Three new compounds with their anti-glioma effects from the roots of Arnebia guttata Bunge

Abstract Three new compounds, arneatas A-C (1–3), together with three known compounds (4–6) were isolated from the roots of Arnebia guttata Bunge. The structures were established on the basis of extensive spectroscopic data including NMR and HRESIMS. All the new compounds (1–3) were tested for their cytotoxic activity against two glioma cell lines (U118-MG and U373-MG) in vitro after treatment for 48 h. Compound 1 exhibited moderate cytotoxic activity against U118-MG and U373-MG glioma cell lines, with IC50 values of 10.4 and 17.5 µM, respectively. Graphical Abstract


Introduction
Arnebia guttata Bunge. is a traditional Chinese medicine for thousands of years, used for promoting blood circulation, measles impervious, eczema, sores, spots, water and fire scalding and removing rashes (Cheng et al. 2018;Xiang et al. 2019;Wang et al. 2021). A.guttata. is mainly distributed in northern China, northwest India, Pakistan, Kashmir, Afghanistan, Central Asia and Siberia of the Soviet Union (Ma et al. 2021). The literature indicates that meroterpenoids (Liao et al. 2018), naphthoquinones (Shen et al. 2002), phenolic acid (Kashiwada et al. 1995), alkaloids (Roeder and Rengel 1990) extracted from A.guttata. are the major functional compounds and exhibit important biological activities such anti-inflammatory, anti-tumor, antibacterial, antiviral, hepatoprotective, immune regulation and anticancer activities (Zhan et al. 2015;Liu et al. 2020). As part of an on going investigation on the discovery of natural functional agents from A.guttata., we examined the chemical constituents of the dry roots of this plant and isolated three new terpenoid derivatives arneatas A-C (1-3), and three known compounds arnebinone (4) (Yao et al. 1991), euchroquinol C (5) (Li et al. 2012) and heliotropin one A (6) (Guntern et al. 2001). These known compounds have some biological activities. Arnebinone showed the inhibitory effect to prostaglandin biosynthesis (Yao et al. 1983). Euchroquinol C about it anti-HCV and anti-bacterial activities was test by Li et al, and showed no activity (Li et al. 2012). Heliotropin one A demonstrated anti-fungal activities against Cladosporium cucumerinum and Candida albicans as well as anti-bacterial activity against Bacillus subtili (Guntern et al. 2001). In this paper, we elucidated the structure and anti-glioma activity of the isolated new compounds.

