Wound healing potential of formulated ointment by using virgin coconut oil in combination with Ethanol extract of Artocarpus lakoocha Roxb. leaves
Ibrenna G. Manik1, Jansen Silalahi1*, Yuandani2, Denny Satria3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sumatera Utara,
Medan 20222, North Sumatera, Indonesia.
2Department of Pharmacology, Faculty of Pharmacy, University of Sumatera Utara,
Medan 20222, North Sumatera, Indonesia.
3Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Sumatera Utara,
Medan 20222, North Sumatera, Indonesia.
*Corresponding Author E-mail: jansen@usu.ac.id
ABSTRACT:
Wound healing is a complex process involving many cells consisting of four phases namely hemostasis, inflammation, proliferation, and remodeling. The usage of natural resources that have pharmacological features can hasten the wound's physiological healing procedure. The example of natural resources is virgin coconut oil (VCO) and ethanol extract of Artocarpus lakoocha Roxb. leaves (EEAL) which have the potential for wound healing. This study aimed to examine the combination of VCO and EEAL in an ointment formulation for wound healing in male Wistar rats. The ointment was divided into five formulas with different concentrations of VCO and EEAL and underwent stability and homogeneity evaluation. In vivo animal testing was divided into five groups according to the number of formulas. The wound healing activity was evaluated by using white male rats in the aspect of wound healing percentage and epithelial diameter from a histopathology study. The results showed that all formulas are stable and F3 by using the ratio of 3:1 (VCO: EEAL) has the best wound healing activity in the aspect of wound healing percentage and epithelial diameter compared to the other groups. In conclusion, our findings suggest that the VCO and EEAL in ointment preparation have great potential for wound healing activity.
KEYWORDS: Artocarpus lakoocha Roxb., Extract, Virgin coconut oil, Ointment, Wound healing.
INTRODUCTION:
Wound healing is a complicated process that involves the participation of a large number of cells and may be broken down into four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Platelets engage in the earliest stages of the wound healing process, which is referred to as the hemostasis phase. Fibroblasts serve as a source of cytokine secretions and growth factors during the inflammatory phase of the disease, which helps to activate the body's defensive system.
During periods of proliferation and remodeling, fibroblasts play an essential role in the granulation and reorganization of the tissues that make up the extracellular matrix1-5. One of the medicinal plants that are famous in Indonesia is Mobe or Artocarpus lakoocha Roxb and goes by the common name monkey jack. Artocarpus lakoocha Roxb. is a member of the family Moraceae which the large populations can be found in the tropical regions of South and Southeast Asia, particularly in the countries of Nepal, Sri Lanka, India, Myanmar, Indonesia, Vietnam, and Thailand6. Anti-inflammatory, antiviral, anticancer, antibacterial, and anti-HIV are only a few of its many pharmacological properties. Anthelmintic agents in Thailand have traditionally been made from the dried aqueous extract of its heartwood7.
Virgin coconut oil (VCO) is derived from mature fresh coconuts (Cocos nucifera) by using the cold-pressure technique. VCO contains several phytosterols that have anti-inflammatory potential. The component of triglyceride in coconut oil has no antibacterial or antiviral properties, whereas partially hydrolyzed VCO does. Unlike triglycerides and diglycerides, free fatty acids and monoglycerides are antibacterial and antiviral. Lauric acid and monolaurin (monoglyceride of lauric acid) are antiviral and antibacterial by liquefying and destroying the virus and bacterial cell membrane lipid layers8. The presence of bacteria in the wound area can slow down the healing process. The end goal of healing a wound is to get the skin to work again and keep an infection from happening. This study aimed to examine the wound healing activity of a combination of VCO and ethanol extract of Artocarpus lakoocha Roxb (EEAL) leaves in the ointment on excision wounds in male Wistar rats.
MATERIALS AND METHODS:
Materials:
The materials that were used include Artocarpus lakoocha Roxb. leaves from Toba Samosir regency, North Sumatera, Indonesia and was identified by the Indonesian Institute of Science (No: 2027/IPH.1.01/If.07/VIII/2017), Lanolin, Paraffin, and Ethanol (95%). All reagents and ointment bases (Lanolin and paraffin) were obtained from Sigma-Aldrich.
