New caffeoyl derivatives with potent DPPH radical scavenging activity from Elephantopus tomentosus

Abstract Eight new caffeoyl derivatives, elephantomentosides A–H (1 − 8), together with ten known ones (9 − 18), were isolated from the whole plant of Elephantopos tomentosus L. Their structures were elucidated using detailed spectroscopic analysis. Structurally, compounds 1 − 8 are composed of β-D-glucopyranose, and almost all of the substituent positions are at the C-1′ and C-4′ of glucopyranose. The anti-inflammatory and antioxidant activities of all isolated compounds were evaluated in vitro. Compounds 9–10, 13–15, and 17–18 exhibited significant DPPH scavenging capacity with IC50 values in the range of 10.01–25.07 μM, in comparison with Vc (IC50, 17.98 μM).


Introduction
Elephantopos tomentosus L., which belongs to the genus Elephantopus (Asteraceae), is a perennial herb, mainly distributed in tropical areas [1].There are more than 30 species of this genus in the world, and only two of them exist in China, which are E. tomentosus L. and E. scaber L. E. tomentosus is a common medicinal material of Li nationality in China.It is used to treat epistaxis, jaundice, gonorrhea, beriberi, edema, carbuncle, swelling, boils, snakebite and other diseases [2].Previous phytochemical studies revealed the presence of various components such as sesquiterpenoids, triterpenoids and other constituents, which possessed anti-tumor, antibacterial, and antiinflammatory activities [3][4][5][6][7][8].In the course of searching for bioactive constituents from medicinal plants in China, some caffeoyl derivatives with potential anti-inflammatory activity isolated from E. scaber have attracted our attention [9][10].In order to enrich the active compounds for further pharmacological research, the chemical components of the whole plant of E. tomentosus were investigated, leading to the isolation of eight new caffeoyl compounds (1-8) and ten knowns (9-18) (Figure 1).Herein, we report the structural elucidation and bioactive evaluation of these compounds.
Compound 3 showed the same molecular formula as 2 by its positive ion HRESIMS data (m/z 421.1472 Comparing the NMR data of 3 with those of 2, the results showed that they are almost the same, except for the butan-2-ol group in 3 compared to an isobutanol group in 2. The conclusion was supported by the 1 H-1 H COSY cross-peaks of H-1/H-2/H-3 and H-1/H-4.In addition, the HSQC and HMBC spectra (Figure 2) of 3, further confirmed these observations, leading to the assignment of its structure as shown.Thus, the structure of 3 was determined and designated as elephantomentoside C.
Compound 4 with the molecular formula was determined as C 23 H 26 O 9 from the negative ion HRESIMS (m/z 445.1497 [M − H] − ), with a calculated unsaturation number of 11.The 1 H and 13 C NMR data of 4 were similar to those of 1, except that a Z-hex-3-en-1-ol moiety in 1 was replaced by a 2-phenylethanol group in 4 [12], which was confirmed by 1 H-1 H COSY cross-peaks of H-7/H-8 and the HMBC correlations from H 2 -8/H 2 -7 to C-1, and from H-1 0 to C-8, together with their chemical shifts.Thus, the structure of compound 4 was determined and designated as elephantomentoside D.
Compound 5 gave a molecular formula of C 22 H 24 O 9 , as indicated by HRESIMS.Comparison of NMR data between 5 and 4 demonstrated replacement of the 2- phenylethanol unit in 4 by a benzyl alcohol group in 6, in addition to the absence of the methylene group at the aglycone part.The deduction was confirmed by the HMBC correlation from H 2 -7 to C-1, in combination with their molecular formula and chemical shifts.Thus, the structure of 5 was determined and designated as elephantomentoside E.
Compound 6 was assigned the molecular formula of C 24 H 26 O 10 as deduced from HRESIMS at m/z 473.1488 [M − H] -and NMR data, requiring 12 degrees of unsaturation.By comparing 1 H and 13 C NMR data of 6 and 5, it was found that 6 had one more acetyl group.The HMBC correlation between d H 4.19 (H-6 0 a), 4.12 (H-6 0 b) and d C 172.6 (C-8) showed that acetyl group was located at C-6 0 .Thus, the structure of 6 was determined and designated as elephantomentoside F.
Compound 7 had the molecular formula C 25 H 28 O 10 that was established by HRESIMS.Comparison of the NMR spectroscopic data between 7 and 4 demonstrated that the 2-phenylethanol moiety in 4 was replaced by the eugenol moiety [13].The conclusion was confirmed by 2D NMR spectroscopic data analysis of 7, especially by the 1 H-1 H COSY cross-peaks of H-7/H-8/H-9 and H-5/H-6, as well as the HMBC correlations of H-8/C-4, H 2 -7/C-4, H 3 -10/C-2, and H-1 0 /C-1.Thus, the structure of 7 was determined and designated as elephantomentoside G. Compound 8 had the molecular formula C 26 H 30 O 11 as indicated by HRESIMS data.By comparing 1 H and 13 C NMR data of 8 and 7, it was found that the aglycone of 8 had one more methoxyl signal.The HMBC correlation between d H 3.84 (H-11) and d C 154.2 (C-6) showed that methoxyl signal was located at C-6.Thus, the structure of 8 was determined and designated as elephantomentoside H.
All isolated compounds were evaluated for their DPPH radical scavenging activity.Vitamin C (Vc) was used as a positive control, and the IC 50 values are summarized in Table 3.The results demonstrated that 9-10, 13-15, and 17-18 exhibited significant DPPH scavenging capacity with IC 50 values in the range of 10.01-25.07lM.Among them, compounds 13-14 showed slightly better scavenging capacity than Vc.In addition, compounds that have no cytotoxicity were tested for their anti-inflammatory activities using LPS-induced RAW 264.7 cells.L-NAME was used as a positive control.Unfortunately, the tested compounds did not exhibit significant anti-inflammatory effect at a concentration of 50 lM (Table S2, Supplementary material).
In summary, eight new caffeoyl derivatives, elephantomentosides A-H (1-8), together with ten known analogues (9-18) were isolated from the whole plant of E. tomentosus L. Structurally, new compounds are composed of b-D-glucopyranose, and almost all of the substituent positions are at the C-1 0 and C-4 0 of glucopyranose.Notably, we obtained three sets of isomers (2/3, 9/10/13/14/15/16, and 11/12) from the same plant.The isolation of these isomers is a huge challenge because they are highly oxygenated and have similar structures.Moreover, in bioactivity assays, seven compounds exhibited significant DPPH scavenging capacity.As potent antioxidants, these caffeoyl derivatives were thought to be the active ingredients of this medicinal plant.This investigation may contribute to the discovery of potential antioxidants and broaden the application field of E. tomentosus L. 17.98 ± 0.58 a Values were expressed as the means ± SD based on three independent experiments.b Vc is the positive control.