Results and discussion
Compound 1 was obtained as yellow oil. Its molecular formula was established to be C 14 Figure S6) of 1 showed two carbonyl carbons (d C 170.4, 187.0) and six olefinic carbons (d C 153.0, 144.1, 135.7, 127.5, 117.9 and 116.3).
The 1 H-1 H COSY spectrum (Supplementary Figure S7) indicated the presence of one coupling fragment H-6/H 2 -7/H-8. The HMBC correlations (Supplementary Figure S9) from H 3 -10 to C-8 (d C 117.9)/C-9 (d C 135.7), H 3 -11 to C-8/C-9, H 2 -7 to C-6 (d C 67.8)/C-8 established that an isopentenyl unit was attached to C-6. Cross-peaks from H 3 -13 to C-12 (d C 170.4) and C-6 in the HMBC spectrum suggested that one acetyl group was linked to C-6. Considering the degree of unsaturation in compound 1, the remaining four olefinic carbons (d C 153.0, 144.1, 116.3 and 127.5) and one carbonyl carbon (d C 187.0) were properly assigned to a cyclopentadienone unit. The HMBC correlations from H 3 -14 to C-3 (d C 117.9) and C-4 (d C 187.0) indicated that one methyl was substituted at C-3 in cyclopentadienone unit. Thus, the structure of compound 1 was completely determined as shown in Figure 1 and named as arneata A.
Compound 2 was isolated as yellow oil. Its molecular formula was determined to be C 17 H 20 O 3 on the basis of the positive ion HRESIMS peak at m/z 273.1496 [M þ H] þ (Calcd for [C 17 H 21 O 3 ] þ 273.1491), indicative 8 degrees of unsaturation. An absorption band at 270 nm in the UV spectrum of compound 2 was indicative of aquinone skeleton (Yao et al. 1991 . With the aid of the HSQC spectra, 13 C-NMR analysis of 2 revealed 17 carbon signals composed of one methoxy, two methyls, two methylenes, one methine, two olefinic methylenes, two olefinic methines, four olefinic quaternary and two carbonyls. These data were very similar to those of arnebinone B previously reported in Arnebia euchroma (Yao et al. 1991). Compared with arnebinone B, one methoxy was absent in the corresponding 1 H-and 13 C-NMR spectrum of 2. In the HMBC spectrum (Supplementary Figures S2 and S16), the methoxy signal had correlations with C-3 (d C 158.6) and C-4 (d C 181.9), indicating its connection to C-3. Finally, the planar structure of compound 2 was completely established as shown in Figure 1 and named as arneata B.
Compound 3 was isolated as yellow oil and assigned the molecular formula C 17 H 20 O 3 by HR-ESIMS, which showed a positive ion peak at m/z 273.1496 ([M þ H] þ ). The structure of compound 3 is very similar to that of compound 2. The difference between compound 3 and 2 was the position of methoxy substitution. In the HMBC spectrum (Supplementary Figures S3 and S23), the methoxy signal had correlations with C-2 (d C 158.6) and C-1 (d C 182.0), indicating its connection to C-2. Finally, the planar structure of compound 3 was completely established as shown in Figure 1 and named as arneata C. The effects of arneatas A-C on cancer cell growth were examined. Arneatas B-C were inactive against U118-MG and U373-MG glioma cell lines, while arneata A showed moderate cytotoxic activity against U118-MG and U373-MG glioma cell lines, with IC 50 values of 10.4 and 17.5 mM, respectively, compared with the positive control temozolomide (8.4 lM for U118-MG and 10.7 l M for U373-MG).

General experimental procedures
Optical rotation data are obtained using the Perkin Elmer 341 digital polarimeter (Perkin Elmer, Norwalk, CT, U.S.A.). The UV and IR spectra were recorded on Shimadzu UV-2550 (Kyoto, Japan) and Spetrum 100 (Perkinelmer,U.S.A.). The NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer (chemical shift values expressed in d, TMS as internal standard). High resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a LTQ Obitrap XL spectrometer (Thermo Fisher Scientific, Boston, Massachusetts, U.S.A.). In silica gel (100-200, 200-300 and 300-400 mesh; gel (CHP 20P, 75-150lm, Tokyo Mitsubishi Chemical Co., Japan) and column chromatography (CC) were performed on China Qingdao Ocean Chemical Group Co., Ltd. In addition, TLC was performed using a precoated silica gel GF254 plate (Yantai ZhifuHuangwu Silica Gel Development Pilot Plant, China). All solvents used were analytical grade (Beijing Chemical Works, Beijing, China).

Cytotoxicity assay
The human malignant glioma U118-MG and U373-MG cell lines were obtained from the Institute of Biochemistry and Cell Biology (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated foetal bovine serum, 100 units/mL penicillin and 100 lg/mL streptomycin in a humidified atmosphere containing 5% CO 2 at 37 C. All media and sera were purchased from Gibco Life Technologies (Gibco, USA). Then, the cells were treated by exposure to varying concentrations of the indicated agents for the indicated times. The control cultures received the same amount of dimethyl sulfoxide as that of the treated culture.

Conclusion
In our investigation on the root of A. guttata., three new compounds (1-3) and three known compounds (4-6) were obtained. The isolated three new compounds were tested for their cytotoxic activity against two glioma cell lines (U118-MG and U373-MG) in vitro after treatment for 48 h. Compound 1 exhibited moderate cytotoxic activity against U118-MG and U373-MG glioma cell lines, with IC 50 values of 10.4 and 17.5 mM, respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.