UHPLC-ESI-QTOF-MS/MS profiling of saponins and tyrosinase inhibitory activity of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves from Vietnam

Abstract

In this study, a rapid and sensitive UHPLC-ESI-QTOF-MS/MS method was established to characterize and differentiate saponin profiles in the leaves of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda collected in Vietnam. This is the first report on the chemical composition of I. cochinchinensis and I. annamensis using this advanced analytical approach. A total of 63 saponins were tentatively identified across all samples, with I. cochinchinensis, I. annamensis, and I. rotunda containing 39, 30, and 34 compounds, respectively. Among these, seven saponins were newly reported from I. cochinchinensis, including two novel structures corresponding to compound 9 at a retention time (RT) of 11.83 minutes, one novel structure corresponding to compound 15 at RT 12.09 minutes, and four novel structures corresponding to compound 46 at RT 13.71 minutes. Notably, compound 46 was also detected in I. annamensis, whereas no new saponins were identified in I. rotunda. Saponin profiles allowed clear differentiation among the three species. Additionally, we evaluated the tyrosinase inhibitory activity of methanolic leaf extracts at 100 μg mL⁻¹. I. annamensis exhibited the highest inhibition (40.70% ± 1.84), followed by I. cochinchinensis (24.40% ± 1.27) and I. rotunda (14.43% ± 1.53). These findings not only expand the phytochemical database of Vietnamese Ilex species but also highlight the potential of I. annamensis as a promising source of natural tyrosinase inhibitors for cosmetic and dermatological applications.

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Introduction

Ilex, also known as holly, is the only genus still existing in the monogenetic family Aquifoliaceae and estimated to be at least 669 known Ilex species.¹ According to Plants of the World Online (POWO), Ilex is the second-largest genus of dioecious plants in the world after Diospyros L. which has 730 recognized species.² Numerous species in this genus were reported for potential pharmacological uses such as antimicrobial, anti-inflammatory, antioxidant activity, which could be used as anti-obesity, anti-diabetic agents. Thus, these species could be applied to treat a wide range of medical conditions.^1–7 The chemical compounds performing these functions were mentioned as terpenoids, saponins, polyphenols, flavonoids and phenolic glycosides.^3,5

Among them, Ilex rotunda has been officially listed in the Chinese Pharmacopoeia 2020 and traditionally used by the Yao ethnic group for treating cardiovascular conditions. Modern pharmacological studies have reported its potential to reduce coronary artery flow, regulate heart rate, enhance hypoxia tolerance, and exhibit antiarrhythmic properties. A recent study identified 105 chemical compounds from the bark of I. rotunda using UPLC-QTOF-MS/MS (Chen et al., 2021).⁸ However, the chemical profile of I. rotunda cultivated in Vietnam remains poorly documented. Additionally, including I. rotunda in a comparative study with the lesser-known Ilex species provides valuable insights into differences in phytochemical composition and associated bioactivities.

Recently, UHPLC coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS/MS) has become a powerful tool for rapid screening and identification of complex phytochemicals in natural products. For example, LC-QTOF-MS/MS has been successfully used to characterize 80 compounds from Acer species,⁹ 65 metabolites in various berries,¹⁰ 171 constituents in lotus plants,¹¹ 74 secondary metabolites from fruits, leaves, and flowers of Forsythia suspensa,¹² etc. Our research team also applied this technique to study the chemical composition of Hedera helix and Hedera nepalensis in Vietnam.^13–15

Melanin is primarily responsible for determining skin color and pigmentation. Approximately 10% of the skin cells in the deepest layer of the epidermis are involved in the production of melanin. When the skin is exposed to UV light, the process of melanogenesis is triggered by an enzyme called tyrosinase.^16–18 This inspired scientists and researchers to concentrate on the discovery, synthesis, isolation, and characterisation of novel, highly effective tyrosinase inhibitors for use in the food,¹⁹ cosmetics,²⁰ and medicinal industries.²¹ Tyrosinase inhibitors such as kojic acid and hydroquinone, although originally derived from natural sources such as fungi and plants, are commonly synthesized for commercial applications, and their use has been associated with skin irritation.¹⁶ Therefore, researchers are actively searching for alternatives, such as naturally derived chemicals for use in safer skin whitening products.

Although numerous studies have investigated the phytochemical composition and bioactivities of various Ilex species,^22–27 research on Ilex cochinchinensis and Ilex annamensis remains limited. In this study, we developed a straightforward, rapid, and sensitive UHPLC-ESI-QTOF-MS/MS method to identify and differentiate saponins from the leaf extracts of I. cochinchinensis, I. annamensis, and I. rotunda collected in Vietnam. Furthermore, the tyrosinase inhibitory activity of the leaf extracts from all three species was evaluated for the first time. These findings not only expand the phytochemical and biological knowledge of Ilex species in Vietnam but also support their potential applications in the development of natural tyrosinase inhibitors.

Results and discussion

Structural characterization of saponins by UHPLC-ESI-QTOF-MS/MS

The crude methanol extracts from leaves of Ilex cochinchinensis and Ilex annamensis were analyzed by UHPLC-ESI-QTOF-MS/MS in positive and negative ionization modes to comprehensively characterize the saponins in each part of the two species. The identification information was summarized in Table 1. The appearance of the saponins in each sample was presented in Table 2. Deprotonated ions [M − H]⁻ and solvent adduct ions [M + HCOO]⁻ can be observed in the negative ESI mode, while protonated ions [M + H]⁺ and ammoniated ions [M + NH₄]⁺ can be obtained in the positive ESI mode. Additionally, fragments representing dehydration and HCOOH loss from these aglycones can be easily observed in positive ESI mode. To facilitate structural elucidation, UPLC-ESI-QTOF-MS/MS data were interpreted in combination with previously reported fragmentation patterns of structurally related saponins. Previous reports, such as the comprehensive MS analysis of matesaponins from Ilex paraguariensis,²⁸ the dereplication of triterpenoid saponins from Pulsatilla chinensis,²⁹ and the recent characterisation of Hedera nepalensis saponins³⁰ have demonstrated characteristic fragmentation pathways: (i) preferential cleavage of glycosides at C-28 due to the labile ester linkage, (ii) stepwise loss of C-3 linked monosaccharides, and (iii) complementary evidence from both positive and negative ion modes. By following these established MS-based strategies, we tentatively identified the saponins in our study and provided supporting references in the revised manuscript.

