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Evaluation of wound healing activity of Acacia caesia in rats
Accepted Manuscript Title: Evaluation of wound healing activity of Acacia caesia in rats Author: Sembian Suriyamoorthy Kalidass Subramaniam Jeevan Raj Durai. S Femina Wahaab Pemila Edith Chitraselvi. R. PII: DOI: Reference:
S2213-9095(15)00004-X http://dx.doi.org/doi:10.1016/j.wndm.2015.03.001 WNDM 50
To appear in: Received date: Revised date: Accepted date:
21-5-2014 3-12-2014 18-3-2015
Please cite this article as: Suriyamoorthy S, Subramaniam K, S JRDi, Wahaab F, R. PECi, Evaluation of wound healing activity of Acacia caesia in rats, Wound Med. (2015), http://dx.doi.org/10.1016/j.wndm.2015.03.001 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.
*Manuscript
Evaluation of wound healing activity of Acacia caesia in rats Sembian Suriyamoorthy, Kalidass Subramaniam*, Jeevan Raj Durai. S, Femina Wahaab,
Department of Biotechnology
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Pemila Edith Chitraselvi. R.
School of Biotechnology and Health Sciences, Karunya University, Karunya Nagar,
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[email protected].
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Coimbatore-641114, Tamil Nadu, India
Tel: 91 422 2614300 Ext. 4499
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Fax: 91 422 2615615 ABSTRACT
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Acacia caesia (L) is a leguminous perennial climbing shrub belonging to the family
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Mimosaceae, and is native to south-east Asia. Despite the traditional practice of using the bark
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juice for wound healing there is no scientific report on the wound healing properties of this plant. Ethno pharmacologically the bark of Acacia caesia has significant prohealing activity. The
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higher levels of alkaloids, flavonoids, glycosides and saponins noted in the bark extracts of Acacia caesia could be responsible for the medicinal properties of this plant. Secondary metabolites that serve as the defensive agents are produced under stressed condition by the plants.Wound healing activity of Acacia caesia bark ethanolic extract were investigated by excision and incision wound healing models in Wistar male rats in comparison with reference standard Betadine. Wounds treated with topical application of 5% W/W Acacia caesia ethanolic extract were found to heal faster as evidenced by observing the rate of epithelialization and percentage of wound contraction.
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The increase in tensile strength of treated wounds may be due to the increase in collagen and synthesis of new extracellular matrix molecules Hexosamine and Uronic acid, which acts as a ground substratum. Tensile strength was doubled in 5% W/W Acacia caesia ethanol extract
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treated tissue. Wound tissues formed on days 4,8,12 and 16 were used to estimate DNA, total protein, total collagen, hexosamine and uronic acid. It is observed in ACEE treated groups that
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the amount of protein, hexosamine, collagen and uronic acid increased from day 4 to day 12 and
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thereafter there was a gradual decrease until the 16th day.
Key Words: Acacia caesia, Wound Healing, Period of Epithelialization, Tensile strength.
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INTRODUCTION:
Wounds are physical injuries that result in an opening or breaking of the skin. Wound
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healing is essential for the restoration of disturbed functional status and disrupted anatomical
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continuity of the skin. It is a complex multifactorial process that results in contraction, closure of
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the wound and restoration of a functional barrier (1).Current estimates indicate that nearly six million people suffer from chronic wound (2) causing great physiological and mental
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trauma.Wound healing is the interaction of a complex cascade of cellular and biochemical actions leading to the restoration of structural and functional integrity with regain of strength of injured tissue. It involves continuous cell- cell interaction and cell- matrix interactions that allow the process to proceed in different overlapping phases and processes including inflammation, wound contraction, reepithelialization, tissue remodeling and formation of granulation tissue with angiogenesis (3). Problems like resistance and pollution associated with irrational use of orthodox medicines have necessitated the use of plants as a source of effective and safer alternative for human ailments. Use of plants on curing wound and skin repair is in practice from days
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immemorable. For thousands of years nature has provided as a source of medicinal agents and number of modern drugs have been isolated from them, many based on their use in traditional medicine (4).The process of wound healing can be promoted by several plant products, which are
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composed of active principles like triterpenes, alkaloids, flavonoids (5) and biomolecules (6). Polyherbal formulations are better than the pure chemical wound healing formulations
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because the crude poly herbal formulation have various phyto constituents which have
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antimicrobial, anti-inflammatory properties and antioxidant potential. Hence phytoherbal formulation used in the treatment of wound owes assured safety and high efficacy.Hence
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scientific investigation is needed to explore the pharmacological activities of medicinal plants and to elucidate the claims made about them in folklore medicines.
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With the advent of the modern molecular techniques in biomedical sciences,the potential
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of many traditional unknown medicinal plants used for treating wounds and burns are analyzed
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and scientific investigations on the mechanism of wound healing is gaining momentum worldwide. The hidden traditional knowledge of plants used in curing wounds by the folkloric
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medicinal practitioners in countries like India, Africa, China and South East Asian countries is actively being investigated across the global scientific community. Sufficient literature informs drugs used for wound healing are invariably derived from plants; plant based wound therapy not only accelerates healing process but also maintains safety. More than 70% of wound healing pharma products are of plant origin. The plant based materials are used as first aid, antiseptic, coagulants and wound wash. Many phytopharmaceutical laboratories are now concentrating their effort to identify the active constituents and mode of action of various medicinal plants. Acacia caesia (L) is a leguminous perennial climbing shrub belonging to the family Mimosaceae, and is native to south-east Asia. The information collected from traditional
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medicine practitioners and literature shows the stem bark forms froth when rubbed with water and thus used as soap, while its decoction is used as lice killer (7). The juicy extract from the stem bark is well-known among the Mizo tribes of north-east India as remedy to gastrointestinal
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infections (8).The leaves were used as vegetable and used in the treatment of asthma, skin diseases (9,10), menstrual disorder (11-14) and scabies (15). Due to these values, it is exploited
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severely by the local public in western districts of Tamilnadu and it becomes a rare sighted
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species in the natural habitats of Western Ghats (16).
The medicinal value of this plant lies in bioactive phytochemical constituents that
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produce definite physiological action on the human body (13).Literature reports on Acacia caesia (L) inform the presence of secondary metabolites like alkaloids, flavonoids, glycosides
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and saponins. Acacia caesia extracts exhibited good antimicrobial and demonstrated good
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antioxidant activity (10). Despite these beneficial activity reports of Acacia caesia,and the
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presence of various active principles in this plant,there is a lacuna of scientific documentation on the wound healing property of Acacia caesia.To fill this research gap, we have investigated this
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study and report here a firsthand documentation of our findings on wound healing properties of Acacia caesia.
