Effect of Green Coffee Beans Extract Ointments for Wound Healing

Some interventions, such as coffee powder, are applied on wound to facilitate healing process. Coffee has several properties that can facilitate wound healing. Application of Robusta coffee bean ointment 22.5 % on full thickness wound could increase fibroblast. Povidone iodine 10% has remained popular as wound antiseptic. This study aimed to evaluate the effect of green coffee bean extract in lower concentration for healing wound in rats. This study used male Sprague Dawley rats, 10 rats in each group. The skin incision was held in right back of rats with a diameter of 1.5 cm under lidocaine anesthetic subcutaneously. Group I was not given intervention, group II was given liquid povidone iodine 10%, and group III was given 15% green coffee bean extract in vaseline base. Interventions were held every day without wound debridement. Wound size was measured at day 0, 7 and 16. Animals were sacrificed at day 7 (proliferative phase) and 16 (remodeling phase). Sections were stained using Haematoxilyn-eosin. Histopathology assessment of wound tissue is on reepithelization and presence of: inflammatory cells, fibroblast, collagen, and vascularization. Green coffee extract could increase wound contraction and accelerated wound healing. Histopathology result on day 7 showed that green coffee extract had high response inflammatory cells and fibroblast. The provision of 15% green coffee bean extract ointment can accelerate wound healing with higher inflammation response and fibroblast in proliferative phase .