Ethanol extract of Artocarpus lakoocha Roxb leaves (EEAL) preparation:
The extraction process was conducted by the maceration method. The air-dried and powdered leaves of Artocarpus lakoocha Roxb. (500g) were macerated with ethanol 96% (7.5 L) for 6 days. By using a rotary evaporator, the EEAL solution was yielded as crude extract9,10.
Formulation of VCO and EEAL ointment:
A combination of VCO and EEAL ointments was prepared as per the composition given in Table 1. In this method, the constituents of the base like lanolin and paraffin were placed together and then mixed and stirred gently during the cooling stage to form the ointment base. The active component was incorporated into the base of the ointment by grinding the active ingredient in a mortar and pestle.
Table 1: Ointment formulation
|
Formula |
Ointment base (g) |
VCO (g) |
EEAL (g) |
|
F1 |
100 |
- |
- |
|
F2 |
95 |
5 |
0 |
|
F3 |
95 |
3.75 |
1.25 |
|
F4 |
95 |
2.5 |
2.5 |
|
F5 |
95 |
1.25 |
3.75 |
|
F6 |
100 |
- |
- |
Homogeneity and stability testing::
The homogeneity test was carried out by applying an ointment to an object glass slide, then observing the presence or absence of any granules The freshly prepared ointment samples were assessed for an appearance by visual assessment with the unaided eyes. Furthermore, observations were conducted on the 7th day, 14th day, 21st day, and 28th day. The appearance (color and odor) was observed weekly for 3 months for any changes in 25oC temperature and 60% humidity11-14.
Animal preparation:
The Animal House of the Faculty of Pharmacy at Universitas Sumatera Utara provided us with healthy adult male Wistar rats weighing 200-250g. Polycarbonate cages with a 12-hour day/night cycle were utilized to house the rats at normal temperatures. Food and water were provided without restriction to the rats. This study was carried out following AREC's ethical standards (No: 00612/KEPH-FMIPA/2020).
In vivo wound healing evaluation:
Thirty male Wistar rats were used for 5 groups with 6 male rats in each group. The rats were acclimatized for 7 days, then anesthetized using ketamine HCl (70mg/kg BW) via IP injection, then the hair was shaved on the back of the rat to be wound until smooth and then cleaned with cotton with 70% alcohol. The back of the rat was wounded by using a 2cm punch biopsy. A punch biopsy is pressed against the skin then rotated while pressing and pulled up until the tissue is cut15-17. The wound was treated by applying the ointment with different formulas (F1-F5) twice a day, in the morning and evening. The wound healing activity observation was conducted by using visual observation of the wound by measuring the diameter of the wound and calculating the percentage reduction in the diameter of the excision wound in rats. Wound observations were carried out once a day, on the 0th, 7th, 14th, and 21st days wounds are considered healed if the diameter of the wound is zero.
Histopathology analysis:
The wound sites from the rats were then excised and fixed in formalin. The section between 3-5µm in thickness was stained with hematoxylin and eosin. Image analysis was used to determine the percentage of wound re-epithelialization that had occurred).
RESULT:
Homogeneity and stability testing from VCO and EEAL Ointment:
From the results of the homogeneity test that was carried out on the ointment with the combination of VCO and EEAL, all the ointment preparations there are no granules on the glass object, then the ointment preparation is said to be homogeneous (Table 2). The results of observations of stability for 3 months were carried out by observing the changes that occurred in the ointment starting in the form of color and odor. There are no physical changes in the ointment, so it can be concluded that the ointment combination between VCO and EEAL in all formulas made is stable (Table 3).
Table 2: Homogeneity results from each formulation
|
Formula |
Homogeneity |
|
F1 |
Homogenous |
|
F2 |
Homogenous |
|
F3 |
Homogenous |
|
F4 |
Homogenous |
|
F5 |
Homogenous |
In vivo wound healing results:
The purpose of this study was to see if the ointment treatment for wound healing in male Wistar rats worked better when VCO and EEAL were used together. Rats with wounds on their backs were treated twice daily with several formulas of ointment. The wound's diameter was monitored weekly until it had healed entirely. Each week, the wound's diameter was measured to determine how well it was healing and calculated as a percentage of wound healing (figure 1 and Table 4).