MATERIALS AND METHODS
Plant material collection and extraction Fresh barks of Acacia caesia were collected from Pannaipuram, Theni district, Tamilnadu, India. The barks were air-dried, pulverized to a coarse powder in a mechanical grinder, passed through a 40 mesh sieve. 25 gm of powder was extracted with 250 ml of ethanol in soxhlet extractor. The extract was decanted, filtered with Whatman No.1 filter paper and concentrated at reduced pressure below 40º C through rotary vapour to obtain cream of extract.
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The Acacia caesia ethanolic extract (ACEE) was stored in a closed container until further evaluation of wound healing activity. In vitro antioxidant assay
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The free radical scavenging activity of Acacia caesia ethanolic extract was measured by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) using the modified method of McCune et al.,(17). The
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superoxide anion radical scavenging activity of Acacia caesia ethanolic extract was measured by
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the method of Robak et al., (18). The hydroxyl radical scavenging activity of Acacia caesia ethanolic extract was measured by studying the competition between deoxyribose and test
reaction) (19).
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Preparation of formulation and standard used
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compound for hydroxyl radicalsgenerated by Fe+3- ascorbic acid-EDTA-H2O2 system (Fenton
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Two types of formulations with different concentrations 2 % and 5 % w/w of ACEE were
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prepared by tituration method in a ceramic mortar and pestle (20). Reference Standard (Betadine ointment) was compared with the formulations to check the wound healing potential in different
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animal models. Animals used
Wistar male rats weighing approximately 150 g - 175 g were used in the study. The rats were acclimatized to the laboratory environment for a period of 7 days. The rats were housed at 24± 2oC at a relative humidity of 40-45 % and light dark cycles of 11 and 13 h respectively. Animals were provided with rodent diet and water ad libitum. The animal study protocol was subjected to the scrutiny of Institutional Ethical Committee and was cleared by the same before beginning the experiment (IAEC/KU/BT /12/010).
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Acute dermal toxicity Acute dermal toxicity study was carried out to determine the dose as per OECD guidelines No. 402 (21). ACEE ointment with highest concentration (5 % w/w) was applied on the shaved
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dorsal region of the rats. Grouping of Animals
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Two wound models were used in our study:
Incision wound model
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The animal models contain four groups each.
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Excision wound model
The groups were:
: Control (Treated with Simple ointment base (SOB)) (n=6)
Group 2
: Standard group treated with Betadine ointment (n=6)
Group 3
: Treated with 2 % ACEE ointment (n=6)
Group 4
: Treated with 5 % ACEE ointment (n=6)
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Excision wound model
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Group 1
The animals in each group were anaesthetized by the open mask method with anaesthetic ether. Depilations of the rats were done on the dorsal side. The excision wound was inflicted by cutting away a 100 mm2 full thickness of skin from predetermined shaved area (22). Excision wound was left undressed. Topical application of the drugs to the divided groups viz. SOB, Reference Standard (Betadine ointment), ACEE 2 % w/w ointment and ACEE 5 % w/w ointment respectively were done once daily until the wound healed completely. Wound contraction and epithelialization period were monitored. Biochemical parameters were done to estimate total protein, DNA, hexosamine, total collagen and uronic acid in the granulation tissues of control
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and experimental wounds. Histopathological analysis of the tissue from the healed wound was also done (23). Incision wound model
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The animals in each group were anaesthetized and one paravertebral long incision of 4 cm length on the depilated dorsal side was made through the skin till the cutaneous muscle. Aseptic
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measures were not taken throughout the experiment (24). After incision was made the parted
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skin was kept together and stitched with black silk surgical thread (No.000) at 1 cm intervals using curved needle (No.11). Good closure of the wound was made by tightening the edges of
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the thread and was left undressed. ACEE ointments along with SOB and reference standard were applied once daily topically to their respective groups. The sutures were removed
on the 9
th
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Parameters evaluated for wound healing
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day. Tensile strength of the cured skin was measured using tenisiometer (25).
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Measurement of wound contraction
The periphery of the excision wound was traced on a transparent paper on every 4th day, till the
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wound got healed. Wound area was measured by retracing the wound on a millimeter scale graph paper .The evaluated surface area was used to calculate the percentage of wound contraction (26).
% of wound contraction = {(Initial wound size – Specific day wound size) / Initial wound size} X 100.
Epithelialization period The wound was monitored for complete epithelialization. It was measured in days from wounding day (0 thday) till the Escher totally separated itself with the raw wound left behind.
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Tensile strength of incision wound model The degree of wound healing is directly proportional to the gain in tensile strength of the tissue.
wounds was compared with control and reference standard treated groups. Biochemical estimations
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Tensile strength was measured on the 10 thday. The tensile strength of ACEE ointment treated
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Protein was estimated by the method of Lowry et al., (27). As per Burton, (28) estimation of
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DNA was done. An Uronic acid level was estimated by the method of Schiller et al., (29). Hexosamine and collagen were estimated by the method of Woessner (30).
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Histopathological examination
The cross sectional full thickness skin specimens from each group were collected at the end of
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the experiment to evaluate the histopathological analysis. Samples were fixed in 5 % buffered
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formalin, processed and blocked with paraffin and then sectioned into 5 µm sections and stained
epidermal remodeling.
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Statistical analysis
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with hematoxylin and eosin (HE) stains. The tissues were examined by light microscope for
Results obtained from three wound models have been expressed as Mean ± SEM. The data was evaluated by one way ANOVA followed by Dunnett’s T-Test, P < 0.05 and P < 0.01 was considered as significant. RESULTS
In vitro antioxidant assay The DPPH radical significantly reduced by Acacia caesia ethanolic extract and standard with increasing concentrations. Acacia caesia ethanolic extract showed potent hydroxyl radical scavenging activity which may be due to the presence of various phytochemicals. Acacia caesia
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ethanolic extract increases superoxide anion radical scavenging activity as that of the standard (Figure 1, 2 and 3). Wound contraction studies
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Reduction of wound area of different groups until the 16th day for excision wound model was calculated and depicted in Figure 4. Control groups showed least rate of wound healing. Faster
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rate of wound healing was seen in groups treated with 5 % w/w ACEE ointment. Reference
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standard heals the wound almost equivalent to the groups treated with 2% w/w ACEE ointment. Epithelialization period
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The epithelialization was observed from the first day. The epithelialization time was found to be lesser or quicker in group treated with 5 % w/w ACEE ointment than the two groups treated with
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2 % w/w ACEE ointment and Reference standard treated groups (Figure 5).
