Chemical Constituents of Euphorbia stracheyi Boiss (Euphorbiaceae)

Euphorbia stracheyi Boiss was used for hemostasis, analgesia, and muscular regeneration in traditional Chinese medicine. To study the chemical constituents of E. stracheyi, the ethyl acetate part of the methanol extract of the whole plant was separated by silica gel, sephadex LH-20 column chromatography, and semi-preparative HPLC. The isolation led to the characterization of a new lathyrane type diterpenoid, euphostrachenol A (1), as well as eleven known compounds (2–11), including a lathyrane, three ingenane-type and two abietane-type diterpenoids, two ionones, and two flavonoids. The structures of these compounds were established using 1D- and 2D-NMR experiments, mass spectrometry, and X-ray crystallographic experiments. The MTT method was used to determine the cytotoxic activity of five cancer cell lines (Leukemia HL-60, lung cancer A-549, liver cancer SMMC-7721, breast cancer MCF-7, and colon cancer SW480) on the isolated compounds. However, only compound 4 showed moderate cytotoxicity against these cell lines, with IC50 values ranging from 10.28 to 29.70 μM, while the others were inactive. Our chemical investigation also confirmed the absence of jatrophane-type diterpenoids in the species, which may be related to its special habitat.


Introduction
With nearly 2000 species, the genus Euphorbia (Euphorbiaceae) is the third largest genus in flowering plants, second only to Astragalus (Fabaceae) and Psychotria (Rubiaceae) [1]. Plants of Euphorbia are a rich source of structurally diverse macrocyclic and polycyclic diterpenoids, with more than twenty skeleton types, including casbane, jatrophane, daphnane, tigliane, lathyrane, myrsinane, premyrsinane, cyclomyrsinane, paraliane, pepluane, and ingenane [2,3]. The frameworks of these diterpenoids are further modified by different functional group substitutions at different positions and numbers, such as hydroxyl, epoxy, ether, acyl, polyester, and carbonyl groups, which makes the structure of these compounds even more complex and diversified. So far, more than 700 diterpenoids have been isolated from the genus Euphorbia, showing anti-tumor, multi-drug-resistance-reversing, anti-inflammatory, antibacterial, and pesticidal activities [2,3]. In 2012, the approval of ingenol 3-angelate (ingenol mebutate) by the U.S. Food and Drug Administration and European Medicines Agency for the treatment of actinic keratosis, a precancerous skin condition, attracted more interest in these types of diterpenoids [4].
Plant secondary metabolites are often lineage-specific, and the occurrence and content of secondary metabolites in a certain plant species could be determined by genetic and environmental factors. Thus, phylogenetically related plants in different habitats may harbor distinctive secondary metabolite profiles. For example, Salvia officianlis L. and S. miltiorrhiza are native to the Mediterranean and East Asia, respectively. While the former mainly contains tricyclic diterpenoids, the latter accumulates nor-diterpene quinones named tanshinones [5]. These differences could influence their pharmacological performances, since both species are medicinally highly valuable. Furthermore, the availability of closely related species with different secondary metabolite constituents allows us to elucidate the biosynthetic pathway of these natural products through a comparative genomics analysis [6]. Clearly, this phenomenon is important and should be given much attention in phytochemical research.
Euphorbia stracheyi Boiss is a perennial noxious weed mainly distributed in the western region of China, including Qinghai, Tibet, Sichuan, and Yunnan province [7]. While the nearcosmopolitan nature of Euphorbia is well acknowledged, E. stracheyi is one of the few plants that can grow in high-altitude alpine meadow regions (from 1000 to 4900 ASL). E. stracheyi roots were used as traditional medicine for hemostasis, analgesia, and muscular regeneration [6]. As such, extensive phytochemical investigations have been performed on this plant, which involved the isolation of flavonoids, coumarins, phenylpropanoids, ionones, steroids, triterpenoids, and its main components, diterpenoids [8][9][10][11][12][13][14]. The cytotoxic activity of the isolated compounds has been evaluated [10,11,13]. The structures of some common compounds are shown in Figure 1. Interestingly, although jatrophane-type diterpenoids are widely occurring in plants of Euphorbia, to date there are no compounds of this type isolated from the species. In order to further study the chemical composition of the species, the methanol extract of the whole plant of E. stracheyi was investigated in the present study, which led to the isolation and characterization of a new lathyrane diterpenoid, which was designated euphostrachenol A (1), together with eleven known compounds, including a lathyrane (2), three ingenane (3)(4)(5) and two abietane diterpenoids (6, 7), two ionones (8,9), and two flavonoids (10,11). The structures of the isolated compounds were determined through extensive spectroscopic analysis and X-ray crystallographic experiments. In the present study, we report the isolation, structure elucidation, and in vitro cytotoxic activity of these compounds.

