INTRODUCTION:

Second-degree burn is one of acute traumas that has complexities in its healing process. Delay in burn wound healing may occur due to disruption in the migration and function of neutrophils⁽¹^,²⁾. Several studies mentioned that peripheral neutrophils in a patient with burn wounds experience a disruption in the migration and function⁽³^,⁴^,5).

Impaired migration and function of neutrophils result in an extended period of inflammation phase in the burn wounds. Therefore, the burn wounds become chronic and more susceptible to infection. However, other studies mentioned that the number of neutrophils in lung, kidney, and brain tissues from rat burn models fluctuates in the course of seven days after burn injury^(1,6^,⁷⁾. On the other hand, the number of neutrophils in skin tissues remains high up until the seventh day after burn injury⁽¹⁾. An increase in the number of neutrophil migrations will trigger micro vascular plugging and increase the severity of burns⁽⁸⁾

The apoptosis of neutrophils are crucial to be managed during the inflammation phase because the presence of high neutrophils after 48 hours can cause an extended period of inflammation⁽⁹^,¹⁰^,¹¹⁾. High level of neutrophils in a wound after 48 hours may slow down healing process due to the increase in myeloperoxidase (MPO) enzyme that causes tissue necrosis and scar formation⁽¹²⁾. Management of neutrophils quantity in a tissue can be done by applying appropriate topical therapy to the wound⁽¹²^,¹³⁾. One of local natural ingredients with immunomodulation properties that has not been widely used for wound treatment is Cyclea barbata Miers⁽¹⁵⁾.

Cyclea barbata Miers (CBM) is a plant endemic to Southeast Asia that has many local names. The most common name for Cyclea barbata Miers is Cincau hijau (Green Grass Jelly)⁽¹⁶⁾. The part of CBM mostly used by the society, either for food or medicine, is the leaves⁽¹⁵⁾. The extract of CBM leaves is often consumed as a healthy food. Traditionally, CBM leaves extract is utilized to treat fever, diarrhea, and hypertension⁽¹⁶⁾. Other studies also mentioned that CBM leaves extract can protect gastric mucosa that has been exposed to aspirin⁽¹⁷⁾. This protective property of CBM may be enabled by antioxidant compounds contained in CBM leaves⁽¹⁸⁾.

Katrin et al. stated that the highest antioxidant activity is present in the fifth fraction that contains alkaloid, polyphenol, glycoside, and tannin⁽¹⁸⁾. Another study also stated that the strongest antioxidant contained in CBM is a flavonoid compound, it was identified as flavonol 3-O-glycoside⁽¹⁹⁾. In another article, Mackaman (2014) stated that CBM leaves also contain a polysaccharide compound which is pectin⁽²⁰⁾. Pectin monosaccharide is a d-galacturonic acid. A previous study mentioned that 40% and 50% doses of CBM leaves extract can lower the IL1 level and increase the number of macrophages in second-degree burn wounds⁽²¹⁾.

The potential of CBM asan alternative material capable to improve inflammation process provided an opportunity to identify the compounds contained in CBM leaves extract and their effect in the migration and apoptosis of neutrophils in burn wounds. The novelty of this study is the type and mass of compounds identified in CBM leaves extract. Another new finding of this study is the effect of CBM leaves extract on the migration and apoptosis of neutrophils.

MATERIALS AND METHOD:

Materials:

Cyclea barbata Miers (CBM) leaves were acquired from Materia Medika, a medicinal plant association, in Batu, Indonesia. Hydrogels used in this study was a standard therapy for burn wounds, which is hidroactive gel®⁽²²⁾. Meanwhile, the third topical therapy was NaCl 0.9% purchased from Otsuka. Standard compounds of isoquercetin were obtained from Chemfaces (China) with the catalog number of CFN98753. Meanwhile, d-galacturonic acid was obtained from Santa Cruz (USA) with the catalog number of SC-257277. Acetonitrile and formic acid that were used for the mobile phase of LCMS/MS were purchased from Merck (Germany). Meanwhile, H2O that was used in the LCMS/MS analysis was obtained from laboratory preparation using an Evoqua equipment with the conductivity of 0.055µS. Neutrophils migration and apoptosis were examined by using immunofluorescence assay technique. The antibody being used, CD18 PE-conjugated antibody, was obtained from Bioss (USA) bs-0177R-Cy7. Apoptotic cells were examined using FITC conjugated annexyn V that was acquired from Biorbyt (USA) orb15096. Meanwhile, the cell nucleus was stained using DAPI.