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Measurement of tensile strength
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Comparison of the tensile strength of the healed skin of the animals in different groups is shown in Figure 6. Minimum tensile strength was noticed in untreated group. Among the tensile
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strength of the tissues treated with other ointments; Reference standard and 2 % w/w ACEE exerts more or less the same strength. The groups treated with 5 % w/w ACEE ointment have the highest tensile strength. It is informed that 5% extract of Acacia caesia bark possesses excellent wound healing property by seeing the tensile strength. Biochemical estimations
The total protein, DNA, hexosamine, total collagen and uronic acid in the granulation tissues of control and experimental wounds are depicted in Figure 7, 8, 9, 10 &11. ACEE treatment significantly increases the DNA content from day 4 to day 12. Increasing amount of protein,
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hexosamine, collagen and uronic acid from day 4 to day 12 was observed in ACEE treated groups. There after there is a gradual decrease until the 16th day. Histopathological examination
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Results of the histopathological examination were depicted in Figure 12. The histopathological examination of the control tissue showed Skin with underlying granulation tissue, Skin shows
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underlying granulation and collagen in the tissue treated with standard Betadine. Acacia caesia
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extract treated tissue showed high amount of fibro collagenous tissue. The tissue treated with ACEE ointments have led to reduce scar formation and exhibits enhanced fibroblast
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proliferation, angiogenesis, keratinization and epithelialization as compared to reference standard and untreated control group.
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DISCUSSION
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Wound healing is a complex process and differs pathologically making it complex to discuss.
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The results were obtained for different parameters in different groups. ACEE when topically applied on rats showed convincing wound healing suggesting that it enhances various stages of
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the healing process. Secondary metabolites produced by the plants have been used to find new drug molecules; these plant origin compounds have historically served as templates for the development of many important classes of drugs. (31).The wound healing action of ACEE may be probably due to the synergistic or individual activity of the phytoconstituents present in Acacia caesia. Earlier literature reports on plant extracts have shown that phytochemical constituents like flavonoids (32), triterpenoids (33) and tannins (34) are known to enhance the wound healing process. These phytoconstituents include various chemical families like alkaloids, essential oils, flavonoids, tannins, terpenoids, saponins and phenolic compounds. It appears that different mechanisms like free radical scavenging, metal chelation as well as immune
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modulation of plant extracts may act at different levels individually or in combination to bring about the wound healing effects of medicinal plants (35). Wound healing is a natural process which could be delayed by reactive oxygen species
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and/or by microbial infection (36). In recent years, oxidative stress has been implicated in a variety of degenerative processes and diseases; these include acute and chronic inflammatory
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conditions such as wounds (37). Wound healing process includes cell proliferation, suppression
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of inflammation and contraction of the collagen tissue (38). Antioxidant activity helps in the
matrix, thus enhancing the healing process (39).
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release of oxygen radicals, thus controlling microbial infection and clearing the wound fibrin
The role of antioxidants from plant extracts in wound healing has been reported widely
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(40). Many plant extracts and medicinal herbs have shown potent antioxidant activity.
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Flavonoids, the main components of many plant extracts, act as powerful free radical scavengers
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(41). Positive correlation has been demonstrated between antioxidant activity and phenolic content of plant extracts (42). The free radical scavenging activity of plant flavonoids help in the
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healing of wounds (43). It is reported that radical-scavenging capacity of plant extract could be responsible for wound healing (44). The DPPH assay done here for the ethanolic extract of Acacia caesia showed higher antioxidant activity. The ethanolic extract of Acacia caesia has good radical-scavenging hence the accelerated wound healing of the tissues treated with ACEE may be due to the radical scavenging activity thus its control of microbial infection. It can be extrapolated that Acacia caesia can be used to control and cure wounds infected with microbes, nosocomial infections like bed sores and control multidrug resistant strains like Pseudomonas aeruginosa and staphylococcus aureus.
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Healing of wound starts from the moment of injury and can continue through varying periods of time, depending on the extent of wounding and the process of wound healing can be broadly categorized into three stages: Inflammatory phase, proliferative phase and finally the
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remodeling phase which ultimately determine the strength and appearance of the healed tissue. Wound healing process holds several steps which involve hemostasis, coagulation,
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inflammation, formation of granulation tissue, matrix formation, remodeling of connective
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tissue, collagenization and acquisition of wound strength (45).In excision wound, all three phases coexist together. The results of the excision wound model study manifest that the percentage of
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wound contraction is 95.32 ± 1.21 % in Betadine treated group on the 17th day and in control it is 85.32 ± 1.41 % on the 21st day; whereas it is 5 % in Acacia caesia ethanol extract (ACEE)
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treated group 97.9± 1.35 %. Wound contraction is obtained on the 16th day itself this much
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accelerated while comparing control and Betadine treated groups. It is inferred that the skin
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restoration or re-epithelialization of control group and Betadine applied group were respectively 21 days and 17 days while in the group treated with Acacia caesia ethanol extract, the restoration
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occurs in 16 days.
Re-epithelialization is a process of restoring the epidermis and involves proliferation and migration of keratinocytes. Cell proliferation is an essential event during re-epithelialization, so proliferating keratinocytes ensure an adequate supply of cells to migrate and cover the wound. Accelerated dermal and epidermal regeneration in Acacia caesia ethanol extract treated rats also confirmed that the extract had a positive effect towards cellular proliferation, granular tissue formation and epithelialization. Synthesis of ECM is also a keyfeature of wound healing. Dermal reconstruction is characterized by the formation of granulation tissue, which includes cell proliferation, ECM deposition, wound contraction and angiogenesis (46).
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Earlier reports promised that plant productsare potential agents for wound healing and are largely preferred because of their wide spread availability, non-toxicity, effectiveness and absence of unwanted side effects as crude preparations (47). Topically administered drugs are
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effective in faster wound contraction because of the larger availability at the wound site (48). From this study, we confirm that the rate of wound contraction was significantly higher
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and period of epithelialization was shorter in Acacia caesia ethanol extract treated rats. In
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conclusion, topical administration of Acacia caesia ethanol extract accelerates scar formation and promotes various stages of wound healing such as fibroplasia, collagen synthesis, wound
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contraction and epithelialization.
The biochemical evaluation of the wound animal model revealed the amplified DNA
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content in the treated wounds, which indicates cellular hyperplasia. Similarly there is an increase
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the granulation tissues.
