The Activity of Hydrolyzed Virgin Coconut Oil to Increase Proliferation and Cyclooxygenase-2 Expression towards on NIH 3T3 Cell Line in Wound Healing Process

ID Design Press, Skopje, Republic of Macedonia Open Access Macedonian Journal of Medical Sciences. 2019 Oct 15; 7(19):3164-3168. https://doi.org/10.3889/oamjms.2019.804 eISSN: 1857-9655 Basic Science The Activity of Hydrolyzed Virgin Coconut Oil to Increase Proliferation and Cyclooxygenase-2 Expression towards on NIH 3T3 Cell Line in Wound Healing Process 1* 2 1 1 Jansen Silalahi , Yuandani Yuandani , Dian Ika Perbina Br Meliala , Linda Margata , Denny Satria 3 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Sumatera Utara, Jalan Tri Dharma No. 5, 2 Kampus USU, Medan, 20155, Indonesia; Department of Pharmacology, Faculty of Pharmacy, Universitas Sumatera Utara, 3 Jalan Tri Dharma No. 5, Kampus USU, Medan, 20155, Indonesia; Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Sumatera Utara, Jalan Tri Dharma No. 5, Kampus USU, Medan, 20155, Indonesia Abstract Citation: Silalahi J, Yuandani Y, Meliala DIPBR, Margata L, Satria D. The Activity of Hydrolyzed Virgin Coconut Oil to Increase Proliferation and Cyclooxygenase-2 Expression towards on NIH 3T3 Cell Line in Wound Healing Process. Open Access Maced J Med Sci. 2019 Oct 15; 7(19):3164-3168. https://doi.org/10.3889/oamjms.2019.804 Keywords: Cell proliferation; COX-2; HVCO; NIH 3T3; Percent of the wound closed *Correspondence: Jansen Silalahi. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Sumatera Utara, Jalan Tri Dharma No. 5, Kampus USU, Medan, 20155, Indonesia. E-mail: Received: 13-Jun-2019; Revised: 20-Jul-2019; Accepted: 21-Jul-2019; Online first: 14-Oct-2019 Copyright: © 2019 Jansen Silalahi, Yuandani Yuandani, Dian Ika Perbina Br Meliala, Linda Margata, Denny Satria. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0) AIM: This study aims to determine the effect of hydrolysed virgin coconut oil (HVCO) to increase cell proliferation, COX-2 expression of NIH 3T3. METHODS: The sample used was Virgin Coconut Oil (VCO). VCO was partially hydrolysed using lipase from Rhizomucor miehei (active on sn-1,3 position) to produce hydrolysed VCO (HVCO) composed of free fatty acids, 2-monoglycerides. Then acid value was determined. The effect of HVCO on proliferation was evaluated using the MTT method. Wound healing assay was established by a cell migration method, and COX-2 expression was determined using RT-PCR. RESULTS: Acid value is 135.89 ± 0.12 mg NaOH/g oil and free fatty acids (FFA) is 48.50 ± 0.06%. The effect of HVCO 62.5 µg/mL on cell proliferation after 24h, 48h, and 72h incubation found as viable cells are 109.24 ± 0.52%; 118.26 ± 0.91% and 106.59 ± 0.74%. Percent of wound closed after 24 h and 48 h incubation are 69.94 ± 0.54% and 100.00 ± 0.00%, and expression of COX-2 increased from 1 (control) to 1.83 (HVCO). CONCLUSION: The results suggest that HVCO is effective to increase cells proliferation and hence wound healing process. Funding: The investigation was financially supported by the Ministry of Research, Technology and Higher Education, Indonesia through “Hibah Penelitian Dasar Unggulan Perguruan Tinggi” research grant 2019 and the Research Center University of Sumatera Utara through “Hibah Penelitian Guru Besar” research grant, 2018 Competing Interests: The authors have declared that no competing interests exist Introduction Wound healing is a process involving many cells consisting of four stages, namely hemostasis, inflammation, proliferation, and remodelling [1]. In the hemostasis, stages are the beginning of the wound healing process by involving platelets [2]. During the inflammatory phase, fibroblasts function as cytokine secretions, and growth factors to activate the body's defence system [3]. During the proliferation and remodelling phases, fibroblasts are important for granulating and reorganising tissues of the extracellular matrix [3]. Wound healing is associated with bacterial contamination in the wound area. The ultimate goal of wound healing is to restore the functional properties of the skin and prevent infection [4]. The COX enzyme consists of 2 isoenzymes such as COX-1 (COX-3 = COX-1 variants), and COX-2. COX-2 plays a role in the process of angiogenesis [5]. The expression of COX-2 affects the process of angiogenesis, migration, and proliferation of fibroblasts which is very important in wound healing [6]. The expression of COX-2 affects the process of angiogenesis, migration, and proliferation of fibroblasts [6]. VCO is obtained from the flesh of mature fresh coconuts fruit (Cocos nucifera) processed at low _______________________________________________________________________________________________________________________________ 3164 https://www.id-press.eu/mjms/index Silalahi et al. The Activity of Hydrolyzed Virgin Coconut Oil to Increase Proliferation and Cyclooxygenase-2 Expression towards on NIH 3T3 Cell Line _______________________________________________________________________________________________________________________________ temperature or without heating [4]. VCO contains phytosterol that can be beneficial as antiinflammation. Coconut oil as triglyceride does not have antimicrobial and antiviral activities, but when VCO is partially hydrolysed, it will generate free fatty acids and monoglycerides [7]. The combination of free fatty acids and monoglycerides are proved to be an antibacterial and antiviral agent, whereas diglycerides are not [8], [9]. Lauric acid and monolaurin (monoglyceride of lauric acid) are antibacterial and antiviral through several mechanisms including by liquefying and damaging the lipid layer structure in virus and cell membrane of bacteria [8], [9]. This study aims to determine the effect of HVCO in the wound healing process by measuring the activity of cell proliferation, COX-2 expression, and cell migration in NIH 3T3 cells. Material and Methods Materials Virgin coconut oil (VCO) (Palem Mustika®, Indonesia), NIH 3T3 fibroblasts were purchased from Parasitology Laboratory, Faculty of Medicine, Gadjah Mada University. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal bovine serum and kept at 37°C with a CO2 supply of 5%. Lipase from R. miehei 20.000 U/g (Sigma), and reagents used were buffer Tris-HCl, nuclease-free water, sodium hydroxide, concentrated hydrochloric acid, distilled water, n-hexane, sodium sulfate anhydrous, potassium hydrogen phthalate, phenolphthalein, ethanol, and demineralized water, DMSO, phosphate buffer saline (PBS), and fetal bovine serum (FBS). All chemicals and reagents commonly used in COX-2 expression assay. anhydrous was added to absorb the water residue. The combined extract was allowed to stand for 15 min, filtered, and the n-hexane was evaporated using the water bath and resulted in HVCO, and then the acid value was determined. HVCO was then used to determine biomarkers in the woun (...truncated)