Table 1 Identification of the chemical constituents of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves by UPLC-Q-TOF-MS/MS

No.	RT	Precursor Mass	m/z Calculated	Error	Ion	Chemical name	Putative structure	MS/MS	Formula
1	11.08	711.3985	711.3956	−4.12	[M + HCOO]^−	2,3,19,23-Tetrahydroxy-12-ursen-28-oic acid; (2α,3β,19α,20β)-form, 28-O-β-D-glucopyranosyl ester	A1–Glc	665.3876 [M − H]^−	C36H58O11
								503.3356 [M − H–Glc]^−
2	11.12	751.4632	751.4633	0.07	[M + H]^+	20-Taraxastene-3,28-diol; 3β-form, 28-carboxylic acid, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-xylopyranoside]	Glc–Xyl–A2	457.3684 [M + H–Glc–Xyl]^+	C41H66O12
3	11.13	665.3909	665.3901	−1.22	[M + H]^+	3,19,24-Trihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Glu–A3	489.3561 [M + H–Glu]^+	C36H56O11
4	11.24	857.4574	857.4535	−4.57	[M + HCOO]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucopyranoside, 28-O-β-D-glucopyranosyl ester	Glc–A4–Glc	811.4497 [M − H]^−	C42H68O15
								649.3924 [M − H–Glc]^−
								487.3403 [M − H–2Glc]^−
5	11.41	825.4299	825.4273	−3.19	[M − H]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Glu–A4–Glc	663.3718 [M − H–Glc]^−	C42H66O16
								487.3434 [M − H–Glc–Glu]^−
6	11.44	695.4039	695.4007	−4.67	[M + HCOO]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucopyranoside	Glc–A4	649.3938 [M − H]^−	C36H58O10
								487.3412 [M − H–Glc]^−
7	11.74	827.4447	827.4429	−2.16	[M + HCOO]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Ara–A4–Glc	781.4348 [M − H]^−	C41H66O14
								619.3820 [M − H–Glc]^−
								487.3438 [M − H–Glc–Ara]^−
8	11.82	667.4069	667.4057	−1.74	[M + H]^+	2,3,19,24-Tetrahydroxy-12-oleanen-28-oic acid; (2α,3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A5–Glc	505.3551 [M + H–Glc]^+	C36H58O11
9	11.83	959.5243	959.5216	−2.86	[M + HCOO]^−	12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside] OR 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Glc–Rha–Ara–A27	913.5132 [M − H]^−	C47H78O17
							Or	751.4598 [M − H–Glc]^−
								605.4026 [M − H–Glc–Rha]^−
								473.3607 [M − H–Glc–Rha–Ara]^−
10	11.93	809.4355	809.4324	−3.89	[M − H]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Glu–A6–Glc	647.3771 [M − H–Glc]^−	C42H66O15
								471.3454 [M − H–Glc–Glu]^−
11	11.94	709.3821	709.3799	−3.07	[M + HCOO]^−	3,19-Dihydroxy-12-ursene-24,28-dioic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A7–Glc	663.3744 [M − H]^−	C36H56O11
								501.3195 [M − H–Glc]^−
12	12.01	668.4373	668.4374	0.11	[M + NH4]^+	3,19,24-Trihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A3–Glc	489.3584 [M + H–Glc]^+	C36H58O10
13	12.06	973.5047	973.5008	−3.98	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Glc–Ara–A6–Glc	927.4918 [M − H]^−	C47H76O18
								765.4394 [M − H–Glc]^−
								603.3873 [M − H–2Glc]^−
								471.3451 [M − H–2Glc–Ara]^−
14	12.07	711.3991	711.3956	−4.96	[M + HCOO]^−	3,19,23,30-Tetrahydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A8–Glc	665.3872 [M − H]^−	C36H58O11
								503.3348 [M − H–Glc]^−
15	12.09	813.4647	813.4637	−1.29	[M + HCOO]^−	12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Glc–Ara–A27	767.4548 [M − H]^−	C41H68O13
								605.4023 [M − H–Glc]^−
								473.3617 [M − H–Glc–Ara]^−
16	12.17	784.4840	784.4847	0.92	[M + NH4]^+	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside]	Glc–Ara–A9	605.4081 [M + H–Glc]^+	C41H66O13
								473.3653 [M + H–Glc–Ara]^+
17	12.28	693.3877	693.3850	−3.89	[M + HCOO]^−	3,19,23-Trihydroxy-12,20(30)-ursadien-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A10–Glc	647.3764 [M − H]^−	C36H56O10
								485.3245 [M − H–Glc]^−
18	12.35	1265.6171	1265.6166	−0.36	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside], 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester		1219.6023 [M − H]^−	C59H96O26
								911.5003 [M − H–Rha–Glc]^−
								749.4495 [M − H–Rha–2Glc]^−
								603.3924 [M − H–2Rha–2Glc]^−
								471.3465 [M − H–2Rha–2Glc–Ara]^−
19	12.45	695.4008	695.4007	−0.21	[M + HCOO]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A4–Glc	649.3926 [M − H]^−	C36H58O10
								487.3403 [M − H–Glc]^−
20	12.53	957.5087	957.5059	−2.91	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Rha–Ara–A9–Glc	911.4975 [M − H]^−	C47H76O17
								749.4451 [M − H–Glc]^−
								603.3886 [M − H–Glc–Rha]^−
								471.3451 [M − H–Glc–Rha–Ara]^−
21	12.56	1103.5644	1103.5638	−0.53	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester	Rha–Ara–A6–Glc–Rha	1057.5583 [M − H]^−	C53H86O21
								749.4476 [M − H–Glc–Rha]^−
								603.3897 [M − H–Glc–2Rha]^−
