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In-Vitro wound healing activity of Herbal topical formulation on H9C2 Heart cells

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Abstract

The Need of Traditional Medical treatments are gradually increasing day by day because of raising awareness about our deep rooted traditions, adverse effects of current treatment methods etc. On the other side, healing wounds will be considered as an art of Medicine. With new technologies, we are still facing many down drifts in healing the wounds especially in patients with diabetes. So the authors', tried to develop a new Poly-herbal formulation and introduced the current formulation for the treatment of diabetic ulcers especially foot ulcers. Here we carried out the In-vitro Wound healing Scratch assay on H9C2 Heart cells, the results show significant healing activity and area of wound closure when compared to the existing control. Thus the study suggests, that the current formulation has been successful in treating diabetic wounds through In-vitro studies and further studies are needed on a large scale involving clinical trials to explore this more.
www.siddhapapers.org Siddha Papers 2016 (1)(1)
ISSN 0974-2522 Research Article
In-Vitro wound healing activity of Herbal topical formulation
on H9C2 Heart cells
Thomas M. Walter1, S. Merish2
1Asst Professor, Department of Gunapadam, Government Siddha Medical College,
Palayamkottai, Tirunelveli, walter@siddhawalter.org
2CRRI, Government Siddha Medical College, Palayamkottai, Tirunelveli.
merish@siddhawalter.org
ABSTRACT
The Need of Traditional Medical treatments are gradually increasing day by day
because of raising awareness about our deep rooted traditions, adverse effects of current
treatment methods etc. On the other side, healing wounds will be considered as an art of
Medicine. With new technologies, we are still facing many down drifts in healing the wounds
especially in patients with diabetes. So the authors’, tried to develop a new Poly-herbal
formulation and introduced the current formulation for the treatment of diabetic ulcers
especially foot ulcers. Here we carried out the In-vitro Wound healing Scratch assay on
H9C2 Heart cells, the results show significant healing activity and area of wound closure
when compared to the existing control. Thus the study suggests, that the current formulation
has been successful in treating diabetic wounds through In-vitro studies and further studies
are needed on a large scale involving clinical trials to explore this more.
Keywords
Wound healing, Scratch wound assay, Diabetic foot ulcer, Herbal wound treatment,
Siddha Medicine, In-vitro assay.
INTRODUCTION
Wound healing is a complex and dynamic process of replacing the missing cellular
structures and tissue layers. Healing is the interaction of complex cascade of cellular events
that generates resurfacing, reconstitution and restoration of strength of injured tissue. In
wound healing, Inflammation, Proliferation and maturation are the major three classical steps.
The poly coded herbal formulation is mainly composed of rich wound healing activity drugs,
formulated by the expert committee of Walters Siddha Research Centre (WSRC), Tirunelveli.
The same poly coded formula was prepared as per SOP (Standard operative Procedure) and
also carried out Institutional clinical trials with respect to ethical committee approval from
WSRC.
MATERIALS AND METHODS
Exponentially growing H9C2 cells were trypsinized and seeded at a density of
200,000 cells per well into 12-well plates for 24 h incubation (90% confluence). The
scratch wounds were made by a sterile 10L pipette tip through a pre-marked line. After
removal of the resulting debris from five lineal scratches, H9C2 monolayer was subsequently
rinsed three times with PBS followed by incubated with PBS containing indicated
concentrations of HA for 30 min. After rinsed with PBS, cell were further incubated in 400 m
H2O2 for 24 h and rinsed three times with PBS. The wound areas were displayed by taking
images just above the interchanges between scratched wound areas and pre-marked lines.
