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Abstract
Torbangun (Coleus amboinicus) leaves are a common medicinal plant in Indonesia, notably used by the Bataknese society as a lactagogue. Multiple studies have found that torbangun leaves are loaded with flavonoids and phenolic compounds, some of which have the potential to act as a photoprotective agent. This study aims to determine the ability of torbangun leaf extract in sunscreen cream to control the amount of melanin in the skin exposed to ultraviolet B (UVB) rays. This study is an experimental investigation employing a randomized post-test-only control group design. The subjects of this study were 28 male rats (Rattus norvegicus), which were divided into seven groups: the first two groups served as the negative and positive controls, while the other five groups were treated with torbangun leaf extract sunscreen cream (TLESC) at different concentrations. This study found that the application of TLESC at a concentration of 12.5% managed to control the average amount of melanin to 9.5%, whereas the base cream resulted in an average melanin content of 56.84%. There was a significant mean difference among all groups (p<0.05). It can be concluded that TLESC was as effective as standard sunscreen in controlling the amount of melanin in the subjects' skin.
Introduction
As a tropical country, Indonesia, which stretches along the equatorial line, has sun exposure all year round. Data from The National Aeronautics and Space Administration (NASA) Earth Observation (NEO) indicate that Indonesia has a potential for exposure to an ultraviolet (UV) index between 8 and 16 1 . The World Health Organization suggests that a UV index higher than 11 poses a high risk of causing UV damage and recommends the usage of sunscreen for a UV index higher than 2 due to the relatively high potential for solar erythema incidence after exposure 2 . One of the ultraviolet rays, ultraviolet B (UVB), plays a substantial role in vitamin D synthesis in humans. Unlike ultraviolet A from the sun, which reaches the earth's surface in its entirety, only 10% of UVB reaches the earth's surface, while the rest is reflected by the ozone layer. Despite this large proportion difference, UVB has an erythemogenic effect a thousand times more potent compared to UVA, which leads to sunburn and delayed tanning. This delayed tanning occurs due to UVB stimulating the melanocytes to synthesize melanin, hence increasing the amount of melanin in the skin 3 .
Aside from all that, Indonesia also has a large multitude of medicinal plants. One such plant is torbangun (Coleus amboinicus). Known in the West as Mexican mint or Indian borage, torbangun leaves are customarily used as a lactagogue in Bataknese society, while in Ayurveda, it is oftentimes used as a remedy for respiratory, digestion, or skin conditions 4 . These multitudes of uses are due to the presence of flavonoids, alkaloids, polyphenols, tannins, glycosides, and saponins in the torbangun leaves. Multiple studies have also shown that some flavonoids and phenolic compounds have photoprotective properties, whether through antioxidant, anti-inflammatory, or other properties 5 , 6 .
The fact that torbangun leaves contain some flavonoids and phenolic compounds that have photoprotective properties opens the possibilities of formulating torbangun leaf extract into sunscreen form.
Methods
This study is an experimental study employing a randomized post-test-only control group design. The extraction process of Torbangun leaves and the formulation of the sunscreen cream were conducted in the Biomolecular Laboratory of Prima Indonesia University, while the experiments were conducted in the Pharmacology and Toxicology Laboratory of the Faculty of Pharmacy at the University of North Sumatra. This study was conducted from August to September 2022.
Materials
The specimen used in this study was the leaves of the Torbangun plant, procured from a local farmer in North Sumatra, and identified by Herbarium Medanense at the University of North Sumatra. The Herbarium Medanense of the Faculty of Mathematics and Natural Science at the University of North Sumatra confirmed the specimen as Torbangun (Coleus amboinicus) leaves.
Animal subject
The experimental subjects in this study were rats ( Rattus norvegicus ) of the Wistar strain. The rats included in this study were male, aged between 3 to 4 months old, weighed 150-200 grams, and were healthy. The sample size for this study was calculated using the Federer formula: (n-1)(t-1)≥15, where n is the sample size and t is the number of interventions conducted. Hence, the number of samples needed for this study was 28.
The subjects were then grouped into 7 groups, each consisting of 4 subjects. These seven groups represent the 7 different interventions that the subjects received during the experiment but all received the same induction/stimulus. The first group was the negative control, while the second group was the positive control. The remaining five groups received Torbangun leaves extract sunscreen cream (TLESC) with different extract concentrations (2.5%, 5%, 7.5%, 10%, and 12.5%).
