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EFFECTS OF ETHANOL CINCAU LEAVES EXTRACT (Cyclea barbata Miers) ON AUTOLITIC ESCHAR OF BURN WOUND Dina Dewi SLI*, Wibi Riawan**, Ika Setyorini*,Titin Andri Wihastuti*, Ika Fitri Aprilianti *, Retty Ratnawati***, Loeki Enggar Fitri**** *Nursing

College, Faculty of Medicine Universitas Brawijaya ** Department of Biochemistry Faculty of Medicine Universitas Brawijaya *** Department of Physiology Faculty of Medicine Universitas Brawijaya **** Department of Parasitology Faculty of Medicine Universitas Brawijaya Corresponding Author Dina Dewi SL Nursing College of Medical FacultyBrawijaya University Jl. Mayjen Haryono 171 Kampus Sumbersari Malang 65145 Email: [email protected]

ABSTRACT

Eschar is a dead tissue which attaches strongly to the burns and gives risk in burns infection. Autolysis of eschar is done by macrophages. Migration of macrophages into the wound area is affected by interleukin-1. Ethanol extract of cincau or green grass jelly leave or Cyclea barbata Miers (CBM) contain pectin, flavonoids and alkaloids. These compounds influence the inflammatory process. The purpose of this study was to reveal the effect of ethanol cincau leaves extract on the expression IL1 and the number of macrophages in second degree burn wounds tissue. This study used 30 rats, which were divided into six groups, the control group which was treated with NaCl 0.9% (K), silver sulfadiazine group (P1), hydrogels group (P2), cincau extract 40% group (P3), cincau extract 50% group (P4), and cincau extract 60% group (P5). Treatment of burn wounds was conducted over 4 days. On day 5th, burn wounds tissue was taken, then immunohistochemistry staining was applied. One way ANOVA test on the expression of IL1 showed a differences among groups (P=0,001). As well as on the number of macrophages (P=0,001). Post hoc tests on the expression of IL1 showed cincau extract 40% dan 50% treated groups significantly differ with control group but not with hydrogel treated group (P2). Similar result showed cincau extract 40% and 50% treated groups significantly differ from the control but not with SSD (P1) and Hydrogel (P2) treated groups. It can be concluded that ethanol extract of cincau leaves can decreased IL1 expression and increased the number of macrophages which have important role in second degree burn eschar autolysis. Keywords: Green grass jelly leaves (Cyclea barbata Miers), Macrophages, Burn Wound, Cincau, IL1

INTRODUCTION Burn is one type of trauma that can be suffered from people in all age ranges and genders (1). Although the incidence of burn decreases in global, but the prevalence of burn in Indonesia is still quite high (1). Previous research in Cipto Mangun kusumo Hospital showed that burn had high morbidity and mortality numbers (2). Moreover, burn require special treatment from the early phase to the late phase The degree of burn is divided based on the depth of the skin layer which suffered from the injury, the 1stdegree (superficial), 2nd

degree (partial thickness), and 3rddegree (full thickness) (3). Second degree burn is divided into 2A and 2B stages. In the 2B-degree burn, almost the whole of dermis is damage (two-thirds of layer are damage). Appendices skin such as hair follicles, sweat glands, and sebaceous glands partially intact. In addition, the 2B degree burn often encountered thin eschar. Eschar is dead tissue or denatured proteins that are strongly attached to the tissue and increase the risk of infection (4). The 2B-degree burn healing normally takes around 2 weeks (4).