Sample preparation and 2D-DIGE-based proteomic analysis:
The detail experimental procedures have been described in our previous publications
(Lai et al., 2010). Briefly, H9C2 cells in normal growth medium at 80% confluence were
used for proteomic analysis. For total cellular protein analysis, cells with various treatments
were washed in chilled 0.5 × PBS and scraped in 2-DE lysis buffer containing 4% (w/v)
CHAPS, 7 M urea, 2 M thiourea, 10 mMTrisHCl, pH 8.3, 1 mM EDTA. Lysates were
homogenized by passage through a 25-gauge needle 10 times, insoluble material was
removed by centrifugation at 13,000 rpm for 30 min at 4 ◦C, and protein concentrations were
determined by using Coomassie Protein Assay Reagent (BioRad). Before performing 2D-
DIGE, protein samples were labelled with N-hydroxysuccinimidyl ester-derivatives of the
cyanine dyes Cy2, Cy3 and Cy5 following the protocol described previously (Wu et al.,
2012). Briefly, 150g of protein sample was minimally labelled with 375pmol of either Cy3 or
Cy5 for comparison on the same 2-DE. To facilitate image matching and cross-gel statistical
comparison, a pool of all samples was also prepared and labeled with Cy2 at a molar ratio of
2.5 pmol Cy2 per g of protein as an internal standard for all gels. Thus, the triplicate samples
and the internal standard could be run and quantify on multiple 2-DE. The labeling reactions
were performed in the dark on ice for 30 min and then quenched with a 20-fold molar ratio
excess of free l-lysine to dye for 10 min. The differentially Cy3- and Cy5-labeled samples
were then mixed with the Cy2-labeled internal standard and reduced with dithiothreitol for 10
min. IPG buffer, pH 310 nonlinear 2%, v/v (GE Healthcare) was added and the final volume
was adjusted to 450 L with 2D-lysis buffer for rehydration. The rehydration process was
performed with immobilized non-linear pH gradient (IPG) strips (pH 310, 24 cm) which
were later rehydrated by CyDyelabeled samples in the dark at room temperature overnight (at
least 12 h). Isoelectric focusing was then performed using a Multiphor II apparatus (GE
Healthcare) for a total of 62.5 kV-h at 20 C. Strips were equilibrated in 6 M urea, 30% (v/v)
glycerol, 1% SDS (w/v), 100 mMTrisHCl (pH 8.8), 65 mMdithiothreitol for 15 min and
then in the same buffer containing 240 mMiodoacetamide for another 15 min. The
equilibrated IPG strips were transferred onto 26 cm ×20 cm 12.5% poly-acrylamide gels
casted between low fluorescent glass plates. The strips were overlaid with 0.5% (w/v) low
melting point agarose in a running buffer containing bromophenol blue. The gels were run in
an Ettan Twelve gel tank (GE Healthcare) at 4.5 W per gel at 10 ◦C until the dye front had
completely run off the bottom of the gels. Afterward, the fluorescence 2-DE was scanned
directly between the low fluorescent glass plates using an Ettan DIGE Imager (GE
Healthcare). This imager is a charge-coupled device-based instrument that enables scanning
at different wavelengths for Cy2-, Cy3-, and Cy5-labeled samples. Gel analysis was
performed using DeCy-der 2-D Differential Analysis Software v7.0 (GE Healthcare) to co-
detect, normalize and quantify the protein features in the images. Features detected from non-
protein sources (e.g. dust particles and dirty backgrounds) were filtered out. Spots displaying
aaverage-fold increase or decrease in abundance with a p-value < 0.05 were selected for
protein identification.
Protein staining, in-gel digestion and MALDI-TOF MS analysis
Colloidal coomassie blue G-250 staining was used to visualize CyDye-labeled protein
features in 2-DE followed by excised interested post-stained gel pieces for MALDI-TOF MS
identification. The detailed procedures for protein staining, in-gel digestion, MALDI-TOF
MS analysis and the algorithm used for data processing were described in our previous
publication (Lai et al., 2010). The spectrometer was also calibrated with a peptide calibration
standard (Bruker Daltonics) and internal calibration was performed using trypsin autolysis
peaks at m/z 842.51 and m/z 2211.10. Peaks in the mass range of m/z 8003000 were used to
generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL
database (v57.12) with 513,877 entries using Mascot software v2.2.06 (Matrix Science,
London, UK). The following parameters were used for the search: Rodent; tryptic digest with
a maximum of 1 missed cleavage; carbamido-methylation of cysteine, partial protein N-
terminal acetylation, partial methionine oxidation and partial modification of glutamine to
pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on
significant MASCOT Mowse scores (p < 0.05), spectrum annotation and observed versus
expected molecular weight and pI on 2-DE.