Histopathology analysis:
Histopathological observations were carried out to determine changes in the size of the epithelium observed using a microscope. Epithelial thickening was observed after wound healing on day 21st. The samples observed were part of the skin that was previously part of the wound that was made at the beginning of the treatment and given treatment with each formula. Observations were made using a microscope with a magnification of 10x and the length of the epithelium was measured using Optic Lab 3.0 software and measured in three parts. The results are shown in figure 2 and Table 5.
Table 3: Stability results of formulated ointment
|
No |
Formula |
Observation |
|||||||||
|
0th day |
7th day |
14th day |
21st day |
28th day |
|||||||
|
Colour |
Odour |
Colour |
Odour |
Colour |
Odour |
Colour |
Odour |
Colour |
Odour |
||
|
1 |
F1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
2 |
F2 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
3 |
F3 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
4 |
F4 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
5 |
F5 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
Figure 1: Representative photographs of macroscopic appearance wound excised on rats
Table 4: Percentage of wound healing (n=6)
|
Day |
Formula |
Percentage of wound healing (%) ± SD |
|
0th |
F1 |
0.00 ± 0.00 |
|
F2 |
0.00 ± 0.00 |
|
|
F3 |
0.00 ± 0.00 |
|
|
F4 |
0.00 ± 0.00 |
|
|
F5 |
0.00 ± 0.00 |
|
|
7th |
F1 |
22.02 ± 0.57* |
|
F2 |
32.72 ± 0.23* |
|
|
F3 |
40.67 ± 3.16* |
|
|
F4 |
37.07 ± 1.83* |
|
|
F5 |
27.57 ± 0.53* |
|
|
14th |
F1 |
45.52 ± 3.12* |
|
F2 |
59.23 ± 2.98* |
|
|
F3 |
72.26 ± 2.73* |
|
|
F4 |
67.56 ± 2.69* |
|
|
F5 |
56.12 ± 2.38* |
|
|
21st |
F1 |
100.00 ± 0.00 |
|
F2 |
100.00 ± 0.00 |
|
|
F3 |
100.00 ± 0.00 |
|
|
F4 |
100.00 ± 0.00 |
|
|
F5 |
100.00 ± 0.00 |
Figure 2: Histopathology analysis (Re-epithelialization). Scale bar = 500 µm
Table 5: Average epithelial diameter (n=6) (*p<0.05 vs F1)
|
Formula |
Epithelial Diameter (μm) ± SD |
|
F1 |
62.60 ± 0.56 |
|
F2 |
82.56 ± 0.71* |
|
F3 |
102.57 ± 0.58* |
|
F4 |
92.49 ± 0.45* |
|
F5 |
72.64 ± 0.63* |
DISCUSSION:
The wound-healing property of EEAL and VCO may be contributed to the quicker process of wound healing. This was confirmed based on the results of observations of changes in wound healing, F3 formula which consists of the ratio 3:1 for VCO: EEAL showed better wound changes compared to the other formulas (figure 1). Wound changes can be seen on day fourteen which showed the size of the wound is smaller and higher wound healing percentage than the other groups. Changes in the size and shape of the wound showed healing activity in the wound so that on the 21st day each group had healed. Several active substances in VCO can be used to accelerate wound healing such as lauric and oleic acids. EEAL also contains several active substances such as tannins and flavonoids which play a key role in protecting wounds from bacterial growth so that it can help accelerate wound healing and increase the number of capillary blood vessels in the formation of fibroblast cells18-20. VCO and EEAL play an important role in the growth of epithelial tissue, with the compounds possessed can help the healing process, especially in the inflammatory phase where the growth and increase in blood vessels can help the regeneration process of damaged tissue with the formation of new fibroblasts in the epithelium21,22. Inflammation, granulation tissue development, and extracellular matrix remodeling all occur simultaneously throughout the wound healing process, which is a highly coordinated series of events. These processes are characterized by the participation of several cellular activities, including migratory, proliferative, adhesion, phenotypic differentiation, and others23,24. Clot development begins almost immediately after an injury, and the early stages of wound repair entail inflammation and the creation of ground material. Proteoglycans, which are nonfibrillar, heterogeneous extracellular matrix components, make up the majority of the ground material. Together with proteoglycans, glycosaminoglycans have been proven to be involved in the wound-healing event25,36. The interaction of the skin with the many different binding proteins is the cause of the skin's biological activity. During the process of tissue repair, inflammatory cells encourage the proliferation and migration of endothelial cells, which results in the formation of new blood vessels27. These connective tissue cells then produce extracellular matrices, including collagen, which leads to the re-epithelialization of injured tissue28-31.
