Anti-Lymphangiogenic Terpenoids from the Heartwood of Taiwan Juniper, Juniperus chinensis var. tsukusiensis

Abstract

To look in-depth into the phytochemical and pharmacological properties of Taiwan juniper, this study investigated the chemical profiles and anti-lymphangiogenic activity of Juniperus chinensis var. tsukusiensis. In this study, four new sesquiterpenes, 12-acetoxywiddrol (1), cedrol-13-al (2), α-corocalen-15-oic acid (3), 1,3,5-bisaoltrien-10-hydroperoxy-11-ol (4), one new diterpene, 1β,2β-epoxy-9α-hydroxy-8(14),11-totaradiene-3,13-dione (5), and thirty-three known terpenoids were successfully isolated from the heartwood of J. chinensis var. tsukusiensis. The structures of all isolates were determined through the analysis of physical data (including appearance, UV, IR, and optical rotation) and spectroscopic data (including 1D, 2D NMR, and HRESIMS). Thirty-four compounds were evaluated for their anti-lymphangiogenic effects in human lymphatic endothelial cells (LECs). Among them, totarolone (6) displayed the most potent anti-lymphangiogenic activity by suppressing cell growth (IC₅₀ = 6 ± 1 µM) of LECs. Moreover, 3β-hydroxytotarol (7), 7-oxototarol (8), and 1-oxo-3β-hydroxytotarol (9) showed moderate growth-inhibitory effects on LECs with IC₅₀ values of 29 ± 1, 28 ± 1, and 45 ± 2 µM, respectively. Totarolone (6) also induced a significant concentration-dependent inhibition of LEC tube formation (IC₅₀ = 9.3 ± 2.5 µM) without cytotoxicity. The structure–activity relationship discussion of aromatic totarane-type diterpenes against lymphangiogenesis of LECs is also included in this study. Altogether, our findings unveiled the promising potential of J. chinensis var. tsukusiensis in developing therapeutics targeting tumor lymphangiogenesis.

1. Introduction

Lymphangiogenesis is the process of new lymphatic vessels developing from pre-existing lymphatic vasculature. It plays a role in diverse physiological scenarios, such as maintaining homeostasis, supporting the immune system, contributing to embryonic development, and aiding in wound healing. Conversely, in pathological contexts, this process is often associated with issues like organ graft rejection, lymphedema, and even cancer spread [1]. Based on the annual statistics reported by the World Health Organization, cancer is a primary contributor to mortality and a significant impediment to raising life expectancy in every nation [2]. It is worth noting that around 90% of cancer-related deaths are caused by metastatic tumor spread. Tumor lymphangiogenesis has consequently emerged as a pivotal prognostic role for cancer patients [3]. Given this understanding, developing targeted cancer therapies with anti-lymphangiogenic activity is a promising strategy to enhance patient survival rates.

The Juniperus species (Cupressaceae) are coniferous plants encompassing evergreen trees and shrubs, widely distributed throughout the cold temperate regions of the Northern Hemisphere to Tropical Africa [4]. The aromatic nature of Juniperus is due to pine oil and resin within the plant. These compounds contribute to the unique scent and flavor associated with juniper berries. Furthermore, the high resin content makes Juniperus wood more resistant to decay and infestation, making it a valuable material in woodworking projects. Some characteristic phytochemicals have been isolated from Juniperus, including acetophenones, monoterpenes, sesquiterpenes, diterpenes, flavonoids, lignans, and phenylpropanoides, mainly terpenoids [5,6,7,8,9,10,11,12,13,14,15,16,17]. The representative terpenoids in Juniperus include α-pinene, camphene, β-pinene, sabinene, myrcene, limonene, imbricatolic acid, junicedral, trans-communic acid, and isocupressic acid [18,19]. The cytotoxic lignan compounds, podophyllotoxin and deoxypodophyllotoxin, could also be found in some Juniperus species [20]. One of the most famous Juniperus plants, J. communis Linn., is widely used as a diuretic, antiseptic, leucorrhea, and treating gastrointestinal problems in folk medicine [4,21]. It also presents various pharmacological potentials for anti-inflammatory [22,23,24,25], antifungal [24,25,26], hepatoprotective [18,27], neuroprotective [19], anti-diabetic and anti-hyperlipidemic [28,29], and anti-proliferative activities [30,31,32,33,34]. Due to the distinctive phytochemical composition and promising pharmacological activities exhibited by Juniperus plants, our interest in researching them has been sparked. Our previous studies on the J. chinensis var. tsukusiensis revealed some characteristic Juniperus sesquiterpenoids [35,36]. However, the biological activity of J. chinensis var. tsukusiensis has never been studied, which prompted us to further explore its bioactive ingredients.