								471.3474 [M − H–Glc–2Rha–Ara]^−
22	12.58	841.4589	841.4586	−0.40	[M + HCOO]^−	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester	A11–Glc–Rha	795.4497 [M − H]^−	C42H68O14
								487.3412 [M − H–Glc–Rha]^−
23	12.67	1119.5582	1119.5587	0.48	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]		1073.5495 [M − H]^−	C53H86O22
								911.5000 [M − H–Glc]^−
								749.4452 [M − H–2Glc]^−
								603.3868 [M − H–2Glc–Rha]^−
								471.3451 [M − H–2Glc–Rha–Ara]^−
24	12.70	971.4869	971.4852	−1.78	[M + HCOO]^−	3,11,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,11β,19α,20β)-form, 28→20 lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside]		925.4784 [M − H]^−	C47H74O18
								763.4247 [M − H–Glc]^−
								617.3700 [M − H–Glc–Rha]^−
								485.3243 [M − H–Glc–Rha–Ara]^−
25	12.74	955.4943	955.4903	−4.23	[M − H]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-6-O-methyl-β-D-glucuronopyranoside], 28-O-β-D-glucopyranosyl ester	Ara–methyl Glu–A6–Glc	793.4388 [M − H–Glc]^−	C48H76O19
								661.3918 [M − H–Glc–Ara]^−
								471.3497 [M − H–Glc–Ara–methyl Glu]^−
26	12.77	713.3583	713.3571	−1.72	[M − H]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-sulfate, 28-O-β-D-glucopyranosyl ester	sulfate–A6–Glc	551.3015 [M − H–Glc]^−	C36H58O12S
								471.3478 [M − H–Glc–SO3H]^−
27	12.83	647.3805	647.3795	−1.51	[M + H]^+	3,23-Dihydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-β-D-glucuronopyranoside	Glu–A13	471.3453 [M + H–Glu]^+	C36H54O10
28	12.84	839.4426	839.4429	0.38	[M + HCOO]^−	3,11,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,11β,19α,20β)-form, 28→20 lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside]	Rha–Glc–A12	793.4352 [M − H]^−	C42H66O14
								485.3269 [M − H–Glc–Rha]^−
29	12.89	809.4360	809.4324	−4.51	[M − H]^−	Hederagenin bisdesmosides; diglycosides, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Glu–A14–Glc	647.3787 [M − H–Glc]^−	C42H66O15
								471.3458 [M − H–Glc–Glu]^−
30	13.01	665.3929	665.3901	−4.22	[M + HCOO]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinopyranoside	Ara–A4	619.3844 [M − H]^−	C35H56O9
								487.3445 [M − H–Ara]^−
31	13.03	955.4909	955.4903	−0.67	[M + HCOO]^−	3-Hydroxy-19-oxo-19,20-seco-13(18)-ursen-28,20-olide; (3β,20S)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside]		909.4812 [M − H]^−	C47H74O17
								747.4304 [M − H–Glc]^−
								601.3720 [M − H–Glc–Rha]^−
								469.3346 [M − H–Glc–Rha–Ara]^−
32	13.06	695.4017	695.4007	−1.50	[M + HCOO]^−	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A11–Glc	649.3926 [M − H]^−	C36H58O10
								487.3412 [M − H–Glc]^−
33	13.09	649.3969	649.3952	−2.66	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinoside	Ara–A6	603.3872 [M − H]^−	C35H56O8
								471.3456 [M − H–Ara]^−
34	13.17	784.4838	784.4847	1.17	[M + NH4]^+	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Ara–A9–Glc	635.4134 [M + H–Ara]^+	C41H66O13
								473.3646 [M + H–Ara–Glc]^+
35	13.23	763.4288	763.4269	−2.53	[M − H]^−	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]	Rha–Ara–A16	617.3636 [M − H–Rha]^−	C41H64O13
								485.3184 [M − H–Rha–Ara]^−
36	13.26	663.3767	663.3744	−3.41	[M − H]^−	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Glu–A4	487.3419 [M − H–Glu]^−	C36H56O11
37	13.27	911.5036	911.5004	−3.48	[M − H]^−	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Glc–Ara–A23–Glc	749.4453 [M − H–Glc]^−	C47H76O17
								587.3956 [M − H-2Glc]^−
								455.3503 [M − H-2Glc–Ara]^−
38	13.29	987.4827	987.4801	−2.64	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside], 28-O-β-D-glucopyranosyl ester	Ara–Glu–A6–Glc	941.4704 [M − H]^−	C47H74O19
								779.4177 [M − H–Glc]^−
								647.3763 [M − H–Glc–Ara]^−
								471.3472 [M − H–Glc–Ara–Glu]^−
39	13.38	957.5077	957.5059	−1.87	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Rha–Ara–A6–Glc	911.4966 [M − H]^−	C47H76O17
								749.4442 [M − H–Glc]^−
								603.3867 [M − H–Glc–Rha]^−
								471.3485 [M − H–Glc–Rha–Ara]^−
40	13.43	665.3916	665.3901	−2.27	[M + H]^+	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Glu–A11	489.3596 [M + H–Glu]^+	C36H56O11
41	13.43	693.3861	693.3850	−1.58	[M + HCOO]^−	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-β-D-glucopyranoside	Glc–A16	647.3795 [M − H]^−	C36H56O10
								485.3267 [M − H–Glc]^−
42	13.43	839.4448	839.4429	−2.25	[M + HCOO]^−	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside]	Rha–Glc–A16	793.4345 [M − H]^−	C42H66O14
								485.3241 [M − H–Glc–Rha]^−
43	13.48	971.4856	971.4852	−0.44	[M + HCOO]^−	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]		925.4753 [M − H]^−	C47H74O18