General experimental procedures
IR data were obtained using a Nicolet IS5 FT-IR spectrophotometer (Thermo Scientific, Madison, WI, USA).The optical rotations of the compounds were measured on an Anton Paar MCP 200 Automatic Polarimeter (Anton Paar GmbH, Graz, Austria) and UV spectra were recorded on a Thermo Genesys-10S UV-vis spectrophotometer (Thermo Scientific, Madison, WI, USA).The HRESIMS were recorded using the Q-TOF analyzer of a SYNAPT HDMS system (Waters, Milford, MA, USA). 1 H NMR, 13 C NMR, HSQC, HMBC, COSY and NOESY spectra were acquired with Bruker AVANCE III 500 MHz or 600 MHz spectrometer (Karlsruhe, Germany) using the solvent signals (CD 3 OD) as references.Silica gel (60-100, 100-200 and 200-300 mesh) used for column chromatography (CC) were supplied by Qingdao Marine Chemical Plant (Qingdao, China).A YMC-Pack ODS-A semi-preparative HPLC column (250 � 20 mm and 250 � 10 mm, 5 lm, YMC, Tokyo, Japan) or a Cosmosil-packed PBr column (5 lm, 10 � 250 mm, YMC, Kyoto, Japan) on a Waters 2535 instrument equipped with a 2489 UV detector (Waters, MA, USA) was used for purification.The mouse leukemia cells of monocyte macrophage (RAW 264.7) were obtained from the National Biomedical Experimental Cell Resource Bank (Beijing, China).Biological reagents were from Sigma-Aldrich Company (St. Louis, USA).

Plant material
The

DPPH free radical scavenging activity
The 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH�) was dissolved in methanol (3.94 mg, 50 ml) and added to 100 ll methanol solution of the tested compound (6.25-100 lM), methanol (negative control) or Vitamin C (Vc, positive control) in each well of a flat-bottomed 96-well microliter plate.The reaction between DPPH� and the tested compounds was then monitored at k ¼ 517 nm using a microplate reader at 20 � C in the dark after 30 min [21].All samples were made three multiple holes to take the average value.The IC 50 values were calculated.

NO inhibition in LPS-induced RAW 264.7 cells
As described in the literature [22], the anti-inflammatory activities were determined by inhibiting the production of nitric oxide (NO) in LPS stimulated RAW264.7 cells using the Griess method.The RAW264.7 cells were inoculated into a 96-well plate (2 � 10 5 cells/well), stimulated with LPS (100 ng/ml), then added into the medium with or without different concentrations of the compounds to be tested and incubated under 37 � C for 24 h, while NG-Nitro-L-arginine Methyl Ester (L-NAME) acted as the positive control.After that, the medium was collected, and Griess reagent was used to measure the NO production level with absorbance at 540 nm by using a microplate reader.

Statistical analysis
All experiments were performed in triplicate (n ¼ 3), and data are expressed as mean value and standard deviation.Statistical significance was analyzed using ANOVA and Dunnett's multiple comparison test by the GraphPad Prism (version 8).A p value of less than 0.05 (p < 0.05) was considered statistically significant.IC 50 values were calculated using the GraphPad Prism software.
dried whole plants of Elephantopus tomentosus were collected from RongXian, Guangxi Province of China in May 2018, and identified by Prof. Yunfeng Huang (Institute of Chinese Medicine Resources, Guangxi Institute of Chinese Medicine & Pharmaceutical Science, Guangxi, China).The voucher specimen (SCI-NCL003130-3) was deposited in the National Compounds Library of Traditional Chinese Medicines (NCL-TCM), Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC), China.

Table 3 .
Free radical scavenging activity of isolated compounds 1 − 18 a .