In this study, a series of isolation and purification procedures were implemented, resulting in five new compounds (1–5) (Figure 1) and thirty-three known terpenoids (6–38) from the heartwood of J. chinensis var. tsukusiensis. Terpenoids are renowned for their cytotoxic effects on various types of tumors. Interestingly, in the previous investigation, it was discovered that diterpenes possess anti-lymphangiogenic properties, which is a new concept in targeting tumor cell metastasis [37,38,39]. To further explore the relationship between terpenoids and their anti-lymphangiogenic activity, as well as to expand the pharmacological profile of terpenoids, we evaluated the anti-lymphangiogenic activity of the isolated compounds in our study. Among these compounds, 6, 7, 8, and 9 demonstrated significant anti-lymphangiogenic activity.

2. Results

2.1. Structure Elucidation

Compound 1 was obtained as a colorless, viscous oil with optical rotation. Its molecular formula was determined as C₁₇H₂₈O₃ from HRESIMS data (m/z 281.2101 [M+H]⁺ (calcd. for 281.2117)), implying four degrees of unsaturation. The infrared spectroscopy (IR) spectrum showed typical absorptions of C=O (1742 cm⁻¹) for the ester group and hydroxy group (3433 cm⁻¹). The ¹H NMR spectrum (

Table 1. ¹H NMR data of compounds 1–5 in CDCl₃.

Position	1 a	2 a	3 b	4 b	5 b
	δH (mult, J in Hz)	δH, mult (J in Hz)	δH, mult (J in Hz)	δH, mult (J in Hz)	δH, mult (J in Hz)
1			8.03 (s)	7.05 (d, 7.8)	3.64 (d, 4.6)
2	1.23 (m) 1.49 (m)	1.62 (m)		7.06 (d, 7.8)	3.38 (d, 4.6)
3	1.48 (m) 1.72 (m)	1.23 (m) 1.88 (m)	2.55 (t, 8.0)
4	1.32 (m) 1.79 (m)	1.24 (m) 1.80 (m)	2.79 (t, 8.0)	7.06 (d, 7.8)
5		1.9 (t, 7.6) (β)		7.05 (d, 7.8)
6			7.07 (d, 8.0)		1.38 (dd, 13.3, 4.0) (β) 1.81 (m) (α)
7	5.41 (dd, 8.8, 6.0)	2.26 (d, 4.4)	7.12 (d, 8.0)	2.63 (sex, 7.2)	2.58 (m) (β) 2.94 (m) (α)
8	2.06 (dd, 14.0, 8.8) (β) 2.46 (dd, 14.0, 6.0) (α)			1.58 (m) 1.84 (m)
9		1.71 (dt, 5.6, 1.6) (β) 1.82 (m) (α)		1.28 (m) 1.32 (m)
10	1.33 (m) 1.62 (m)	1.35 (m) (β) 1.44 (m) (α)		3.26 (dd, 10.0, 2.4)
11	1.24 (m) 1.41 (m)	1.42 (m) 1.76 (m)	3.33 (sept, 6.8)		7.01 (d, 10.0)
12	3.99 (s)	1.31 (s)	1.23 (d, 6.8)	1.08 (s)	6.28 (d, 10.0)
13	1.07 (s)	9.42 (s)	1.23 (d, 6.8)	1.11 (s)
14	1.04 (s)	1.41 (s)	2.26 (s)	1.21 (d, 7.2)
15	1.20 (s)	0.84 (d, 7.2)		2.29 (s)	3.22 (sept, 7.2)
16					1.23 (d, 7.2)
17	2.10 (s)				1.21 (d, 7.2)
18					1.20 (s)
19					0.98 (s)
20					0.71 (s)

^a Data measured at 300 MHz. ^b Data measured at 400 MHz.