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in the total protein content representing the active synthesis and deposition of matrix proteins in
In incision wound model on the 10th day the tensile strength of the tissues in control
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group was 389.87± 10.71 %, and the tissues in Betadine treated group was 653.39 ± 10.52 % while in 5 % Acacia caesia ethanol extract treated tissue it was 713.74 ± 10.47 %. It is observed that the tensile strength was doubled in Acacia caesia ethanol extract treated rat tissue in comparison with untreated control group tissue. The increase in tensile strength of treated wounds may be due to the increase in collagen concentration, Hexosamine, Uronic acid and stabilization of the fibers by increase in protein content (35, 49).The synthesis of new extracellular matrix is improved by the matrix molecules Hexosamine and Uronic acid which act as ground substratum. The collagen synthesized is laid down at the wound site and becomes cross linked to form fibers. Collagen not only deliberates
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strength and integrity to the tissue matrix but also plays a vital role in homeostasis and in epithelialization at the later phase of healing. The wound healing process is mainly accelerated by the regulated biosynthesis and
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deposition of new collagens and their subsequent maturation (50).Hydroxyproline, the main constituent of collagen serves as a marker of collagen biosynthesis at the wound site. The amount
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of collagen content in granulation tissues of control and experimental wounds clearly suggests
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that ACEE enhances collagen synthesis and deposition. The amount of collagen may be increased in total as a result of increased cell division. When compared with control, Acacia
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caesia ethanol extract has an increased level of Hexosamine; this has helped in its proper deposition and alignment of collagen. In ACEE treated wounds, the levels of hexosamine and
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uronic acid increased until day 8 post wounding and decreased thereafter. Similar trend was
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reported earlier that there is an increase in the levels of these components during the early stages
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of wound healing, following which normal levels are restored (51). In our laboratory we are involved in the isolation and evaluation of major active
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principles present in the plant material and test their efficiency against various multidrug resistant microbial strains like Methicillin Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii . Further studies with purified phytoconstituents of Acacia caesia ethanol extract are in progress to identify the complete mechanism of wound healing activity.
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40 30 20 10 0
Acacia caesia 0
50
100
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Standard 150
Conc (µg/ml)
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DPPH Radical %
DPPH Free Radical Scavenging Assay
Standard.
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Figure1: DPPH Free Radical Scavenging Assay of Acacia caesia in comparison with
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100
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50 0 0
50
100
Standard Acacia caesia
150
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Conc (µg/ml)
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Superoxide Radical %
Superoxide Radical Scavenging Assay
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Figure 2: Superoxide Radical Scavenging Assay of Acacia caesia in comparison with Standard.
Hydroxyl Radical %
Hydroxyl Radical Scavenging Assay
100
50
Standard Acacia caesia
0
0
50
100
150
Conc (µg/ml)
Figure 3: Hydroxyl Radical Scavenging Assay of Acacia caesia in comparison with Standard.
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Percentage of Wound Healing
BETADINE 8th DAY
ACEE 5%
12th DAY
16th DAY
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4th DAY
ACEE 2%
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CONTROL
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100 90 80 70 60 50 40 30 20 10 0
Figure 4: Graphical representation of percentage of wound contraction on various days
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of control and treated groups. Values are expressed as Mean ± SEM.
Period of Epithelialization in Days
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15
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20
d
25
10
5
0
CONTROL
BETADINE
ACEE 2%
ACEE 5%
Figure 5: Graphical representation of period of epithelialization in days of control and treated groups. Values are expressed as Mean ± SEM.
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Tensile Strength (g) 800 700
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600 500
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400 300
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200 100 0 BETADINE
ACEE 2%
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CONTROL
ACEE 5%
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Figure 6: Tensile strength of incision wound of control and treated groups. Values are expressed as Mean ± SEM.
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Total Protein mg/100 mg wet tissue 8
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7
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9
6 5 4 3 2 1 0
CONTROL
BETADINE 4th DAY
8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 7: Total protein content of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
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DNA mg/100 mg wet tissue
BETADINE 4th DAY
ACEE 2%
8th DAY
ACEE 5%
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CONTROL
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9 8 7 6 5 4 3 2 1 0
12th DAY
16th DAY
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Figure 8: DNA of wound tissue of different groups on various days. Values are
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expressed as Mean ± SEM.
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Hexosamine (µg/100mg dry tissue) 1000
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800
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1200
600 400 200 0
CONTROL
4th DAY
BETADINE 8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 9: Hexosamine content of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
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Total Collagen (mg/100mg dry tissue) 9 8 7 6
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5 4 3
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2 1 0 BETADINE 8th DAY
ACEE 5%
12th DAY
16th DAY
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4th DAY
ACEE 2%
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CONTROL
Figure 10: Total collagen of wound tissue of different groups on various days. Values
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are expressed as Mean ± SEM.
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Uronic Acid (µg/100mg dry tissue) 120
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100
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140
80 60 40 20 0
CONTROL
4th DAY
BETADINE 8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 11: Uronic acid of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
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Figure 12: Photomicrographs of histological study of different groups.
(A) Control (H&E400×),
(B) Wound treated with Std. Betadine (H&E 400×), (C) Wound treated with Acacia caesia ethanolic extract 2 % Ointment (H&E 400×) and (D) Wound treated Acacia caesia ethanolic extract 5 % Ointment (H&E 400×)
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16. Sathishkumar P, Evaluation of populations of the folkloremedicinal plant, Acacia caesia (L.) Willd. in the Western Ghatsof India for ecomorphological and phytochemical traits. Ph.D.,thesis, Bharathiar University, Coimbatore, India, 2010. 17. Mccune LM, Jhons T, Antioxidant activity in medicinal plantsassociated with the symptoms of diabetes mellitus used bythe indigenous peoples of the North American boreal forest. Journal of Ethnopharmacology, 2002; 82: 197-205.
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18. Robak J, Gryglewski RJ, Flavonoids are scavengers of superoxideanions. Biochemical Pharmacology, 1988; 37: 837-841. 19. Srinivasan R, Chandrasekar MNJ, Nanjan MJ, Suresh B, Antioxidant activity of
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21. OECD, Guidelines for the Testing of Chemicals/Section 4: Health Effects: Acute Dermal Toxicity Test No. 402, 1987.
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22. Saha K, Mukherjee PK, Das J, Pal M, Saha BP, Wound healing activity of Leucas lavandulaefolia Rees. Journal of Ethnopharmacology, 1997; 56: 139-144.
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corniculata whole plant extract in rats. Indian Journal of Pharmaceutical Sciences, 2004;
24. Udupa D, Kulkarni R, Udupa SL, Effect of Tridax procumbens extracts on wound
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26. Sadaf F, Saleem R, Ahmed M, Ahmed SI, Navaid-ul-zafer, Healing potential of cream containing extract of Sphaeranthus indicus on dermal wounds in guinea pigs. Journal of Ethnopharmacology, 2006; 107: 161-163. 27. Lowry OH, Rosebrough NJ, Farr Al, Randall RT, Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 1951; 193: 265-276.
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28. Burton K, A study of the conditions and mechanism of thediphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical Journal, 1956; 62: 315323.
