Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium Fortunei

International Journal of Molecular Sciences

Article Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei

Chun-Hao Chang 1, Semon Wu 2, Kai-Cheng Hsu 3,4, Wei-Jan Huang 5,6 and Jih-Jung Chen 7,8,9,*

1 Institute of Biopharmaceutical Sciences, School of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112, Taiwan; [email protected] 2 Department of Life Science, Chinese Culture University, Taipei 110, Taiwan; [email protected] 3 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan; [email protected] 4 Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan 5 Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan; [email protected] 6 Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan 7 Department of Pharmacy, School of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112, Taiwan 8 Faculty of Pharmacy, National Yang-Ming University, Taipei 112, Taiwan 9 Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan * Correspondence: [email protected]; Tel.: +886-2-2826-7195; Fax: +886-2-2823-2940

Abstract: Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8- methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6-16). Citation: Chang, C.-H.; Wu, S.; Hsu, K.-C.; Huang, W.-J.; Chen, J.-J. Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6- Dibenzofuran, 4-Chromanone, methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory Acetophenone, and Dithiecine activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 µM, respectively, against A549 and MCF-7 Derivatives: Cytotoxic Constituents cells. The colony-formation assay demonstrated that compound 1 (5 µM) obviously decreased A549 from Eupatorium fortunei. Int. J. Mol. and MCF-7 cell proliferation, and Western blot test conﬁrmed that compound 1 markedly induced Sci. 2021, 22, 7448. https://doi.org/ apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways. 10.3390/ijms22147448 Keywords: Eupatorium fortunei; dibenzofuran; cytotoxity; apoptosis Academic Editor: Toshio Morikawa

Received: 26 May 2021 Accepted: 7 July 2021 1. Introduction Published: 12 July 2021 Eupatorium fortunei Turcz. (Asteraceae) is a perennial herb widely distributed in China. E. fortunei Publisher’s Note: MDPI stays neutral The aerial part of (Chinese name: Pei-Lan) has been used as a traditional medicine with regard to jurisdictional claims in for the treatment of various diseases such as flu, poor appetite, constipation, nausea, and published maps and institutional affil- siriasis [1]. Diverse monoterpenoids [2–6], triterpenoids [2,7], sesquiterpenoids, stereoiso- iations. mer, coumarins [7], pyrrolizidine alkaloids [7,8], benzofuran [4,9], and their derivatives were isolated from this species in the past studies. Many of these isolated compounds showed anti-inflammatory [5], anti-bacterial [3,4], anti-cancer [6], and anti-diabetic [7] activities. In our study on the cytotoxic constituents of Chinese herbal medicines, many species have been screened for cytotoxic effect, and E. fortunei has been found to be an Copyright: © 2021 by the authors. b,d 1 Licensee MDPI, Basel, Switzerland. active species. Two new dibenzo[ ]furan derivatives, eupatodibenzofuran A ( ) and This article is an open access article eupatodibenzofuran B (2), a new 4-chromanone, 6-acetyl-8-methoxy-2,2-dimethylchroman- distributed under the terms and 4-one (3), a new acetophenone derivative, eupatofortunone (4), a new dithiecine derivative, conditions of the Creative Commons eupatodithiecine (5), and eleven known compounds (6–16) have been isolated and con- Attribution (CC BY) license (https:// firmed from the aerial part of E. fortunei. This report describes the structural elucidation creativecommons.org/licenses/by/ of five new compounds 1–5 and the inhibitory activities of 1 against non-small-cell lung 4.0/). cancer and breast cancer cells.

Int. J. Mol. Sci. 2021, 22, 7448. https://doi.org/10.3390/ijms22147448 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17

Int. J. Mol. Sci. 2021, 22, 7448 This report describes the structural elucidation of five new compounds 1–5 and2 of 16the inhibitory activities of 1 against non-small-cell lung cancer and breast cancer cells.

2. Results and Discussion 2. Results and Discussion 2.1.2.1. General General SeparationSeparation of of the the EtOAc-soluble EtOAc-soluble fraction fraction of aof MeOH a MeOH extract extract of the of aerialthe aerial part ofpartE. of E. fortuneifortuneiby by silica silica gel gel chromatography chromatography and and preparative preparative thin-layer thin-layer chromatography chromatography (TLC) (TLC) affordedafforded ﬁve five new new (1 –(51)–5 and) and eleven eleven known known compounds compounds (6–16 (6)– (Figure16) (Figure1). 1).

FigureFigure 1. 1.The The chemical chemical structures structures of new of new compounds compounds1–5 and 1–5known and known compounds compounds6–16 isolated 6–16 isolated from E.from fortunei E. fortunei. .