Extraction technique:

The extraction method of CBM was maceration technique⁽²¹⁾. First step of extraction procedure is weighing the CBM powder. 100gr CBM powder macerated in 1000mL, then shaken until thoroughly mixed and precepted for 24 hours. Put the solution into a rotary evaporator tube and rotate it in a water bath at 90°C degrees. Rotate the tube for 1.5 to 2 hours. Let the ethanol evaporate and the CBM extract will remain in the jar. Then save the extract in a temperature of 5 degrees⁽²³⁾.

ANIMAL MODEL:

This experimental study used white wistar rats as the animal model. They were obtained from the Pharmacology Laboratory of Medical Faculty in Universitas Brawijaya. This study used 30 male rats weighing 280-320grams. The rats were separated into group A and B. Fifteen group A rats were euthanized 48 hours after burn injury. Group A was then divided into three treatment groups, i.e. five rats treated with CBM leaves extract (AP), five with hydrogels (AQ), and five with NaCl 0.9% (AR). Meanwhile, 15 group B rats were euthanized 72 hours after burn injury. Group B was also divided into three treatment groups, i.e. five rats treated with CBM leaves extract (BP), five with hydrogels (BQ), and five with NaCl 0.9% (BR). During treatment, the rats were cared in individual cages, fed standard food, and given distilled water ad libitum. This study had been approved by the Ethical Clearance Team of the Medical Faculty of Universitas Brawijaya with the identification number of 347/EC/KEPK/10/2017.

BURN WOUND CREATION AND TREATMENT:

Burn wounds on the back of the rats were produced using 2mm thick iron plate measuring 2x2cm. Prior to burning, the rat’s back was shaved and cleaned with alcohol. The iron plate was heated on a fire for 8 minutes until it reached 80ºC temperature. Afterwards, the plate was placed on the rat’s back for 6 seconds until a second degree burn wound was obtained as shown in Figure 1. Burn wound treatment was carried out immediately, i.e. rinsing the wound with NaCl 0.9% and applying topical therapies appropriate to each treatment groups. Burn wound was treated every 24 hours. Wound care is given once a day. Group A was given 2 times of wound care in 48 hours, then the rats were euthanized and burn tissue was taken for examination. Whereas in group B, rat were given 3 times of wound care within 72 hours, then the rats were euthanized and the wound tissue was taken for examination

Figure 1. 2^nd Degree of burn wound, characterized by pale skin (non-vascularized tissue) and rough skin texture

IMMUNOFLUORESCENCEASSAY:

Initially, burn wound tissues were stored as paraffin blocks and embedded to polysilane slides. On the first day, deparaffinization was conducted. After that, antigen retrieval was perfomed using ph 6 citrate buffer and the slides were put into 95°C water bath for 20 minutes. Sequentially, the slides were rinsed with PBS and PBS Triton X-100. After incubation in PBC for 30 minutes, the slides were incubated with antibody for one night in 4°C temperature. Lastly, the slides were rinsed with PBS and covered with the mounting and cover glass. CD18 and annexinV expressions were observed in a dark room using Olympus immunofluorescence microscope type IX21. Migrated neutrophils in the tissue were observed by counting the CD18 expression, i.e. the red color. Meanwhile, apoptotic neutrophils were noted by spotting the orange or yellow color which is the combination of red (CD18) and green (annexin-V). Observation was conducted in five fields of view with 400x magnification. The color intensity was calculated using ImageJ image processing software from NCBI website.