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34. Rane M, Madhura, Mengi A, Shusma, Comparative effectof oral administration and topical application of alcoholic extract of Terminalia arjuna bark on incision and excisionwounds in rats. Fitoterapia, 2003; 74: 553-558. 35. Somashekar Shetty , Saraswati Udupa , Laxminarayana Udupa, Evaluation of Antioxidant and Wound Healing Effects of Alcoholicand Aqueous Extract of Ocimum
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sanctum Linn. in Rats. Evidence-Based Complementary and Alternative Medicine, 2008; 5(1):95–101. 36. Bodeker G, Hughes MA, Wound healing, traditional treatments and research policy.
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Plants for food and medicine, 1998, pp: 245–359. Kew, London: Royal Botanic Gardens. 37. Soliman Yusufoglu H, Ibrahim Alqasoumi S, Anti inflammatory and wound healing
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38. Houghton PJ, Hylands PJ, Mensah AY, Hensel A, Deters AM, In vitro tests and
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40. Tran VH, Hughes MA, Cherry GW, The effects of polyphenolic extract from Cudrania
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cochinchinenesis on cell response to oxidative damage caused by H2O2 and xanthine oxidase. Abstract 27, First Joint Meeting of Chinese and Europian Tissue Repair Society, 1996,pp: 22–27, Xi’an, China 41. Tran VH, Antioxidant and free radical scavenging effects of some polyphenolic extracts from medicinal plants on wound healing. Abstract, PAN/Asian European Tissue Repair Society Post-satellite Meeting, 1997, pp:2–3, Hanoi, Vietnam 42. Elzaawely AA, Tawata S, Antioxidant activity of phenolic rich fraction obtained from Convolvulus arvensis L. leaves grown in Egypt. Asian Journal of Crop Science, 2012; 4(1) 32–40.
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43. Havsteen BH, The biochemistry and medical significance of the flavonoids. Pharmacology and Therapeutics, 2002; 96:67–72. 44. Martin, A, The use of antioxidants in healing. Dermatologic Surgery, 1996; 22: 156–160.
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S2213-9095(15)00004-X http://dx.doi.org/doi:10.1016/j.wndm.2015.03.001 WNDM 50
To appear in: Received date: Revised date: Accepted date:
21-5-2014 3-12-2014 18-3-2015
Please cite this article as: Suriyamoorthy S, Subramaniam K, S JRDi, Wahaab F, R. PECi, Evaluation of wound healing activity of Acacia caesia in rats, Wound Med. (2015), http://dx.doi.org/10.1016/j.wndm.2015.03.001 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.
*Manuscript
Evaluation of wound healing activity of Acacia caesia in rats Sembian Suriyamoorthy, Kalidass Subramaniam*, Jeevan Raj Durai. S, Femina Wahaab,
Department of Biotechnology
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Pemila Edith Chitraselvi. R.
School of Biotechnology and Health Sciences, Karunya University, Karunya Nagar,
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[email protected].
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Coimbatore-641114, Tamil Nadu, India
Tel: 91 422 2614300 Ext. 4499
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Fax: 91 422 2615615 ABSTRACT
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Acacia caesia (L) is a leguminous perennial climbing shrub belonging to the family
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Mimosaceae, and is native to south-east Asia. Despite the traditional practice of using the bark
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juice for wound healing there is no scientific report on the wound healing properties of this plant. Ethno pharmacologically the bark of Acacia caesia has significant prohealing activity. The
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higher levels of alkaloids, flavonoids, glycosides and saponins noted in the bark extracts of Acacia caesia could be responsible for the medicinal properties of this plant. Secondary metabolites that serve as the defensive agents are produced under stressed condition by the plants.Wound healing activity of Acacia caesia bark ethanolic extract were investigated by excision and incision wound healing models in Wistar male rats in comparison with reference standard Betadine. Wounds treated with topical application of 5% W/W Acacia caesia ethanolic extract were found to heal faster as evidenced by observing the rate of epithelialization and percentage of wound contraction.
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The increase in tensile strength of treated wounds may be due to the increase in collagen and synthesis of new extracellular matrix molecules Hexosamine and Uronic acid, which acts as a ground substratum. Tensile strength was doubled in 5% W/W Acacia caesia ethanol extract
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treated tissue. Wound tissues formed on days 4,8,12 and 16 were used to estimate DNA, total protein, total collagen, hexosamine and uronic acid. It is observed in ACEE treated groups that
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the amount of protein, hexosamine, collagen and uronic acid increased from day 4 to day 12 and
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thereafter there was a gradual decrease until the 16th day.
Key Words: Acacia caesia, Wound Healing, Period of Epithelialization, Tensile strength.
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INTRODUCTION:
Wounds are physical injuries that result in an opening or breaking of the skin. Wound
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healing is essential for the restoration of disturbed functional status and disrupted anatomical
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continuity of the skin. It is a complex multifactorial process that results in contraction, closure of
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the wound and restoration of a functional barrier (1).Current estimates indicate that nearly six million people suffer from chronic wound (2) causing great physiological and mental
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trauma.Wound healing is the interaction of a complex cascade of cellular and biochemical actions leading to the restoration of structural and functional integrity with regain of strength of injured tissue. It involves continuous cell- cell interaction and cell- matrix interactions that allow the process to proceed in different overlapping phases and processes including inflammation, wound contraction, reepithelialization, tissue remodeling and formation of granulation tissue with angiogenesis (3). Problems like resistance and pollution associated with irrational use of orthodox medicines have necessitated the use of plants as a source of effective and safer alternative for human ailments. Use of plants on curing wound and skin repair is in practice from days
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immemorable. For thousands of years nature has provided as a source of medicinal agents and number of modern drugs have been isolated from them, many based on their use in traditional medicine (4).The process of wound healing can be promoted by several plant products, which are
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composed of active principles like triterpenes, alkaloids, flavonoids (5) and biomolecules (6). Polyherbal formulations are better than the pure chemical wound healing formulations
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because the crude poly herbal formulation have various phyto constituents which have
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antimicrobial, anti-inflammatory properties and antioxidant potential. Hence phytoherbal formulation used in the treatment of wound owes assured safety and high efficacy.Hence
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scientific investigation is needed to explore the pharmacological activities of medicinal plants and to elucidate the claims made about them in folklore medicines.