Burn healing process begins from the inflammatory phase, which is consisted of the initial inflammatory and late inflammatory (5). Inflammation usually occurs in three days up to seven days (6). In the late inflammatory phase the proinflammatory mediators such as IL-1 decreased, it is intended to achieve a resolution of inflammation. In the late inflammatory phase, the dominant cells are macrophages. The main function of macrophages is to clean foreign particles from the body, such as bacteria, necrotic tissue, and other foreign objects (7). The increase in both the number and the activity of macrophages would help elimination of necrotic tissue in burns (eschar). This mechanism is referred to as autolysis debridement (8). The Increase in number migration of macrophages into the wound site would improve the process of autolysis debridement. Enhanced macrophage migration can be improved by keeping the wound surface moisture, so that the wound treatment material required in maintaining the moisture balance in the wound surface (8). Topical therapy which is often used for burn treatment is NaCl 0.9%, silver sulfadiazine (SSD) and hydrogel. NaCl 0.9% is an isotonic fluid that is used to irrigate and compress the wound, but NaCl 0.9% has a weakness when apply as compress because it is volatile (9). Silver sulfadiazine cream 1% is water soluble which is bactericidal, but has lacked to penetrate (8). Besides that silver sulfadiazine has several side effects such as leucopenia, argiria and crystalluria (10). Hydrogel is a wound treatment material include in an absorptive dressings type (11). Hydrogels are macro-molecules that can absorb liquid, reduce pain, and give a cooling effect (14,15). The main component of hydrogels is carboxyl-methyl cellulose (CMC). The mechanism of hydrogel is through soften necrotic tissue, absorb exudates and retain moisture wound base (16). Unfortunately, hydrogel has a weakness such as the low capacity of community that cause by the limited availability and high prices. The effort to overcome this problem is by providing alternative wound topical

therapy, that have a prime ability and low price. One natural herbal that have close ingredient with hydrogel is green grass jelly or cincau leaves (Cyclea barbata Miers (cbm). Cincau leaves have been used as a remedy for fever in chronic wounds and high blood pressure. It can soften the stool for long time. Cincau commonly grows in moist and shady areas (15). This plant is easily cultivated and grows wild. Substances that contained in the Cincau are alkaloids, flavonoids, vitamin A, polysaccharide pectin (18, 19, 20, 21). Alkaloids and flavonoids are antioxidant compounds that function as an immunostimulant to macrophages and scavenger of the free radicals in burn (20,21). Polysaccharide pectin can configure gels that have the ability to absorb the surrounding fluid. Cincau gel is hydrophilic and capable to serve cool effect and retain moisture wound area as well as CMC (23). So that leaves grass jelly has the potential to be developed as an alternative treatment for burn. MATERIAL AND METHODE This research used a true experimental posttest only control group design. The samples were 30 male rats (Rattus norvegicus) which divided into 6 groups, there were control group, group that was treated with NaCl 0.9% (K), group that treated with Silver Sulfadiazine/SSD (P1), group that was treated with a hydrogel (P2), and 3 treatment groups that were treated with the ethanol extract of cincau leaves (Cyclea barbata Miers) with concentration of 40% (P3), 50% (P4), and 60% (P5) respectively.

Second burn degree form First, anesthesia was performed intramuscular in the lower abdomen of experimental rats using ketamine with a dose of ±5mg diluted in 0.5ml water for injection. Then was injected to each mouse. Upper right back area was determined as induced area for second degree burn, after that the hair on that area was cleaned and shaved. The iron plate 2 x 2 cm with 2 mm thickness was heated until the temperature reaches 80

° C. This process was done for ± 8 minutes using a bunsen flame that is already set on axis 1 cm high. Iron plate then was attached to the determined area in the back of rat for 6 seconds with minimal pressure. After iron plate was appointed, then wound was bathed with 0.9% NaCl for 60 seconds. Wound dried using sterile gauze and bandaged. Burn wound care After washing hand with soap and placing the pad and the caring set close to wound car, the rat was holding and setting in position as comfortable as possible by placing the wound on the upper part to facilitate the wounds handling. First wound irrigated with 0.9% NaCl in order to facilitate the dressing easy to be opened. By sterile gloves the wound was cleaned using 0.9% NaCl and dried with sterile gauze. Furthermore, the wound was applied using treatment materials and then bandaged.

Green Cincau Extract Dry cincau leaves were mashed in a blender until smooth. Next, the cincau leaves powder weighed until 100 gr and put in a 1 lt glass Erlenmeyer. Soaked with ethanol to a volume of 1000 ml, then shaken until thoroughly mixed (± 30 min) and precipitated for 24 hours. Next, the upper layer of ethanol mixture was taken, then put in a 1 lt evaporation flask and fitted on the evaporator. After that, a water bath filled with water to the brim, then all the tools were installed, including a rotary evaporator. The water bath set to 900C and connected to electricity. Ethanol solution was allowed to separate with the existing active substance in the flask and wait until the flow stops dripping pumpkin ethanol container (± 1.5 to 2 hours to 1 pumpkin). Extraction results included in a plastic bottle and stored in a freezer.