CONCLUSION:
The formulated ointment by using a combination of VCO and EEAL and applied topically, effectively increased the wound healing ability in 14 days which was significant as compared to the ointment base. The results of the study suggest that a combination between VCO and EEAL in the ointment has significant wound healing activity with the best ratio combination of VCO and EEAL (3:1). The formulated ointment from VCO and EEAL could be made use of clinically, in the healing of wounds. However, the implementation of these results needs further investigation in the aspect of molecular expression and clinical evaluation.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank the Faculty of Pharmacy Universitas Sumatera Utara for the laboratory facilities.
REFERENCES:
1. Wild T. et al. Basics in nutrition and wound healing. Nutrition. 2010; 26(9):862-866. doi.org/10.1016/j.nut.2010.05.008
2. Rodrigues HG. et al. Oral administration of linoleic acid induces new vessel formation and improves skin wound healing in diabetic rats. Plos One. 2016; 12(5):e0179071. doi.org/10.1371/journal.pone.0165115
3. Ridiandries A. Bursill C. Tan J. Broad-spectrum inhibition of the CC-Chemokine class improves wound healing and wound angiogenesis. International Journal of Molecular Sciences. 2017; 18(1):155. doi.org/10.3390/ijms18010155
4. Sasidharan S. et al. Wound healing potential of Elaeis guineensis Jacq leaves in an infected albino rat model. Molecules. 2010; 15(5):3186-3199. doi.org/10.3390/molecules15053186
5. Arndt S, et al. Cold atmospheric plasma (CAP) changes gene expression of key molecules of the wound healing machinery and improves wound healing in vitro and in vivo. Plos One. 2013; 8(11):e79325. doi.org/10.1371/journal.pone.0079325
6. Jagtap UB. Bapat VA. Artocarpus: a review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology. 2010; 129(2):142-166. doi.org/10.1016/j.jep.2010.03.031
7. Gautam P. Patel R. Artocarpus lakoocha Roxb: An overview. European Journal of Complementary and Alternative Medicine. 2014;1(1):10-14. doi.org/10.1016/j.nut.2010.05.008
8. Loung FS. Silalahi J. Suryanto D. Antibacterial activity of enzymatic hydrolyzed of virgin coconut oil and palm kernel oil against Staphylococcus aureus, Salmonella thypi and Escherichia coli. International Journal of PharmTech Research. 2014;6(2):628-633
9. Sinaga SM. et al. Phytochemical screening and antihyperglycemic activity of ethanolic extract of Coriandrum sativum L. leaf. Rasayan Journal of Chemistry. 2019; 12(4):1992-1996. doi.org/10.31788/RJC.2019.1245451
10. Haro G. et al. Evaluation of antioxidant activity and minerals value from watercress (Nasturtium officinale R.Br). Rasayan Journal of Chemistry. 2018; 11(1):232-237. doi.org/10.7324/RJC.2018.1112011
11. Togatorop B. et al. Effect of different polymer and oleic acid enchancer in Nifedipine matrix transdermal patch formulation and evaluation. Rasayan Journal of Chemistry. 2019; 11(2):516-521. doi.org/10.31788/RJC.2018.1122070
12. Tripathi S. et al. Formulation and characterization of Virgin Coconut Oil Emulsion (VCOE) for treatment of Alzheimers disease. Research Journal of Pharmaceutical Dosage Forms and Technology.2018; 10(2): 49-54. doi.org/10.5958/0975-4377.2018.00009.5.
13. Suryani S. et al. Preparation and evaluation of physical characteristics of Vitamin E nanoemulsion using virgin coconut Oil (VCO) and olive oil as oil phase with variation concentration of tween 80 surfactant Research Journal of Pharmaceutical Dosage Forms and Technology. 2020; 13(7): 3232-3236. doi.org/10.5958/0974-360X.2020.00572.7.
14. Hariyadi DM. et al. Virgin coconut oil Emulgel: Effect of VCO and carbopol 940 concentration on characterization and antibacterial activity. Research Journal of Pharmacy and Technology. 2022; 15(5):2087-2. doi.org/10.52711/0974-360X.2022.00345.