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With the advent of the modern molecular techniques in biomedical sciences,the potential
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of many traditional unknown medicinal plants used for treating wounds and burns are analyzed
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and scientific investigations on the mechanism of wound healing is gaining momentum worldwide. The hidden traditional knowledge of plants used in curing wounds by the folkloric
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medicinal practitioners in countries like India, Africa, China and South East Asian countries is actively being investigated across the global scientific community. Sufficient literature informs drugs used for wound healing are invariably derived from plants; plant based wound therapy not only accelerates healing process but also maintains safety. More than 70% of wound healing pharma products are of plant origin. The plant based materials are used as first aid, antiseptic, coagulants and wound wash. Many phytopharmaceutical laboratories are now concentrating their effort to identify the active constituents and mode of action of various medicinal plants. Acacia caesia (L) is a leguminous perennial climbing shrub belonging to the family Mimosaceae, and is native to south-east Asia. The information collected from traditional
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medicine practitioners and literature shows the stem bark forms froth when rubbed with water and thus used as soap, while its decoction is used as lice killer (7). The juicy extract from the stem bark is well-known among the Mizo tribes of north-east India as remedy to gastrointestinal
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infections (8).The leaves were used as vegetable and used in the treatment of asthma, skin diseases (9,10), menstrual disorder (11-14) and scabies (15). Due to these values, it is exploited
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severely by the local public in western districts of Tamilnadu and it becomes a rare sighted
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species in the natural habitats of Western Ghats (16).
The medicinal value of this plant lies in bioactive phytochemical constituents that
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produce definite physiological action on the human body (13).Literature reports on Acacia caesia (L) inform the presence of secondary metabolites like alkaloids, flavonoids, glycosides
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and saponins. Acacia caesia extracts exhibited good antimicrobial and demonstrated good
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antioxidant activity (10). Despite these beneficial activity reports of Acacia caesia,and the
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presence of various active principles in this plant,there is a lacuna of scientific documentation on the wound healing property of Acacia caesia.To fill this research gap, we have investigated this
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study and report here a firsthand documentation of our findings on wound healing properties of Acacia caesia.
MATERIALS AND METHODS
Plant material collection and extraction Fresh barks of Acacia caesia were collected from Pannaipuram, Theni district, Tamilnadu, India. The barks were air-dried, pulverized to a coarse powder in a mechanical grinder, passed through a 40 mesh sieve. 25 gm of powder was extracted with 250 ml of ethanol in soxhlet extractor. The extract was decanted, filtered with Whatman No.1 filter paper and concentrated at reduced pressure below 40º C through rotary vapour to obtain cream of extract.
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The Acacia caesia ethanolic extract (ACEE) was stored in a closed container until further evaluation of wound healing activity. In vitro antioxidant assay
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The free radical scavenging activity of Acacia caesia ethanolic extract was measured by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) using the modified method of McCune et al.,(17). The
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superoxide anion radical scavenging activity of Acacia caesia ethanolic extract was measured by
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the method of Robak et al., (18). The hydroxyl radical scavenging activity of Acacia caesia ethanolic extract was measured by studying the competition between deoxyribose and test
reaction) (19).
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Preparation of formulation and standard used
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compound for hydroxyl radicalsgenerated by Fe+3- ascorbic acid-EDTA-H2O2 system (Fenton
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Two types of formulations with different concentrations 2 % and 5 % w/w of ACEE were
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prepared by tituration method in a ceramic mortar and pestle (20). Reference Standard (Betadine ointment) was compared with the formulations to check the wound healing potential in different
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animal models. Animals used
Wistar male rats weighing approximately 150 g - 175 g were used in the study. The rats were acclimatized to the laboratory environment for a period of 7 days. The rats were housed at 24± 2oC at a relative humidity of 40-45 % and light dark cycles of 11 and 13 h respectively. Animals were provided with rodent diet and water ad libitum. The animal study protocol was subjected to the scrutiny of Institutional Ethical Committee and was cleared by the same before beginning the experiment (IAEC/KU/BT /12/010).
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Acute dermal toxicity Acute dermal toxicity study was carried out to determine the dose as per OECD guidelines No. 402 (21). ACEE ointment with highest concentration (5 % w/w) was applied on the shaved
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dorsal region of the rats. Grouping of Animals
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Two wound models were used in our study:
Incision wound model
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The animal models contain four groups each.
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Excision wound model
The groups were:
: Control (Treated with Simple ointment base (SOB)) (n=6)
Group 2
: Standard group treated with Betadine ointment (n=6)
Group 3
: Treated with 2 % ACEE ointment (n=6)
Group 4
: Treated with 5 % ACEE ointment (n=6)
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Excision wound model
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Group 1
The animals in each group were anaesthetized by the open mask method with anaesthetic ether. Depilations of the rats were done on the dorsal side. The excision wound was inflicted by cutting away a 100 mm2 full thickness of skin from predetermined shaved area (22). Excision wound was left undressed. Topical application of the drugs to the divided groups viz. SOB, Reference Standard (Betadine ointment), ACEE 2 % w/w ointment and ACEE 5 % w/w ointment respectively were done once daily until the wound healed completely. Wound contraction and epithelialization period were monitored. Biochemical parameters were done to estimate total protein, DNA, hexosamine, total collagen and uronic acid in the granulation tissues of control
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and experimental wounds. Histopathological analysis of the tissue from the healed wound was also done (23). Incision wound model
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The animals in each group were anaesthetized and one paravertebral long incision of 4 cm length on the depilated dorsal side was made through the skin till the cutaneous muscle. Aseptic
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measures were not taken throughout the experiment (24). After incision was made the parted
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skin was kept together and stitched with black silk surgical thread (No.000) at 1 cm intervals using curved needle (No.11). Good closure of the wound was made by tightening the edges of
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the thread and was left undressed. ACEE ointments along with SOB and reference standard were applied once daily topically to their respective groups. The sutures were removed
on the 9
th
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Parameters evaluated for wound healing
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day. Tensile strength of the cured skin was measured using tenisiometer (25).
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Measurement of wound contraction
The periphery of the excision wound was traced on a transparent paper on every 4th day, till the
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wound got healed. Wound area was measured by retracing the wound on a millimeter scale graph paper .The evaluated surface area was used to calculate the percentage of wound contraction (26).
% of wound contraction = {(Initial wound size – Specific day wound size) / Initial wound size} X 100.
Epithelialization period The wound was monitored for complete epithelialization. It was measured in days from wounding day (0 thday) till the Escher totally separated itself with the raw wound left behind.
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Tensile strength of incision wound model The degree of wound healing is directly proportional to the gain in tensile strength of the tissue.
wounds was compared with control and reference standard treated groups. Biochemical estimations
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Tensile strength was measured on the 10 thday. The tensile strength of ACEE ointment treated
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Protein was estimated by the method of Lowry et al., (27). As per Burton, (28) estimation of
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DNA was done. An Uronic acid level was estimated by the method of Schiller et al., (29). Hexosamine and collagen were estimated by the method of Woessner (30).
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Histopathological examination
The cross sectional full thickness skin specimens from each group were collected at the end of
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the experiment to evaluate the histopathological analysis. Samples were fixed in 5 % buffered
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formalin, processed and blocked with paraffin and then sectioned into 5 µm sections and stained
epidermal remodeling.