Extraction of torbangun leaves extract
The Torbangun leaves extraction process began by adding 150 grams of powdered Torbangun leaves to a container followed by 1.5 liters of 96% ethanol. This mixture was mixed continuously for 6 hours and stored in a dark chamber for 18 hours. After 18 hours, the mixture was passed through a filter, producing the first filtrate and residue. The residue was then reused for the second maceration, adding only 0.75 liters of 96% ethanol, and producing the second filtrate. Both filtrates were then mixed, and the ethanol was removed by evaporation using a rotary evaporator at a temperature of 40ÂşC, resulting in a thick and high concentration of Torbangun leaves extract.
Phytochemical Analysis of Torbangun Leaves Extract
Phytochemical analysis was conducted in this study to determine the phytochemical contents of torbangun leaves. The analysis was conducted both qualitatively (screening) and quantitatively.
Phytochemical screening was performed to ascertain the presence of alkaloids, steroids, terpenoids, saponins, flavonoids, tannins, and glycosides in the torbangun leaves. The quantitative analysis of torbangun leaves was performed to determine the total flavonoid content and total phenolic contents.
Alkaloid Screening
Alkaloid screening in this study was carried out using four different methods/reagents: Bouchardat’s, Mayer’s, Dragendorff’s, and Wagner’s. This began by mixing 1 ml of HCl with 9 ml of aquadest and adding 500 mg of torbangun leaf extract into it. This mixture was then heated for 2 minutes in a water bath and filtered afterward to produce the filtrate. The filtrate was then divided into 4 separate test tubes, labeled A, B, C, and D. Test tube A was for Bouchardat’s, B for Mayer’s, C for Dragendorff’s, and D for Wagner’s. Into each test tube, 2 drops of its respective reagent were added. The presence of alkaloids was indicated by a yellow-orange precipitate in test tube A, a white to yellowish-white precipitate in test tube B, a reddish-blackish precipitate in test tube C, or a reddish-brown precipitate in test tube D.
Flavonoid Screening7
Flavonoid screening in this study was carried out using four different methods/reagents: 5% FeCl 3 , Mg(s) + HCl(p), 10% NaOH, and H 2 SO 4 (p). Into four different test tubes, 2 mL of the extract was added and labeled A, B, C, and D. Into tube A, 2 drops of 5% FeCl 3 were added, while into tube B, a few magnesium turnings and a few drops of concentrated HCl were added. Meanwhile, into tube C, 2 mL of NaOH was added, followed by HCl. And into tube D, a few drops of concentrated H 2 SO 4 were added. For tube A, the presence of flavonoids was signified by a blackish-red coloration; for tube B, it was signified by a red coloration; for tube C, after NaOH was added, an intense yellow coloration appeared, and the presence of flavonoids was signified by the dilution of the color by adding acid; and for tube D, it was signified by an orange coloration.
Steroid Screening7
Into 2 mL of torbangun extract, 2 mL of chloroform, and 2 mL of H 2 SO 4 were added. The solution was shaken well. This resulted in the formation of a red chloroform layer and greenish-yellow fluorescence in the acid layer, indicating the presence of steroid compounds.
Triterpenoid7
Into 1 mL of torbangun extract, ethanol, acetic anhydride, and a few drops of H2SO4 were added. The formation of a pink to violet color indicates the presence of triterpenoids.
Saponin7
To the torbangun extract, 10 mL of distilled water was added and shaken vigorously to create a stable and persistent froth. If the froth persists even after adding HCl, then saponins are present.
Tannin7
Into the torbangun extract, a few drops of 1% FeCl 3 were added. The presence of brownish-green or blue-black coloration signifies the presence of tannins.
Glycoside7
Into a test tube, a small amount of the torbangun extract was added, followed by 2 drops of Molisch’s reagent and mixed well. A few drops of H 2 SO 4 were then cautiously added along the walls of the test tube to prevent mixing. The formation of a purple ring indicates the presence of glycosides.
