Exploring the Cosmeceutical Potential of Six Malaysian Indigenous Tropical Fruit Trees: Radical Scavenging, Anti‐Tyrosinase, and Photoprotective Activities Evaluation

ABSTRACT

Malaysian indigenous tropical fruit trees have been reported to possess various biological activities, but remain largely underutilized. This study aims to investigate the skin‐lightening cosmeceutical potential of six Malaysian indigenous tropical fruit trees, namely Averrhoa bilimbi, Baccaurea motleyana, Bouea macrophylla, Cynometra cauliflora, Garcinia celebica, and Syzygium malaccense. Herein, crude ethanol and its fractionated extracts (hexane, chloroform, and ethyl acetate) from the leaves and twigs were evaluated for total phenolic content (TPC), radical scavenging, tyrosinase inhibitory activity, and sun protection factor (SPF) values. The results revealed that B. macrophylla leaf ethyl acetate fractionated extract exhibited the highest TPC (577.47 ± 16.15 mg GAE/g extract), very strong radical scavenging activity (EC₅₀ < 50 µg/mL) across DPPH•, ABTS^•+, and superoxide anion (O₂ ^•−) assays, as well as the highest SPF value (28.64 ± 0.51). Liquid chromatography‐mass spectrometry (LC‐MS) analysis identified the presence of m‐trigallic acid (1), cis‐coutaric acid (2), and ellagic acid (3), which may contribute to its noticeable bioactivities. Meanwhile, B. macrophylla twig ethanol extract exhibited the strongest mushroom tyrosinase inhibitory activity (IC₅₀ 55.51 ± 1.99 µg/mL). Collectively, these findings suggest that the twig ethanol extract and leaf ethyl acetate fractionated extract of B. macrophylla are promising natural ingredients for potential skin‐lightening cosmeceutical applications.

B. macrophylla leaf ethyl acetate fractionated extract exhibits the highest TPC, very strong radical‐scavenging activities, and the highest SPF value of 28.64 ± 0.51. LC‐MS analysis identifies m‐trigallic acid, cis‐coutaric acid, and ellagic acid, which may contribute to its bioactivities. The present work highlights B. macrophylla as a promising natural ingredient for skin‐lightening cosmeceutical applications.

1. Introduction

The skin, as the largest organ of the human body, comprises approximately 16% of body mass and exhibits a wide range of tone, from darker brown to lighter white. The variation in skin tone is primarily due to melanin, a dark‐colored pigment responsible for the coloration of the skin, eyes, and hair. Additionally, melanin acts as a protective barrier, shielding the skin from harmful ultraviolet (UV) radiation. The process of melanin production is known as melanogenesis, which takes place in the melanosome within melanocytes located in the basal layer of the epidermis. Melanogenesis is a complex process in the skin epidermis, as it can be influenced by both intrinsic (e.g., hormones and inflammation) and extrinsic (e.g., UV radiation and chemical exposure) factors at different stages. For example, prolonged exposure to UV radiation leads to the overproduction of reactive oxygen species (ROS), causing an imbalance between ROS levels and antioxidants, which consequently results in oxidative stress that stimulates melanogenesis [1]. An exacerbated melanogenesis could lead to dermatological conditions associated with hyperpigmentation, such as melasma, post‐inflammatory pigmentation, and senile lentigo, resulting in darker regions on the skin.

Although cosmetic products, such as foundations and concealers, can temporarily mask skin hyperpigmentation, they do not address the underlying cause. Current skin‐lightening agents, particularly synthetic compounds (e.g., hydroquinone and kojic acid), have been widely incorporated into cosmetic formulations due to their anti‐melanogenic properties. Hydroquinone has long been regarded as the gold standard for skin‐lightening; however, prolonged use or high concentrations may cause epidermal disruption, reduced skin thickness, and skin irritation [2]. Similarly, kojic acid, as a well‐known tyrosinase inhibitor to prevent melanogenesis, exhibits limited stability during storage, which may restrict its application in cosmetic formulations [3]. To address these concerns, natural‐based cosmeceutical products with skin‐lightening effects from bioactive components have been developed to conceal and reduce pigments in a relatively safe approach. The bioactive components could be phytochemicals, vitamins, fermented substances (e.g., hyaluronic acid), and animal‐derived biomolecules (e.g., collagen) [4, 5]. These components generally possess good bioavailability and can penetrate the skin through intercellular, transcellular, and appendageal pathways, thereby addressing skin issues at the cellular level. In the cells, the bioactive components could inhibit melanogenesis via several mechanisms, for example, downregulation of the expression of melanogenesis‐related genes, inhibition of tyrosinase activity, neutralization of ROS, protection against UV, etc. [6, 7]. The therapeutic benefits of skin‐lightening cosmeceuticals have contributed to the rapid expansion of the market, which is projected to reach $13.3 billion by 2030 [8]. Although plant‐derived bioactive compounds are generally perceived as safer and healthier alternatives, their phytochemical composition and biological activity may vary depending on the plant source and extraction method. Hence, further research is needed to identify the plant‐derived bioactive compounds with potent skin‐lightening activity, improved safety, and consistent efficacy for cosmeceutical applications.

Malaysia is a tropical country located near the equator, where the climate is characterized by abundant sunshine and high humidity. In light of this, the Malaysian rainforests are believed to be the oldest and among the most biologically diverse forests in the world. The indigenous trees have been a primary source of traditional medicine throughout human history. According to Food and Agriculture Organisation of the United Nations [9], indigenous trees refer to the native tree species that have evolved in the same area, region, or biotope, where the forest stand is growing, and are adapted to the specific ecological conditions predominant at the time of the establishment of the stand. The literature search (Table 1) reveals that the indigenous tropical fruit trees in Malaysia exhibit various pharmacological and biological properties, including antioxidant, anti‐tyrosinase, anti‐obesity, anticancer, and anti‐fungal activities. Leaves are continuously exposed to environmental stresses; particularly intense sunlight under tropical conditions in Malaysia, which may contribute to the accumulation of secondary metabolites, such as phenolic associated with antioxidant and photoprotective properties. Similarly, twigs may accumulate various defensive phytochemicals in response to environmental and biotic stresses, making them promising sources of bioactive compounds. Therefore, the leaves and twigs were selected to investigate their potential skin‐protective and skin‐lightening activities. Despite numerous biological activities being reported, studies investigating the potential skin‐lightening applications of these trees remain limited. Therefore, the leaves and twigs were selected as the plant materials for further investigation of their potential skin‐lightening activities.

TABLE 1.

Reported biological activities of selected indigenous tropical fruit trees (years 2009‐2024).