Instruments and Materials
X-ray crystallographic analyses was performed on a Bruker APEX DUO X-ray single crystal diffractometer (Bruker, Rheinstetten, Germany). Optical rotations were recorded on a Jasco P1020 spectropolarimeter. IR spectra were measured on a NICOLET iS107 FTIR spectrometer (Thermo Fisher, Massachusetts, USA). NMR spectra were obtained on Bruker AVANCE III 500 MHz and AV 600 MHz superconducting NMR spectrometers (Bruker, Rheinstetten, Germany) with TMS as the internal standard. High-resolution MS data were obtained on an Agilent 1290 UPLC/6540 Q-TOF mass spectrometer in positive mode. UV spectroscopic data were recorded on a Shimadzu-210A double-beam spectrophotometer. Agilent 1290 UPLC/6540 Q-TOF (Agilent, Santa Clara, USA) was used to undertaken liquid chromatography/quadrupole time-of-flight mass spectrometry data.
The whole plants of E. stracheyi (flowering) were collected in August 2020 from Gongshan County, Nujiang Prefecture, Yunnan Province. The plant sample was identified by Mr. Yan-Bo Huang of Shanghai Chenshan Botanical Garden. The age of the selected plants was two years, and the material was dried and extracted with methanol.

X-ray Crystallographic Analyses
A light, colorless crystal of 1 was used for the X-ray crystallographic analysis on a Bruker APEX DUO diffractometer equipped with Cu Kα radiation (λ = 1.54178 Å). To solve and determine the structures and absolute configurations of 1, the ShelXT 28 program with Intrinsic Phasing was used [15].

1.
Cell inoculation: A single cell suspension was obtained from a culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and 3000-15,000 cells per well were inoculated onto a 96-well flat-bottomed microtiter plate at a volume of 100 µL per well. The cells were inoculated 12-24 h in advance for culture.

2.
Addition of compounds to be tested: Dimethyl sulfoxide (DMSO) solutions containing different concentrations of the tested compound (diluted from 40 µM) were added to a final volume of 200 µL per well, and three replicates were set for each group of treatments.

3.
Color development: After incubation at 37 • C for 48 h, the culture solution was discarded for adherent cells, and 20 µL of MTS solution and 100 µL of culture solution were added to each well, respectively; 100 µL of culture supernatant was discarded for suspended cells and 20 µL of MTS solution was added to each well. Incubation was continued for 2~4 h. Then, to measure the light absorption values, 3 blank controls (120 µL of the mixture of MTS solution and culture solution) were set. 4.
Colorimetric: A wavelength of 492 nm was selected, and the light absorption value of each well was read by MULTISKAN FC. The results were recorded, and the data were processed and plotted against the compound number as the horizontal coordinate and the cell inhibition rate as the vertical coordinate.

5.
Positive control compounds: Two positive compounds, DDP and Taxol, were used in each experiment, and the cell growth curves were plotted with the concentration as the horizontal coordinate and the cell survival rate as the vertical coordinate.