Total Flavonoids Content7
500 µL of torbangun extract was added into a test tube, followed by 100 µL of 10% AlCl 3 , 100 µL of potassium acetate (CH 3 COOK), and 4.3 mL of distilled water; the mixture was then vortexed and stored at room temperature for 30 minutes. The absorbance was measured at the wavelength of 415 nm. The total flavonoid content was determined according to the standard quercetin curve and measured as milligrams of quercetin equivalent per gram (mgQE/g).
Total Phenolic Content7
200 µL of torbangun extract was added into a test tube followed by 1 mL of 10% Folin-Ciocalteu reagent. It was allowed to sit for one minute before adding 3 mL of Na 2 CO 3 . The mixture was vortexed and stored in a dark room at room temperature for two hours. The absorbance was measured at the wavelength of 760 nm. The total phenolic content was determined according to the standard gallic acid curve and measured as milligrams of gallic acid equivalent per gram (mgGAE/g).
All the phytochemical analyses were performed by an independent third party, the Organic Chemistry Laboratory of the Faculty of Mathematics and Natural Sciences at North Sumatera University 7 .
Preparation of Torbangun Leaves Extract Sunscreen Cream
The preparation of the torbangun leaves extract sunscreen cream began with separately preparing the oil and water phases. The oil phase was prepared by mixing cetyl alcohol, stearic acid, and propylparaben in a porcelain crucible on top of a water bath until completely liquefied (maintaining a stable temperature of 70-75°C). The water phase included methylparaben, half part glycerin, triethanolamine (TEA), and distilled water (aquadest), also heated in a crucible on a water bath until they were completely liquefied (maintaining a stable temperature of 70-75°C). The other half of the glycerin was used to dissolve the torbangun leaves extract. The oil and water phases were then combined in a mortar and homogenized by grinding with a pestle until a cream mass was formed. Finally, the torbangun leaves extract was added and homogenized using the same technique, resulting in the torbangun leaves extract sunscreen cream (TLESC) 7 . The cream formulation is present in Table 1 .
Before the TLESC was used in the experiment, some physical evaluations of the cream were performed. These evaluations consisted of the organoleptic test, pH test, and spreadability, which were performed before the storage cycle test and after each cycle. The cycle consisted of storing the cream for 24 hours at a low temperature (4°C), followed by 24 hours of storage at a high temperature (40°C); this process was repeated 3 times. The organoleptic test, which uses the human senses, consists of assessing color (visual), smell (olfactory), consistency, and form (tactile).
Evaluation of Torbangun Leaves Extract Sunscreen Cream's Effect on The Amount of Melanin
The evaluation of the TLESC effect on the amount of melanin in the experiment subjects' skin was conducted after the intervention and induction phase (applying control creams/TLESC and exposing the experiment subjects to UVB) was completed.
The intervention and induction phases began by acclimatizing all experiment subjects. After that, a patch with a size of 3x3 cm was created on the back of each experiment subject by shaving it. Into that patch, interventions were carried out. In the first group, a base cream was applied, while in the second group, Vaseline® Healthy White was applied. In the other five groups, TLESC with different concentrations was applied. After the application of the interventions, all experiment subjects were exposed to UVB light for 6 hours per day for 4 weeks.
After the intervention and induction phases were completed, the experiment subjects were left without any intervention for 24 hours after the last exposure to UVB light to remove any acute UVB exposure effects. After 24 hours, a biopsy (5 mm in diameter) was taken from each experiment subject. The biopsy was then fixed by soaking it in formaldehyde phosphate for 24 hours. After that, trimming was performed to remove unnecessary parts. The tissue was then dehydrated by serially soaking it in alcohol with concentrations of 50%, 70%, 90%, 96%, and 100% for two hours at each concentration. The dehydrated tissue was then cleared by soaking it in a clearing agent (xylene) for 24 hours, which produced a transparent tissue. The transparent tissue was infiltrated twice with 60°C pure liquid paraffin for one hour each time, then embedded in liquid paraffin for 24 hours to create a paraffin block with the tissue embedded in it. The paraffin block was then serially cut using a microtome with a 3 µm thickness, and the best cut was placed on top of an object glass and incubated for 2 hours at 60°C.