Species (Local name)	Part	Bioactive extract/ Component	Biological activity	Reference
A. bilimbi L. (Belimbing buluh)	Leaf	Ethanol extract	Antioxidant activity determined by ABTS•+ assay (EC50 = 51.91 ± 3.76 mg/mL)	Th and Silprasit [10]
			Mushroom tyrosinase inhibitory activity (EC50 = 8.66 ± 1.61 mg/mL) via uncompetitive inhibition
B. motleyana (Müll.Arg.) Müll.Arg. (Rambai)	Fruit	Methanol extract	Antioxidant activity determined by β‐carotene/linoleic acid system (71.17 ± 5.63%)	Ikram, et al. [11]
	Leaf	Ethanol extract	Antioxidant activity determined by DPPH• (IC50 = 41.36 ± 1.79 µg/mL) and ABTS•+ assays (IC50 = 34.07 ± 2.51 µg/mL)	Fitri, et al. [12]
			Anti‐proliferative activity against pancreatic cancer (PANC‐1) cells via 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) assay (IC50 at 72 h = 20.75 ± 2.13 µg/mL)
			Immunohistochemistry demonstrated an increase in p53 protein expression and downregulates Bcl‐2 protein expression.
B. macrophylla Griff. (Kundang)	Peel	Hydro‐ethanol extract contains gallic acid and ellagic acid	Mushroom tyrosinase inhibitory activity (IC50 = 1.66 ± 0.84 mg/mL)	Maneechai, et al. [13]
			Antioxidant activity determined by DPPH• (IC50 = 56.55 ± 3.63 µg/mL), ABTS•+ (TEAC 0.19 ± 0.01 mg Trolox/mg extract), linoleic acid peroxidation (IC50 = 0.12 ± 0.05 mg/mL), and ferric reducing antioxidant power (FRAP) assays (EC1 = 0.019 ± 0.001 mg/mL)
			Anti‐inflammatory effect in nitric oxide inhibition assay using mouse monocyte macrophage (RAW 264.7) cells (% inhibitory of nitric oxide = 24.76 ± 3.73%, at 0.5 mg/mL)
			Non‐cytotoxic activity in sulforhodamine B (SRB) assay using human fibroblast cells (cell viability > 90%, at concentration ranging from 0.0001 to 1.0000 mg/mL)
	Twig	Ethyl acetate extract	Antioxidant activity determined by DPPH• assay (IC50 = 4.89 µg/mL)	Rudiana, et al. [14]
C. cauliflora L. (Nam‐nam)	Leaf	Ethyl acetate extract	Antioxidant activity determined by DPPH• assay (IC50 = 0.049 ± 0.25 mg/mL)	Ado, et al. [15]
			Enzyme inhibition of tyrosinase (IC50 = 0.034 ± 0.011 mg/mL), 𝛼‐glucosidase (IC50 = 0.030 ± 0.004 mg/mL), and acetylcholinesterase (IC50 = 0.031 ± 0.012 mg/mL)
	Twig	Essential oil	Antioxidant activity determined by DPPH• assay (IC50 = 37.12 ± 2.84 µg/mL)	Samling, et al. [16]
			Antimicrobial properties using broth microdilution assay (MIC = 120.0 µg/mL) against Bacillus cereus, Staphylococcus aureus, Methicillin‐resistant Staphylococcus aureus (MRSA), Proteus mirabilis, Escherichia coli and Candida albicans
G. celebica L. (Manggis hutan)	Leaf	Chloroform extract	Antioxidant activity determined by DPPH• assay (IC50 = 386.05 ± 19.22 µg/mL)	Mustafa, et al. [17]
			Alzheimer's disease inhibition in the acetylcholinesterase inhibiting (IC50 = 13.70 ± 1.56 µg/mL), and butyrylcholinesterase inhibiting assays (IC50 = 32.20 ± 0.36 µg/mL)
		Ethyl acetate extract	Antioxidant activity determined by FRAP assay (EC50 = 2.969 µg/mL)
	Bark	Ethanol extract contains flavonoid, triterpenoid, alkaloid, and saponin	Antioxidant activity determined by DPPH• (IC50 = 10.49 µg/mL) and ABTS•+ assays (IC50 = 13.92 µg/mL)	Ismail, et al. [18]
S. malaccense L. (Jambu bol)	Peel	Methanol extract contains high anthocyanins	Antioxidant activity determined by DPPH• (47.22 µMol Trolox equivalent antioxidant capacity/g dry weight), and FRAP assays (0.19 ± 0.01 mM ferreous sulfate/g dry weight)	Nunes, et al. [19]
	Leaf	Methanol extract	Antimicrobial effects (MIC ≤ 0.3 mg/mL) against gram‐positive bacteria namely Candida albicans, Staphylococcus epidermidis, and Streptococcus pyogenes as well as gram‐negative bacteria, Pseudomonas aeruginosa	Quenon, et al. [20]
		Ethyl acetate extract	Antioxidant activity determined by DPPH• (IC50 = 34.35 µg/mL) and hydrogen peroxide assays (IC50 = 12.03 µg/mL)	Itam and Anna [21]

This study was initiated to investigate the skin‐lightening potential of the leaves and twigs from six selected indigenous tropical fruit trees, that is, Averrhoa bilimbi, Baccaurea motleyana, Bouea macrophylla, Cynometra cauliflora, Garcinia celebica, and Syzygium malaccense. In this endeavour, the leaves and twigs of these plants were extracted using ethanol, followed by extract fractionation by hexane, chloroform, and ethyl acetate. Phytochemical analysis of the extracts was conducted by determining the total phenolic content (TPC) using the Folin‐Ciocalteu colorimetric method. Since the excess ROS production indirectly contributes to melanogenesis, the radical scavenging activities of extracts were evaluated using three different assays, namely 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH•), 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS^•+), and superoxide anion (O₂ ^•−) radical scavenging assays. Additionally, the inhibitory effects of the extracts on tyrosinase (the rate‐limiting enzyme in melanogenesis) were determined through a mushroom tyrosinase inhibition assay. Subsequently, the photoprotective effect of the extracts was evaluated by calculating their SPF values from the UV absorption spectra. Spearman's correlation analyses were conducted to examine the relationships among the results of the different assays. Finally, the remarkable bioactive extract was selected and subjected to liquid chromatography‐mass spectrometry (LC‐MS) to identify the chemical constituents that are responsible for the observed activities. To the best of our knowledge, the anti‐tyrosinase activity of B. motleyana, G. celebica, and S. malaccense is reported here for the first time.

2. Results and Discussion

2.1. Extraction Yield of Plant Samples

In this study, four solvents, that is, ethanol, hexane, chloroform, and ethyl acetate, were selected for fractional extraction based on their different polarity indices, ensuring a diverse phytochemical profile. As shown in Table 2, extraction using hexane generally produced a higher yield, indicating a higher abundance of non‐polar compounds in the hexane fraction, compared to the semi‐polar (ethyl acetate) and polar (ethanol) fractions. In contrast, the ethyl acetate fractionated extracts generally showed the lowest yield across all extracts. Exceptions were observed for B. macrophylla (leaf and twig), C. cauliflora (leaf), and G. celebica (twig), where the hexane‐ or chloroform‐fractionated extract exhibited a lower yield. The semi‐polar nature of ethyl acetate makes it selective toward moderately polar compounds and less efficient at extracting a broad spectrum of compounds, resulting in a lower overall yield [22].

TABLE 2.

Yield of selected indigenous tropical fruit trees.

Species (Common name)	Part	Extract	Initial weight (g)	Yield (g)	Percentage yield (%) a
A. bilimbi (Belimbing buluh)	Leaf	Ethanol	382.00	23.79	6.23
		Hexane	21.54	9.31	43.22
		Chloroform	21.54	0.63	2.92
		Ethyl acetate	21.54	0.48	2.23
	Twig	Ethanol	160.00	13.66	8.54
		Hexane	11.94	0.70	5.86
		Chloroform	11.94	0.71	5.95
		Ethyl acetate	11.94	0.11	0.92
B. motleyana (Rambai)	Leaf	Ethanol	150.00	10.34	6.89
		Hexane	9.29	3.16	34.02
		Chloroform	9.29	2.15	23.14
		Ethyl acetate	9.29	0.08	0.86
	Twig	Ethanol	300.00	3.75	1.25
		Hexane	2.71	0.74	27.31
		Chloroform	2.71	0.18	6.64
		Ethyl acetate	2.71	0.05	1.85
B. macrophylla (Kundang)	Leaf	Ethanol	250.00	23.39	9.36
		Hexane	21.89	4.68	21.38
		Chloroform	21.89	1.08	4.93
		Ethyl acetate	21.89	3.38	15.44
	Twig	Ethanol	300.00	11.27	3.76
		Hexane	9.61	0.27	2.81
		Chloroform	9.61	0.25	2.60
		Ethyl acetate	9.61	1.65	17.17
C. cauliflora (Nam‐nam)	Leaf	Ethanol	300.00	32.96	10.99
		Hexane	27.49	3.12	11.35
		Chloroform	27.49	0.32	1.16
		Ethyl acetate	27.49	2.43	8.84
	Twig	Ethanol	350.00	47.71	13.63
		Hexane	44.76	1.25	2.79
		Chloroform	44.76	0.10	0.22
		Ethyl acetate	44.76	1.42	3.17
G. celebica (Manggis hutan)	Leaf	Ethanol	200.00	14.50	7.25
		Hexane	13.00	1.59	12.23
		Chloroform	13.00	4.43	34.08
		Ethyl acetate	13.00	0.12	0.92
	Twig	Ethanol	200.00	19.16	9.58
		Hexane	17.66	0.17	0.96
		Chloroform	17.66	2.18	12.34
		Ethyl acetate	17.66	0.37	2.10
S. malaccense (Jambu bol)	Leaf	Ethanol	200.00	24.04	12.02
		Hexane	22.85	3.87	16.94
		Chloroform	22.85	4.14	18.12
		Ethyl acetate	22.85	0.19	0.83
	Twig	Ethanol	350.00	4.32	1.23
		Hexane	3.12	0.98	31.41
		Chloroform	3.12	0.03	0.96
		ethyl acetate	3.12	0.07	2.24

^a Percentage yield of ethanol extract was calculated based on the initial weight (dried sample); percentage yield of the fractionated extracts was calculated based on the ethanol extract.

2.2. TPC of the Plant Extracts

Phenolic compounds are the largest and most diverse class of secondary metabolites found in plants, which contribute significantly to radical scavenging, anti‐inflammatory, anti‐aging, and antiproliferation properties. The accumulation of the phenolic compounds is recognized as part of the plant's defense mechanisms against various stressors, including light stress, microbial invasion, and pathogenic attacks. As shown in Table 3, the TPC values ranged from 3.44 ± 0.58 to 577.47 ± 16.15 mg GAE/g extract, following the order: ethyl acetate fractionated extracts > ethanol extracts > chloroform fractionated extracts ≈ hexane fractionated extracts. Ethyl acetate is a semi‐polar solvent that comprises both a polar ester group and a non‐polar ethyl group, enabling it to extract both polar (e.g., gallic acid) and non‐polar (e.g., α‐tocopherol) phenolic compounds. B. macrophylla leaf ethyl acetate fractionated extract exhibited the highest TPC (577.47 ± 16.15 mg GAE/g extract). This finding contrasts with the results of Rudiana, et al. [23], who reported a considerably lower TPC for the ethyl acetate extract (97.90 ± 12.67 mg GAE/g extract) from the same plant. This variation may be attributed to differences in extraction methods. In the present study, the ethyl acetate fractionated extract was obtained from a crude ethanol extract, whereas Rudiana et al. [23] directly macerated the ground plant material in ethyl acetate as a crude extract. Based on the literature, the potential phenolic compounds in B. macrophylla leaves are flavonoids, such as myricetin, mearnsitrin, kaempferol, afzelin, quercetin, quercitrin, myricitrin, and naringenin [24, 25]; some of which exhibit antioxidant activities (vide infra).