INTRODUCTION
extract in ointmnet base on wound healing with histology evaluation for epithelization, inflammatory cells, Roasting coffee beans powder has been used as fibroblast, collagen and vascularization on proliferative traditional medicine in wounds healing. Many local health phase (day 7) and remodelling phase (day 16). provides reported coffee powder sprinkled on wound does not cause infection (1). Experimental study with rats METHODS has showed result that roasted coffee beans extract ointment base dose 22,5 % could increase inflammatory Extraction cells, fibroblast, and blood vessels in wound area on day 4 This study used robusta green coffee beans that dried in (2). The chemical constituent of coffee include phenolic the oven for 40°C for 24 hours and than crushed by compounds and their derivatives (such as chlorogenic blender. Extraction used ethanol 70% as solvent with ratio acids), alkaloid (especially caffeine), diterpenoid alcohols 1:3. Rotary evaporator was used to dried the extract. We (such as cafestol and kahweol) (3). Chlorogenic acids has used vaseline as base for made 15% ointment of green been shown to accelerate the skin wound healing and coffee extracts. Fifteen grams of extract diluted by a little burn healing (1,3). There are a different chlorogenic acids ethanol and added Vaseline up to 100 grams in weight. amount in green coffee and roasted coffee, green coffee has higher amount of chlorogenic acids than roasting Wound Model coffee beans (3).
This study has been approved by Ethic Committee of One of antiseptic that has been widely used is povidone Medical and Health Science Faculty of Jambi University. iodine. Povidone iodine has been used in wound for many We used rats strain Sprague dawney, male, age 2-3 decades. In aqueous medium, more free iodine is released months, weight 150-200 grams and healthy. In each left into solution from the povidone iodine complex. Iodine is back of rats was made incissions with diameter size 1,5cm a substance that has germicidal activity. The use of using scalpel and scissor under anesthesia with lidocaine povidone iodine for cleansing, irrigating and dressing subcutaneously around incision area. We used ten rats for wounds in controversial. Side effects of iodine in prolong each groups: 5 rats for subgroup day 7 and 5 rats for day 16 use lead to skin irritation and staining (4). Many studies observation. In group I as control, there were no applied showed that povidone iodine did not effectively promote dressing on the wound. Groups II were applied povidone good wound healing (5).
iodine solution 10% and groups III were applied with 15% Cutaneous wound is repaired by three basic mechanism: ointment of green coffee extracts on the wound. connective tissue matrix deposition, contraction and Treatment were held every day without wound epithelization (6,7). When cutaneous tissue bleeded, the debridement. platelet release clotting factors, growth factors and Wound Contraction and Wound Healing cytokines. These give signal for neutrophils and macrophages to enter the wound site (8). This phase is Wound area were measured on day 7 and day 16 by tracing called inflammatory phase. Neutrophils and macrophages the wound area using transparant graph paper. responsible for cleaned out the wound from foreign The evaluated wound surface was used to calculate the materials, bacteria and damaged tissue (9). Once the percentage of wound contraction (12). Wound healing has wound site is cleaned out, fibroblast migrate to wound complete if wound contraction percentage is 100%. area to begin the proliferative phase. Fibroblast produce a Duration of complete wound healing by counting the new collagen matrix. Fibroblast undergo a phenotypical number of days from day 0 up to day when wound differentiation into myofibroblast that have contracting contraction percentage has been 100%. forces to bring the wound edges together (10). New blood vessels enter the wound repair area to supply oxygen and Percentage wound contraction was determinated by nutrients. This process is called angiogenesis or equation neovascularization (6). The new collagen matrix then becomes cross linked and organized during the final Histopathology Evaluation remodeling phase. In the process of collagen remodelling, Histopathology evaluation held on day 7 and day 16. Five collagen degradation occurs by collagenase enzymes. rats in each group were sacrificed on day 7 and day 16 after Parallel to all of the mentioned events above, the the incissions to histopathological assesment using epithelial-coating cells proliferate and migrate from the haematoxylin eosin (HE) staining. Histological assessment border of the wound in an attempt to close it. This is called used semi-quantitative methods. They were read by one reepithelization. Reepithelization occurs through the Anatomical Pathologist with blind method. Scoring for action of specific growth factors and cytokines that are epithelization: 0=thickness of cut edges, 1=migration of produced by macrophages, platelets and keratinocytes. cells <50%, 2=migration of cells ≥50%, 3=bridging the Once the epithelial bridge is complete, enzyme are excision, 4=keratinization. Scoring for inflammation cells, released to dissolve the attachment at the base of the scab fibroblast, vascularization and collagen: 0=absent, 1=mild resulting in removal (6,11).
ST, 2=mild DL/GT, 3=moderate DL/GT, 4=marked DL/GT. The three phase of wound healing are not mutually (ST=surrounding Tissue, DL=Demarcation Line, GT= excluding, but rather overlap over time. Inflammatory Granulation Tissue) (13,14). phase start within the fisrt 24 hours. Inflammatory cells Statistical Analysis can extended in wound for 12 days. Proliferative phase start after wound site is cleaned out and can unfold up to Statistical analysis for each parameter mean values ± SEM 14 days after the onset of lesion. This phase is continued of score histopathology, wound contraction and by remodeling phase that can last for one year or more (6) percentage duration of complete wound healing were This study is to determine the effect of green coffee beans represented in table and compared using non-parametric Kruskall-Wallis Test followed by Mann Whitney test. The treatment groups. level of significance was set at p<0,05.

Histopathology Evaluation
Histopathology evaluation among the groups on day 7 and RESULTS day 16were seen in Table 2 and Figure 2.

Wound Contraction and Complete Wound Healing
Histopathology results of cutaneous wound on days 7 were The wound healing effect of 15% ointment of green coffee found that inflammatory cells in green coffee extract group extracts in wound contraction and complete healing is was highest than the others. Also, fibroblast in green shown in Table 1 and Figure 1. Wound contraction in coffee extract group was highest among the others. group of green coffee extract were highest among the Vascularization in green coffee extract group were lowest others, followed by no treatment group and povidone among the groups. Collagen level in green coffee extract iodine group. There was significant difference on wound group were not different with povidone iodine group but it contraction among the groups (p=0,003). Based on Mann were higher than no treatment group on day 7, there had Whitney Test, that significant difference was between no epithelization yet in all groups. Myofibroblast, a type of green coffee extract group and povidone iodine group fibroblast, were responsible for wound contraction. Green (p=0,001).
coffee extract group have highest wound contraction ( Table 1 and Figure 1) and highest fibroblast ( Table 2) among the others.