Cytotoxicity
The isolated compounds were screened and evaluated for their cytotoxicities against five human cancer cell lines (namely, human promyelocytic Leukemia cells HL-60, human lung cancer cells A-549, human liver cancer cells SMMC-7721, human breast cancer cells MCF-7, and human colon cancer cells SW480), with DDP and Taxol as two positive controls. Only compound 4 showed moderated cytotoxic activity against HL-60, A-549, SMMC-7721, MCF-7, and SW480 cell lines, with IC 50 values of 10.5 ± 0. 18

Discussion
Euphorbia plants are known for their irritant milky latex [27] and characteristic and chemically diverse diterpenoids. Among the macrocyclic and polycyclic diterpenoids from Euphorbia, jatrophane types are the most common [28][29][30][31]. To date, the diterpenoids reported from E. stracheyi have included ent-kaurane, ent-abietane, ent-atisane, ent-isopimarane, labdane, tigliane, lathyrane, and ingenane-type diterpenoids [10][11][12][13][14], but no jatrophanes were isolated from the species. In the present study, besides the new compound euphostrachenol A, one lathyrane-type, three ingenane-type, and two abietane-type diterpenoids were isolated from E. stracheyi, all of which are known compounds and were discovered from other species of Euphorbia, suggesting a similar diterpenoid profile of these species. Importantly, our phytochemical investigation of E. stracheyi reported here also yielded no jatrophane-type diterpenoids, in accordance with the previous results. This type of diterpenoid is found exclusively in the Euphorbiaceae family, and it has a 5/12 bicyclic pentadecane skeleton, in which the absence of a cyclopropane ring is the most obvious structural feature that differentiates it from other macrocyclic and polycyclic diterpenoids from Euphorbia, such as tigliane, lathyrane, and ingenane types. The absence of jatrophanes in the species may be related to the special environment in which E. stracheyi lives, namely, the high-altitude region, which is often associated with high loads of UV radiation and low temperatures. Jatrophane-type diterpenoids exhibit many different activities, including antitumor, antiviral, antifungal, and anti-inflammatory effects [2,3]. Many of them showed promising P-glycoprotein, a membrane protein that pumps anticancer drugs out of cells, exhibits inhibitor activity, and could be developed as a new drug to reverse multidrug resistance [2,3]. Thus, the pharmacological value of E. stracheyi may be influenced. Furthermore, the lack of jatrophanes implied that its biosynthetic pathway may be lost in the plant of E. stracheyi. Currently, jatrophanes were suggested to be derived from lathyrane-type diterpenoids by combined transcriptomic, genomic, and metabolomic investigation of E. peplus, a species harboring many jatrophane-type diterpenoids [32]. Thus, comparative genomic and transcriptomic analysis between these two species could provide useful clues for uncovering the biosynthesis of jatrophane-type diterpenoids.
Notably, ionones are rarely isolated from the species of Euphorbia [2]; however, they were reported constituents of E. stracheyi [8,12]. We also reported two ionones from E. stracheyi, in agreement with previous studies. Ionones are degraded from carotenoids that play an important role in helping plants adapt to high UV light and low temperatures. During photosynthesis, carotenoids are known to protect chlorophylls and bacteriochlorophylls from sensitizing deleterious photodestructive reactions [33]. The detection of ionones in E. stracheyi in the present and previous studies strongly suggested that this type of secondary metabolite should be the result of high-altitude adaptation of the species. Further work should be undertaken to reveal whether other Euphorbia plants adapted to the harsh environments of Tibetan peaches also harbor ionones.
Notably, the planar structure of compound 1 shows high similarity to those of kansuingols A and B, except for a β-D-glucose group and a hydroxyl group present at C-15 and C-19 of kansuingol A, as well as a β-D-glucose group at C-19 of kansuingol B, respectively [17]. Notably, the relative conformations of these three compounds are also different for methyl-16, methyl-20, and hydroxyl-7. While the relative conformations of compound 1 were supported by X-ray diffraction analysis, those of kansuingols A and B were not. After checking the key ROESY correlations that determined the relative conformation assignment of methyl-16, methyl-20, and hydroxyl-7 in kansuingols A and B, we found that there are no ROESY correlations to support the claimed β-orientation of methyl-16 in both compounds. Therefore, the structures of kansuingols A and B remain to be determined.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available in the main article and the Supplementary Materials.