TABLE 3.

TPC, radical scavenging, and mushroom tyrosinase inhibition activities of selected indigenous tropical fruit trees.

				EC50 (µg/mL)			IC50 (µg/mL)
Species/Control	Part	Extract	TPC (mg GAE/g extract)	DPPH•	ABTS•+	O2 •−	Mushroom tyrosinase
A. bilimbi	Leaf	Ethanol	57.08 ± 2.09 hijklm	220.73 ± 17.11 f	59.26 ± 2.92 e	>500.00	>250.00
		Hexane	31.38 ± 0.93 klmnop	819.14 ± 18.35 a	169.30 ± 10.30 b	>500.00	>250.00
		Chloroform	61.35 ± 2.16 hijkl	619.20 ± 45.76 b	83.14 ± 7.92 d	>500.00	>250.00
		Ethyl acetate	181.42 ± 0.45 e	8.22 ± 0.20 qrs	22.92 ± 0.22 hijklm	220.74 ± 9.62 c	143.03 ± 5.17 bc
	Twig	Ethanol	85.79 ± 3.12 ghi	75.45 ± 3.37 jkl	31.24 ± 0.41 fgh	415.87 ± 8.87 a	>250.00
		Hexane	57.98 ± 0.95 hijklm	294.49 ± 28.60 cd	65.51 ± 1.71 e	>500.00	>250.00
		Chloroform	75.73 ± 2.60 hij	82.66 ± 0.32 ijk	29.87 ± 1.21 fghi	>500.00	>250.00
		Ethyl acetate	382.83 ± 11.26 b	33.78 ± 2.42 lmnopqrs	100.67 ± 6.61 c	140.88 ± 5.84 ef	212.92 ± 28.95 a
B. motleyana	Leaf	Ethanol	63.32 ± 2.50 hijk	67.67 ± 2.22 jklm	12.78 ± 0.53 klmno	>500.00	>250.00
		Hexane	38.47 ± 1.21 jklmnop	72.46 ± 2.64 jkl	14.15 ± 0.46 klmno	>500.00	>250.00
		Chloroform	37.99 ± 1.68 jklmnop	66.87 ± 1.03 jklm	14.71 ± 0.99 jklmno	>500.00	>250.00
		Ethyl acetate	97.47 ± 0.84 gh	14.70 ± 0.08 nopqrs	6.50 ± 0.00 no	140.10 ± 1.61 efg	>250.00
	Twig	Ethanol	85.54 ± 2.55 ghi	49.21 ± 1.71 klmnopqr	10.27 ± 0.34 mno	277.94 ± 30.24 b	>250.00
		Hexane	14.58 ± 0.63 nop	152.46 ± 2.10 g	31.10 ± 1.27 fgh	>500.00	>250.00
		Chloroform	20.97 ± 0.72 lmnop	149.28 ± 6.13 g	29.47 ± 1.41 fghi	>500.00	>250.00
		Ethyl acetate	155.79 ± 3.89 ef	11.24 ± 0.06 opqrs	4.79 ± 0.09 no	118.03 ± 2.08 fgh	>250.00
B. macrophylla	Leaf	Ethanol	82.94 ± 7.70 ghi	20.09 ± 1.87 nopqrs	4.59 ± 0.19 no	127.17 ± 11.38 efgh	97.07 ± 1.48 cd
		Hexane	50.84 ± 4.41 ijklmn	48.98 ± 5.13 klmnopqr	14.80 ± 0.41 jklmno	>500.00	>250.00
		Chloroform	69.49 ± 1.55 hijk	50.44 ± 2.25 klmnopq	14.21 ± 0.84 jklmno	280.61 ± 24.04 b	>250.00
		Ethyl acetate	577.47 ± 16.15 a	6.47 ± 0.21 rs	1.80 ± 0.09°	37.22 ± 1.63 j	>250
	Twig	Ethanol	371.90 ± 40.56 b	11.81 ± 1.17 opqrs	2.68 ± 0.18°	73.85 ± 1.45 ij	55.51 ± 1.99 de
		Hexane	4.66 ± 1.31 p	256.80 ± 12.64 def	8.68 ± 0.19 no	>500.00	>250.00
		Chloroform	29.46 ± 2.39 klmnop	248.11 ± 4.03 ef	10.48 ± 0.17 mno	>500.00	>250.00
		Ethyl acetate	329.38 ± 42.97 c	8.58 ± 0.45 qrs	2.19 ± 0.03°	41.82 ± 4.14 j	72.34 ± 4.55 d
C. cauliflora	Leaf	Ethanol	117.62 ± 11.41 fg	12.84 ± 0.49 nopqrs	7.66 ± 0.24 no	167.36 ± 16.32 de	144.43 ± 8.80 bc
		Hexane	67.05 ± 3.22 hijk	52.69 ± 6.29 klmnop	16.91 ± 2.45 ijklmn	>500.00	149.72 ± 7.18 bc
		Chloroform	76.00 ± 5.18 hij	147.62 ± 20.97 g	24.40 ± 3.82 hijkl	>500.00	>250.00
		Ethyl acetate	240.82 ± 6.64 d	4.98 ± 0.59 s	11.28 ± 1.27 lmno	120.51 ± 16.79 fgh	>250.00
	Twig	Ethanol	191.21 ± 11.10 e	15.01 ± 1.10 nopqrs	6.01 ± 0.49 no	107.20 ± 10.39 fghi	219.20 ± 13.63 a
		Hexane	47.80 ± 2.77 ijklmno	54.81 ± 3.36 klmn	42.20 ± 1.80 f	>500.00	>250.00
		Chloroform	18.77 ± 0.98 mnop	154.13 ± 2.03 g	34.92 ± 2.71 fgh	>500.00	>250.00
		Ethyl acetate	261.69 ± 14.78 d	4.60 ± 0.38 s	4.77 ± 0.33 no	66.82 ± 6.53 ij	165.68 ± 3.05 ab
G. celebica	Leaf	Ethanol	66.40 ± 1.02 hijk	49.01 ± 1.76 klmnopqr	10.95 ± 0.55 lmno	187.73 ± 3.26 cd	214.33 ± 4.33 a
		Hexane	3.44 ± 0.58 p	308.58 ± 20.86 c	58.06 ± 0.56 e	>500.00	>250.00
		Chloroform	8.31 ± 0.73 op	238.84 ± 8.86 f	41.73 ± 0.97 fg	316.08 ± 2.17 b	>250.00
		Ethyl acetate	74.63 ± 9.80 hij	27.56 ± 1.20 mnopqrs	3.21 ± 0.23 no	107.36 ± 2.60 fghi	>250.00
	Twig	Ethanol	95.74 ± 3.55 gh	19.21 ± 0.36 nopqrs	3.62 ± 0.15 no	99.35 ± 2.42 ghi	>250.00
		Hexane	8.49 ± 1.14 op	>1000.00	337.72 ± 16.47 a	>500.00	>250.00
		Chloroform	5.19 ± 0.25 p	287.21 ± 15.39 cde	61.34 ± 3.69 e	411.20 ± 12.80 a	>250.00
		Ethyl acetate	178.71 ± 3.72 e	10.07 ± 0.31 pqrs	2.29 ± 0.03°	68.47 ± 1.71 ij	>250.00
S. malaccense	Leaf	Ethanol	19.61 ± 0.24 mnop	103.45 ± 2.26 hij	24.20 ± 0.56 hijklm	433.02 ± 6.99 a	>250.00
		Hexane	19.96 ± 1.61 mnop	118.32 ± 5.33 ghi	23.43 ± 1.15 hijklm	217.32 ± 4.78 c	>250.00
		Chloroform	15.64 ± 3.95 nop	145.50 ± 8.19 gh	28.10 ± 1.14 ghij	221.77 ± 1.10 c	>250.00
		Ethyl acetate	155.43 ± 2.62 ef	19.30 ± 0.96 nopqrs	4.68 ± 0.12 no	68.84 ± 2.84 ij	>250.00
	Twig	Ethanol	80.70 ± 2.82 ghi	25.80 ± 1.06 mnopqrs	6.01 ± 0.21 no	94.08 ± 3.31 hi	>250.00
		Hexane	36.64 ± 1.97 jklmnop	53.49 ± 4.30 klmno	12.62 ± 0.20 klmno	191.54 ± 13.00 cd	>250.00
		Chloroform	37.75 ± 2.59 jklmnop	51.57 ± 1.03 klmnop	25.98 ± 0.95 hijk	188.10 ± 3.55 cd	>250.00
		Ethyl acetate	187.66 ± 1.87 e	4.60 ± 0.00 s	3.28 ± 0.02 no	50.40 ± 0.78 j	>250.00
*Ascorbic acid				6.71 ± 0.56 rs	2.45 ± 0.23°
*Gallic acid						146.32 ± 13.82 ef
*Kojic acid							3.42 ± 0.17 e

^a–zSuperscript with different letters within each column indicates significant differences (p < 0.05) among all extracts in this study. [*] Asterisk indicates the positive control.