DISCUSSION
There are four responses to tissue injury: regeneration, normal repair, deficient, and excessive healing. Regeneration is a process to replace injured structure by replacing exactly what was there before the injury. Normal repair is a response where there is are established equilibrium between scar formation and scar remodeling. This is the typical response that most human experiences following injury. Basically, all cutaneous wound heal by three basic mechanism, they are deposition of connective tissue matrix, contraction and epithelization (15).
Days 7 was selected in this study to represent proliferative phase of wound healing. Based on histopathology results n day 7, applying of green coffee extract on skin wound caused higher inflammatory cells and fibroblast level. Inflammatory cells were associated with wound healing for clean the wound area and for stimulating fibroblast migration.  (8,9,16,17). Fibroblast undergo a phenotypical differentiation into myofibroblast under stimulus of Transforming Growth Factor β (TGF-β) that released by Percentage of complete wound healing in group of green macrophages (10,16,18). TGF-β1 and TGF-β2 stimulate coffee extract were highest among the others. All of rats in fibroblast to attach fibrous protein in extracellular matrix this groups had complete wound healing (100%) in 16 days via integrin containing adhesions receptor (15,19,20). This observation. Duration complete wound healing in group binding cause fibroblast to express stress fibers or actin of green coffee extract were fastest among the others filaments in the cytoplasm. The stress fibers and actin (mean 13,6±1,34 days). Wound contraction that observed filament are critical to further fibroblast migration and on day 7 were in mutual with duration complete wound wound contracting forces (10,18). healing. Higher wound contraction would facilitate a Migration of fibroblast to wound area need help from faster wound healing. There were 2 rats (40%) in fibronectin, tenascin C and proteoglycan (7,10). Then, povidone iodine group that haven't had complete wound fibroblast begin to proliferate and produce MMPs (matrix healing in the end of 16  denatured protein and provisional matrix-associated tissue. Fibroblast undergo apoptosis, which could be nitric material not required in the healed wound (17,18,24). oxide induced (10). Simultaneously, fibroblast also produce collagen by TGF β This study showed that green coffee could help skin wound stimulation. Collagen fibril were further organized into healing faster than povidone iodine liquids. Wound fibrous structure, which it is the framework for contraction on day 7 in group that received green coffee epithelialization. Once an abundant collagen matrix has extract had highest wound contraction. Wound model in been deposited in the wound, the fibroblast stop this study is full-thickness wounds that are characterized producing collagen (10,15). Reepithelization of a wound by complete destruction of the epidermis and dermis as by keratinocytes is performed by the combination of the well as deeper structures. Repair of tissue loss is initiated proliferative phase with the migration of cells near the by the formation of granulation tissue that replaces the lesion by growth factor stimulation. Vascularization has defect before epithelial covering can occur. Cutaneous important function to supply oxygen and nutrients wounds close by epithelial resurfacing and wound (7,17,21).

Migration of inflammatory cells to wound area is a crucial
contraction. Dependent on the species, one or the other Day 16 was selected in this study to represent remodeling process dominates the progress of wound repair. For phase. Remodeling phase exhibits a progressive increase example, rodents heal mainly by contraction, whereas in in its collagen fibers level. Finally, collagen remodeling humans, reepithelization accounts for up to 80% of wound occurs. Specific collagenase enzymes clip the molecules at closure (7). a specifics site through all three chains and break it down The result was supported by histopathology finding on day to characteristics three-quarter and one-quarter pieces.
extract phenolic compound, but with no quantification of chlorogenic acids in our extract. Active compound in coffee that have wound healing properties is chlorogenic acids (11,22). Aphenolic In this study, green coffee extract 15% ointment could compound (23). Chlorogenic acids have antioxidative accelerate wound healing by increases inflammatory cells properties to neutralize the effects of oxidative damage to and fibroblast in proliferative phase. Further study related the skin (24), also have antibacterial (25,26), antiviral (27), to the molecular mechanism of wound healing is needed anti inflammatory (28,29). Chlorogenic acid showed a to investigate coffee and it's active compound that suppresive activity of metalloproteinase expression (30), responsible for wound healing.