								763.4231 [M − H–Glc]^−
								617.3689 [M − H–Glc–Rha]^−
								485.3249 [M − H–Glc–Rha–Ara]^−
44	13.54	784.4851	784.4847	−0.49	[M + NH4]^+	3,23-Dihydroxy-12-ursen-28-oic acid; (3β,20α)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Ara–A17–Glc	635.4134 [M + H–Ara]^+	C41H66O13
								473.3608 [M + H–Ara–Glc]^+
45	13.54	1133.5387	1133.5380	−0.62	[M + HCOO]^−	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]		1087.5378 [M − H]^−	C53H84O23
								925.4779 [M − H–Glc]^−
								763.4226 [M − H–2Glc]^−
								485.3238 [M − H–2Glc–Rha–Ara]^−
46	13.71	941.5124	941.5110	−1.49	[M + HCOO]^−	12-ursene-3,11,28-triol; (3β,11α)-form, 11-ketone, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside OR 12-ursene-3,11,28-triol; (3β,11α)-form, 11-ketone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside OR ursolic acid, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside] OR ursolic acid, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside]	Glc–Ara–Rha–A28	895.5014 [M − H]^−	C47H76O16
							Or
								733.4492 [M − H–Glc]^−
							Or	601.4092 [M − H–Glc–Ara]^−
							Glc–Ara–Rha–A23
							Or
								455.3528 [M − H–Glc–Ara–Rha]^−
47	13.77	663.3772	663.3744	−4.16	[M + HCOO]^−	3,19-Dihydroxy-24-nor-4(23),12-ursadien-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	A18–Glc	617.3666 [M − H]^−	C35H54O9
								455.3157 [M − H–Glc]^−
48	13.84	955.4925	955.4903	−2.34	[M + HCOO]^−	3-Hydroxy-12,19-ursadien-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-xylopyranoside], 28-O-β-D-glucopyranosyl ester	Glc–Xyl–A19–Glc	909.4803 [M − H]^−	C47H74O17
								747.4293 [M − H–Glc]^−
								585.3762 [M − H–2Glc]^−
								453.3341 [M − H–2Glc–Xyl]^−
49	13.86	811.4514	811.4480	−4.19	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Glc–Ara–A6	765.4393 [M − H]^−	C41H66O13
								603.3877 [M − H–Glc]^−
								471.3456 [M − H–Glc–Ara]^−
50	13.90	957.5077	957.5059	−1.87	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Rha–Glc–Ara–A6	911.4965 [M − H]^−	C47H76O17
								765.4397 [M − H–Rha]^−
								603.3878 [M − H–Rha–Glc]^−
								471.3451 [M − H–Rha–Glc–Ara]^−
51	14.19	647.3800	647.3795	−0.73	[M − H]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Glu–A6	471.3453 [M − H–Glu]^−	C36H56O10
52	14.32	825.4296	825.4273	−2.83	[M + HCOO]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside]	Ara–Glu–A6	779.4193 [M − H]^−	C41H64O14
								471.3450 [M − H–Ara–Glu]^−
53	14.57	647.3790	647.3795	0.81	[M + HCOO]^−	3,23-Dihydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-α-L-arabinopyranoside	Ara–A13	601.3740 [M − H]^−	C35H54O8
								469.3295 [M − H–Ara]^−
54	14.61	1117.5431	1117.5431	−0.01	[M + HCOO]^−	3,19,20-Trihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]		1071.5322 [M − H]^−	C53H84O22
								909.4851 [M − H–Glc]^−
								747.4310 [M − H–2Glc]^−
								601.3776 [M − H–2Glc–Rha]^−
								469.3345 [M − H–2Glc–Rha–Ara]^−
55	14.82	955.4900	955.4903	0.27	[M + HCOO]^−	3,19,20-Trihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]		909.4796 [M − H]^−	C47H74O17
								747.4296 [M − H–Glc]^−
								601.3745 [M − H–Glc–Rha]^−
								469.3346 [M − H–Glc–Rha–Ara]^−
56	15.12	647.3819	647.3795	−3.67	[M − H]^−	3,19-Dihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Glu–A21	471.3473 [M − H-Glu]^−	C36H56O10
57	15.21	645.4013	645.4003	−1.61	[M − H]^−	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-(2-O-Acetyl-β-D-xylopyranoside)	Acetyl Xyl–A6	471.3465 [M − H-acetyl Xyl]^−	C37H58O9
58	15.32	585.3805	585.3791	−2.34	[M − H]^−	3-Hydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-α-L-arabinopyranoside	Ara–A22	453.3367 [M − H–Ara]^−	C35H54O7
59	15.47	795.4548	795.4531	−2.16	[M + HCOO]^−	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Glc–Ara–A23	749.4451 [M − H]^−	C41H66O12
								587.3922 [M − H–Glc]^−
								455.3508 [M − H–Glc–Ara]^−
60	15.77	631.3860	631.3846	−2.20	[M + HCOO]^−	3-Hydroxy-12,18-ursadien-28-oic acid; 3β-form, 3-O-β-D-xylopyranoside	Xyl–A24	453.3357 [M − H–Xyl]^−	C35H54O7
61	16.08	779.4591	779.4582	−1.19	[M − H]^−	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside]	Glc–Gal–A23	455.3503 [M − H-2Glc]^−	C42H68O13
62	17.31	617.3840	617.3842	0.35	[M − H]^−	3,24-Dihydroxy-12-ursen-28-oic acid; 3β-form, 24-O-(4-hydroxy-E-cinnamoyl)	A25-[24-O-(4-Hydroxy-E-cinnamoyl)]	471.3474 [M − H-hydroxy-cinnamoyl]^−	C39H54O6
63	18.29	617.3852	617.3842	−1.60	[M − H]^−	3,27-Dihydroxy-12-ursen-28-oic acid; 3β-form, 27-O-(4-hydroxy-E-cinnamoyl)	A26-[27-O-(4-Hydroxy-E-cinnamoyl)]	471.3491 [M − H-hydroxy-cinnamoyl]^−	C39H54O6