The tissue slides were then stained using the Montana-Masson method to show the amount of melanin in the skin tissue of the experiment subjects. The slide was then placed under 400 times objective magnification on a microscope and photographed. The number of pixels occupied by the melanin was then counted and used to calculate the amount of melanin by the formula 8 :
Amount of Melanin = (Pixels occupied by Melanin) / (Pixels occupied by Epidermis) Ă— 100%
Statistical Analysis
Data analysis in this study was performed using SPSS for Windows software. ANOVA with the Bonferroni post-hoc test was employed because the data of this study qualified for analysis using ANOVA (data distribution was normal and homogenous).
RESULT
Phytochemical Analysis
The phytochemical analysis of torbangun leaves, performed by the Organic Chemistry Laboratory of the Faculty of Mathematics and Natural Sciences at North Sumatra University, found that torbangun leaves contain compounds such as alkaloids, steroids, triterpenoids, saponins, flavonoids, and tannins ( Table 2 ), with total alkaloid content of 32.36 mgQE/gram (milligram quercetin equivalent per gram) and total phenolic content of 88.98 mgGAE/gram (milligram gallic acid equivalent per gram).
Physical Evaluation of Sunscreen Cream Formulation
The physical evaluation of sunscreen cream formulations found that all formulations were stable, even after undergoing a cycle test. The organoleptic test demonstrated that all formulations retained their color, smell, form, and homogeneity after each cycle. The pH of all cream formulations, before the cycle test, was within the safe range for skin usage, and after each cycle, the pH decreased but remained within the safe range for skin usage. While remaining stable, the spreadability of the sunscreen cream formulation increased after the cycling test.
Evaluation of TLESC Effect on the Amount of Melanin
The evaluation of the effect of TLESC on the amount of melanin in the experiment subject's skin found that the first experimental group subject had the highest amount of melanin, 56.84%, while the lowest amount of melanin was found in the second group, which received Vaseline® Healthy White, with 8.76% melanin. Meanwhile, TLESC has shown differing performance, where the amount of melanin decreased with an increase in TLESC concentration. The 2.5% TLESC average amount of melanin was 39.59%, while the highest concentration, TLESC 12.5%, had an average amount of melanin of only 9.5% ( Table 3 ).
Data normality and homogeneity of variance tests performed on the study's data found that the data were normally distributed (p>0.05) and homogeneous (p>0.05), hence qualifying for ANOVA.
ANOVA carried out on the study’s data found that there was a significant mean difference in the amount of melanin between all the groups. Further post-hoc (Bonferroni) tests found that there was a significant mean difference between the negative control group and the rest of the groups (p<0.05). A similar significant mean difference was also found in the TLESC 2.5%, 5%, and 7.5% groups compared to other groups. However, the TLESC 10% did not show a significant mean difference compared to the TLESC 12.5% (p>0.05), while showing a significant mean difference compared to the other groups (p<0.05). The TLESC 12.5% was the only formulation to show no significant difference compared to the positive control and TLESC 10% (p>0.05), while showing a significant difference compared to the other groups (p<0.05).
Discussion
Phytochemical screening of torbangun leaves in this study found that the leaves contain alkaloids, steroids, triterpenoids, saponins, flavonoids, and tannins ( Table 2 ). This result is in accordance with multiple studies of the phytochemical profiles of torbangun leaves 9 , 10 , 11 , 12 . The study by Matita et al. showed that torbangun leaves contained flavonoids, phenols, and tannins, while Damanik et al. found that alkaloids, triterpenoids, saponins, tannins, and flavonoid compounds were present in the ethanol extract of torbangun leaves 9 , 10 . Meanwhile, the studies by Laila et al . and Gomes et al . focused on the phenolic and flavonoid content of torbangun leaves 11 , 13 . Different extraction and detection methods performed in these studies contribute to the varying results in the phytochemical profile, both qualitatively and quantitatively. Ethanol extraction, which underwent a fractionation process, can produce three different extract fractions: hexane, ethyl acetate, and the aqueous fraction. For instance, in ethanol extracts, qualitative tests detected the presence of alkaloids, flavonoids, triterpenoids, saponins, and tannin compounds 9 . However, in the hexane fraction, tannins and flavonoids were not detected, while in the ethyl acetate fraction, saponins and tannins were not detected 9 . Aside from different extraction methods, different detection techniques among the studies also affected the results. For example, the Laila et al. study determined the phytochemical content of torbangun leaves using Gas Chromatography-Mass Spectrometry (GC-MS), and the Gomes et al. study used High-Performance Liquid Chromatography (HPLC) 11 , 13 . This study, however, was carried out using the maceration technique with ethanol as the solvent, and phytochemical detection was conducted both qualitatively and quantitatively.
