Wound Healing Potential of Rhodomyrtus tomentosa and its Bioactive Compounds- Rhodomyrtone

Aims: The present work was aimed to study the phytochemical composition of a crude ethanolic extract of Rhodomyrtus tomentosa [SERT], and the presence of rhodomyrtone and SERT's in vitro wound healing activity. Introduction: Rhodomyrtus tomentosa is native plant to southern and southeastern Asia, India, east to southern China, Taiwan, Philippines, and south to Malaysia. In the traditional Vietnamese, Chinese and Malaysia, all its part, including leaves, roots, buds, and fruits have been used. A need for a new source of wound healing agent is the call for the investigation of the potential of R. tomentosa as the source of health-promoting agent, specifically as a natural wound healing agent. Methodology: SERT was screen for its phytochemicals and the detection of rhodomyrtone using liquid chromatography–mass spectrometry, /Quadrupole time-of-flight [LC-MS/QTOF] analysis. Cell viability, cell proliferation, and migration assay were performed to examine the SERT effect's in Original Research Article Kamarudin et al.; JPRI, 33(40A): 262-273, 2021; Article no.JPRI.72390 263 vitro wound healing activity on human fibroblast cells [CRL-2522]. Results: The phytochemical study showed the presence of saponins, flavonoids, tannins and steroid in the crude ethanolic extract. The LC-MS analysis of crude ethanolic extract of SERT showed presents of rhodomyrtone which is one of the major compounds in the extract. SERT exhibit proliferative and migratory rate in human fibroblast cells [CRL 2522] in dose-dependent manner, which supports wound healing process. Its bioactive compounds presented wound healing activities at 0.325 up to 2.5 μg/mL. Conclusion: Both SERT and rhodomyrtone portrayed in vitro wound healing activities. Further studies to elucidate the mechanism of action of SERT and rhodomyrtone is recommended.


INTRODUCTION
Rhodomyrtus tomentosa (Aiton) Hassk.) is a shrub of flowering plant and belongs to the family Myrtaceae. It originating from South-East Asia and is distributed in many countries like China, Taiwan, Philippines, Thailand, Indonesia, Vietnam, and Malaysia [1]. R. tomentosa was differently known according to the country. In Malaysia, it is known as "kemunting" or "karamunting", "phruat" in Thailand, "harendong sabrang" in Indonesia and "downey rose myrtle" in most of the western countries [2]. Nearly 80% of the world's population relies on traditional medicines for primary health care [3]. This is due to their role as a source of pharmacologically active compounds [4]. Back then, medicinal plants were used by people of ancient cultures without the knowledge of their active ingredients and this consist of phytochemicals that responsible for medicinal activity of plants which have protected human from various diseases [5]. R. tomentosa has often been used in traditional medicine to treat colic diarrhoea, dysentery, abscesses, haemorrhage, and gynecopathy [6]. Similar traditional usage as Thailand, in Bangladesh and Vietnam, while it is sold as an herbal supplementary product as vitamins in America [7]. A review made by Hamid et al,. stated that the tender leaves has been traditionally used to treat colic, dysentery, abscesses, sepsis and tuberculosis [8].
Phytochemicals can be categorized as primary and secondary metabolites [9]. It is well known that phytochemical compounds were responsible in delivering the medicinal properties of the plant [10]. The major constituents of phytochemical consist of carbohydrates, amino acids, proteins and chlorophylls, while, secondary metabolites generally consist of alkaloids, saponins, steroids, flavonoids and tannins [11]. Among other bioactive compounds present in the R. tomentosa, rhodomyrtone is a potentially great drug that offered pharmacology benefits specifically intended to promote the development and expansion of this chemical compounds for the new lead drug development [12]. Rhodomyrtone has been reported to have antimicrobial and anti-tumor activities as one of the active ingredients in R. tomentosa [13]. As rhodomyrtone is a potential antimicrobial drug, normal human cell toxicity has been examined and methods for detecting food safety have been developed; these studies are being examined elsewhere [14]. A wound is a break in the skin, the first line of defense against infection. Wound healing is a complex and dynamic process, consisting of hemostasis, inflammation, proliferation, and maturation. Any disruption in the overlapping process resulting to failure for skin restoration. Plant with antioxidant, anti-microbial and antiinflammatory exhibit potential for wound healing agent [23] [24]. Most of the commercially available wound care products are generally expensive, which impose a significant financial burden on the patient and the healthcare system [25]. Therefore, there is a need for alternative and cost-effective therapies in wound management. In this study, the phytochemical properties of R. tomentosa were analyzed, detection of rhodomyrtone were performed using LC-MS MS/QTOF and in vitro wound healing potential of R. tomentosa crude leaves ethanolic extract and rhodomyrtone been assessed.