Western Blot Analysis
Cells were cultured at a density of  cells/cm2 per plate. After treatment with H2O2
for 1 h, cells were washed once with cold PBS and then lysed with RIPA buffer (50 mMTris,
150 mMNaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium
orthovanadate, sodium fluoride, EDTA, and leupeptin) and 1 mM phenylmethylsulfonyl
fluoride (PMSF) for 15 min on ice. Soluble proteins were collected by centrifugation at
5500 ×g for 5 min. The protein concentration in each sample was determined using a BCA
protein assay kit (with BSA as a standard). For immunoblotting, 80 μg of protein were loaded
onto a 15% SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred
onto a polyvinylidene difluoride (PVDF) membrane. Adequate transfer of protein was
confirmed by Coomassie Blue staining of the gel and Ponceau Red staining of the
membranes. Equal protein loading was confirmed by probing for β-actin. After blocking with
7% skim milk, the membranes were probed with respective primary antibodies (1: 1000
dilution). The membranes were subsequently probed with horseradish peroxidase-conjugated
anti-rabbit antibody (1 : 1500). Proteins were detected using enhanced chemiluminescence
ECL Western blotting detection reagent, and bands were visualized by exposure to
photographic film. Densito-metric analysis of protein band was performed by using
BandScan 4.0 software.2.8.
Phospho-specific Protein Microarray Analysis
Phospho-specific protein microarray was obtained from Full Moon Biosystems Inc.
Protein microarray analysis was carried out using the protocol provided. The procedure used
to process the array was as follows: 50 μg of cell lysates in 60 μL of reaction solution were
labeled with 3 μL biotin in 10 mg/mL N, N-dimethylformamide. 50 μg of biotin-labeled
proteins were diluted in 6 mL of coupling solution and labeled as “Protein Coupling Mix”
before being applied to the array for conjugation. To prepare the antibody microarray, it was
blocked with blocking solution for 45 min at room temperature, rinsed with Milli-Q grade
water. Then the array was incubated with Protein Coupling Mix on an orbital shaker rotating
at 35 rpm for 2 h at room temperature. Afterwards, the array slide was washed twice with
30 mL of wash solution for 10 min each, rinsed extensively with Milli-Q water, and then
incubated with a Cy3-steptavidin (0.5 mg/mL) for 45 min in the dark at room temperature.
This was followed by rinsing steps with Milli-Q water. After drying by centrifugation, the
slide was scanned on a GenePix 4000B scanner (Axon Instruments, USA) and the images
were analyzed with GenePix Pro 6.0. Fluorescence intensity of each array spot was
quantified, and the mean value was calculated. For each treatment group, a phosphorylation
signal ratio induction or reduction was calculated based on the following equation: where
phosphor A or phosphor B and unphosphoA or unphosphoB were signals of the
phosphorylated and non-phosphorylated proteins from the experimental samples,
respectively.
Simulated Ischemia (SI):
SimulatedIschemia (SI)will be induced byincubatingcardiomyocytes (H9c2 cell line)
with specifiedmodified Krebs-Henseleit buffer (137 mMNaCl, 3.8 mMKCl, 0.49 mMMgCl2,
0.9mM CaCl2, and 4.0 mM HEPES) with 20mM 2-deoxyglucose, 20 mM sodiumlactate and
1 mM sodiumdithionite atpH 6.5. Control buffer composed ofKrebs-Henseleit buffer (137
mMNaCl, 3.8mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2 and 4.0 mM HEPES) supplemented
with20 mM D-glucose, 1 mM sodiumpyruvate. After simulate ischemia will haveachieved,
the ischemic buffer or controlbufferwill be removed and the cells will besubjected to
reperfusion by the addition of 2ml complete medium before furtherincubating at 37°C, 5%
CO2 for 24 hours.
Measurement of cell viability Assay:
The measurement of H9c2 cardio-myoblastviability will be performed by the reductionof
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) bymitochondrial
reductases. At the end ofreperfusion period, the medium will becollected for lactate
dehydrogenase (LDH)activity. Cells will be incubated with 0.01g/ml MTT for 2 hours at
37°C. Then, 1ml of 0.04 M HCl in isopropanol will be added to each well. The converted
dyewill be collected and the optical density will be determined spectro-photometrically at
λ570 nm with background subtraction at λ 650 nm. Cell viability will becalculated as a
percentage of control.