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Table 2 Chemicals constituent differentiation of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves^a

No.	Chemical name	Synonym	ICL	IAL	IRL
a +: appear in the sample, −: not appear in the sample.
1	2,3,19,23-Tetrahydroxy-12-ursen-28-oic acid; (2α,3β,19α,20β)-form, 28-O-β-D-glucopyranosyl ester	20S-Nigaichigoside F1	+	+	−
2	20-Taraxastene-3,28-diol; 3β-form, 28-carboxylic acid, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-xylopyranoside]	x	+	−	−
3	3,19,24-Trihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Ilexpuson G	−	+	−
4	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucopyranoside, 28-O-β-D-glucopyranosyl ester	Ilexoside XXXVII	+	+	−
5	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Ilexoside XXXIX	+	+	−
6	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucopyranoside	Ilexpernoside C	+	+	−
7	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Ilekudinoside D	+	−	−
8	2,3,19,24-Tetrahydroxy-12-oleanen-28-oic acid; (2α,3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Sericoside	−	+	−
9	12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside] OR 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	x	+	−	−
10	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Ilekudinoside B	+	+	+
11	3,19-Dihydroxy-12-ursene-24,28-dioic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	x	−	−	+
12	3,19,24-Trihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	x	+	+	+
13	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Ilexoside II	+	−	−
14	3,19,23,30-Tetrahydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Ilexoside XLIII	+	+	−
15	12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	x	+	−	−
16	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside]	Ilemaminoside B	+	−	−
17	3,19,23-Trihydroxy-12,20(30)-ursadien-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Oblonganoside E	+	−	−
18	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside], 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester	Latifoloside F	−	−	+
19	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Pedunculoside	+	+	+
20	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Latifoloside D	+	+	+
21	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester	Ilekudinoside W	−	−	+
22	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl] ester	x	+	+	+
23	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Latifoloside L	−	−	+
24	3,11,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,11β,19α,20β)-form, 28→20 lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside]	Kudinoside F	−	−	+
25	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-6-O-methyl-β-D-glucuronopyranoside], 28-O-β-D-glucopyranosyl ester	x	+	+	+
26	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-sulfate, 28-O-β-D-glucopyranosyl ester	Ilexoside XXIX	−	+	−
27	3,23-Dihydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-β-D-glucuronopyranoside	x	+	−	−
28	3,11,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,11β,19α,20β)-form, 28→20 lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside]	Ilekudinoside S	−	−	+
29	Hederagenin bisdesmosides; Diglycosides, 3-O-β-D-glucuronopyranoside, 28-O-β-D-glucopyranosyl ester	Ilexoside XLVIII	+	+	+
30	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinopyranoside	Mateside	+	+	−
31	3-Hydroxy-19-oxo-19,20-seco-13(18)-ursen-28,20-olide; (3β,20S)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-arabinopyranoside]	Kudinoside LZ20	+	+	+
32	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Rotungenoside	+	+	−
33	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-α-L-arabinoside	Ziyuglycoside II	−	+	+
34	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Brevicuspisaponin 3	+	−	−
35	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside]	Ilekudinoside R	−	−	+
36	3,19,23-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Ilexpernoside D	+	+	−
37	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Matesaponin 1	+	−	−
38	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside], 28-O-β-D-glucopyranosyl ester	x	−	−	+
39	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside], 28-O-β-D-glucopyranosyl ester	Latifoloside A	+	−	−
40	3,19,24-Trihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Ilexpuson F	−	+	−
41	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-β-D-glucopyranoside	x	−	−	+
42	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside]	Ilekudinoside Q	−	−	+
43	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Kudinoside A	−	+	+
44	3,23-Dihydroxy-12-ursen-28-oic acid; (3β,20α)-form, 3-O-α-L-arabinopyranoside, 28-O-β-D-glucopyranosyl ester	Mateglycoside C	+	−	+
45	3,12,19,20-Tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Kudinoside C	−	−	+
46	12-Ursene-3,11,28-triol; (3β,11α)-form, 11-ketone, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside OR 12-ursene-3,11,28-triol; (3β,11α)-form, 11-ketone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside OR ursolic acid, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside] OR ursolic acid, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside]	x	+	+	−
47	3,19-Dihydroxy-24-nor-4(23),12-ursadien-28-oic acid; (3β,19α)-form, 28-O-β-D-glucopyranosyl ester	Ilexchinenoside D	+	+	−
48	3-Hydroxy-12,19-ursadien-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-xylopyranoside], 28-O-β-D-glucopyranosyl ester	Ilexsaponin K	−	−	+
49	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Ilexside I	+	−	+
50	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	Rotundinoside J	+	−	+
51	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Ilexasprellanoside C	+	+	+
52	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside]	x	−	−	+
53	3,23-Dihydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-α-L-arabinopyranoside	x	−	+	+
54	3,19,20-Trihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Ilekudinoside G	−	−	+
55	3,19,20-Trihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, (28→20)-lactone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside]	Ilekudinoside T	−	−	+
56	3,19-Dihydroxy-12-oleanen-28-oic acid; (3β,19α)-form, 3-O-β-D-glucuronopyranoside	Ilexasprellanoside F	−	+	−
57	3,19-Dihydroxy-12-ursen-28-oic acid; (3β,19α)-form, 3-O-(2-O-Acetyl-β-D-xylopyranoside)	Ilexasprellanoside B	+	−	−
58	3-Hydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, 3-O-α-L-arabinopyranoside	x	+	−	+
59	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside]	x	+	−	−
60	3-Hydroxy-12,18-ursadien-28-oic acid; 3β-form, 3-O-β-D-xylopyranoside	Ilexasprellanoside A	+	+	+
61	3-Hydroxy-12-ursen-28-oic acid; 3β-form, 3-O-[β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside]	x	+	−	−
62	3,24-Dihydroxy-12-ursen-28-oic acid; 3β-form, 24-O-(4-hydroxy-E-cinnamoyl)	Obtusin	−	+	+
63	3,27-Dihydroxy-12-ursen-28-oic acid; 3β-form, 27-O-(4-hydroxy-E-cinnamoyl)	27-Coumaroyloxyursolic acid	+	+	+

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The composition of the sugar chain can be inferred from the loss of specific fragment ions as follows: arabinopyranosyl (Ara) or xylopyranosyl (Xyl) loss is 132 Da, rhamnopyranosyl (Rha) loss is 146 Da, glucopyranosyl (Glc) or galactopyranosyl (Gal) loss is 162 Da, and glucuronopyranosyl (Glu) loss is 176 Da. Moreover, some functional groups including methyl, acetyl, 4-hydroxy-E-cinnamoyl were observed when analyzing the fragments.