) displayed signals of four singlet methyl groups at δ_H 1.04 (3H, s, H-14), 1.07 (3H, s, H-13), 1.20 (3H, s, H-15), and 2.10 (3H, s, OAc), one oxymethylene at δ_H 3.99 (2H, s, H-12), and one trisubstituted olefinic proton at δ_H 5.41 (1H, dd, J = 8.8, 6.0 Hz, H-7). The ¹³C NMR (

Table 2. ¹³C NMR data of compounds 1–5 in CDCl₃.

Position	1 a	2 a	3 b	4 b	5 b
	δC	δC	δC	δC	δC
1	39.8	53.7	135.3	126.8	60.9
2	41.4	41.0	127.7	129.1	54.5
3	18.6	37.8	21.0	135.3	211.0
4	38.1	25.9	24.6	129.1	44.8
5	36.9	58.0	132.6	126.8	38.6
6	155.2	57.6	122.9	144.4	24.0
7	116.3	53.3	132.0	39.7	26.6
8	34.4	73.8	144.6	35.5	153.7
9	73.8	34.6	129.1	29.9	74.2
10	32.7	42.6	136.0	78.8	46.7
11	40.5	30.8	28.5	73.1	144.9
12	70.2	30.2	23.8	23.1	131.3
13	31.7	205.6	23.8	26.5	184.6
14	32.9	19.3	19.4	22.4	140.0
15	27.5	15.4	172.3	20.9	26.6
16	171.3				21.5
17	20.9				21.4
18					28.4
19					20.8
20					13.9

^a Data measured at 75 MHz. ^b Data measured at 100 MHz.

) and DEPT spectra showed seventeen resonances comprising four methyls, seven methylenes, one methine, and five quaternary carbons. From the ¹H and ¹³C NMR spectra, one C=O group (δ_C 171.3, C-16) and one C=C unit [δ_C 116.3 (C-7), 155.2 (C-6)] accounted for two of four degrees of unsaturation. Thus, compound 1 was suggested to be a bicyclic sesquiterpene with an acetyl group. The existence of the acetoxy methyl group was confirmed by the HMBC correlation (Figure 2) from H-17 to C-16 and the absorption of the ester group (1742 cm⁻¹) in the IR spectrum. Comparing the ¹H and ¹³C NMR data of 1 to those of the literature compound, widdrol [6], they shared similar structures, except for the oxidation of C-12 and an additional acetyl group in 1. Further HMBC correlation (Figure 2) between H-12 and C-16 allowed the acetoxy group to be located at C-12. The relative stereochemistry of 1 was assigned using the information provided by the NOESY spectrum (Figure 2) and compared with the literature compound, widdrol [6]. The NOESY correlations (Figure 2) between H-13/H-8α and H-15, and H-12/H-8β, confirmed that OH-9, H-13, and H-15 were in the same phase, while H-12 and H-14 were on the same side. Thus, compound 1 was determined and named 12-acetoxywiddrol.

Compound 2 was isolated as an optical, viscid oil, and displayed a pseudo-molecular ion at m/z 237.1848 [M+H]⁺ (calcd. for C₁₅H₂₅O₂, 237.1855) by HRFABMS with four degrees of unsaturation. Its IR spectrum showed hydroxy and aldehyde groups at 3403 cm⁻¹ and 1711 cm⁻¹, respectively. The ¹H NMR data (Table 1) depicted signals of three methyls at δ_H 0.84 (3H, d, J = 6.9 Hz, H-15), 1.31 (3H, s, H-12), and 1.41 (3H, s, H-14), and an aldehyde group with low field chemical shift at δ_H 9.42 (1H, s, H-1). Further ¹³C NMR (Table 2) and DEPT spectra identified three methyls, five methylenes, three methines, and four quaternary carbons, including one oxygenated quaternary carbon (δ_C 73.8) and one aldehyde (δ_C 205.6). According to the above data, the structure of 2 was assumed to be a three-membered ring sesquiterpene with an aldehyde group. A detailed comparison of the NMR data of 2 to those of 8β,13-dihydroxycedrane [40] revealed that these two compounds were structure analogous, except for the hydroxymethyl group of 8β,13-dihydroxycedrane was changed to an aldehyde group in compound 2. The HMBC correlations (Figure 3) from H-14 to C-5 (δ_C 58.0), C-6 (δ_C 57.6), C-7 (δ_C 53.3), C-13 (δ_C 205.6), and H-13 (δ_H 9.42, 3H, s, H-13) to C-6 indicated that the aldehyde group was located at C-6. In the HMBC spectrum, the correlations of H-12/C-7, C-8 (δ_C 73.8), and C-9 (δ_C 34.6) verified the hydroxy group was attached at C-8. The planar structure of 2 was further supported by HMBC and COSY correlations, as shown in Figure 2. Further NOESY correlations (Figure 3) between H-15/H-10, H-14/H-5, H-9, and H-12/H-13 supported the relative configuration of 2 was the same as 8β,13-dihydroxycedrane [40]. Based on the above data, the structure of compound 2 was determined and named cedrol-13-al.