LCMS/MS Assay:

Preparation of Stock Solution:

The 1000ppm main stock solutions of isoquercetine were prepared in methanol (13,18). Meanwhile, the 1000 ppm main stock solution for d-galacturonic acid (DGA) was prepared in H2O(20). Furthermore, serial solution of 70, 120, 200, 500 and 1000ng/ml of isoquercetin were made. At the same time, serial solution of 50, 100, 500, and 1000ng/ml of d-galacturonic acid were also made. The main stock solutions were stored in -2°Celcius temperature.

Sample Preparation:

The first step of sample preparation was weighing 11mg of extract. After that, the extract was diluted in 1 mililiter of methanol: H20 = 80: 20 solvent and sonicated for five minutes. 1000µL of methanol and H20 were then added to a 100µLaliquotand centrifuged in 1000rpm speed for 10 minutes. Then, the supernatant was collected and filtered using a 0.2µM membrane filter. It was then injected to LCMS/MS for further analysis.

Operating Condition of LCMS/MS:

Sample testing of CBM leaves extract was conducted using LCMS/MS equipment. The column used was the Hypersil Gold measuring 50mm x 2.1mm x 1.9µm. The ultra-high performance liquid chromatography (UHPLC) equipment ACELLA type 1250 manufactured by Thermo Scientific that consisted of vacuum degasser, quartener pump, and thermostatic autosample was controllable from a personal computer through x-calibur 2.1. software. The A solvent was 0.1% formic acid in water while the B solvent was 0.1% formic acid in acetonitrile. A mobile phase gradient with 300 µl/minutes was set with the following arrangement: 0.0-0.75 minutes 5%B, 0.75-2.5 minutes 70%B, 2.5-3.4 minutes 70%B, 3.4-3.5 minutes 5%B, 3.5-5.0 minutes 5%B. The injection volume on the LC was 2 µL. The column was controlled at 30°C. Meanwhile, the autosampler compartment was set to 16°C(24).

The utilization of MS/MS triple Q (quadrupole) mass spectrometer TSQ QUANTUM ACCESS MAX from Thermo Finnigan with ESI (Electrospray Ionization) ionization source was controlled by TSQ Tune software operated in negative mode. The identification of the mass spectra of isoquercetin was at 463m/z as precursor ion and at 300m/z as product ions. Meanwhile, DGA was at 193m/z as precursor ion and 192m/z as product ion.

The ionization conditions of the ESI were as follows: 2.5 kV electrospray voltage; 250°C evaporation temperature; 300°C capillary temperature; 40 psi nitrogen as the sheath gas pressure and 10 psi argon gas as the Aux pressure.

STATISTICAL ANALYSIS:

Statistical analysis using IBM SPSS 23 program was conducted to determine the differences between each group. Statistical significance comparison between the groups were performed using the ANOVA test. The degree of confidence used was 95% with α 0.05⁽²⁴^,²⁵⁾.

RESULTS:

Identification of Isoquercetin and D-galacturonic acid in Cyclea barbata Miers:

On examination of CBM leaves extract, the graph of isoquercetin appeared in the third minute and presented a negative charge. Meanwhile, the graph of d-galacturonic acid appeared in the first minute and was more polar than isoquercetin. The identification result of isoquercetin and d-galacturonic acid compounds is shown in Figure 2 where the number of total ion chromatogram (TIC) was presented. TIC was the result of further in-depth analysis of isoquercetin and DGA. This analysis resulted in the peak of the isoquercetin graph at point 3.1, while the DGA peak was at 0.66. Further analysis on each compounds were presented in extracted ion chromatography (EIC) graph of DGA that was at 193m/z as precursor ion and at 192m/z as product ion. Meanwhile, isoquercetin was at 463m/z as precursor ion and at 300m/z as product ion. Another result of LCMS test on CBM leaves extract was the quantitative analysis of the contents of isoquercetin and DGA. DGA content in CBM leaves extract was found to be 0.114 ng/ml while isoquercetin was 93.83ng/ml.