Measurements of cellular injury:
The cellular injury of H9c2 cardiomyoblastcell line will be measured based on
theextracellular release of lactatedehydrogenase (LDH) and CK, according to theloss of
plasma membrane integrity.
Measurement of Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) Activity:
Coronary heart perfusate will be placed on ice after weighinguntil they will be assayed for
creatine kinase (CK) andlactate dehydrogenase (LDH) activity. The extent of CKand LDH
leakage during the reperfusion period, as anindication of myocardial injury, will be estimated
by thepercentage of CK and LDH accumulative activity at eachminute of the reperfusion time
(115 minutes). In thepresent study, the CK and LDH content will be measuredby
commercially available kits.
For measuring CK activity, 20 μL of the heart perfusatewill be added to 30°C pre-warmed
cuvette containing 1 ml CK reagent and mixed by inversion. After3 min incubation, the
absorbance will be read at 340 nm,versus water as a reference. Absorbancewill be measuredat
30-second intervals for a period of 120 seconds. Theactivity of CK in U/l is the change of
absorbance per minute (ΔA/min).
For LDH measurement, 50 μLof the heart perfusatewill be added to 30°C pre-warmed cuvette
containing 1 mlCK reagent and mixed by inversion. After 3 min incubation,the absorbance
will be read at 340 nm versus water asa reference. Absorbance will be read at 10-second
intervalsfor a period of 60 seconds. The activity of LDH in U/l isthe change of absorbance
per minute (ΔA/min).
Results & Discussion
The test drug shows maximum results in a very little dosage (Fig 1). The In-vitro
scratch healing assay shows, at 50 μg, 100 μg the area of intensity will be at 35644, 17929
respectively when compared to the control 23323. The area of wound closure (Table. 1) will
be raised in 100 μg.
Fig 1. Phase contrast of Wound closure
Fig 1: Wound closure after 24 hours of extract treatment- ( MRI wound healing tool-
ImageJ analysis software )
Test
Area of intensity (Arbitrary
units)
Area of Wound closure
Control
23323
121.930
50 ug
35644
165.922
100ug
17929
142.295
Fig. 2 shows the Wound closure after 24 hours of extract treatment and it is visualized
under MRI wound healing tool- ImageJ analysis software. Where the plotting made by Along
Y axis Area in intensity (arbitrary units), Along X axis Samples. 100ug of extracts
reduced the area of wound suggesting more activity over 50ug.
Fig. 2 Graphical representation of In-vitro Wound Healing Scratch Assay
Conclusion
The present study demonstrated that the herbal topical formulation was effective in
healing the wounds. The in -vitro data indicated that wound healing effects of Test drug
might be due to the regulation and coordination of inflammation, angiogenesis and tissue
regeneration. This study gave us good scientific evidence that theherbal formula is a
promising therapy for diabetic patients with foot ulcers & chronic ulcerations.
Acknowledgement
My sincere acknowledge to our Chief, Walters Siddha Research Centre, for their
timely motivation and funds to carry out this work.
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Article
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The active role of siddha medicine in combating chronic diseases is being widely recognized nowadays. In that way, immobile patients who have bedsores carry a high incidence of morbidity and even mortality. A decubitus ulcer, also called a pressure sore or bed sore, is an open wound on the skin. Pressure sores often occur on the skin covering bony areas. The most common places for a pressure sore to appear include hips, back, ankles, and buttocks. A considerable number of nearly 1300 new cases of decubitus ulcers are occurring on a daily basis globally. This has resulted in deaths amounting up to 34,320 during 2000-2002 in US alone. While going through the classic Siddha literatures, the authors of this paper found out a very promising remedy PPP and decided to carryout Anti-microbial sensitivity testing to prove its efficacy in treating decubitus ulcers. Disc diffusion method was followed with Mueller Hinton Agar as the culture media. The organisms tested were Pseudomonas auroginosa, Staphylococcus aureus, Streptococcus mutans, Klebsiella pneumonia, E.coli, The test results show that the drug PPP is sensitive against Streptococcus mutans and E.coli. The clinical significance of the Anti-microbial study results are discussed in detail.