2.3. Radical Scavenging Activity

The radical scavenging activity is one of the key factors in the skin‐lightening mechanisms. The production of ROS triggers oxidative stress, which subsequently accelerates melanogenesis and causes skin hyperpigmentation [26]. Due to the diverse mechanisms involved in radical scavenging, three different experiments, viz., DPPH•, ABTS^•+, and O₂ ^•− assays, were carried out to comprehensively evaluate the radical scavenging activity of the extracts.

The results of the radical scavenging activity for all extracts are presented in Table 3 as EC₅₀ values, which are the concentration of any sample that effectively induces a response at 50% between the baseline and maximum. The RACI of the extracts was calculated to integrate and compare results from multiple radical scavenging assays into a single standardized index. In Table 4, the extracts are ranked in descending order of RACI, which corresponds to a decrease in overall radical scavenging activity. The semi‐polar (ethyl acetate) and polar (ethanol) solvent extracts generally showed a stronger radical scavenging activity than the less polar (chloroform) and non‐polar (hexane) ones, with the ethyl acetate fractionated extracts of five species filling the top five RACI rankings. The overall radical scavenging activity follows the trend: Ethyl acetate fractionated extracts > ethanol extracts > chloroform fractionated extracts > hexane fractionated extracts. The obtained results are in accordance with the study conducted by Nawaz, et al. [27], which reported that medium polar solvent extracts (especially ethyl acetate) recorded the highest radical scavenging ability in neutralising DPPH and hydroxyl radicals. Similar to the study conducted by See, et al. [28], ethyl acetate extracts exhibited the strongest reducing power among hexane, chloroform, and methanol extracts due to their highest TPC.

TABLE 4.

RACI of the extracts.

Species	Part	Extract	RACI
B. macrophylla	Leaf	Ethyl acetate	+2.49
B. macrophylla	Twig	Ethyl acetate	+2.45
S. malaccense	Twig	Ethyl acetate	+2.40
G. celebica	Twig	Ethyl acetate	+2.30
C. cauliflora	Twig	Ethyl acetate	+2.28
B. macrophylla	Twig	Ethanol	+2.26
S. malaccense	Leaf	Ethyl acetate	+2.22
G. celebica	Twig	Ethanol	+2.08
S. malaccense	Twig	Ethanol	+2.04
G. celebica	Leaf	Ethyl acetate	+2.01
C. cauliflora	Twig	Ethanol	+2.00
B. motleyana	Twig	Ethyl acetate	+1.98
B. macrophylla	Leaf	Ethanol	+1.90
C. cauliflora	Leaf	Ethyl acetate	+1.87
B. motleyana	Leaf	Ethyl acetate	+1.82
C. cauliflora	Leaf	Ethanol	+1.66
G. celebica	Leaf	Ethanol	+1.36
S. malaccense	Twig	Hexane	+1.29
A. bilimbi	Leaf	Ethyl acetate	+1.10
S. malaccense	Twig	Chloroform	+1.07
B. motleyvana	Twig	Ethanol	+0.88
B. macrophylla	Leaf	Chloroform	+0.79
S. malaccense	Leaf	Hexane	+0.72
S. malaccense	Leaf	Chloroform	+0.51
A. bilimbi	Twig	Ethyl acetate	+0.02
A. bilimbi	Twig	Ethanol	−0.35
B. macrophylla	Leaf	Hexane	−0.41
S. malaccense	Leaf	Ethanol	−0.41
B. motleyana	Leaf	Ethanol	−0.44
C. cauliflora	Leaf	Hexane	−0.46
B. motleyana	Leaf	Chloroform	−0.47
B. motlevana	Leaf	Hexane	−0.48
G. celebica	Leaf	Chloroform	−0.58
A. bilimbi	Twig	Chloroform	−0.81
C. cauliflora	Twig	Hexane	−0.94
C. cauliflora	Leaf	Chloroform	−0.94
B. motleyana	Twig	Chloroform	−1.04
B. macrophylla	Twig	Hexane	−1.04
B. macrophylla	Twig	Chloroform	−1.04
B. motleyana	Twig	Hexane	−1.08
C. cauliflora	Twig	Chloroform	−1.16
G. celebica	Twig	Chloroform	−1.63
A. bilimbi	Leaf	Ethanol	−1.84
G. celebica	Leaf	Hexane	−2.13
A. bilimbi	Twig	Hexane	−2.22
A. bilimbi	Leaf	Chloroform	−3.70
A. bilimbi	Leaf	Hexane	−5.99
G. celebica	Twig	Hexane	−12.32

Since the selected Malaysian indigenous tropical fruit trees remain relatively underexplored with respect to their antioxidant properties, published data on their radical scavenging activities are limited. Therefore, comparison with previous studies was only possible for 10 out of the 48 extracts evaluated in the present study. The obtained results were compared with the reported values to evaluate the similarities and differences in their radical scavenging potential. For the remaining extracts, direct comparison was not possible due to the limited availability of relevant literature. Therefore, the present findings contribute additional information regarding the radical scavenging potential of these underexplored Malaysian indigenous tropical fruit trees.

As shown in Tables 3 and 4, B . macrophylla leaf ethyl acetate fractionated extract exhibited the strongest radical scavenging activity, with the RACI of +2.49, and the EC₅₀ obtained from all three radical scavenging assays was below 50 µg/mL (DPPH•, 6.47 ± 0.21; ABTS^•+, 1.80 ± 0.09; O₂ ^•−, 37.22 ± 1.63 µg/mL). According to the classification proposed by Rudiana, et al. [23], samples with EC₅₀ values below 50 µg/mL are categorized as having very strong radical scavenging activity. Therefore, B. macrophylla leaf ethyl acetate fractionated extract was classified as exhibiting “very strong” radical scavenging activity. It is also noteworthy that the EC₅₀ values of this extract were lower than the positive controls in all three radical scavenging assays. Interestingly, the observed EC₅₀ value of B. macrophylla leaf ethyl acetate extract in the DPPH assay (6.47 ± 0.21 µg/mL, Table 3) is significantly lower than the one reported by Rudiana, et al. [23], that is, 20.36 ± 0.44 µg/mL, albeit tested on the same plant species. Apart from the difference in the extraction method (vide supra), the discrepancy in the EC₅₀ values could be attributed to the geographical location and environmental conditions of the collected sample, which influence the plants’ phytochemical composition [29]. Thummajitsakul and Silprasit [30] further supported this finding by reporting genetic variations among three B. macrophylla leaf samples collected from different geographical locations, which possibly contributed to the disparity in radical scavenging activities.

Similar variations were observed for other extracts with available literature data (Table 1). Differences in EC₅₀ values between the present study and previous reports were observed for both DPPH• and ABTS^•+ assays. For example, the DPPH• EC₅₀ value of B. motleyana leaf ethanol extract obtained in the present study (67.67 ± 2.22 µg/mL) differed from the previously reported value (41.36 µg/mL). Similar differences were observed for B. macrophylla twig ethyl acetate fractionated extract (8.58 ± 0.45 vs. 4.89 µg/mL), C. cauliflora leaf ethanol extract (12.84 ± 0.49 µg/mL vs. 0.049 ± 0.250 mg/mL), and other extracts as summarized in Table 1. These variations may be attributed to differences in extraction conditions, plant materials, geographical origin, environmental factors, and assay parameters, which may influence the recovery and activity of antioxidant compounds.

Among the extracts evaluated, the strong radical scavenging activity observed in B. macrophylla extracts may be associated with the presence of bioactive compounds extracted by solvents with different polarities. Due to its polar nature, ethanol can dissolve a wider range of bioactive compounds in B. macrophylla compared to ethyl acetate, and it exhibits the second‐highest radical scavenging activity among extracts from the same plant part. According to the study by Nguyen, et al. [31], several bioactive compounds, including polyphenols, flavonoids, caryophyllene, phytol, and trans‐geranylgeraniol, were identified in B. macrophylla leaf ethanol extracts using gas chromatography‐mass spectrometry (GC‐MS). Some of these compounds have been reported to exhibit radical scavenging activities. For instance, caryophyllene, one of the major compounds (27.48%), contains a methylene π‐bond capable of donating electrons to neutralize free radicals [32]. Similarly, Squalene, another major compound (32.11%) has been shown to maintain the ROS balance by scavenging the excessive O₂ ^•− [33]. The presence of these identified bioactive compounds may contribute to the strong radical scavenging activity observed in B. macrophylla leaf ethanol extract and its fractionated extracts, with an EC₅₀ value of less than 100 µg/mL.