Figure 2. Chromatogram analysis. Graph of total ion chromatogram (TIC) analysis of d-galacturonic acid and isoquercetin (A). Chromatogram of d-galacturonic acid (B). Chromatogram of isoquercetin (C).

Neutrophils Migration:

Neutrophils migration with CD18 marker on the skin tissue with burn wound was expressed in red color. The observation of CD18 expression in each treatment group in the 48^th hour showed that the highest red color expression was present in the group treated with NaCl 0.9%, i.e. 1.28 a.u. On the other hand, the lowest red color expression was present in the group treated with hydrogel, i.e. 0.195 a.u. ANOVA test result showed a significant difference (p>0.05) between the groups. Furthermore, the result of post hoc analysis showed that the group treated with NaCl 0.9% was significantly different from the groups treated with hydrogel and CBM leaves extract. The graph of CD18 expression is presented in Figure 3.

Fig 3. Expression of CD18. A. Expression of CD18 in BP group. B. Expression of CD18 and Dapi in BP group. C. Expression of CD18 in BQ group. D. Expression of CD18 and Dapi in BQ group. E. Expression of CD18 in BR group. F. expression of CD18 and Dapi in BR group. G. mean difference among group.

NEUTROPHIL APOPTOSIS:

Neutrophil apoptosis was observed from the orange color expression on the burn wound tissue. The comparison of orange color expression between each treatment group in the 72^nd hours showed that the lowest orange color expression was present in the group treated with NaCl 0.9%, i.e. 1.28 lnp/mm. On the other hand, the highest orange color expression was present in the group treated with CBM leaves extract, i.e 0.46 lnp/mm. ANOVA test result showed a difference (p>0.05) between the groups. Furthermore, the result of post hoc analysis showed that the group treated with NaCl 0.9% was significantly different from other groups while the group treated with CBM leaves extract was not significantly different from the hydrogel group. The graph of orange color expression is presented in Figure 4.

DISCUSSION:

Identification of Isoquercetin and D-galacturonic acid

This study showed that one of flavonol glycoside compounds contained in CBM leaves extract is quercetin glycoside or isoquercetin. As stated in previous studies, fractionation of CBM leaves with methanol solvent results in the most potent antioxidant, i.e. alkaloid and flavonoid⁽¹⁸⁾. Furthermore, other studies also indicated that flavonoids isolated from CBM extract, identified using acid hydrolysis technique, are flavonol 3-O-glycosides⁽¹⁹⁾. Isoquercetin is one of flavonol compounds commonly found in plants. Several studiessuggested that quercetin glycoside or isoquercetin has better bioavaibility than quercetin⁽²⁷⁾^,⁽²⁸⁾. Another study also mentioned that isoquercetin has anti-inflammatory and antiradical properties⁽²⁹⁾.

This study showed that monomer pectin contained in CBM leaves extract was d-galacturonic acid. This study also found that despite having a low mass in CBM extract, d-galacturonic acid gives a unique property to CBM benefits. Arkapanthu (2005) stated that CBM leaves have distinguished gel formation characteristics, i.e. 1) there is an intermolecular hydrogen bond between galacturonic acid chains; 2) there is a connection between polygalacturonic acid chain and phenol compound; 3) the ionic bond between polygalacturonic acid chains is cation divalent; 4) there is a hydrophobic intermolecular bond between metilated groups on the polygalacturonic acid chains⁽³⁰⁾. Another superiority of CBM is its ability to survive in poor extracting condition. On the same article, Mackaman stated that the ability of CBM as an emulsifying agent depends largely on its protein content which amounts to 6%⁽²⁰⁾. The protein content enables absorption on water or oil surface(20). Besides its anti-inflammatory property, CBM’s ability to maintain humidity is also crucial in promoting the resolution of inflammation⁽³⁰^,³¹⁾.