Several studies have reported comprehensive phytochemical profiles of torbangun leaves 14 , 15 , 16 . Those studies support the findings in this study that the ethanol extract of torbangun leaves contains multiple compounds, such as alkaloids, steroids, triterpenoids, saponins, flavonoids, and tannins 14 , 15 , 16 . In this study, flavonoid detection was carried out using four different methods, but only one managed to detect the presence of flavonoids. However, this result can be accepted as confirming the presence of flavonoid compounds in torbangun leaves. This conclusion was supported by the fact that quantitative analysis of torbangun leaves found a total flavonoid content of 32.36 mgQE/gram. This total flavonoid content was slightly lower than that found by Taher et al., which was 41.4 mgQE/gram, and much lower compared to the findings of Ślusarczyk et al., which were 96.83 ± 1.4 mgQE/gram 14 , 17 .
Apart from total flavonoid content, this study also quantified the total phenolic content of torbangun leaves. The study found that the total phenolic content of torbangun leaves was 88.89 mgGAE/gram. The Ślusarczyk et al. study found that phenolic compounds were the major phytochemical content in torbangun leaves with total phenolic contents of 112.95 ± 0.88 mgGAE/gram 14 . A review article by Rahmawati et al. found that generally, the total phenolic content of torbangun leaves was more than 40 mgGAE/gram, with the highest concentration reaching 313 mgGAE/gram, which was obtained from the hot water extract of torbangun leaves 15 .
The differences in total flavonoid and total phenolic content can be attributed to different extraction techniques, as well as the geographic and climatic conditions where the torbangun plant grows. A study by Gomes et al . found that the total flavonoid and total phenolic content of torbangun varied every month of the year, with the lowest total flavonoid content of 19.34 mgQE/gram in February and the highest total flavonoid content of 49.82 mgQE/gram in December, and the lowest total phenolic content of 79.41 mgGAE/gram in February and the highest total phenolic content of 164.7 mgGAE/gram in July 12 . A study by Ĺšlusarczyk et al. that compared the phytochemical profile of torbangun cultivated in Indonesia and Poland found that torbangun leaves cultivated in Indonesia had lower total flavonoid and total phenolic content compared to those cultivated in Poland 14 . The Ĺšlusarczyk et al . study found that torbangun leaves cultivated in Indonesia had a total flavonoid content of 13.47 mgQE/gram and a total phenolic content of 23.61 mgGAE/gram, while the torbangun leaves cultivated in Poland had a total flavonoid content of 96.83 mgQE/gram and a total phenolic content of 112.95 mgGAE/gram 14 .
This study aims to understand the potency of torbangun leaves extract formulated into cream form in controlling the amount of melanin in the skin of experiment subjects exposed to UVB light. This study found that, even though at lower concentrations (2.5% ~ 10%), TLESC performed better than the base cream in controlling the amount of melanin in the subjects' skin, 12.5% TLESC was the only concentration that performed as well as Vaseline® Healthy White, the positive control ( Table 3 ). This finding showed that TLESC has a photoprotective effect, with a better effect at higher concentrations. Rosmarinic acid and caffeic acid are likely responsible for this photoprotective effect 13 .
Rosmarinic acid, aside from having photoprotective properties, also has anti-radical free properties and is effective as a lipid peroxide antagonist 18 . The study by Sanchez-Campillo et al. found that oral administration of rosmarinic acid showed photoprotective effects in experiment subjects exposed to UVA light for 120 minutes per session with 100 total sessions 19 . Seventy percent of the subjects given rosmarinic acid did not have even slight dysplasia on the skin and the rest 30% only had slight dysplasia, while 80% of the subjects not given rosmarinic acid had moderate skin dysplasia, and the rest had severe skin dysplasia 19 . The same study surmised that this photoprotective effect of rosmarinic acid was due to its antioxidant and anti-inflammatory properties 19 . Another study by Cândido et al . found that rosmarinic acid had a cytoprotective effect against UVB radiation-induced oxidative stress 18 . The mechanism behind these properties is yet to be explained, though the inhibition of reactive oxygen species (ROS) induced by UVB radiation and inflammation marker, along with intracellular signaling pathways inhibition 20 . Another explanation of this melanin inhibition was proposed by Oliveira et al., which hypothesized that rosmarinic acid, at higher concentrations, acts as a competitive inhibitor of the tyrosinase binding site, leading to the formation of o-quinone derivatives and blocking the enzymatic oxidation of L-DOPA to o-dopaquinone, while its antioxidant action reduces o-dopaquinone, limiting the synthesis of dopachrome, hence melanin 21 . Multiple studies also showed that rosmarinic acid was the most prevalent phenolic compound in torbangun leaves, followed by caffeic acid 12 , 14 , 16 , 22 .