In the three Ilex species, the identified saponins were found to contain triterpenoid aglycones primarily belonging to the ursane, oleanane, and taraxastene skeleton types, including (A1) 2,3,19,23-tetrahydroxy-12-ursen-28-oic acid; (2α,3β,19α,20β)-form (23-hydroxytormentic acid), (A2) 20-taraxastene-3,28-diol; 3β-form, 28-carboxylic acid (heterobetulinic acid), (A3) 3,19,24-trihydroxy-12-oleanen-28-oic acid; (3β,19α)-form (spathodic acid), (A4) 3,19,23-trihydroxy-12-ursen-28-oic acid; (3β,19α)-form (rotundic acid), (A5) 2,3,19,24-tetrahydroxy-12-oleanen-28-oic acid; (2α,3β,19α)-form (sericic acid), (A6) 3,19-dihydroxy-12-ursen-28-oic acid; (3β,19α)-form (pomolic acid), (A7) 3,19-dihydroxy-12-ursene-24,28-dioic acid; (3β,19α)-form (Ilexgenin A), (A8) 3,19,23,30-tetrahydroxy-12-ursen-28-oic acid; (3β,19α)-form (30-hydroxyrotundic acid), (A9) 3,19-dihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form (Ilexgenin B), (A10) 3,19,23-trihydroxy-12,20(30)-ursadien-28-oic acid; (3β,19α)-form, (A11) 3,19,24-trihydroxy-12-ursen-28-oic acid; (3β,19α)-form (rotungenic acid), (A12) 3,11,19,20-tetrahydroxy-13(18)-ursen-28-oic acid; (3β,11β,19α,20β)-form, with a 28→20 lactone (γ-kudinlactone), (A13) 3,23-dihydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, (A14) hederagenin, (A15) 3-hydroxy-19-oxo-19,20-seco-13(18)-ursen-28,20-olide; (3β,20S)-form (kudinone G), (A16) 3,12,19,20-tetrahydroxy-13(18)-ursen-28-oic acid; (3β,12β,19α,20β)-form, with a 28→20 lactone (β-kudinlactone), (A17) 3,23-dihydroxy-12-ursen-28-oic acid; (3β,20α)-form, (A18) 3,19-dihydroxy-24-nor-4(23),12-ursadien-28-oic acid; (3β,19α)-form, (A19) 3-hydroxy-12,19-ursadien-28-oic acid; 3β-form (tomentosolic acid), (A20) 3,19,20-trihydroxy-12-ursen-28-oic acid; (3β,19α,20β)-form, with a 28→20 lactone, (A21) 3,19-dihydroxy-12-oleanen-28-oic acid; (3β,19α)-form (siaresinolic acid), (A22) 3-hydroxy-12,18-ursadien-28-oic acid; (3β,20β)-form, (A23) 3-hydroxy-12-ursen-28-oic acid; 3β-form (ursolic acid), (A24) 3-hydroxy-12,18-ursadien-28-oic acid; 3β-form (ilexolic acid), (A25) 3,24-dihydroxy-12-ursen-28-oic acid; 3β-form, (A26) 3,27-dihydroxy-12-ursen-28-oic acid; 3β-form (27-hydroxyursolic acid), (A27) 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, and (A28) 12-ursene-3,11,28-triol; (3β,11α)-form, with an 11-ketone.

All saponins aglycones in Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves were illustrated in Fig. 1. Their structural moieties (aglycones and sugar chains) were shown in Table 3.

Fig. 1 Chemical structures of aglycones in Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves.

Table 3 Structural moieties of identified saponins in Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves

Compound No.	Aglycone	C-3 moiety	C-28/Other moiety
1	A1	—	Glc–
2	A2	Glc–Xyl–	—
3	A3	Glu–	—
4	A4	Glc–	Glc–
5	A4	Glu–	Glc–
6	A4	Glc–	—
7	A4	Ara–	Glc–
8	A5	—	Glc–
9(A)	A27	Glc–Rha–Ara	—
9(B)			—
10	A6	Glu–	Glc–
11	A7	—	Glc–
12	A3	—	Glc–
13	A6	Glc–Ara–	Glc–
14	A8	—	Glc–
15	A27	Glc–Ara–	—
16	A9	Glc–Ara–	—
17	A10	—	Glc–
18	A9		Rha–Glc–
19	A4	—	Glc–
20	A9	Rha–Ara–	Glc–
21	A6	Rha–Ara–	Rha–Glc–
22	A11	—	Rha–Glc–
23	A6		—
24	A12		—
25	A6	Ara–methyl Glu–	Glc–
26	A6	SO3H–	Glc–
27	A13	Glu–	—
28	A12	Rha–Glc–	—
29	A14	Glu–	Glc–
30	A4	Ara–	—
31	A15		—
32	A11	—	Glc–
33	A6	Ara–	—
34	A9	Ara–	Glc–
35	A16	Rha–Ara–	—
36	A4	Glu–	—
37	A23	Glc–Ara–	Glc–
38	A6	Ara–Glu–	Glc–
39	A6	Rha–Ara–	Glc–
40	A11	Glu–	—
41	A16	Glc–	—
42	A16	Rha–Glc–	—
43	A16		—
44	A17	Ara–	Glc–
45	A16		—
46(A)	A28	Glc–Ara–Rha–	—
46(B)			—
46(C)	A23	Glc–Ara–Rha–	—
46(D)			—
47	A18	—	Glc–
48	A19	Glc–Xyl–	Glc–
49	A6	Glc–Ara–	—
50	A6	Rha–Glc–Ara–	—
51	A6	Glu–	—
52	A6	Ara–Glu–	—
53	A13	Ara–	—
54	A20		—
55	A20		—
56	A21	Glu–	—
57	A6	Acetyl Xyl–	—
58	A22	Ara–	—
59	A23	Glc–Ara–	—
60	A24	Xyl–	—
61	A23	Glc–Gal–	—
62	A25	—	(C-24) 4-Hydroxy-E-cinnamoyl-
63	A26	—	(C-27) 4-Hydroxy-E-cinnamoyl-

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Using UHPLC-ESI-QTOF-MS/MS analysis, a total of eleven saponins were identified across all three Ilex species, corresponding to compounds 10, 12, 19, 20, 22, 25, 29, 31, 51, 60, and 63. In addition, fourteen saponins were found exclusively in the leaf extract of Ilex cochinchinensis, including compounds 2, 7, 9, 13, 15–17, 27, 34, 37, 39, 57, 59, and 61. Notably, the chemical structures of compounds 9 and 15 are reported here for the first time. In the methanolic leaf extract of Ilex annamensis, five saponins – compounds 3, 8, 26, 40, and 56 – were tentatively characterized. Furthermore, eighteen additional saponins were detected in two of the three Ilex species examined. Among these, compound 46, found in both I. cochinchinensis and I. annamensis, is newly reported in terms of its chemical structure. These findings significantly expand the current knowledge of saponin diversity and distribution within the Ilex genus and provide a valuable foundation for future pharmacological and phytochemical investigations. Fragmentation pathways of compounds 9, 15, and 46 were shown in Fig. 2–4.

Fig. 2 Fragmentation of two structures of compound 9.

Fig. 3 Fragmentation of compound 15.

Fig. 4 Fragmentation of four structures of compound 46.