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Statistical analysis
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with hematoxylin and eosin (HE) stains. The tissues were examined by light microscope for
Results obtained from three wound models have been expressed as Mean ± SEM. The data was evaluated by one way ANOVA followed by Dunnett’s T-Test, P < 0.05 and P < 0.01 was considered as significant. RESULTS
In vitro antioxidant assay The DPPH radical significantly reduced by Acacia caesia ethanolic extract and standard with increasing concentrations. Acacia caesia ethanolic extract showed potent hydroxyl radical scavenging activity which may be due to the presence of various phytochemicals. Acacia caesia
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ethanolic extract increases superoxide anion radical scavenging activity as that of the standard (Figure 1, 2 and 3). Wound contraction studies
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Reduction of wound area of different groups until the 16th day for excision wound model was calculated and depicted in Figure 4. Control groups showed least rate of wound healing. Faster
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rate of wound healing was seen in groups treated with 5 % w/w ACEE ointment. Reference
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standard heals the wound almost equivalent to the groups treated with 2% w/w ACEE ointment. Epithelialization period
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The epithelialization was observed from the first day. The epithelialization time was found to be lesser or quicker in group treated with 5 % w/w ACEE ointment than the two groups treated with
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2 % w/w ACEE ointment and Reference standard treated groups (Figure 5).
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Measurement of tensile strength
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Comparison of the tensile strength of the healed skin of the animals in different groups is shown in Figure 6. Minimum tensile strength was noticed in untreated group. Among the tensile
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strength of the tissues treated with other ointments; Reference standard and 2 % w/w ACEE exerts more or less the same strength. The groups treated with 5 % w/w ACEE ointment have the highest tensile strength. It is informed that 5% extract of Acacia caesia bark possesses excellent wound healing property by seeing the tensile strength. Biochemical estimations
The total protein, DNA, hexosamine, total collagen and uronic acid in the granulation tissues of control and experimental wounds are depicted in Figure 7, 8, 9, 10 &11. ACEE treatment significantly increases the DNA content from day 4 to day 12. Increasing amount of protein,
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hexosamine, collagen and uronic acid from day 4 to day 12 was observed in ACEE treated groups. There after there is a gradual decrease until the 16th day. Histopathological examination
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Results of the histopathological examination were depicted in Figure 12. The histopathological examination of the control tissue showed Skin with underlying granulation tissue, Skin shows
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underlying granulation and collagen in the tissue treated with standard Betadine. Acacia caesia
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extract treated tissue showed high amount of fibro collagenous tissue. The tissue treated with ACEE ointments have led to reduce scar formation and exhibits enhanced fibroblast
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proliferation, angiogenesis, keratinization and epithelialization as compared to reference standard and untreated control group.
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DISCUSSION
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Wound healing is a complex process and differs pathologically making it complex to discuss.
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The results were obtained for different parameters in different groups. ACEE when topically applied on rats showed convincing wound healing suggesting that it enhances various stages of
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the healing process. Secondary metabolites produced by the plants have been used to find new drug molecules; these plant origin compounds have historically served as templates for the development of many important classes of drugs. (31).The wound healing action of ACEE may be probably due to the synergistic or individual activity of the phytoconstituents present in Acacia caesia. Earlier literature reports on plant extracts have shown that phytochemical constituents like flavonoids (32), triterpenoids (33) and tannins (34) are known to enhance the wound healing process. These phytoconstituents include various chemical families like alkaloids, essential oils, flavonoids, tannins, terpenoids, saponins and phenolic compounds. It appears that different mechanisms like free radical scavenging, metal chelation as well as immune
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modulation of plant extracts may act at different levels individually or in combination to bring about the wound healing effects of medicinal plants (35). Wound healing is a natural process which could be delayed by reactive oxygen species
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and/or by microbial infection (36). In recent years, oxidative stress has been implicated in a variety of degenerative processes and diseases; these include acute and chronic inflammatory
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conditions such as wounds (37). Wound healing process includes cell proliferation, suppression
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of inflammation and contraction of the collagen tissue (38). Antioxidant activity helps in the
matrix, thus enhancing the healing process (39).
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release of oxygen radicals, thus controlling microbial infection and clearing the wound fibrin
The role of antioxidants from plant extracts in wound healing has been reported widely
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(40). Many plant extracts and medicinal herbs have shown potent antioxidant activity.
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Flavonoids, the main components of many plant extracts, act as powerful free radical scavengers
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(41). Positive correlation has been demonstrated between antioxidant activity and phenolic content of plant extracts (42). The free radical scavenging activity of plant flavonoids help in the
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healing of wounds (43). It is reported that radical-scavenging capacity of plant extract could be responsible for wound healing (44). The DPPH assay done here for the ethanolic extract of Acacia caesia showed higher antioxidant activity. The ethanolic extract of Acacia caesia has good radical-scavenging hence the accelerated wound healing of the tissues treated with ACEE may be due to the radical scavenging activity thus its control of microbial infection. It can be extrapolated that Acacia caesia can be used to control and cure wounds infected with microbes, nosocomial infections like bed sores and control multidrug resistant strains like Pseudomonas aeruginosa and staphylococcus aureus.
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Healing of wound starts from the moment of injury and can continue through varying periods of time, depending on the extent of wounding and the process of wound healing can be broadly categorized into three stages: Inflammatory phase, proliferative phase and finally the
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remodeling phase which ultimately determine the strength and appearance of the healed tissue. Wound healing process holds several steps which involve hemostasis, coagulation,
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inflammation, formation of granulation tissue, matrix formation, remodeling of connective
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tissue, collagenization and acquisition of wound strength (45).In excision wound, all three phases coexist together. The results of the excision wound model study manifest that the percentage of
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wound contraction is 95.32 ± 1.21 % in Betadine treated group on the 17th day and in control it is 85.32 ± 1.41 % on the 21st day; whereas it is 5 % in Acacia caesia ethanol extract (ACEE)
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treated group 97.9± 1.35 %. Wound contraction is obtained on the 16th day itself this much
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accelerated while comparing control and Betadine treated groups. It is inferred that the skin
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restoration or re-epithelialization of control group and Betadine applied group were respectively 21 days and 17 days while in the group treated with Acacia caesia ethanol extract, the restoration
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occurs in 16 days.
Re-epithelialization is a process of restoring the epidermis and involves proliferation and migration of keratinocytes. Cell proliferation is an essential event during re-epithelialization, so proliferating keratinocytes ensure an adequate supply of cells to migrate and cover the wound. Accelerated dermal and epidermal regeneration in Acacia caesia ethanol extract treated rats also confirmed that the extract had a positive effect towards cellular proliferation, granular tissue formation and epithelialization. Synthesis of ECM is also a keyfeature of wound healing. Dermal reconstruction is characterized by the formation of granulation tissue, which includes cell proliferation, ECM deposition, wound contraction and angiogenesis (46).