Compound 3 was yielded as a whitish solid. Analysis by HREIMS of 3 indicated a molecular formula of C₁₅H₁₈O₂, representing seven IHDs. The UV spectrum showed maximum absorptions at 213, 231, and 295 nm. The IR absorption bands at 1622, 1600, and 1485 cm⁻¹, as well as the observation of featuring carbon resonances (Table 2), confirmed the existence of an aromatic ring [δ_C 122.9 (C-6), 129.1 (C-9), 132.0 (C-7), 132.6 (C-5), 136.0 (C-10), 144.6 (C-8)] and a double bond [δ_C 127.7 (C-2), 135.3 (C-1)]. The carboxylic acid group was observed both in IR (1682 cm⁻¹) and ¹³C NMR [δ_C 172.3 (C-15)] spectra. The IR spectrum also revealed the double bond (1622 cm⁻¹) and aromatic ring (1600 and 1485 cm⁻¹). The ¹H NMR spectrum (Table 1) of 3 exhibited one methyl group bearing with an aromatic ring at δ_H 2.26 (3H, s, H-14), an isopropyl group at δ_H 1.23 (6H, d, J = 6.8 Hz, H-12 and H-13), 3.33 (1H, sept, J = 6.8 Hz, H-11), an ortho-coupling aromatic ring at δ_H 7.07 (1H, d, J = 8.0 Hz, H-6), 7.12 (1H, d, J = 8.0 Hz, H-7), a trisubstituted olefinic proton at δ_H 8.03 (1H, s, H-1), and two methylene groups at δ_H 2.55 (2H, t, J = 8.0 Hz, H-3), 2.79 (2H, t, J = 8.0 Hz, H-4). The HMBC experiment (Figure 4) for 3 revealed correlations between H-1/C-2, C-8, C-9, and C-10, and H-3/C-1, C-4, and C-10, confirming the 3,4-dihydronaphthalene skeleton of 3. The HMBC correlations between H-14/C-5, C-6, and C-10 verified the methyl group (C-14) was attached to C-5 (Figure 4). The isopropyl group was located at C-8 based on the HMBC correlations between H-11/C-7 and H-13/C-8 (Figure 4). Finally, the carboxylic acid group gave cross-peak to a correlation with H-1, suggesting the carboxylic acid group was bearing with C-2. Therefore, the structure of 3 was defined as shown and named α-corocalen-15-oic acid.