Caffeic acid, a phenolic compound, was the second-highest phenolic compound in torbangun leaves after rosmarinic acid. Like rosmarinic acid, caffeic acid also has photoprotective properties, especially when combined with ferulic acid, which is also present in torbangun leaves 14 , 15 , 23 , 24 . Caffeic acid can protect the skin from UVB light, and ferulic acid is a strong UVB absorber, which is why the combination of the two has a significant photoprotective effect 23 . An in-vitro experimental study by Maruyama et al. found that both ferulic and caffeic acids decreased melanogenesis in a melanoma cell line 25 . The same study also found that ferulic acid and caffeic acid inhibit the conversion of tyrosine to DOPA, and DOPA to dopaquinone, which ultimately inhibits melanogenesis 25 . Ferulic acid also inhibits melanogenesis by directly binding itself to tyrosinase, while direct tyrosinase binding was not observed with caffeic acid 25 .
The study by Terto et al. found that torbangun leaves have a sun protective factor (SPF) of 12.6 13 . The same study also concluded that torbangun leaves extract has high potency as a material for sunscreen to protect skin from damage by UVA and UVB exposure 13 . The findings of Terto et al. support this study's result that TLESC, especially at higher concentrations, can control the amount of melanin in the experiment subjects' skin exposed to UVB light. In the Bonferroni post-hoc test, 12.5% TLESC showed no significant mean difference with Vaseline® Healthy White, the positive control (p>0.05). This means that 12.5% TLESC performed as well as Vaseline® Healthy White in controlling the amount of melanin in the subjects' skin.
Conclusions
This experimental study was carried out to understand the efficacy of torbangun leaf extracts as a sunscreen cream in controlling the amount of melanin in the skin. The study concluded that torbangun leaf extract sunscreen was effective at preventing the increase of melanin in the skin due to UV exposure, especially at a high concentration (12.5%). The study also found that torbangun leaves contain alkaloids, steroids, triterpenoids, saponins, flavonoids, and tannins, with total flavonoid contents of 32.36 mgQE/gram and total phenolic content of 88.89 mgGAE/gram. The findings in this study, supported by several previous studies, also suggest that torbangun leaves have photoprotective properties, which make them a good candidate for sunscreen material. This is particularly true at higher concentrations, attributed to its high flavonoid and phenolic contents.
Abbreviations
ANOVA - Analysis of Variance 3 - Ferric ChlorideGC-MS - Gas Chromatography-Mass SpectrometryH 2 SO 4 - Sulfuric AcidHCl - Hydrochloric AcidHPLC - High-Performance Liquid ChromatographymgGAE/g - Milligrams of Gallic Acid Equivalent per GrammgQE/g - Milligrams of Quercetin Equivalent per GramNaOH - Sodium HydroxideNASA - The National Aeronautics and Space AdministrationNEO - Earth ObservationROS - Reactive Oxygen SpeciesSPF - Sun Protection FactorSPSS - Statistical Package for the Social SciencesTEA - TriethanolamineTLESC - Torbangun Leaves Extract Sunscreen CreamUV - UltravioletUVA - Ultraviolet AUVB - Ultraviolet B
Acknowledgments
None.
Author’s contributions
All authors significantly contributed to this work, read and approved the final manuscript.
Funding
None.
Availability of data and materials
Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
This study and its protocols have been approved by the Ethics Committee of Health Study of Prima Indonesia University as stated in letter No. 056/KEPK/UNPRI/IV/2022 and declared in accordance to seven WHO 2011 Standards.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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