Immunohistochemistry This study was conducted for 4 days. Rats terminated on the fourth day post wound

creation. Next the scar tissue was taken and identified for IL1 expression and macrophages number using immunohistochemistry staining. IL1 staining used IL1 antibody (H-153): sc7884, and staining of macrophages used macrophages antibody (mac387): sc-66 204 Santa Cruise inc. The percentage of IL1 expression was determined by measuring the area of a brownish color on intra-cellular. IL 1 expression was observe under light microscope. While the macrophage was determined as a round or oval cell with irregular edge, Kidney nucleus (24), brown appearance in the area of cytoplasm of the cell with purplish blue nucleus under light microscope (25,26). The expression of macrophages can be seen in figure 2. Number of IL1 and macrophage expression was observed in 10 visual fields. Further recorded, counted and averaged. Summary of the IL1 expression and macrophage cells mean can be seen in Table 1 and Table 2. Table 1.Mean of IL1 Expression Groups NaCl 0,9% (K) SSD (P1) Hidrogel (P2) Cincau 40% (P3) Cincau 50% (P4) Cincau 60% (P5)

Mean ±SD 22.17 ± 1,47 16.67 ± 3.14 12.67 ± 2.65 12.17 ± 2.13 9.50 ± 1.76 5.50 ± 1.37

Analysis The number of IL1 and macrophages expression was statistically analyzed using IBM SPSS 20 software. Normality test was conducted using Shapiro-Wilk test with α> 0.05. Homogeneity test was done using levene test homogeneity of variances with α> 0.05. If the data normal and homogeneous, it can be continued with parametric test of oneway ANOVA (29) and continued using Tukey HSD post hoc test (30).

K

P1

P3

P2

P4

P5

K

P1

P2

P4

P3

P5

Figure 1.IL1 Expression IL1 Expression from 2nd degree burn tissue that are treated using: (a) NaCl 0.9 % (b) SSD (c) hidrogel (d) extract ethanol of cincau leaves 40 % (e) extract ethanol of cincau leaves 50 % , and (f) extract ethanol of cincau leaves 60 % .

RESULT Normality and homogeneity test obtained p>0,05 showing that data was distributed normally and homogeneous then continued with One Way ANOVA test. ANOVA test of IL1 expression showed there was a significant difference among the groups (P=0.001) as well as on macrophages number (P=0.001). Table 2.Mean of Macrophage Number Groups NaCl 0,9% (K) SSD (P1) Hidrogel (P2) Cincau 40% (P3) Cincau 50% (P4) Cincau 60% (P5)

Figure 2. Macrophage Expression Macrophages Expression from 2nd degree burns tissue that are treated using: (a) NaCl 0.9 %; (b) SSD (c) hidrogel (d) extract ethanol of cincau leaves 40 % (e) extract ethanol of cincau leaves 50 %. and (f) extract ethanol of cincau leaves 60 %.

Based on the post hoc tukey HSD with confident interval 95 %, showed that there was a significance different on the IL1 expression between the p3 and p4 with control group (P=0.001 ; P=0.001). Both groups did not have a difference with the comparison group that used hydrogel as standard therapy (P2). P5 was the lowest expression of IL1 and having the difference to the control and treatment groups (figure 3).

Mean±SD 9.67 ± 2.06 11.00 ± 1.78 12.50 ± 1.87 14.17 ± 2.71 14.67 ± 4.08 19.83 ± 2.04

Figure 4. Mean of IL1 expression There are significant differences on IL1 expression between the group treated with extract ethanol green cincau leaves 40 % (P3) and 50 % (P4) with control groupgroup (k)

The post hoc on the number of macrophages showed a differences between the treatment group P3 and P4 with control group (P=0.001). Both P3 and P4 groups were not different with comparison groups (P1 and P2) that used SSD and hydrogel as standard therapy. The group that treated with cincau extract 60% (P5) had the highest macrophages average. There were significant differences between P5 with the whole group. Macrophage number was showed in figure 4.