Compound 4 was purified as a colorless, viscous oil. Its molecular formula of C₁₅H₂₄O₃ was determined by HRESIMS (m/z 253.1802, calcd. for 253.1804), indicating four degrees of unsaturation. Its IR spectrum depicted a hydroxy group at 3410 cm⁻¹. The ¹³C NMR (Table 2) and DEPT spectra identified four methyls [δ_C 20.9 (C-15), 22.4 (C-14), 23.1 (C-12), 26.5 (C-13)], an aromatic ring [δ_C 126.8 (C-5), 129.1 (C-4), 135.3 (C-3), 144.4 (C-6)], one oxymethine [δ_C 78.8 (C-10)], one oxygenated quaternary carbon [δ_C 73.1 (C-11)], two methylenes [δ_C 29.9 (C-9), 35.5 (C-8)], and one methine [δ_C 39.7 (C-7)]. Further ¹H NMR spectrum (Table 1) confirmed the substituted pattern of the para-substituted aromatic ring [δ_H 7.05 (2H, d, J = 7.8 Hz, H-1 and H-5), 7.06 (2H, d, J = 7.8 Hz, H-2 and H-4)] attaching with a low field methyl group [δ_H 2.29 (3H, s, H-15)]. The COSY correlation (Figure 4) between H-7/H-14 and H-9/H-10 revealed the existence of fragments C-7–C-14 and C-9–C-10, respectively. The cross-peak between H-14 (δ_H 1.21, d, J = 7.2 Hz)/C-7, C-8 (δ_C 35.5), H-7 (δ_H 2.63, sex, J = 7.2 Hz)/C-8, C-9, H-8β (δ_H 1.58, m)/C-9, C-10, and H-13 (δ_H 1.11, s)/C-10, C-11, and C-12 in the HMBC spectrum (Figure 4) suggested the existence of the side-chain group. Further analysis of the HMBC correlations (Figure 4) between H-7/C-1, C-5, and C-6 showed that the location of the side-chain group was connected with the aromatic ring at C-6. According to the carbon and HRESIMS spectra, compound 4 contained two oxygenated carbons (C-10 and C-11) with three oxygen atoms, implying the existence of a hydroxy and a hydroperoxy group. Based on the chemical shift of H-10 (δ_H 3.26), the hydroxy group was attached to C-10, and the hydroperoxy group was connected with C-11, respectively. The structure of 4 was accordingly confirmed and named 1,3,5-bisaoltrien-10-hydroperoxy-11-ol.

Compound 5 was obtained as a colorless crystal with optical rotation. A molecular formula of C₂₀H₂₆O₄ was determined for this compound by HRESIMS (m/z 331.1906, calcd. for 331.1909), with eight degrees of unsaturation. The IR spectrum of 5 showed IR absorption bands at 3417 cm⁻¹ for a hydroxy group, at 1699 cm⁻¹ for one carbonyl group [δ_C 211.0 (C-3)], and at 1669 cm⁻¹ for one conjugated carbonyl group [δ_C 184.6 (C-13)], which was supported by analysis of the ¹³C NMR spectrum (Table 2). The IR spectrum also revealed the presence of C=C double bonds (1631, 990, 840 cm⁻¹). The ¹H NMR spectrum of 5 exhibited three singlet methyl groups [δ_H 0.71 (3H, s, H-20), 0.98 (3H, s, H-19), and 1.20 (3H, s, H-18)], an isopropyl group [δ_H 1.21 (3H, d, J = 7.2 Hz, H-17), 1.23 (3H, d, J = 7.2 Hz, H-16)], and 3.22 (1H, sept, J = 7.2 Hz, H-15)], two oxymethines [δ_H 3.38 (1H, d, J = 4.6 Hz, H-2), 3.64 (1H, d, J = 4.6 Hz, H-1)], and double-bond signals [δ_H 6.28 (1H, d, J = 10.0 Hz, H-12), 7.01 (1H, d, J = 10.0 Hz, H-11)]. The ¹³C NMR and DEPT spectra indicated the presence of five methyls [δ_C 13.9 (C-20), 20.8 (C-19), 21.4 (C-17), 21.5 (C-16), 28.4 (C-18)], two oxymethines with epoxy ring [δ_C 54.5 (C-2), 60.9 (C-1)], one oxygenated quaternary carbon [δ_C 74.2 (C-9)], two pairs of double bonds [δ_C 131.3 (C-12)/144.9 (C-11), 144.0 (C-14)/153.7 (C-8)], two carbonyl groups [δ_C 184.6 (C-13), 211.0 (C-3)], two quaternary carbon [δ_C 44.8 (C-4), 46.7 (C-10)], two methines [δ_C 26.6 (C-15), 38.6 (C-55)], and two methylenes [δ_C 24.0 (C-6), 26.6 (C-7)]. From the above spectroscopic data, 5 was assumed to be a totarane diterpene similar to totarolone [41], except for an additional epoxy group at C-1/C-2, a hydroxy group at C-9, and the aromatic ring in totarolone was changed to a cyclohexa-2,5-dien-1-one ring in 5. The HMBC correlations (Figure 5) between H-20/C-1; H-1/C-2, C-5, and C-10; and H-2/C-3, C-4 indicated C-1 and C-2 were connected with epoxy group. Moreover, the cis disposition of H-1/H-2 was confirmed by the small coupling constant of H-1/H-2 (J = 4.6 Hz). The hydroxy group was located at C-9 from HMBC correlations of H20/C-9 and H-12/C-9. The existence of the cyclohexa-2,5-dien-1-one unit was verified based on the HMBC correlations between H-11/C-8, C-13 and H-12/C-9, C-14. The NOESY correlation (Figure 5) between H-19/H-6β, H-20 and H-5/H-6α, H-18, indicating H-19 and H-20 were in axial position. The NOESY correlations of H-1/H-2 and H-11 suggested that H-1, H-2, and H-11 were in the same phase (Figure 5). Additionally, the epoxy group and OH-9 were at the α-position based on the ring junction afforded by NOESY correlations. Thus, the structure of compound 5 was elucidated as 1β,2β-epoxy-9α-hydroxy-8(14),11-totaradiene-3,13-dione.