An earlier study by Alothman, et al. [34] has shown that the radical scavenging activity is positively correlated with the TPC. The radical scavenging activity of phenolic compounds can be achieved by donating hydrogen atoms or electrons to the radicals. Due to the structure‐activity relationship, the radical scavenging activity of phenolic compounds is affected by the number and position of hydroxyl group(s), the presence of a carbon‐carbon double bond, glycosylation, and the presence of a substituent in the rings [35, 36]. Based on the results presented in Table 3, extracts with higher TPC, especially the ethyl acetate fractionated extract, generally exhibit a higher radical scavenging activity (the statistically calculated correlation between these variables will be discussed later). Therefore, it is suggested that phenolic compounds in these extracts might play an important role in scavenging free radicals.

2.4. Anti‐Tyrosinase Activity

Tyrosinase is the rate‐limiting enzyme in melanogenesis, and its inhibition suppresses the downstream melanogenesis process [37]. Various plant‐derived (e.g., kojic acid, arbutin, chalcones, and quercetagetin) and synthetic (e.g., hydroxypyridinone, azole, and thiazolidine) tyrosinase inhibitors have been extensively studied for their ability to reduce melanin production. Mushroom tyrosinase is widely utilized for evaluating the skin‐lightening effects of samples due to its structural similarity to mammalian tyrosinase [38], cost‐effectiveness, and suitability for high‐throughput investigation. The mushroom tyrosinase inhibitory activities of all the extracts from the six species are presented in Table 3 as IC₅₀ values. Briefly, the ethanol extracts demonstrated the highest inhibitory effects on mushroom tyrosinase, followed by ethyl acetate, hexane, and chloroform fractionated extracts, except for C. cauliflora twigs, for which the strongest one is the ethyl acetate extract. The results revealed that the ethanol extracts showed stronger inhibitory effects against mushroom tyrosinase compared to their corresponding ethyl acetate fractions, suggesting that the enhanced anti‐tyrosinase activity of the ethanol extracts may be attributed to the abundance of polar bioactive compounds.

Previous studies have reported the mushroom tyrosinase inhibitory activities of A. bilimbi and C. cauliflora leaves ethanol extracts with IC₅₀ = 8.66 ± 1.61 and 0.034 ± 0.011 mg/mL, respectively (Table 1). Compared with the previously reported values, both A. bilimbi and C. cauliflora leaf ethanol extracts exhibited weaker inhibitory activity in the current study with IC₅₀ values of >250.00 µg/mL and 144.43 ± 8.80 µg/mL, respectively. The observed differences may be attributed to variations in the geographical origin and plant growth conditions, extraction procedures, and assay conditions. Among the extracts evaluated in the present study, B. macrophylla leaf and twig ethanol extracts exhibited the strongest anti‐tyrosinase activity, with IC₅₀ values of 55.51 and 97.07 µg/mL, respectively. The observed tyrosinase inhibitory activity may be attributed to the presence of flavonoids (e.g., catechins and taxifolin) isolated from B. macrophylla twigs [39, 40]. These compounds likely act via competitive inhibition, as both comprise a common catechol structure, consisting of a benzene ring with two hydroxyl groups, which shares a high structural similarity with the natural substrate L‐DOPA. This structural‐activity relationship had been supported by the molecular docking simulation conducted by Yin, et al. [41], which demonstrated that the benzene ring with phenolic hydroxyl groups of catechins can bind to the active copper ions and putatively interact with the key amino acid residues of tyrosinase's binding site spontaneously. In addition, a study by Maneechai, et al. [13] reported that B. macrophylla peel ethanol extract exhibits tyrosinase inhibitory activity with an IC₅₀ of 1660 ± 840 µg/mL, suggesting that various parts of B. macrophylla contain bioactive compounds capable of inhibiting tyrosinase activity.

2.5. SPF Values for all the Extracts

Solar UV radiation is categorized based on wavelength into long‐wave UVA (315–400 nm), mid‐wave UVB (280–315 nm), and short‐wave UVC (100–280 nm). Among these, most UVA and a portion of UVB radiation can penetrate through the ozone layer and reach the Earth, whereas UVC is entirely absorbed by the ozone layer. Exposure to UVB radiation is a contributing factor to skin hyperpigmentation through an indirect mechanism of ROS generation. Therefore, sunscreens offer skin protection by preventing UV‐induced pigmentation by absorbing, reflecting, or scattering UV radiation.

The effectiveness of a sunscreen is expressed in SPF value, which is defined as the ratio of UV energy required to produce a minimal erythemal dose (MED) in protected skin to unprotected skin. As shown in Table 5, the ethyl acetate fractionated leaf extracts generally exhibited the highest SPF values, particularly B. macrophylla (SPF value 28.64 ± 0.51), followed by C. cauliflora (22.20 ± 1.26) and A. bilimbi (20.57 ± 0.51). According to the classification guideline provided by National Pharmaceutical Regulatory Agency [42], the aforementioned extracts, with SPF values between 15 and 30 are classified as medium‐level sun protection.

TABLE 5.

The SPF values of all the extracts.

Species	Part	Extract	SPF value
A. bilimbi	Leaf	Ethanol	6.49 ± 0.21 hijk
		Hexane	2.94 ± 0.06 rstuvwxy
		Chloroform	9.37 ± 0.54 ef
		Ethyl acetate	20.57 ± 0.51 b
	Twig	Ethanol	3.17 ± 0.13 pqrstuvwx
		Hexane	2.54 ± 0.06 tuvwxyz
		Chloroform	5.16 ± 0.09 klmno
		Ethyl acetate	15.76 ± 0.44 cd
B. motleyana	Leaf	Ethanol	4.11 ± 0.14 mnopqrstu
		Hexane	4.87 ± 0.18 klmnopq
		Chloroform	6.44 ± 0.13 ijk
		Ethyl acetate	4.94 ± 0.16 klmnopq
	Twig	Ethanol	2.05 ± 0.18 vwxyz
		Hexane	1.77 ± 0.13 wxyz
		Chloroform	3.51 ± 0.09 opqrstuvw
		Ethyl acetate	5.10 ± 0.09 klmno
B. macrophylla	Leaf	Ethanol	8.49 ± 0.48 fg
		Hexane	5.82 ± 0.90 jklm
		Chloroform	6.28 ± 0.49 ijkl
		Ethyl acetate	28.64 ± 0.51 a
	Twig	Ethanol	8.06 ± 0.22 fghi
		Hexane	2.27 ± 0.13 uvwxyz
		Chloroform	2.58 ± 0.10 stuvwxyz
		Ethyl acetate	17.16 ± 0.75 c
C. cauliflora	Leaf	Ethanol	4.45 ± 0.09 lmnopqrs
		Hexane	4.66 ± 0.29 klmnopqr
		Chloroform	11.10 ± 1.13 e
		Ethyl acetate	22.20 ± 1.26 b
	Twig	Ethanol	1.39 ± 0.10 xyz
		Hexane	2.65 ± 0.14 stuvwxyz
		Chloroform	3.81 ± 0.28 nopqrstuv
		Ethyl acetate	8.36 ± 1.44 fgh
G. celebica	Leaf	Ethanol	1.88 ± 0.22 wxyz
		Hexane	1.08 ± 0.18 yz
		Chloroform	2.36 ± 0.15 uvwxyz
		Ethyl acetate	7.24 ± 0.19 ghij
	Twig	Ethanol	1.85 ± 0.08 wxyz
		Hexane	0.89 ± 0.09 z
		Chloroform	2.67 ± 0.04 stuvwxyz
		Ethyl acetate	5.64 ± 0.28 jklmn
S. malaccense	Leaf	Ethanol	3.12 ± 0.41 qrstuvwx
		Hexane	4.40 ± 0.23 lmnopqrst
		Chloroform	6.42 ± 0.36 ijk
		Ethyl acetate	15.27 ± 1.00 cd
	Twig	Ethanol	5.03 ± 0.14 klmnop
		Hexane	4.10 ± 0.24 mnopqrstu
		Chloroform	5.45 ± 0.27 jklmn
		Ethyl acetate	14.39 ± 0.22 d

^a‐zSuperscript with different letters within each column indicate significant differences (p < 0.05) among all extracts in this study.

The photoprotective effects of plant extracts mentioned above are largely attributed to the presence of phenolic compounds, particularly flavonoids, which help protect plants against harmful UV radiation [43]. Many bioactive compounds with photoprotective effects have been reported, such as quercetin, myricetin, and kaempferol from B. macrophylla leaf [25]; quercetin and kaempferol derivatives from C. cauliflora leaf [15]; and myricetin and apigenin derivatives from A. bilimbi leaf [44, 45, 46]. This is supported by Rajasekar, et al. [47], who highlighted flavonoids as one of the key chromophores with a π‐conjugated system suitable for efficient electronic transitions when absorbing light at shorter wavelengths. Similarly, Ghazi [48] reported that the aromatic ring structure in phenolic compounds allows them to absorb UVA and UVB radiation within the 200–400 nm range. Through absorption of UV radiation, phenolic compounds may reduce UV‐induced oxidative stress and contribute to melanogenesis inhibition.

