et al. The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues. Proceedings of the National Academy of Sciences 2009. 39. Teixeira AS, Andrade SP. Glucose-induced inhibition of angiogenesis in the rat sponge granuloma is prevented by aminoguanidine. Life Sci 1999;64(8):655-62. 40. Erejuwa OO, Sulaiman SA, Wahab MS. Fructose might contribute to the hypoglycemic effect of honey. Molecules 2012;17(2):1900-15. 41. Busserolles J, Gueux E, Rock E, Mazur A, Rayssiguier Y. Substituting honey for refined carbohydrates protects rats from hypertriglyceridemic and prooxidative effects of fructose. J Nutr 2002;132(11):3379-82.
M
547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568
Ac ce p
te
d
569
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569 570 Table 1 Features Evaluating Angiogenic Potential along with their mathematical formulation and physiological importance Mathematical Formulation
Physiological Importance
ip t
Name of Angiogenic Feature
It measures the percent contribution of vascular and avascular tissues in the given field of view. It is a global measure quantifying the degree of vasoproliferative response of the angiogenic drug being administered. It quantifies the total length of the vascular tree in the field of view.
us
cr
% Vascular Area
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M
an
Length of Vessel Skeleton
Vessel Branch Point Density Vessel End Point Density Major Vessel Density Minor Vessel Density
It is a first order derived feature from the vessel skeleton. It quantifies the degree of branching of the vascular network. It is a first order derived feature from the vessel skeleton. It quantifies the degree of transition of the vessels from vessels to the capillary plexus. It quantifies the major micro-vessel density in the field of view (Vessels > 200 µm). It quantifies the minor micro-vessel density in the field of view (Vessels ≤ 200 µm).
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Table 2 Minimum inhibitory concentration of honey
cr
ip t
Zone of Inhibition (in mm) E. coli P. aeruginosa 23.1±0.3 16.6±0.5 22.4±0.4 13.3±0.5 22.0±0.1 8±0.1 17.3±0.3 5.1±0.1 14.9±0.1 ND 14.2±0.4 ND 8.1±0.2 ND ND ND ND ND
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70% 60% 50% 40% 30% 15% 10% 5% H20
S. aureus 22.9±0.1 20.9±0.8 20.2±0.4 19.0±0.1 8.9±0.1 7.0±0.1 5±0.1 ND ND
us
Honey sample
Ac ce p
570 571 572 573
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574 575 576 577 578 579 580 581 582 583 584
Figure 1. Schema for Image Analytics and Quantitative Feature Extraction from CAM Images
585
depicting their trends under different interventional agents. These are: (a) % Vascular
586
Area, (b) Length of Vessel Skeleton, (c) Vessel Branch Point Density, (d) Vessel End
587
Point Density, (e) Major Vessel Density and (f) Minor Vessel Density. The increasing
588
trend of these features indicates increasing pro-angiogenic potential and increased
589
complexity and extent of the underlying vascular structures.
Figure 2: (a) Catalase activity of different honey dilutions at different intervals, (b) DPPH radical scavenging activity of different honey dilutions
ip t
Figure 3: Stereo-zoom microscopic images (40x) of angiogenesis in CAM developmet under (a) positive control (b) negative control (c) honey. Arrow indicated healing agent impregnated disc
an
us
cr
Figure 4: Notch box plots of quantitative features for assessment of CAM assay
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590
Figure 5: Immunohistochemical photomicrographs (20x) of skin biopsies (normal and
592
leg wound periphery): (a)–(c) depicted expression of HIF1α in normal (a), before (b),
593
15th days after (c) topical intervention of honey: (d)–(f) demonstrated VEGFA
594
expression in in normal (d) skin and in peripheral biopsies of before (e) and after said
595
days of interventions (f); (g)–(i) depicted expression of VEGFR2 in normal skin (g), pre-
596
intervention (h) and postintervention (i). Expressions of HIF1α, VEGFA and VEGFR2
597
substantially low in pre intervention sample (a,d,g) in comparison to post intervention
598
and normal biopsies.
te
Ac ce p
599
d
591
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599
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b
0sec
60 sec
DPPH Radical Scavanging Activity 120
60 % v/v honey dilution in PBS 40 % v/v honey dilution in PBS 20 % v/v honey dilution in PBS 10 % v/v honey dilution in PBS 5 % v/v honey dilution PBS
100 80 60 40 20 0 60%
120 sec
40%
ip t
% of Inhibition
control (Raw honey)
20%
cr
Catalase Activity
Absorbance at 620 nm.
599 600 601 602a 603 0.7 0.6 604 0.5 605 0.4 606 0.3 607 0.2 608 0.1 609 0 610 611 612 613
10%
standard honey dil.
5%
Honey dilutions in PBS
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Time (sec.)
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613
b
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a
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c
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ip t cr us an M Ac ce p
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d
613
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613 15 days Post
Normal
a
b
d
e
g
h
Pre
c
cr
ip t
HIF1-α
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f
d te
i
Ac ce p
VEGFR2
M
VEGF A
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