Compound 9 yielded [M + HCOO]⁻ ion at m/z 959.5243 and [M + NH₄]⁺ ion at 932.5582, corresponding to molecular formula C₄₇H₇₈O₁₇. In the negative mode, compound 9 provided fragments at m/z 751, 605, and 473, indicated the consecutive loss of Glc, Rha, and Ara. Additionally, in the positive mode, fragment ions at m/z 753, 607, and 475 demonstrated that the sugar chain Glc–Rha–Ara was connected to the C-3 position of the aglycone. Moreover, the aglycone ion at m/z 475 suggested an additional 18 Da compared to a reported aglycone of other Ilex species, of which name was 12-ursene-3,21,28-triol; (3β,21α)-form (this aglycone had m/z at 457 in the positive mode). The 18 Da difference was consistent with the presence of a hydroxyl group (–OH), indicating that the aglycone of compound 9 may be a tetrol, supporting the possibility of the aglycone being named 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form. As a result, compound 9 was tentatively identified as 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside] (9A) or 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranoside] (9B), of which structures have been published for the first time.

Compound 15 yielded a solvent adduct ion [M + HCOO]⁻ at m/z 813.4647 and an ammoniated ion [M + NH₄]⁺ at m/z 786.4983, corresponding to molecular formula C₄₁H₆₈O₁₃. In the negative mode, compound 15 showed fragments at m/z 605 and 473. In the positive mode, the fragment ions were at m/z 607 and 475. These data indicated the consecutive loss of a Glc and an Ara at C-3 position of the aglycone. Furthermore, this aglycone was similar with compound 9, which was possibly named as 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form. Hence, compound 15 was tentatively determined as 12-ursene-3,11,16,28-tetrol; (3β,11α,16α)-form, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside], of which structure was firstly reported.

Compound 46 yielded a solvent adduct ion [M + HCOO]⁻ at m/z 941.5124 and an ammoniated ion [M + NH₄]⁺ at m/z 914.5482, corresponding to molecular formula C₄₇H₇₆O₁₆. In the negative mode, compound 46 showed fragments at m/z 733, 601, and 455. In the positive mode, the fragment ions were at m/z 735, 603, and 457. These fragmentation patterns indicated the sequential loss of glucose (Glc), arabinose (Ara), and rhamnose (Rha) units from the C-3 position of the triterpenoid aglycone. Two possible aglycones with similar [M − H]⁻ ions at m/z 455.3528 were considered: 12-ursene-3,11,28-triol (3β,11α-isomer, with an 11-ketone group) and ursolic acid. Based on the MS/MS data, compound 46 was tentatively characterized as one of the following isomeric saponins: 12-ursene-3,11,28-triol (3β,11α), 11-ketone, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside] (46A); 12-ursene-3,11,28-triol (3β,11α), 11-ketone, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside] (46B); ursolic acid, 3-O-[β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside] (46C); or ursolic acid, 3-O-[β-D-glucopyranosyl-(1→3)-[α-L-arabinopyranosyl-(1→2)]-α-L-rhamnopyranoside] (46D). Compound 46 was identified in both Ilex cochinchinensis and Ilex annamensis, and all four of these proposed structures have been reported for the first time. The MS/MS spectra of these isomers were highly similar due to subtle differences in glycosidic linkage positions, stereochemistry, and the identical molecular formula of the aglycones, which do not produce distinctive fragment ions. Consequently, the four isomers could not be unambiguously differentiated based on MS/MS data alone.

Tyrosinase inhibitory activity evaluation

The tyrosinase inhibitory activity of leaf extracts from Ilex annamensis (IAL), Ilex cochinchinensis (ICL), and Ilex rotunda (IRL) was evaluated at a concentration of 100 μg mL⁻¹. The results (Table 4) revealed that IAL exhibited the strongest inhibitory effect at 40.70% ± 1.84, followed by ICL at 24.40% ± 1.27, while IRL demonstrated the weakest activity at 14.43% ± 1.53. Although these values are considerably lower than that of the positive control kojic acid (88.36% ± 0.57), they indicate that certain Ilex leaf extracts possess moderate natural tyrosinase inhibitory potential. The variation in activity among the three species suggests notable differences in their phytochemical profiles, particularly in bioactive constituents linked to melanogenesis regulation.

Table 4 Tyrosinase inhibitory activity of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves extracts at concentration of 100 (μg mL⁻¹)^a

Sample	Tyrosinase inhibition (%)
a According to Tukey's HSD test, all samples exhibited statistically significant differences (p ≤ 0.05).
Ilex annamensis leaves	40.70 ± 1.84
Ilex cochinchinensis leaves	24.40 ± 1.27
Ilex rotunda leaves	14.43 ± 1.53
Positive control (kojic acid)	88.36 ± 0.57

---

Saponin profiling via UHPLC-ESI-QTOF-MS/MS provided insights into these differences. Despite exhibiting the lowest tyrosinase inhibition, IRL contained 34 saponins, but none were novel. ICL, with moderate inhibition, contained 39 saponins, including three previously unreported structures. Interestingly, IAL—which showed the highest inhibitory effect—had 30 saponins, one of which was also found among the three newly identified compounds in ICL. This suggests that not only the number but also the specific structural features of saponins may significantly impact biological activity. The stronger inhibition observed in IAL implies the presence of particularly effective saponins or synergistic interactions with other bioactive compounds not yet fully characterized.

The presence of one novel saponin shared between IAL and ICL might partially explain their higher activities compared to IRL. Moreover, the comparatively lower activity of IRL despite having a similar number of saponins underscores the importance of compound identity and potency over mere quantity. These findings support a positive relationship between the presence of unique or structurally favorable saponins and tyrosinase inhibition. Future work should focus on the targeted isolation and functional validation of these compounds to better understand their mechanisms of action and potential as safer, plant-derived skin-whitening agents.

Experimental

Chemicals and reagents

Deionized water for HPLC; HPLC grade acetonitrile, HPLC grade methanol, analytical grade formic acid (≥98%) were obtained from Scharlau (Barcelona, Spain). L-3,4-dihydroxyphenylalanine (L-DOPA) and kojic acid were purchased from Sigma-Aldrich Pty Ltd. Tyrosinase from mushroom was purchased from BOSF, China. Potassium dihydrogen phosphate (KH₂PO₄) and dimethyl sulfoxide (DMSO) were obtained from Guangdong Guanghua Sci-Tech Co., Ltd.

