Cytotoxic Terpenoids from the Roots of Dracocephalum taliense

A chemical investigation of methanol extract from the roots of Dracocephalum taliense led to the isolation of a new aromatic abietane diterpenoid, 12-methoxy-18-hydroxy-sugiol (1), and one highly-oxygenated ursane triterpenoid, 2α,3α-dihydroxy-11α,12α-epoxy-urs-28,13β-olide (2), together with 15 known natural products (3–17). Among these, compounds 1–13 and 15–17 were detected for the first time in the genus of Dracocephalum. The structures of all of these isolates were determined by extensively spectroscopic analyses. In the anti-inflammatory assay, compounds 1 and 2 had no obvious inhibitory activity on the release of cytokine IL-2 in lipopolysaccharide-induced RAW 264.7 macrophages. However, compound 2 exhibited significant cytotoxic activity against cell lines HepG2 (IC50 = 6.58 ± 0.14 μM) and NCI-H1975 (IC50 = 7.17 ± 0.26 μM).


Introduction
Terpenoids represent the most widespread and largest class of natural products, with more than 80,000 metabolites in a greater family that also includes steroids and carotenoids [1]. Most terpenoids serve a variety of roles in mediating mutualistic and antagonistic interactions among organisms in the natural world [2]. In flowering plants, terpenoids also constitute a large group of naturally-occurring specialized metabolites, especially as common substances in the Lamiaceae family. For example, abundant diterpenoids with diverse structural scaffolds and important pharmaceutical functions have been discovered in the genus Isodon [3][4][5]. The genus Salvia is a rich source of structurally-diverse abietane and clerodane diterpenoids [6]. A series of uniquely defensive sesterterpenoids have been found in the species of Leucosceptrum canum and Colquhounia coccinea var. mollis by our previous phytochemical investigation [7][8][9][10]. As a result, the chemical and structural diversity of terpenoids and their biological activities are interesting for research.
Dracocephalum is a herbaceous genus belonging to the Lamiaceae family and is widely distributed in China. Some plants in this genus, including D. moldavica and D. tanguticum, are broadly used in traditional medicine for gastropathy, tracheitis, and cardiovascular disease in Xinjiang and Tibet [11,12]. Typical metabolites in the plants of this genus were flavonoids and terpenoids [13][14][15][16]. Monoterpenes limonene and α-terpineol might be responsible for antinociceptive properties in the essential oil of D. kotschyi [17]. Icetexane diterpenoids, as the main constitutes in D. komarovi and D. kotschyi, showed moderate trypanocidal activity [16,18,19]. D. taliense is a perennial plant with thick cylindric roots and bluish violet flowers on short stalks. It grows in sandy deposits, cliffs, and rocky slopes. It is endemic in distribution in Dali and Shangri-la regions of Yunnan Province (Figure 1). The whole plant of D. taliense is used by local people to treat liver disorders, which mainly exhibited good effects on hepatitis and icterus, and also applied for adjusting the stomach [20,21]. However, there is nearly effects on hepatitis and icterus, and also applied for adjusting the stomach [20,21]. However, there is nearly no literature reporting the chemical constituents of this plant. Here, we present the isolation and structural elucidation of the chemical components from the roots of D. taliense and their cytotoxic and anti-inflammatory activities.

Structural Elucidation of Compounds
Compound 1 (Figure 2) was obtained as a light yellow oil. Its molecular formula is C 21 Figure S1) exhibited signals for a tetra-substituted benzene ring with protons at δ H 6.95 and 7.78, two singlet methyls at δ H 1.  Figure S2) demonstrated 21 carbon resonances which were assigned to five methyls, five methylenes (with one oxygenated), four methines, and seven quaternary carbons (with one carbonyl carbon at δ C 197.2). These 20 typically skeletal carbons indicated that compound 1 was a characteristic abietane-type diterpenoid [22,23]. In the heteronuclear multiple bond coherence (HMBC) spectrum of 1 (Figure 3), the olefinic proton signal at δ H 7.78 (H-14) showed correlations to the carbonyl carbon at δ C 197.2 (C-7) and the quaternary carbon at δ C 162.3 (C-12), and the methoxyl signal exhibited correlation with the quaternary carbon at δ C 162.3, indicating the carbonyl moiety and methoxyl group substituted in C-7 and C-12, respectively. The HMBC correlations of the oxygenated methylenes at δ H 3.61 and 3.81 to δ C 36.2 (C-3), 39.2 (C-4), 51.1 (C-5), and methyl carbon at δ C 27.2 indicated oxygenation of either Me-18 or Me-19. From the 2D rotational nuclear Overhauser effect spectroscopy (ROESY) spectrum of 1, the correlation between δ H 1.27 (Me-20) and 1.02 (Me-19), δ H 3.61 (H-18b) and 1.94 (H-5) were observed that confirmed the oxygenation of Me-18. Thus, the chemical structure of 1 was confirmed and named 12-methoxy-18-hydroxy-sugiol.

Plant Material
The roots of D. taliense were collected from Shangri-la in Yunnan Province in August 2016 and identified by Dr. Chunlei Xiang. An authentic sample (SYAU-2016-0246) was kept at the College of Bioscience and Biotechnology, Shenyang Agricultural University.

Cytotoxic Assay
The in vitro cytotoxic activity of compounds 1 and 2 were determined by the MTS method [36]. The tested human cancer cell lines, including human lung adenocarcinoma cell line (NCI-H1975), human hepatocellular carcinoma cell line (HepG2), and human breast adenocarcinoma cell line (MCF-7), were seeded in 96-well plates, and then the plates were incubated for 24 h at 37 • C in a 5% CO 2 incubator. Subsequently, the compounds were added at a dosage of 0, 0.128, 0.256, 0.512, 1, 2, 5, 10, 20, 40, and 80 µM. After 72 h, MTS was added to the culture medium and the absorbance at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Each sample was carried out in triplicate. The evaluation of IC 50 values were calculated with the GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA) software.

Anti-Inflammatory Assay
The murine macrophage RAW 264.7 cell line was cultured in DMEM medium supplemented with 10% heated-inactivated fetal bovine serum in a 37 • C, 5% CO 2 incubator. Before the anti-inflammatory assay, test compounds were assessed for their cytotoxicity against the RAW 264.7 cell line and were found to be non-toxic at the tested concentrations (40, 20, 10, 5, and 0 µM). Anti-inflammatory activity was assessed by enzyme-linked immunosorbent assay (ELISA, BD Biosciences, Mountain View, CA, USA) using commercial interleukin-2 (IL-2) detecting kits as previously described [37].

Statistical Analysis
The results were presented as mean values ± SD (standard deviations) of the three replicates.
Supplementary Materials: Supplementary materials are available online. NMR spectra data (Figures S1-S12) of compounds 1 and 2 are available in the Supplementary Material.