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

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.


Introduction
Lymphangiogenesis is the process of new lymphatic vessels developing from preexisting 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.
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  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.

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 1 H NMR spectrum (Table 1) displayed signals of four singlet methyl groups at δH 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 δH 3.99 (2H, s, H-12), and one trisubstituted olefinic proton at δH 5.41 (1H, dd, J = 8.8, 6.0 Hz, H-7).The 13 C NMR (Table 2) and DEPT spectra showed seventeen resonances comprising four methyls, seven methylenes, one methine, and five quaternary carbons.From the 1 H and 13 C NMR spectra, one C=O group (δC 171.3, C-16) and one C=C unit [δC 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 1 H and 13 C 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.

Structure Elucidation
Compound 1 was obtained as a colorless, viscous oil with optical rotation.Its molecular formula was determined as C 17 H 28 O 3 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 1 H NMR spectrum (Table 1) 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 13 C NMR (Table 2) and DEPT spectra showed seventeen resonances comprising four methyls, seven methylenes, one methine, and five quaternary carbons.From the 1 H and 13 C 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 1 H and 13 C 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 C 15 H 25 O 2 , 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 1 H 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 13 C 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.

Cell growth of LECs (%)
Figure 6.Anti-lymphangiogenic effects of compounds in human LECs.LECs were treated with the indicated compounds at a concentration of 50 µM for 48 h, and anti-lymphangiogenic effects were elucidated in a cell growth assay (n = 3).Data were expressed as the mean ± SEM.Table 3. Anti-lymphangiogenic activity of selected compounds in human LECs.
To confirm the anti-lymphangiogenic effects of the active compounds, we proceeded  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.
Plants 2023, 12, 3828 9 of 13 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 (IC 50 = 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.

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.

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].

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.

Figure 6 .
Figure 6.Anti-lymphangiogenic effects of compounds in human LECs.LECs were treated with the indicated compounds at a concentration of 50 µM for 48 h, and anti-lymphangiogenic effects were elucidated in a cell growth assay (n = 3).Data were expressed as the mean ± SEM.

Plants 2023 , 13 Figure 7 .
Figure 7. Effects of compound 6 on tube formation and cytotoxicity in human LECs.(A) Cells were treated with compound 6 (10 and 20 µM) for 8 h, and tubular morphogenesis was recorded by an inverted phase-contrast microscope (n = 3).ImageJ 1.54a software was used to quantify the length of capillary-like tubes.(B) LECs were treated with compound 6 (10 and 20 µM), then the cytotoxicity was evaluated by the LDH assay (n = 3).Data are expressed as the mean ± SEM. * p < 0.05 compared with the control group.

Figure 7 .
Figure 7. Effects of compound 6 on tube formation and cytotoxicity in human LECs.(A) Cells were treated with compound 6 (10 and 20 µM) for 8 h, and tubular morphogenesis was recorded by an inverted phase-contrast microscope (n = 3).ImageJ 1.54a software was used to quantify the length of capillary-like tubes.(B) LECs were treated with compound 6 (10 and 20 µM), then the cytotoxicity was evaluated by the LDH assay (n = 3).Data are expressed as the mean ± SEM. * p < 0.05 compared with the control group.

b δ H (mult, J in Hz) δ H , mult (J in Hz) δ H , mult (J in Hz) δ H , mult (J in Hz) δ H , mult (J in Hz)
a Data measured at 300 MHz.b Data measured at 400 MHz.
a Data measured at 75 MHz.b Data measured at 100 MHz.

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