%

Figure 5. Mean of macrophage expression There are significant differences on macrophage expression between the group treated with extract ethanol green cincau leaves 40 % (P3) and 50 % (P4) with control groupgroup (k)

Discussion In this study, analysis of wound tissue done on the 4th day after injury. In the 4th day after injured is appropriate time to know the existence of a pro-inflammatory cytokin and macrophages migration (5, 27, 28). On the 4th day post-injury the proinflammatory cytokines such as IL1 has a role in the migration of macrophages, although this cytokine physiologically started to decline (29, 30, 31). The declining of IL1 showed in the results, IL1 expression decreased in the whole group. Furthermore there was a significance decrease in the groups that treated with cincau leaf extract 40% (P3) and 50% (P4) compared to the control group. Expression of IL1 between the P3 and P4 with the group treated with standard therapy hydrogels (P2) did not have a difference. The

similarity of IL-1 expression among P2, P3, P4 groups, indicates that the compounds in the cincau leaves (polysaccharide pectin) have the same effect as a component in the hydrogel (carbomethyl cellulose). According Zohdi et al., Treatment for injury by using a hydrogel can decrease IL1 on the 7th day of burns (32). Similar conditions shown in this study, the decrease in the expression of IL1 occurred in the group treated using hydrogels (P2). The decreased expression of IL1 also found in the group treated with cincau leaves extract. Cincau leaves contain antioxidant such as flavonoid that might influence the reduction of IL1 (33). More specifically Kusmardiani et al., revealed that flavonoids contained in the leaves of green grass jelly is a flavonol 3oglikosida (35). Previous study by Chen et al., revealed that flavonoids in Lonicera japonica can reduce pro-inflammatory cytokines in the wound excision day 1 and 9 postmanufacture of wound (33). In another study mentioned that the flavonoids (kaempferol) can help the healing of burns by suppressing pro-inflammatory cytokines (34) Cincau (Cyclea barbata) leaves are a herb that contains antioxidants like flavonoids and alkaloids (33) Flavonoid contained in the cincau leaves also has the ability to increase the migration of macrophages in the injured tissue. The same opinion was delivered by Yuneda (2011) that flavonoids can increase the number of macrophages that migrate along the gum after tooth revocation (34). Macrophage migration may also be affected by the humidity of the wound surface. Moist wound conditions will increase cell migration into the wound area, at this time, monocytes migration from the intravascular would infiltrate and differentiate into macrophages in the wound tissue (35). This process leads to an increase in the number of macrophages in the tissue. Moisture in the wound area can be affected by topical therapy that able to bind wound exudate fluid (hydrophilic dressing). Cincau leaves also contain pectin which is a hydrophilic polysaccharide (36).

The results showed that the group that was treated with ethanol extract of leaves of green grass jelly 40% and 50% (P3 and P4) have no different with the group treated with the standard treatment SSD and hydrogel (P1 and P2) in the migration of macrophages. Extreme conditions occurred in the group treated with the ethanol extract of green grass jelly leaves 60% (P5). P5 group showed the highest increase in macrophage migration and have significant differences with the group treated with the standard treatment SSD and hydrogel (P1 and P2). Macrophage migration influenced by component adhesion molecules, one of them is intra-cellular adhesion molecule (ICAM1). ICAM1 is activated by proinflammatory cytokines such as IL1, IL6, interferon-ɤ, and TNF-α (38). These cytokines induces adhesion molecules that influence macrophages to migrate into the tissue (38). There is a discrepancy in the present study, which is between the expression of IL1 and the number of macrophages. The decline IL1 in the group treated with cincau extract using 60% (P5) is not consistent with the increase number of macrophage migration in the same group. This discrepancy could be explained by other research findings which showed that the wound surface moist conditions can activate ICAM1 (38,39). Wang et al (2014); Klein and Guha,(2014) said that treatment on the wound using wound care moisture can increase the ICAM1. At the same journal also mentioned the increase in ICAM1 followed by an increase in the migration of macrophages (38,39).

Conclusion This study conclude that topical treatment with the ethanol extract of cincau leaves can decrease the expression of IL1 and increase the number of macrophages in burns wound tissue. Flavonoids are components contained in the ethanol extract of cincau leaves has an influence on the decreased expression of IL1 and increased migration of macrophages. Cincau leaves also contain pectin hydrophilic

polysaccharide that is capable of binding liquid wound exudate to maintain surface moisture, so can facilitate the migration of macrophages to the network. References

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