Fig 4. Expression of Apoptosis CD18. A. Expression of Apoptosis CD18 in CP group. B. Expression of Apoptosis CD18and Dapi in CP group. C. Expression of Apoptosis CD18 in CQ group. D. Expression of Apoptosis CD18and Dapi in CQ group. E. Expression of Apoptosis CD18 in CR group. F. Expression of Apoptosis CD18 and Dapi in CR group. G. Mean Difference among the group

The effect of CBM leaves extract on the quantity of neutrophils in burn wounds:

This study showed that there was a decrease in CD18 expression in groups treated with CBM leaves extract and hydrogel. CD18 is a marker for neutrophil adhesion in the tissue, CD18 expression will increase in the inflammatory phase in accordance with an increase in the number of neutrophils in the inflammatory area⁽³²^,³³⁾. The neutrophils in the burn wound tissue started to increase in the 6^th hour, reached a peak in the 24^th hour, decreased in the 48^th hour, and increased again on the 7^th day⁽¹⁾. This study also showed the difference in the 48^th hours between the group treated with NaCl 0.9% and the group treated with CBM leaves extract and hydrogel. Hydrogel is a standard treatment material applied on burn wounds during the early stages. On the beginning of inflammation phase, hydrogel reduces excessive inflammatory responses⁽³⁴^,³¹⁾. Excessive neutrophil control is very important to avoid the incidence of sepsis in burn patients, studies on burn patients showed that venous neutrophils in burn patients experience a disruption in mobility and function, and spontaneously increase within 1 or 2 days before sepsis⁽³^,⁴⁾. Isoquercetin as a member of flavonol glycoside acts as an anti-inflammatory agent that reduces polymorphonuclear cells such as neutrophils⁽³⁶⁾. Isoquercetin also reduces the quantity of neutrophils on allergic lung tissue. The same study also stated that isoquercetin lowers leucocytes more than quercetin does⁽²⁸⁾.

The effect of CBM leaves extract on neutrophils apoptosis in burn wounds:

Migrated neutrophils in the tissue will perform phagocytosis on a bacteria or foreign body in the tissue. Afterwards, neutrophils will experience apoptosis. The apoptotic body will send signals to the monocyte as the second defense on the inflammatory phase⁽³⁶^,³⁷^,³⁸⁾. When the functions of neutrophils and macrophages are completed, the tissue will undergo inflammation resolution. This study showed a difference in apoptotic neutrophils expression between the group treated with NaCl 0.9% and the groups treated with CBM leaves extract and hydrogels. The number of apoptotic neutrophils increased in the 72nd hour in the groups treated with CBM leaves extract and hydrogel. Hydrogel is a standard modern treatment for burn wounds during the early stages⁽²²⁾. Several articles mentioned that hydrogel is a biomaterial that may promote neutrophil’s function as a phagocytic cell that is indicated by the increase in MMPs and MPO⁽³⁰^,³⁹⁾. Isoquercetin as an anti-inflammatory agent can also increase the quantity of apoptotic neutrophils. Another study stated that quercetin as a flavonol can inhibit lipopolysaccharide (LPS) that prevents spontaneous apoptosis in neutrophils⁽⁴⁰^,⁴¹⁾.

CONCLUSION:

Based on the results of this study, it can be concluded that CBM leaves contain isoquercetin and d-galacturonic acid. Both compounds contribute to the modulation of inflammation phase. This study also found that CBM leaves extract can decrease neutrophils migration in burn wound tissues, similar to hydrogel as the standard therapy. CBM leaves extract also exhibits antiradical potential that enables the increase of neutrophils apoptosis, which is also shown in the group treated with hydrogel. The application of CBM leaves extract as an alternative therapy candidate for wound healing process requires further in-depth study to obtain supportive evidences.

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Received on 30.10.2019 Modified on 24.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3059-3064.

DOI: 10.5958/0974-360X.2020.00543.0