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The awareness and use of Traditional Siddha Indian Medicine is on the increase in this globalized world. The specialty lies in its therapeutic potential in curing chronic diseases such as Arthritis, dermatological conditions, life style disorders etc. The usage of NSAID in the treatment of painful musculo-skeletal conditions often results in adverse effects such as gastric irritation, renal damage etc. On the other hand, poly herbal medicines are safe, effective, time-tested and devoid of drastic side-effects. Siddha Medicine has many such herbal medicines indicated for the treatment of Vatha (arthritis). This research paper deals with the In-vitro Anti-inflammatory screening of such a medicine documented in Classic Siddha text, ‘Yugi Muni Vaithiya Kaaviyam’ and specially indicated for ‘Kudaithiri Vaatham’ – which can be correlated to a kind of Rheumatism attended with pain all over the body especially in the loins. In our study we reproduced an inflammatory state by treating THP-1 cells (human myelomonocytic leukemia) with pro-inflammatory stimuli, such as LPS obtaining an up-regulation in the expression and in the activity of nitrate level. Our results show a significant increase in the expression and activity of Tissue nitrite level when cells were treated with the Test drug in different concentrations.
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Results of an ethnobotanical study of wound healing treatments among the tribal people ofTirunelveli hills in southern India are presented. A total of 46 plants belonging to 44 genera and 26 familieshave been documented for their therapeutic use against wounds and related injuries such as cuts, burns, bruisescaused by external injury, boils, sores, abscess and wounds created during delivery. Leaves were the mostfrequently utilized plant part and most herbal remedies are prepared as paste and applied externally; in somecases medicinal preparations were also administered orally. Of the plants collected in the present study none ofthe plants have been reported to have such specific wound healing compounds except Areca catechu andScoparia dulcis. The present study suggested that further clinical experimentation is needed to scientificallyevaluate these widely used herbal remedies for possible bioactive effects.
HPIM,a textbook of Siddha Vaidya Tiratu,fifth edition,published by Department of Indian Medicine & Homeopathy CONTROL 50ug/ml 100 ug
  • K N Dr
  • Kuppusamy
  • Hpim Mudhaliyar
  • Dr K S Uttamarayan
Dr.K.N.Kuppusamy,Mudhaliyar,HPIM,Dr.K.S.Uttamarayan,HPIM,a textbook of Siddha Vaidya Tiratu,fifth edition,published by Department of Indian Medicine & Homeopathy CONTROL 50ug/ml 100 ug/ml 0
HPIM, a textbook of Pharmacopoeia of Hospital of Indian Medicine
  • V Dr
  • Narayana
Dr.V.Narayana swami, HPIM, a textbook of Pharmacopoeia of Hospital of Indian Medicine,second edition, published by Tamilnadu Siddha Medical Board.
Styptic And Wound Healing Properties of Siddha Medicinal Plants -A Review
  • S Merish
  • M Tamizhamuthu
  • Thomas M Walter
S. Merish, M. Tamizhamuthu, Thomas M. Walter, Styptic And Wound Healing Properties of Siddha Medicinal Plants -A Review, Int J Pharm Bio Sci 2014 April ;
The role of RIP2 in p38 MAPK activation in the stressed heart published online February 29
  • Yasuhiro Jacquet
  • Sarawut Nishino
  • Pierre Kumphune
  • James E Sicard
  • Clark
Jacquet, Yasuhiro Nishino, Sarawut Kumphune, Pierre Sicard, James E. Clark, The role of RIP2 in p38 MAPK activation in the stressed heart published online February 29, 2008 J. Biol. Chem.
Yugi Muni Vaithiya Kaaviyam publication of Thamarai Noolagam
  • Rc Mohan
RC Mohan. 2002, Yugi Muni Vaithiya Kaaviyam publication of Thamarai Noolagam, Chennai.
Dictionary Based on Indian Medical science
  • Tv Sambasivam Pillai
TV Sambasivam pillai. 1991, Dictionary Based on Indian Medical science, Second edition, Vol. 2, published by Directorate of Indian Medicine and Homeopathy, Chennai, India.