Sample preparation

Sample collection. Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda leaves were collected from Da Lat City, Lam Dong Province, Vietnam. The botanical identification of specimens was done by Dr. Luong Van Dung from Dalat University, Vietnam. Voucher specimens of Ilex cochinchinensis (NaPro0224a), Ilex annamensis (NaPro0224b), and Ilex rotunda (NaPro0224c) were deposited in the Institute of Advanced Technology, Vietnam Academy of Science and Technology (VAST). After collection, the fresh samples were cleaned in water to remove sand and soil, left to dry naturally, cut into small pieces, and stored at room temperature until used.

Sample extraction. The leaves of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda (about 100 g each) were separately subjected to extraction with methanol for 24 hours at room temperature. The extracts were concentrated by removing the solvent using an R-300 Rotary Vacuum Evaporator (BÜCHI, Switzerland). After three times of extraction, the crude extracts were obtained. These samples were prepared at concentration of 200 ppm (in methanol) and 1000 ppm (in DMSO) for UHPLC-QTOF-MS/MS analyses and bioactivity evaluation, respectively.

UHPLC-QTOF-MS/MS analysis

An AB SCIEX X500_R QTOF mass spectrometer (AB SCIEX, USA) connected to an ExionLC™ UHPLC system (AB SCIEX, USA) system by an electrospray ionization (ESI) interface in both negative and positive ion modes, was used for analyses. A Hypersil GOLD C18 column (150 × 2.1 mm, 3 μm) (Thermo Fisher Scientific, USA) was used for sample separation at 25 °C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The elution conditions were as follows: 0–1 min, 2% B; 1–20 min, 2–98% B; 20–25 min, 98% B. The flow rate was 0.4 mL min⁻¹. The sample volume injected was set at 2.0 (μL).

The MS and MS/MS operating parameters were set as follows: the ion source temperature, 500 °C; curtain gas, 30 psi; nebulizer gas (GS 1), 45 psi; heater gas (GS 2), 45 psi. For the negative mode, ion spray voltage was set at −4.5 kV, the declustering potential (DP) was −80 V, the collision energy (CE) was performed at −20 eV, and the collision energy spread (CES) was 10 eV. For the positive mode, ion spray voltage was set at 5.5 kV, the DP was 80 V, the CE was 20 eV, and the CES was 10 eV.

The data were recorded with a SCIEX OS software version 1.2.0.4122 (AB SCIEX, USA).

Tyrosinase inhibitory activity

Tyrosinase inhibitory activity was assayed according to the method described by Wang et al. (2014)³¹ with some modifications. In summary, 20 μL of the sample was mixed with 40 μL of 50 mM KH₂PO₄ buffer and 80 μL of 150 U per mL enzyme solution. After incubation for 10 minutes at 37 °C, 60 μL of 10 mM L-DOPA was added, and the contents were re-incubated for 20 minutes before measuring the OD values at 475 nm wavelength. Kojic acid used as positive control.

Statistical analyses. The evaluation of tyrosinase inhibitory activity was calculated following the formula below:³²

%Inhibition = (ΔA_control − ΔA_sample)/ΔA_control × 100%

where ΔA_control and ΔA_sample were the change of the absorbance without and with the test sample (data was recorded at 475 nm for tyrosinase inhibitory activity).

The data of the bioactivity assays was represented as the means ± standard deviation. One-way ANOVA followed by Tukey's HSD was used to test for differences in mean values between different samples. These tests were performed by Rstudio (version 1.4.1717) software.

Conclusions

This research successfully employed UHPLC-ESI-QTOF-MS/MS to identify and differentiate saponin profiles in the leaf extracts of Ilex cochinchinensis, Ilex annamensis, and Ilex rotunda collected in Vietnam. A total of 63 saponins were tentatively identified, with 39 compounds from I. cochinchinensis, 30 from I. annamensis, and 34 from I. rotunda. Notably, seven saponins were newly reported from I. cochinchinensis, including two novel structures corresponding to compound 9 at a retention time (RT) of 11.83 minutes, one novel structure corresponding to compound 15 at RT 12.09 minutes, and four novel structures corresponding to compound 46 at RT 13.71 minutes. Among these, compound 46 was also detected in I. annamensis, whereas no new saponins were identified in I. rotunda. The distinct chemical profiles enabled clear differentiation among the three species. In addition, tyrosinase inhibitory assays revealed that I. annamensis exhibited the highest activity (40.70 ± 1.84%), followed by I. cochinchinensis (24.40 ± 1.27%) and I. rotunda (14.43 ± 1.53%). Interestingly, the number of identified saponins did not directly correlate with tyrosinase inhibition activity, suggesting that bioactivity may depend more on specific saponin structures rather than total saponin content. These findings contribute valuable insight into the phytochemical diversity and biological potential of Ilex species in Vietnam and suggest that I. annamensis may serve as a promising natural source for the development of tyrosinase inhibitors in cosmetic and dermatological applications. While the findings are promising, several limitations should be considered, including tentative structural assignments without NMR confirmation, the assessment of tyrosinase inhibition at only a single concentration for comparative screening, and the need for further investigation to clarify the relationship between specific saponin constituents and bioactivity. Future studies should focus on the isolation and structural elucidation of novel saponins using NMR spectroscopy, followed by bioactivity evaluation on pure compounds, in order to confirm new compounds and bioactive candidates through MS-guided approaches.

Author contributions

Pham Hong Ngoc: performed UHPLC-ESI-QTOF-MS/MS analysis, conducted tyrosinase inhibitory assays and statistical analysis, and contributed to manuscript writing. Tran Chieu An: conducted a literature review of previously reported phytochemicals, performed UHPLC-ESI-QTOF-MS/MS analysis, and contributed to manuscript writing. Luong Van Dung: collected plant samples from the field and performed taxonomic identification. Le Tien Dung: co-supervised the research, contributed to manuscript revision and project coordination. Phung Van Trung: supervised the overall project, coordinated all research activities, and critically revised the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

All UHPLC-ESI-QTOF-MS data, including TICs, MS and MS/MS spectra supporting the first report of saponins in this work, are provided in the supplementary information (SI). Supplementary information: MS and MS/MS data for all detected peaks are summarized in Table 1. See DOI: https://doi.org/10.1039/d5ra05096k.

Acknowledgements

This research was funded by Institute of Advanced Technology – Vietnam Academy of Science and Technology (VAST) under the grant number CSCL19.03/24-25. This research was also supported by Vietnam Academy of Science and Technology (VAST) under grand Excellent Research Group Development Program NCXS02.03/25-26.

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