Collection and Identification of Plant Materials
Rhodomyrtus tomentosa leaves were collected from Bukit Batu Putih, Negeri Sembilan, Malaysia and authenticated at the Forest Research Institute Malaysia (FRIM)] with the voucher identification number of PID 050319-05. Leaves were washed with water and oven dried (40 C) for three days [26]. Dried leaves were ground in a mechanical blender into powder and stored at -20 C until use.

Preliminary Phytochemical Analysis
The sample was dissolved in various solvents and the preliminary phytochemical tests were carried out using Harborne [27].About 10 mg of extract mixed with 5mL of ammoniacalchloroform and 2.5mL of chloroform. After filtration, the supernatant was shaken with drops of 0.5M sulfuric acid. The appearance of a creamy precipitate indicated the presence of alkaloids. 10mg extract vigorously shaken with 1mL ethyl ether and 3mL 2N hydrochloride solution (HCl). Precipitate formation indicated the presence of saponins. 5 mg of extract was dissolved in 10 mL of 70 percent ethanol. The sample was then diluted 1: 2 (v/v) with sterile distilled water. Following that, three drops of a 10% (w/v) ferric chloride solution were added. The presence of tannins was indicated by the presence of a blue to black precipitate. About 5mg of the extract was dissolved in 5mL of absolute ethanol and treated with a few drops of concentrated HCl and 0.2 g of magnesium ribbon. The occurrence of a a pink-red color was indicative of the flavonoids. Steroids and terpenoids were detected using the Liebermann-Burchard reaction. A solution containing 5mg of extract dissolved in chloroform was filtered. The filtrate (2 mL) was added to 2mL of acetic anhydride and 50% concentrated sulfuric acid. A blue-green ring indicated the presence of steroids while a red color indicated the presence of terpenoids.

Standardize Compound of SERT
The detection of rhodomyrtone as was as described by Rofiee et al., [28] Rhodomyrtone in the SERT were detected using LC-MS-QTOF. About  Rhodomyrtone's chromatographic profiles were analysed using the precise mass data identified.

Migration Assay
The migration capabilities of HDF cells were assessed using scratch wound assay, which measures the expansion of a cell population on surfaces as described by Fronza et al. [31]. Allantoin was used as a positive control and help for wound healing, remover of necrotic tissue and promoter of epithelial stimulation and it has been used in pharmaceutical preparations for more than 70 years [32]. The rate of migration refers to speed of cell migration within certain time duration [33].

Statistical Analysis
The result was expressed as a mean ± SD, and the significance of results was analyzed using GraphPad Prism software [GraphPad Prism 8, USA]. Results obtained were compared with control and treated groups using One way ANOVA. Differences between the groups were considered as statistically significant at * <0.05, * * < 0.01, and * * * < 0.001 versus control group.