By comparing the spectroscopic data ([a]_D, UV, IR, NMR, and MS) of known compounds with the literature data, the known diterpenes were identified to be totarolone (6) [41], 3β-hydroxytotarol (7) [42], 7-oxototarol (8) [41], and 1-oxo-3β-hydroxytotarol (9) [41], 5,6-dehydrosugiol methyl ether (10) [43], sandaracopimaric acid (11) [35], 3,18-dihydroxypimara-8(14),15-diene (12) [44], secoabietane dialdehyde (13) [43], communic acid (14) [43], and (12R, 13S)-dihtdroxylabda-8(17),14-dien-19-oic acid (15) [45]. Additionally, the known sesquiterpenes were identified to be 2-himachalen-6-ol (16) [36], 3-himachalen-6-ol (17) [36], chinensiol (18) [36], ar-himachalene (19) [36], 12-hydroxy-α-longipinene (20) [35], 15-hydroxyacora-4(14),8-diene (21) [35], cedrol (22) [35], 8α,12-cedranediol (23) [46], 8-cedren-13-ol (24) [8], epicedrane-diol (25) [47], cedrol-13-oic acid (26) [48], 13-acetoxycedrol (27) [48], widdrol (28) [35], (+)-α-bisabolol (29) [7], sesquithuriferol (30) [8], 15-hydroxyallo-cedrol (31) [7], thujopsenal (32) [35], hinokiic acid (33) [35], mayurone (34) [49], 4α,5α-epoxymayurone (35) [50], 8β-hydroxythujopsan-9-one (36) [51], nootkatone (37) [52], and γ-cuparanol (38) [53].

2.2. Anti-Lymphangiogenic Effects of Compounds Isolated from J. chinensis var. tsukusiensis

The role of lymphangiogenesis in tumor development, progression, and metastasis has been well-documented in various human cancers. Therefore, our study focused on examining the anti-lymphangiogenic effects of 34 compounds in human lymphatic endothelial cells (LECs). As shown in Figure 6, compounds 6–9 showed growth-inhibitory effects on LECs (the percentage of LECs growth < 60%). These active isolates were further evaluated for their IC₅₀ values of anti-lymphangiogenic activity (

Table 3. Anti-lymphangiogenic activity of selected compounds in human LECs.

Compound	IC50 (µM)
totarolone (6)	6 ± 1
3β-hydroxytotarol (7)	29 ± 1
7-oxototarol (8)	28 ± 1
1-oxo-3β-hydroxytotarol (9)	45 ± 2
Rapamycin #	<10

LECs were treated with compounds 6–9 for 48 h, and anti-lymphangiogenic effects were elucidated in a cell growth assay (n = 3). Data were expressed as the mean ± SEM. ^# Rapamycin was used as a positive control.