2.6. Spearman's Correlation Analysis

The Spearman's correlation analysis between the datasets reveals various correlations among the measured variables (Figure 1). A strong positive correlation (0.81–0.86) was observed among the DPPH•, ABTS^•+, and O₂ ^•− radical scavenging assays, indicating that the bioactive compounds in indigenous tropical fruit tree extracts exhibit consistent radical‐neutralising capacities across differently charged radicals. Moreover, a moderate to strong negative correlation (–0.78 to –0.63) was observed between TPC and the radical scavenging assays, indicating that higher TPC is associated with lower EC₅₀ values. A moderate positive correlation (0.61) was also observed between TPC and SPF, whereas a weak negative correlation (−0.29) was observed between TPC and the mushroom tyrosinase inhibitory assay, indicating that higher TPC is associated with higher SPF and lower IC₅₀ values, respectively. Collectively, these correlations suggest that phenolic compounds are important contributors to the radical scavenging, photoprotective, and mushroom tyrosinase inhibitory activities of the extracts. This is consistent with previous reports demonstrating that phenolic compounds effectively neutralize various free radicals, including superoxide anions, hydroxyl radicals, and other reactive species [36, 49, 50]; and contribute to photoprotective and tyrosinase inhibitory activities [51, 52].

FIGURE 1.

The Spearman's correlation between radical scavenging EC₅₀, TPC, mushroom tyrosinase IC₅₀, and SPF values. The intensity of color indicates the strength of correlation. The asterisk (*) indicates p < 0.05. DPPH•: 2,2‐diphenyl‐1‐picrylhydrazyl; ABTS^•+: 2,2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid): O₂ ^•−: Superoxide anion; TPC: Total phenolic content; SPF: Sun protection factor.

2.7. Identified Compounds in Selected Bioactive Extract

B. macrophylla leaf ethyl acetate fractionated extract was selected for LC‐MS analysis due to the highest TPC level, very strong radical scavenging capacity (particularly in ABTS^•+ and O₂ ^•− assays), and the highest SPF value (Tables 3 and 5). To minimize the influence of background signals and potential solvent‐derived contaminants, methanol, and water blanks were analyzed under the same chromatographic and mass spectrometric conditions as the samples. The corresponding blank chromatograms are presented in Supporting Information, Figures S1 and S2, while the features detected in the blank samples are summarized in Supporting Information, Tables S1 and S2. Features detected in either blank were compared with the sample features and excluded from subsequent compound annotation to minimize the potential misidentification of background or contaminant signals. A total of 379 compounds were initially detected in B. macrophylla leaf ethyl acetate fractionated extract (Supporting Information, Table S3 and Figure S3). Following annotation using the METLIN database with a confidence threshold of >90%, 77 candidate compounds were retained, of which 29 showed tentative name matches. Subsequent application of a stringent mass error criterion (database mass difference between −2 and +2 ppm), resulted in the annotation of four compounds, including three phenolics and one lipid. As shown in Table 6, the phenolic compounds include m‐trigallic acid (1), cis‐coutaric acid (2), and ellagic acid (3), whereas the lipid component was identified as sphinganine (4). To the best of our knowledge, these compounds were first identified in this species through MS.

TABLE 6.

The chemical constituents of B. macrophylla leaf ethyl acetate fractionated extract.

Class	No.	Compound	Chemical structure	m/z	Mass
Phenolics	1	m‐Trigallic acid (C21H14O13)		475.0509	474.0437
	2	cis‐Coutaric acid (C13H12O8)		319.0428	296.0536
	3	Ellagic acid (C14H6O8)		303.0130	302.0058
Lipid	4	Sphinganine (C18H39NO2)		302.3053	301.2980

Compounds 1 and 3 are gallic acid derivatives belonging to the tannin class. As a subset of phenolic, tannins act as potent antioxidants due to their benzene rings and phenolic hydroxyl groups, which stabilize free radicals via hydrogen atom transfer (HAT). Both compounds are reported to show antioxidant capacity across DPPH•, ABTS^•+, and FRAP assays [53, 54]. Similarly, compound 2, a hydroxycinnamic acid derivative belonging to the phenolic acid class, has shown antioxidant activity in DPPH• and ABTS^•+ assays [55]. Hence, these constituents may contribute to the very strong radical scavenging activity observed in B. macrophylla leaf ethyl acetate fractionated extract. As discussed in the section on SPF values for all the extracts, the remarkable SPF value of B. macrophylla leaf ethyl acetate fractionated extract was likely attributed to the conjugated aromatic structures of compounds 1–3, which can absorb UVB effectively.

In addition, compound 4 can serve as a precursor for ceramide biosynthesis. Ceramides are the major lipid components in the skin's intercellular lipid matrix (mortar) between keratinocytes (brick). Ceramides play a multifunctional role in skin, including maintaining skin barrier integrity, preventing transepidermal water loss (TEWL), and protecting against pathogenic infiltration [56]. Ceramides also modulate inflammatory response and help maintain an optimal microbial environment for skin [57]. Moreover, they function as signalling molecules in various pathways, regulating cell differentiation, proliferation, and apoptosis [58]. Collectively, the presence of compound 4 enhances the value of B. macrophylla leaf ethyl acetate fractionated extract as a multifunctional ingredient for cosmeceuticals.

2.8. Protein Sequence and Multiple Alignments

The BLAST similarity search against the PDB databases identified 5M8T as a structurally homologous protein to the human tyrosinase target sequence obtained from UniProt (entry ID: P14679). 5M8T is a crystal structure of human tyrosinase‐related protein 1 solved by x‐ray diffraction, with a resolution of 2.35 Å. The homologous structure achieved the highest GMQE score of 0.70, and its sequence is 43.78% identical to that of the human tyrosinase. This homologous structure represents the most probable template for the homologous modeling. The generated structure has a Global score of 0.74 ± 0.05, a value close to 0, indicating that the model exhibits very high global structural quality [59]. The energy of the generated human tyrosinase structure was minimized using SwissPDB Viewer before quality assessment. Figure 2A shows the minimized human tyrosinase.

FIGURE 2.

(A) Generated human tyrosinase structure. 2D interactions of the human tyrosinase with m‐trigallic acid (B), cis‐coutaric acid (C), ellagic acid (D), sphinganine (E), and kojic acid (F).

2.9. Quality Assessment of Homology Models

Verify 3D analysis was used to assess the protein models’ stereochemical quality, in which a high‐quality protein model should have ≥ 80% of residues with scores ≥ 0.2 in the 3D/1D profile [60]. The results of the analysis showed that 94.94% of the residues have an average 3D‐1D score ≥ 0.1 in the 3D/1D profile. In addition, the generated human tyrosinase structure shows an ERRAT score of 93.75%, surpassing the generally accepted range of >50 for a high‐quality model [61]. Lastly, the stereochemical quality and the geometry of protein models were evaluated by a Ramachandran plot using PROCHECK. The plot shows that 86.3% of the residues are in the additionally allowed region, 11.5% in the allowed region, 1.1% in the generally allowed region, and 1.1% in the generally disallowed region. Since the majority of residues are located in the additionally allowed region, it indicates that the backbone dihedral angles (φ and ψ) of the homology model adopt stable, energetically favorable conformations. In summary, all the results collectively suggested that the generated human tyrosinase structure is of high quality and reliable.

2.10. Active Site Predictions

The coordinates of the predicted active site on the human tyrosinase are 120.1058 × 277.5851 × 215.1064, which comprises the His180, Asp199, His202, Glu203, Phe347, His363, Asn364, His367, Ile368, Met374, Ser375, Gln376, Val377, and Ser380 residues. The predicted active site is in agreement with the previous study by Dolinska and Sergeev [62], in which the three calculated residues, that is, Phe347, His367, and Ser375, were also reportedly in contact with the common substrates L‐tyrosine, L‐3,4‐dihydroxyphenylalanine (L‐DOPA), 5,6‐dihydroxyindole‐2‐carboxylic acid, and 5,6‐dihydroxyindole. Therefore, this active site was selected for the molecular docking simulations.

2.11. Molecular Docking Simulations

The binding interactions between human tyrosinase and the identified phytochemicals (1–4) and kojic acid were calculated using molecular docking, and the results are shown in Table 7 and Figure 2B–F. All phytochemicals and kojic acid demonstrated negative calculated affinity with the human tyrosinase protein, indicating that the interactions are thermodynamically feasible. It is worth mentioning that phytochemicals 1–3 exhibited lower calculated affinity than kojic acid, suggestive of their potentially higher biological activity.

TABLE 7.

Calculated affinities between human tyrosinase and selected ligands.