Phytochemicals Screening of SERT
The secondary metabolites screening showed the presence of saponins, flavonoids, tannins, phenolics and steroids in the crude ethanolic extract of Rhodomyrtus tomentosa [ Table 1]. However, terpenoids and alkaloids were not detected in the extracts. The phytochemical analysis is not in agreement with previous study that showed present of terpenoid but absent of flavonoid in the ethanol extract of Rhodomyrtus tomentosa [5]. This can be justified by different type of solvent extraction and method extraction. In previous study, present of saponin from ginseng able to accelerate neurovascularization [34], while flavanoids have been proven to enhance synthesis of collagen and increase rate of wound contraction [35] [36]. While tannins and phenolics contribute to action as antiinflammatory, antioxidant and been claimed responsible in enhancing wound healing [37].

Detection of Rhodomyrtone in SERT
The total ion chromatograms [TICs] of SERT and rhodomyrtone standard in positive ion mode are shown in Fig. 1. Rhodomyrtone is one of the major compounds identified with highest peak which belonged to acylphloroglucinols group.  [40]. Further research on rhodomyrtone proved the antibacterial properties and clinical management in Acne Vulgaris [41]. Recently, it was discovered that rhodomyrtone did not exhibit any toxicity in invertebrate or vertebrate models [42].

Cell viability of SERT and Rhodomyrtone
Cell viability assay was performed to determine non toxic concentration of the SERT to be used in the proliferation and migration assay. Based on Fig. 2, concentration that showed the highest percentage of cell viability within the range were selected for the migration assay were 15.62, 31.25,61.5 and 125 µg/mL. While for the rhodomyrtone standard from the cell viability results portray the best range were 0.32, 0.62, 1.25 and 2.5 µg/mL. In any study, utilization of medicinal plants require a basic screening to know the safe and non toxic dose before any experiment been carried out [43]. Based on above results, the non toxic dose for SERT was from 3.90 µg/mL up to 250 µg/mL, on the other hand, rhodomyrtone non toxic dose ranging from 0.325 µg/mL to 2.5 µg/mL. In another study, cell viability of T. ferdinandiana fruits and seedcoats extract of intestinal and hepatic cells range was higher than this study, which were between 3650 and 14400 μg/mL and its abundant compound ellagic acid varied from 1190 to 2390 μg/mL [44].The divergent findings could be explained by the cell line used, the extraction method used, or the plant part tested.

Cell Proliferation of SERT and Rhodomyrtone
Cell proliferation was performed utilising the Microculture Tetrazolium Test [MTT] assay, where the reduction of tetrazolium salts represents the number of viable cells. Based on Fig. 3 the graphical representation of cell proliferation after treatments with 62.5 µg/mL SERT in comparison with positive control [allantoin 30 µg/mL] at 24, 48 and 72hr. Rhodomyrtone standard showed similar pattern with SERT to increase cell proliferation after treatment. Only one representative for the best concentration that showed increased cell proliferation was chosen for both SERT and rhodomyrtone standard. Ability of cell to proliferate play a role whereby, it shows the bioactive compounds in the SERT and the specific compound responsible to accelerate the wound healing process. Obtained results were in contrast with finding by Chorachoo et al., [45] that showed anti proliferative effect at 2-32 µg/mL of rhodomytone. A possible explanation is that the cells used in their study were keratinocytes, whereas the cells used in this study were fibroblasts. In another study, human fibroblast cell proliferation significantly increased when treated with Scrophularia striata at 10, 50 and 100 μg/mL after 48h and with at 50, 100 and 200 μg/ml after 72h [46].

Migration Assay of SERT and Rhodomyrtone
Normally, the cells migrate towards empty spaces. Study by Chen claimed that the rate of migration is not significantly affected by the cell confluency [47].

CONCLUSION
Both the crude ethanolic extract of Rhodomyrtus tomentosa [SERT] and rhodomyrtone were found to be capable of promoting wound healing via stimulation of human dermal fibroblasts. The mechanism of action of SERT in wound healing is unclear and further studies are needed to elucidate the mechanism of action of SERT and rhodomyrtone in wound healing process.