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Earlier reports promised that plant productsare potential agents for wound healing and are largely preferred because of their wide spread availability, non-toxicity, effectiveness and absence of unwanted side effects as crude preparations (47). Topically administered drugs are
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effective in faster wound contraction because of the larger availability at the wound site (48). From this study, we confirm that the rate of wound contraction was significantly higher
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and period of epithelialization was shorter in Acacia caesia ethanol extract treated rats. In
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conclusion, topical administration of Acacia caesia ethanol extract accelerates scar formation and promotes various stages of wound healing such as fibroplasia, collagen synthesis, wound
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contraction and epithelialization.
The biochemical evaluation of the wound animal model revealed the amplified DNA
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content in the treated wounds, which indicates cellular hyperplasia. Similarly there is an increase
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the granulation tissues.
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in the total protein content representing the active synthesis and deposition of matrix proteins in
In incision wound model on the 10th day the tensile strength of the tissues in control
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group was 389.87± 10.71 %, and the tissues in Betadine treated group was 653.39 ± 10.52 % while in 5 % Acacia caesia ethanol extract treated tissue it was 713.74 ± 10.47 %. It is observed that the tensile strength was doubled in Acacia caesia ethanol extract treated rat tissue in comparison with untreated control group tissue. The increase in tensile strength of treated wounds may be due to the increase in collagen concentration, Hexosamine, Uronic acid and stabilization of the fibers by increase in protein content (35, 49).The synthesis of new extracellular matrix is improved by the matrix molecules Hexosamine and Uronic acid which act as ground substratum. The collagen synthesized is laid down at the wound site and becomes cross linked to form fibers. Collagen not only deliberates
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strength and integrity to the tissue matrix but also plays a vital role in homeostasis and in epithelialization at the later phase of healing. The wound healing process is mainly accelerated by the regulated biosynthesis and
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deposition of new collagens and their subsequent maturation (50).Hydroxyproline, the main constituent of collagen serves as a marker of collagen biosynthesis at the wound site. The amount
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of collagen content in granulation tissues of control and experimental wounds clearly suggests
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that ACEE enhances collagen synthesis and deposition. The amount of collagen may be increased in total as a result of increased cell division. When compared with control, Acacia
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caesia ethanol extract has an increased level of Hexosamine; this has helped in its proper deposition and alignment of collagen. In ACEE treated wounds, the levels of hexosamine and
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uronic acid increased until day 8 post wounding and decreased thereafter. Similar trend was
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reported earlier that there is an increase in the levels of these components during the early stages
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of wound healing, following which normal levels are restored (51). In our laboratory we are involved in the isolation and evaluation of major active
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principles present in the plant material and test their efficiency against various multidrug resistant microbial strains like Methicillin Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii . Further studies with purified phytoconstituents of Acacia caesia ethanol extract are in progress to identify the complete mechanism of wound healing activity.
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40 30 20 10 0
Acacia caesia 0
50
100
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Standard 150
Conc (µg/ml)
cr
DPPH Radical %
DPPH Free Radical Scavenging Assay
Standard.
us
Figure1: DPPH Free Radical Scavenging Assay of Acacia caesia in comparison with
an
100
M
50 0 0
50
100
Standard Acacia caesia
150
d
Conc (µg/ml)
te
Superoxide Radical %
Superoxide Radical Scavenging Assay
Ac ce p
Figure 2: Superoxide Radical Scavenging Assay of Acacia caesia in comparison with Standard.
Hydroxyl Radical %
Hydroxyl Radical Scavenging Assay
100
50
Standard Acacia caesia
0
0
50
100
150
Conc (µg/ml)
Figure 3: Hydroxyl Radical Scavenging Assay of Acacia caesia in comparison with Standard.
Page 15 of 26
Percentage of Wound Healing
BETADINE 8th DAY
ACEE 5%
12th DAY
16th DAY
an
4th DAY
ACEE 2%
us
CONTROL
cr
ip t
100 90 80 70 60 50 40 30 20 10 0
Figure 4: Graphical representation of percentage of wound contraction on various days
M
of control and treated groups. Values are expressed as Mean ± SEM.
Period of Epithelialization in Days
Ac ce p
15
te
20
d
25
10
5
0
CONTROL
BETADINE
ACEE 2%
ACEE 5%
Figure 5: Graphical representation of period of epithelialization in days of control and treated groups. Values are expressed as Mean ± SEM.
Page 16 of 26
Tensile Strength (g) 800 700
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600 500
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400 300
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200 100 0 BETADINE
ACEE 2%
an
CONTROL
ACEE 5%
M
Figure 6: Tensile strength of incision wound of control and treated groups. Values are expressed as Mean ± SEM.
d
Total Protein mg/100 mg wet tissue 8
Ac ce p
7
te
9
6 5 4 3 2 1 0
CONTROL
BETADINE 4th DAY
8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 7: Total protein content of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
Page 17 of 26
DNA mg/100 mg wet tissue
BETADINE 4th DAY
ACEE 2%
8th DAY
ACEE 5%
us
CONTROL
cr
ip t
9 8 7 6 5 4 3 2 1 0
12th DAY
16th DAY
an
Figure 8: DNA of wound tissue of different groups on various days. Values are
M
expressed as Mean ± SEM.
d
Hexosamine (µg/100mg dry tissue) 1000
Ac ce p
800
te
1200
600 400 200 0
CONTROL
4th DAY
BETADINE 8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 9: Hexosamine content of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
Page 18 of 26
Total Collagen (mg/100mg dry tissue) 9 8 7 6
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5 4 3
cr
2 1 0 BETADINE 8th DAY
ACEE 5%
12th DAY
16th DAY
an
4th DAY
ACEE 2%
us
CONTROL
Figure 10: Total collagen of wound tissue of different groups on various days. Values
M
are expressed as Mean ± SEM.
d
Uronic Acid (µg/100mg dry tissue) 120
Ac ce p
100
te
140
80 60 40 20 0
CONTROL
4th DAY
BETADINE 8th DAY
ACEE 2% 12th DAY
ACEE 5%
16th DAY
Figure 11: Uronic acid of wound tissue of different groups on various days. Values are expressed as Mean ± SEM.
Page 19 of 26
ip t cr us an M d
Ac ce p
te
Figure 12: Photomicrographs of histological study of different groups.
(A) Control (H&E400×),
(B) Wound treated with Std. Betadine (H&E 400×), (C) Wound treated with Acacia caesia ethanolic extract 2 % Ointment (H&E 400×) and (D) Wound treated Acacia caesia ethanolic extract 5 % Ointment (H&E 400×)
Page 20 of 26
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ip t
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