). As shown in Table 3, totarolone (6) exhibited the most potent anti-lymphangiogenic activity by suppressing LEC growth (IC₅₀ = 6 ± 1 µM), with rapamycin as the positive control. 3β-Hydroxytotarol (7), 7-oxototarol (8), and 1-oxo-3β-hydroxytotarol (9) showed moderate growth-inhibitory effects on LECs with IC₅₀ values of 29 ± 1, 28 ± 1, and 45± 2 µM, respectively.

To confirm the anti-lymphangiogenic effects of the active compounds, we proceeded with the capillary tube formation assay. As illustrated in Figure 7, compound 6 induced the promising anti-lymphangiogenesis property by disrupting LECs tube formation in a concentration-dependent manner (IC₅₀ = 9.3 ± 2.5 µM). Furthermore, it was observed that compound 6 did not increase the levels of lactate dehydrogenase (LDH) in LECs. The results indicate that compound 6 exerts significant anti-lymphangiogenic effects without cytotoxic fashion.

3. Discussion

In this study, compounds isolated from the heartwood of J. chinensis var. tsukusiensis can be categorized into sesquiterpenoids and diterpenoids. Among the tested compounds, only aromatic totarane-type diterpenes depicted anti-lymphangiogenic effects in human LECs. An in-depth discussion of the structure–activity relationship (SAR) in tested compounds was raised. Totarolone (6) showed better anti-lymphangiogenic activity than 7-oxototarol (8), suggesting that the substituted position of the carbonyl group is crucial for anti-lymphangiogenic activity. Surprisingly, 3β-hydroxytotarol (7), a compound reduction by the 3-carbonyl group in totarolone (6), exhibits lower anti-lymphangiogenic activity than totarolone (6). Moreover, 3β-hydroxytotarol (7) provided better anti-lymphangiogenic activity than 1-oxo-3β-hydroxytotarol (9), implying that 3-carbonyl substituent on aromatic totarane-type diterpene may attenuate the anti-lymphangiogenic activity.

4. Material and Methods

4.1. General Experiment Procedures

Optical rotations were measured on a Jasco DIP-1000 Digital polarimeter (Jasco, Kyoto, Japan), and IR spectra (neat) were acquired with a Perkin-Elmer 983G spectrometer (Perkin-Elmer, Waltham, MA, USA). The 1D (¹H, ¹³C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra were obtained from a Burcher DMX-300 spectrometer (Bruker Inc., Bremen, Germany) operated at 300 (¹H) and 75 MHz (¹³C), Varian Unityplus-400 spectrometer (Varian, Inc. Vacuum Technologies, Lexington, MA, USA) operated at 400 (¹H) and 100 MHz (¹³C). The HRESIMS data were generated at the Mass Spectrometry Laboratory by Orbitrap QE Plus Mass Spectrometry (Thermo Fisher Scientific, Inc., Bremen, Germany). Extracts were chromatographed on silica gel (70–230 mesh, 230–400 mesh, Merk, Darmstadt, Germany, ASTM) and purified with a semipreparative normal-phase HPLC column (Merck LichroCART 250 mm × 10 mm, 7 µm, LiChrosorb Si 60, Merck, Darmstadt, Germany) taken on LDC Analytical-III.

4.2. Plant Material

The heartwood of Juniperus chinensis Linn. var. tsukusiensis Masam. was collected in Chingshui Mountain, Hualien, Taiwan, in October 1990 and identified by Dr. Sheng-yYou Lu at the Taiwan Forestry Research Institute.

4.3. Extraction and Isolation

The dried heartwood of J. chinensis var. tsukusiensis (4 kg) was extracted at room temperature with MeOH (80 L) four times (4 days for each time) to yield an MeOH extract that was partitioned between n-hexane and H₂O (1:1) to provide an n-hexane-soluble (230 g) and an H₂O-soluble layer. The n-hexane-soluble layer was subjected to column chromatography (silica gel; 2.3 kg; started from 100% n-hexane, then washed with EtOAc in gradient mode, and finally washed with 100% EtOAc, 100% acetone, and 100% methanol, respectively) to yield 15 subfractions (Fr. A–~Fr. O). Fr. J was subjected to an HPLC column (silica gel; n-hexane/dichloromethane/EtOAc = 75/5/20) to obtain compound 2 (21.3 mg). Fr. L was separated with an HPLC column (silica gel; n-hexane/dichloromethane/EtOAc = 60/20/20) to gain compounds 4 (3.4 mg) and 5 (4.0 mg). Fr. M was purified on an HPLC column (silica gel; n-hexane/dichloromethane/EtOAc = 40/20/40) to furnish compound 1 (11.5 mg). Fr. N was re-chromatographed on an HPLC column (silica gel; n-hexane/dichloromethane/EtOAc = 20/20/60) to afford compound 3 (3.2 mg). The purification process of known compounds can be retrieved from the Supplementary Information (Table S1).