Ligand/Phytochemical	PubChem CID	Calculated Affinity (kcal/mol)
m‐Trigallic acid (1)	90470472	−8.098
cis‐Coutaric acid (2)	102445591	−6.589
Ellagic acid (3)	5281855	−6.509
Sphinganine (4)	91486	−5.592
Kojic acid	3840	−5.520

3. Conclusions

Generally, this study reveals that the ethyl acetate fractionated extracts were the most active across all biological assays tested, except for the mushroom tyrosinase assay. Notably, B. macrophylla leaf ethyl acetate fractionated extract demonstrated the highest TPC, the strongest overall radical scavenging activities, and exhibited the highest SPF values. Significant negative correlations were observed between TPC and DPPH• radical scavenging assay as well as between TPC and O₂ ^‐• radical scavenging assays, suggesting an important role of phenolic compounds in scavenging the neutral and oxygen radicals. The LC‐MS analysis of B. macrophylla leaf ethyl acetate fractionated extract revealed the presence of phenolics (1–3) which may contribute to its observed radical scavenging and photoprotective properties. Whilst the identified lipid (4) in this fractionated extract suggests an additional benefit for skin health. Molecular docking analysis further showed that the phenolics (1–3) exhibited more favourable predicted binding affinities toward tyrosinase than kojic acid, suggesting their potential contribution to tyrosinase inhibition. Conversely, in the mushroom tyrosinase assay, B. macrophylla twig ethanol extract showed the most potent activity. These results highlight the promising potential of B. macrophylla twig ethanol and leaf ethyl acetate fractionated extracts as natural ingredients in cosmeceutical formulations for addressing skin hyperpigmentation and maintaining skin health. For future analysis, comprehensive in vitro cell‐based studies, including cell viability assays, cellular tyrosinase inhibition assays, and mechanistic analyses using qPCR, are necessary to ascertain the potential of these extracts in cosmeceutical formulations. Complementary in vivo models, such as zebrafish, would provide further validation of their efficacy and safety for potential cosmeceutical applications.

4. Experimental Section

4.1. Chemicals and Reagents

Methanol, n‐hexane, chloroform, ethyl acetate, and Folin‐Ciocalteu's phenol reagent were acquired from Merck KGaA, Darmstadt, Germany. The ABTS, DPPH, nicotinamide adenine dinucleotide (NADH), nitro blue tetrazolium chloride (NBT), phenazine methosulfate (PMS), potassium peroxodisulfate, dimethyl sulfoxide (DMSO), kojic acid, mushroom tyrosinase enzyme, L‐3,4‐dihydroxyphenylalanine (L‐DOPA), gallic acid, ascorbic acid, sodium carbonate, sodium phosphate monobasic, sodium phosphate dibasic, and tris(hydroxymethyl)aminomethane (tris base) were acquired from Sigma‐Aldrich, Massachusetts, United States. Sodium sulphate was acquired from Chemiz (M) Sdn. Bhd., Selangor, Malaysia. Ethanol was acquired from R&M Chemicals, Selangor, Malaysia.

4.2. Plant Sample Collection and Identification

The leaves and twigs of selected indigenous tropical fruit trees, that is, A. bilimbi L., B. motleyana (Müll.Arg.)Müll.Arg., C. cauliflora L., G. celebica L. and S. malaccense (L.) Merr. & L.M. Perry was collected from Rimba Ilmu Botanical Garden, Universiti Malaya, Kuala Lumpur. The material of Bouea macrophylla Griff., was collected from the Faculty of Science, Universiti Malaya, Kuala Lumpur. All plant materials were identified by Dr. Sugumaran Manickam from Biologic Environmental, 24‐26 Wickham Street, Perth, 6004, Western Australia, Australia, and Dr Yong Kien Thai from the Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Malaysia. Voucher specimens of the samples (Table 8) were deposited in the Herbarium Universiti Malaya (KLU), UM Agroforestry, Universiti Malaya, Kuala Lumpur, Malaysia.

TABLE 8.

Sample collections of the selected indigenous tropical fruit trees.

Species	Voucher Specimen	Date of collection
A. bilimbi	KLU 50209	24th October 2023
B. macrophylla	KLU 50347	20th March 2024
B. motleyana	KLU 50258	7th March 2024
C. cauliflora	KLU 50210	24th October 2023
G. celebica	KLU 50260	29th February 2024
S. malaccense	KLU 50259	7th March 2024

4.3. Ethanol Extraction and Fractionation

The ethanol extraction and fractionation were carried out according to the method described by Prabakaran, et al. [63], with minor modifications. All the leaves and twigs of the six species were cleaned thoroughly with water upon collection and air‐dried at room temperature (27°C–30°C) for five days. This was then followed by oven drying (Memmert GmbH + Co. KG, Germany) at 60 °C for a maximum of 3 h to remove all remaining moisture from the samples. Approximately 150–382 g of dried plant materials were then ground into fine powder using a laboratory blender (Conair Waring, USA) and extracted with ethanol at a solid‐to‐solvent ratio of 1:6 (w/v) for three days. The crude extract was filtered and concentrated at 40 °C using a rotary evaporator (BÜCHI Labortechnik AG, Switzerland) to obtain the ethanol extract. A portion of the ethanol extract was transferred into a clean vial and kept for further analysis, while the remaining extract was fractionated with hexane. The hexane partitioning was repeated until the hexane layer became colorless, indicating exhaustive extraction of the hexane‐soluble constituents. The hexane‐insoluble residue was partitioned between chloroform and water (1:1, v/v), and the water layer was then further partitioned between ethyl acetate and water (1:1, v/v). Lastly, the hexane‐soluble fraction, chloroform layer, and ethyl acetate layer were concentrated using a rotary evaporator (BÜCHI Labortechnik AG, Switzerland) to obtain the hexane, chloroform, and ethyl acetate fractionated extracts. All the ethanol and fractionated extracts were weighed and stored at −20 °C for less than two weeks prior to bioactivity evaluation.

4.4. Determination of TPC

The determination of TPC was performed using the Folin‐Ciocalteu colorimetric method as described by Prabakaran, et al. [63] with slight modification. Into a 96‐well plate, 10 µL of extract (31.25–20000.00 µg/mL) and 25 µL of Folin‐Ciocalteu phenol reagent were added into each well. After 5 min of incubation at room temperature (27°C–30°C) in the dark, 25 µL of sodium carbonate (w/v 20%) and 140 µL of distilled water were added to each well. The reaction mixtures were incubated for 30 min at room temperature (27°C–30°C) in the dark, then their absorbance was measured at 760 nm using a Tecan M200 Infinite Pro microplate reader. Gallic acid (31.25–1000.00 µg/mL) was used as a positive control, and a standard curve was plotted (y = 0.002841x + 0.7031, R² = 0.9541). The TPC (mg GAE/g extract) of the extract was calculated according to the equation:

$$C\left(\mathrm{mgGAE}/\mathrm{gextract}\right)=C_1\times \left(V/M\right)$$

Where, C is the TPC, C₁ is the concentration of gallic acid obtained from the standard curve in mg/L, V is the volume of extract added into each well in L, and M is the mass of extract in each well in g.

4.5. Free Radical Scavenging Assays

4.5.1. DPPH Radical Scavenging Assay

The DPPH• assay was conducted according to the method described by Mat Saad, et al. [64]. On a 96‐well plate, 50 µL of extract (30.00–1000.00 µg/mL) was added into each well containing 150 µL DPPH• solution (0.3 mM). Ascorbic acid (9.77–313.00 µg/mL) was used as a positive control. As for the untreated control, methanol was added to the DPPH• solution instead of the extract. After 30 min of incubation at room temperature (27°C–30°C) in the dark, the absorbance of the reaction mixture in each well was measured at 515 nm using a Tecan M200 Infinite Pro microplate reader. The DPPH• scavenging effect (%) of extracts was calculated according to the equation:

$$DPPH•scavengingeffect\left(\%\right)=\left(\frac{A_{control}-A_{sample}}{A_{control}}\right)\times 100\%$$

Where, A_sample is the absorbance of the mixture containing extract and A_control is the absorbance of the mixture containing methanol instead of the extract.

4.5.2. ABTS Cation Radical Scavenging Assay

The ABTS^•+ assay was carried out according to the method described by Mat Saad, et al. [64]. Briefly, the ABTS^•+ solution was prepared by mixing an equal ratio of 7.00 mM ABTS and 2.45 mM potassium peroxodisulfate, and incubated for 16 to 18 h at room temperature (27°C–30°C) in the dark. The ABTS^•+ solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 734 nm. Each well was charged with 20 µL of extract (0.39–400.00 µg/mL) and 180 µL of the ABTS^•+ solution. Ascorbic acid (0.40–12.50 µg/mL) was used as a positive control. As for the untreated control, 20 µL of methanol was added to the ABTS^•+ solution in place of the extract. The absorbance of the reaction mixture in each well was measured at 734 nm using a Tecan M200 Infinite Pro microplate reader after 20 min of incubation in the dark. The ABTS^•+ scavenging effect (%) of extracts was calculated according to the equation:

$$\mathrm{ABT}{\mathrm{S}}^{•+}scavengingeffect\left(\%\right)=\left(\frac{A_{control}-A_{sample}}{A_{control}}\right)\times 100\%$$

Where, A_sample is the absorbance of the mixture containing extract and A_control is the absorbance of the mixture containing methanol instead of the extract.