15. Vijayabhaskar K. et al. Wound healing activity of Bauhinia purpurea in albino Wistar rats. Asian Journal of Research in Pharmaceutical Sciences. 2011; 1(2): 47-49.
16. Sharma RK. et al. Assessment of wound healing activity of roots of Bauhinia variegata Linn. by excision and incision model in albino rats. Asian Journal of Research in Pharmaceutical Sciences. 2015; 5(3): 145-152.
17. Purohit SK. et al. Evaluation of wound healing activity of ethanolic extract of Azadirachta indica Leaves in male albino rats. Asian Journal of Research in Pharmaceutical Sciences. 2013; 3(2): 73-75.
18. Hayatullina Z. et al. Virgin coconut oil supplementation prevents bone loss in osteoporosis rat model. Evidence Based Complementary Alternative Medicine. 2012; 2012:237236. doi.org/10.1155/2012/237236
19. Ariffin NHM. Hasham R. Potential dermatological application on Asian plants. Biotechnology and Bioprocess Engineering. 2016; 21:337-354. doi.org/10.1007/s12257-015-0750-4
20. Velnar T, et al. The wound healing process: an overview of the cellular and molecular mechanisms. Journal of International Medical Research. 2009; 37(5):1528-1542. doi.org/10.1177/147323000903700531
21. Martin P. Nunan R. Cellular and molecular mechanisms of repair in acute and chronic wound healing. British Journal of Dermatology. 2015; 173(2):370-378. doi.org/10.1111/bjd.13954
22. Wilkinson HN. Hardman MJ. Wound healing: cellular mechanisms and pathological outcomes. Open Biology. 2020; 10(9):200223. doi.org/10.1098/rsob.200223
23. Umeh VN. Et al. Wound-healing activity of the aqueous leaf extract and fractions of Ficus exasperata (Moraceae) and its safety evaluation on albino rats. Journal of Traditional and Complementary Medicine. 2014; 4(4):246-252. doi.org/10.4103/2225-4110.139105
24. G/Giorgis SG. et al. Wound healing activity of 80% methanolic crude extract and solvent fractions of the Leaves of Justicia schimperiana (Hochst. ex Nees) T. Anderson (Acanthaceae) in mice. Journal of Experimental Pharmacology. 2022;14:167-183. doi.org/10.2147/JEP.S340177
25. Mapoung S. Skin wound-healing potential of polysaccharides from medicinal mushroom Auricularia auricula-judae (Bull.). Journal of Fungi (Basel). 2021; 7(4):247. doi.org/10.3390/jof7040247
26. Marchianti ACN. Gel formulations of Merremia mammosa (Lour.) accelerated wound healing of the wound in diabetic rats. Journal of Traditional and Complementary Medicine. 2019; 11(1):38-45. doi.org/10.1016/j.jtcme.2019.12.002
27. Amminbavi D and Lakshmi NP. Assessment of in vitro wound healing potential of Hibiscus leaf extract emulgel. Asian Journal of Pharmaceutical Research. 2020; 10(2):67-72. doi.org/10.5958/2231-5691.2020.00013.1.
28. Cruz MA. et al. Collagen from marine sources and skin wound healing in animal experimental studies: a systematic review. Marine Biotechnology. 2021; 23(1):1-11. doi.org/10.1007/s10126-020-10011-6.
29. Subha S. et al. Study of in vivo wound healing activity of Dyschoriste littoralis extract in rats models. Research Journal of Pharmacology and Pharmacodynamics .2017; 9(4): 181-185. doi.org/ 10.5958/2321-5836.2017.00031.3.
30. Majumdar M. et al. Study of wound healing activity of different formulations of Nigella sativa seed extract. Research Journal of Pharmacy and Technology. 2016; 9(12):2097-2105. doi.org/10.5958/0974-360X.2016.00427.3
31. Akilandeswari S. et al. Wound healing activity of Sida cordifolia in rats. Research Journal of Pharmacy and Technology. 2020; 13(12):6147-6149. doi.org/10.5958/0974-360X.2020.01072.0.
Received on 03.08.2022 Modified on 12.10.2022
Accepted on 18.12.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(8):3530-3534.
DOI: 10.52711/0974-360X.2023.00583