4.4. Spectroscopic Data of Compounds

4.4.1. 12-Acetoxywiddrol (1)

Viscid oil. [α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$: +71.3 (c 1.0, CHCl₃). IR v_max: 3433 (OH), 1742 (C=O) cm⁻¹. ¹H NMR (CDCl₃, 300 MHz): see Table 1. ¹³C NMR (CDCl₃, 75 MHz): see Table 2.

4.4.2. Cedrol-13-al (2)

Viscid oil. [α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$: −35.3 (c 1.9, CHCl₃). IR v_max: 3403 (OH), 1711 (C=O) cm⁻¹. ¹H NMR (CDCl₃, 300 MHz): see Table 1. ¹³C NMR (CDC1₃, 75 MHz): see Table 2. HRESIMS m/z: 237.1848 [M+H]⁺ (calcd. for C₁₅H₂₅O₂, 237.1855).

4.4.3. α-Corocalen-15-oic Acid (3)

Whitish solid. IR v_max: 3300–2500 (OH), 1682 (C=O), 1622 (C=C), 1600, 1485 (aromatic ring) cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): see Table 1. ¹³C NMR (CDC1₃, 100 MHz): see Table 2. HRESIMS m/z: 231.1831 [M+H]⁺ (calcd. for C₁₅H₁₉O₂, 231.1835).

4.4.4. 1,3,5-Bisaoltrien-10-hydroperoxy-11-ol (4)

Viscid oil. [α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$: +6.0 (c 0.3, CHCl₃). IR v_max: 3410 (OH), 1514 (aromatic ring) cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): see Table 1. ¹³C NMR (CDCl₃, 100 MHz): see Table 2. HRESIMS m/z: 253.1802 [M+H]⁺ (calcd. for C₁₅H₂₅O₃, 253.1804).

4.4.5. 1β,2β-Epoxy-9α-hydroxy-8(14),11-totaradiene-3,13-dione (5)

Colorless crystal. [α]$\begin{array}{c}25\\ \mathrm{D}\end{array}$: +79.4 (c 0.4, CHCl₃). IR v_max: 3417 (OH), 1699, 1669 (C=O), 1631 (C=C) cm⁻¹. ¹H NMR (CDCl₃, 400 MHz): see Table 1. ¹³C NMR (CDC1₃, 75 MHz): see Table 2. HRESIMS m/z: 331.1906 [M+H]⁺ (calcd. for C₂₀H₂₇O₄, 331.1909).

4.5. Anti-Lymphangiogenic Assay

The methods employed for cell culture, cell growth, tube formation, and cytotoxicity assessments of human lymphatic endothelial cells were consistent with our previous work [54].

5. Conclusions

Phytochemical investigation of the heartwood of J. chinensis var. tsukusiensis led to 38 compounds, including 5 new compounds and 33 known compounds in this study. The chemical components of Juniperus species have been well-studied; however, more studies focus on J. communis rather than J. chinensis var. tsukusiensis. The phytochemical findings in this study not only contribute to identifying additional natural sources of terpenoids but also enhance our understanding of the chemical profiles of J. chinensis var. tsukusiensis. Additionally, 34 isolates from J. chinensis var. tsukusiensis screened their anti-lymphangiogenic effects in human lymphatic endothelial cells. It is noticeable that this is the first report on the anti-lymphangiogenic activities of J. chinensis var. tsukusiensis and demonstrates the potential of totarolone (6), 3β-hydroxytotarol (7), 7-oxototarol (8), and 1-oxo-3β-hydroxytotarol (9) to develop therapeutics against tumor lymphangiogenesis.