4.5.3. Superoxide Anion Radical Scavenging Assay

The superoxide anion (O₂ ^•−) scavenging effect was determined according to the method described by Mat Saad, et al. [64]. Tris‐hydrochloric acid (HCl) buffer (16 mM, pH 8.0) was used as the solution for this assay. The reaction mixture, which contained 40 µL of extract (15.60–500.00 µg/mL), 80 µL of NADH (195 µM), 80 µL of NBT (63 µM), and 80 µL of PMS (10 µM) was incubated at room temperature (27°C–30°C) in the dark for 5 min before the absorbance of the reaction mixture in each well was measured at 560 nm using a Tecan M200 Infinite Pro microplate reader. Gallic acid (15.60–500.00 µg/mL) was used as a positive control. As for the untreated control, 40 µL of Tris‐HCl buffer was added to the well instead of the extract. The O₂ ^•− scavenging effect (%) of the extract was calculated by using the equation:

$$O_2^{•-}scavengingeffect\left(\%\right)=\left(\frac{A_{control}-A_{sample}}{A_{control}}\right)\times 100\%$$

Where, A_sample is the absorbance of the mixture containing extract and A_control is the absorbance of the mixture containing Tris‐HCl buffer instead of extract.

4.6. Relative Antioxidant Capacity Index (RACI)

The RACI was calculated according to the method described previously by Mat Saad, et al. [64] and Senarath Mudalige Don, et al. [65]. Briefly, the EC₅₀ values of the extracts that were determined from the radical scavenging assays were transformed and compared into standard scores.

4.7. Mushroom Tyrosinase Inhibition Assay

The mushroom tyrosinase inhibition assay was carried out as described by Mat Saad, et al. [64], using 5 mM L‐DOPA as substrate. Briefly, into a 96‐well plate, 40 µL of extract (7.81–250.00 µg/mL), 40 µL of mushroom tyrosinase enzyme in 0.1 M sodium phosphate buffer (pH 6.8) and 40 µL of 5 mM L‐DOPA in 0.1 M sodium phosphate buffer (pH 6.8) were added into each well containing 80 µL of sodium phosphate buffer (0.1 M, pH 6.8). Kojic acid (3.13–100.00 µg/mL) was used as a positive control. As for the untreated control, sodium phosphate buffer was added instead of the extract, while the blank used sodium phosphate buffer as a substitute for L‐DOPA and tyrosinase enzyme. After incubating for 10 min in the dark at room temperature (27°C–30°C), the absorbance was measured at 475 nm using a Tecan M200 Infinite Pro Microplate. The percentage of tyrosinase inhibition (%) of the extract was calculated using the equation:

$$\begin{array}{ccc}\hfill Tyrosinaseinhibitionactivity\left(\%\right)& & =\left(\frac{A_{control}-A_{sample}}{A_{control}}\right)\hfill \\ & & \times 100\%\hfill \end{array}$$

Where, A_sample is the absorbance of the mixture containing extract and A_control is the absorbance of the mixture containing sodium phosphate buffer instead of extract.

4.8. In Vitro SPF Determination

The in vitro SPF assessment was conducted according to the method by Reis Mansur, et al. [66], with minor modifications. The extracts were dissolved in ethanol at a concentration of 10 mg/mL and subsequently diluted to reach a final concentration of 100 µg/mL. The UV absorbance spectra were measured at a range of 290–320 nm, with an interval of 5 nm. The measurements were taken using a Genesys 150 UV–visible spectrophotometer with a standard 1 cm path length quartz cuvette. Ethanol was used as a blank throughout the assay. The SPF value was calculated according to the equation:

$$SP{F}_{spectrophotometric}=CF\times \sum _{290}^{320}EE\left(\lambda \right)\times I\left(\lambda \right)Abs\left(\lambda \right)$$

Where, CF is the correction factor, EE(λ) represents the Erythemogenic effect of radiation at wavelength λ, I(λ) is the intensity of solar light at wavelength λ, and Abs(λ) is the absorbance of the extract at wavelength λ.

4.9. LC‐MS Analysis

The LC‐MS analysis was performed using an Agilent 1290 Infinity LC system coupled to an Agilent 6520 Accurate‐Mass Quadrupole Time‐of‐Flight (Q‐TOF) mass spectrometer equipped with a dual electrospray ionisation (ESI) source. Agilent Eclipse XDB‐C18 narrow‐bore column (150.0 mm x 2.1 mm, 3.5‐micron) was used and maintained at 25°C. A volume of 3.0 µL sample was injected via the autosampler. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.5 mL/min. Using ESI positive mode, the mass spectrometry was performed with a capillary voltage of 4000 V, fragmentor voltage of 125 V, skimmer of 65 V, gas temperature of 300°C, drying gas flow rate of 10 L/min, and nebulizer gas pressure of 45 psi. The mass spectrum was scanned over a range of 100 to 3200 m/z, using reference ions at 121.0508 and 922.0097 m/z to ensure accurate mass measurements.

4.10. Identification of Constituents

The identification of the bioactive constituents of the selected bioactive extract was done by comparing the obtained MS spectra against the METLIN Metabolite Personal Compound Database and Library. For compounds not present in the database, the software's molecular formula generator was used. Compounds were considered identified if they achieved a METLIN score > 90% with a deviation of ±2, either from the database match or the molecular formula generator.

4.11. Statistical Analysis

The data were presented as means ± standard deviation of three independent experiments, except for the mushroom tyrosinase assay, which was based on two independent experiments. The means of different groups were compared using one‐way ANOVA, followed by Tukey's post hoc test. The 50% effective concentration (EC₅₀) values and statistical analysis were obtained using the GraphPad Prism software, version 7.00 (GraphPad Software Inc., CA, USA). A value of p < 0.05 was considered statistically significant. The Spearman's correlation analysis among the results obtained from different assays was expressed as a correlation coefficient value (r), which was obtained using the GraphPad Prism software, version 7.00 (GraphPad Software Inc., CA, USA).

4.12. Multiple Alignments, Homology Modeling, and Refinement

Since the experimental human tyrosinase structure is not available in the PDB, it was then constructed by homology modeling using the Swiss‐Model server (https://swissmodel.expasy.org/) as described previously [67]. The FASTA sequence of the human tyrosinase was obtained from UniProt (entry ID: P14679). Suitable templates for modeling were identified using BLAST, and target templates were aligned with MAFFT. Subsequently, the experimentally generated homologous structure with the highest global model quality estimate (GMQE) score was selected as the template. Following construction of the protein based on the selected template, energy minimization of the resulting model was performed using SwissPdbViewer with the GROMOS96 force field.

4.13. Model Validation and Stereochemical Properties Evaluation

The protein structure was evaluated using the SAVES online server that contains Ramachandran Plot, ERRAT, VERIFY3D, and PROVE programs (https://saves.mbi.ucla.edu/).

4.14. Active Site Prediction

The binding site and the target protein cavity were computed using the active site prediction of the protein server PrankWeb (https://prankweb.cz/), as described previously [2]. The active site with the highest probability was chosen for the subsequent analyses.

4.15. Molecular Docking Calculations

Molecular docking calculations were conducted to predict the affinity and interactions between the identified phytochemicals and the generated human tyrosinase using Autodock Vina 1.2.5 in the Swiss Dock 2024 online toolkits, as described previously, with some modifications [68, 69, 70]. Briefly, the SMILES of the ligands (identified phytochemicals and kojic acid) was obtained from the PubChem database and inserted into the SwissDock online toolkits, followed by the insertion of the human tyrosinase generated from homology modeling. The grid box was set to enclose the active site predicted in the previous section. The sampling exhaustivity was set to the maximum and all docked conformations were ranked in the order of increasing calculated affinity. The conformation with the lowest calculated affinity without any unfavorable bump was chosen as the most probable docked conformation. The docking protocol has been validated in our previous study [71]. The Discovery Studio and Chimera software were used to visualize the 2D and 3D interactions, respectively.

Author Contributions

Wei Lee Chong: conceptualization, methodology, investigation, and writing – original draft. Zhi Ning Teoh: methodology, and investigation. Nurul Ainanajwa Ramli: methodology and investigation. Chun Hoe Tan: conceptualization, methodology, resources, writing – review and editing, and validation. Sugumaran Manickam: conceptualization, methodology, resources, writing – review and editing, and validation. Siew Huah Lim: conceptualization, methodology, resources, writing – review and editing, and validation. Kae Shin Sim and Hazwani Mat Saad: conceptualization, methodology, writing – review and editing, funding acquisition, resources, validation, and supervision.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: cbdv71752‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.