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2.2. Structure Elucidation of the New Compounds

Compound 1 was obtained as colorless needles. Its molecular formula C22H18O4 was deduced from a sodium adduct ion at m/z 369.11041 [M + Na]+ (calcd 369.11028) in the HRESIMS spectrum. The presence of hydroxyl (3439 cm−1) and conjugated carbonyl (1645 cm−1) groups were revealed from the IR spectrum. Analysis of the 1H and 13C NMR data of 1 showed the signals for an acetyl group [δH 2.41 (3H, s, Ac-2); δC 26.2 (COMe), 203.9 (COMe)], a chelated hydroxyl group [δH 12.65 (1H, s, OH-3)], a methyl group [δH 2.62 (3H, s, Me-6); δC 15.1 (Me-6)], a 2-hydroxy-4-methylphenyl group [δH 2.44 (3H, s, Me-40), 4.97 (1H, s, OH-20), 6.93 (1H, br d, J = 7.5 Hz, H-50), 6.96 (1H, br s, 0 0 0 0 0 0 H-3 ), 7.25 (1H, d, J = 7.5 Hz, H-6 ); δC 21.4 (Me-4 ), 116.1 (C-3 ), 121.7 (C-5 ), 122.4 (C-1 ), 0 0 0 130.2 (C-6 ), 140.4 (C-4 ), 152.7 (C-2 )], two ortho-coupled aromatic protons [δH 7.22 (1H, d, J = 7.5 Hz, H-8), 7.33 (1H, br d, J = 7.5 Hz, H-7); δC 125.1 (C-8), 128.6 (C-7)], and two singlet aromatic protons [δH 7.07 (1H, s, H-4), 7.65 (1H, s, H-1); δC 99.8 (C-4), 125.4 (C-1)] (Table1). The position of each substituent was supported by ROESY correlations between 0 0 H-1 (δH 7.65)/Ac-2 (δH 2.41), Me-6 (δH 2.62)/H-7 (δH 7.33), OH-2 (δH 4.97)/H-3 (δH 0 0 0 0 6.96), H-3 (δH 6.96)/Me-4 (δH 2.44), and Me-4 (δH 2.44)/H-5 (δH 6.93) and by HMBC correlation between ‘Ac-2 (δH 2.41)/C-2 (δC 116.6)’, ‘OH-3 (δH 12.65)/C-2 (δC 116.6), C-4 0 0 (δC 99.8)’, ‘Me-6 (δH 2.62)/C-5a (δC 155.9), C-7 (δC 128.6)’, ‘OH-2 (δH 4.97)/C-3 (δC 116.1)’, 0 0 0 and ‘Me-4 (δH 2.44)/C-4 (δC 140.4), C-5 (δC 121.7)’ (Figure2). The full assignment of 1H and 13C NMR resonances was supported by 1H–1H COSY, HSQC, ROESY (Figure2A ), and HMBC (Figure2B) spectral analyses. Thus, the structure of 1 was elucidated as 1-(3- hydroxy-9-(2-hydroxy-4-methylphenyl)-6-methyldibenzo[b,d]furan-2-yl)ethenone, named eupatodibenzofuran A.

Table 1. 1H and 13C NMR data of 1 and 2.

1 2 a b a b Position δH (J in Hz) δC δH (J in Hz) δC 1 7.65, s 125.4 7.59, s 125.1 2 116.6 116.0 3 163.2 162.9 4 7.07, s 99.8 7.05, s 99.6 4a 161.2 161.3 5a 155.9 155.4 6 121.8 120.5 7 7.33, br d (7.5) 128.6 7.27, br d (7.6) 127.9 8 7.22, d (7.5) 125.1 7.17, d (7.6) 125.0 9 127.7 130.6 9a 121.9 121.7 9b 116.3 117.8 10 122.4 125.4 20 152.7 156.7 30 6.96, br s 116.1 6.91, br s 111.5 40 140.4 139.8 50 6.93, br d (7.5) 121.7 6.95, br d (7.5) 121.4 60 7.25, d (7.5) 130.2 7.28, d (7.5) 131.0 COMe-2 203.9 203.7 COMe-2 2.41, s 26.2 2.40, s 26.2 OH-3 12.65, s 12.62, s Me-6 2.62, s 15.1 2.60, s 15.1 OH-20 4.97, s OMe-20 3.70, s 55.5 Me-40 2.44, s 21.4 2.49, s 21.7 a b Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses; Recorded in CDCl3 at 125 MHz. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 17

2.2. Structure Elucidation of the New Compounds

[δH 7.07 (1H, s, H-4), 7.65 (1H, s, H-1); δC 99.8 (C-4), 125.4 (C-1)] (Table 1). The position of each substituent was supported by ROESY correlations between H-1 (δH 7.65)/Ac-2 (δH 2.41), Me-6 (δH 2.62)/H-7 (δH 7.33), OH-2′ (δH 4.97)/H-3′ (δH 6.96), H-3′ (δH 6.96)/Me-4′ (δH 2′ 152.7 156.7 2.44), and Me-4′ (δH 2.44)/H-5′ (δH 6.93) and by HMBC correlation between ‘Ac-2 (δH 2.41)/C-23′ (δC 116.6)’, 6.96, ‘OH-3 br s(δ H 12.65)/C-2 116.1(δC 116.6), C-4 (δC 6.91,99.8)’, br ‘Me-6 s (δH 2.62)/C-5a 111.5 (δ C 155.9), 4C-7′ (δC 128.6)’, ‘OH-2 ′ (δH 4.97)/C-3 140.4′ (δC 116.1)’, and ‘Me-4 ′ (δH 2.44)/C-4′ (δC 139.8140.4), C-5′ (δC5 ′121.7)’ (Figure 6.93, br 2). d (7.5)The full assignment 121.7 of 1H and 6.95, 13C brNMR d (7.5) resonances was 121.4 supported 6by′ 1H–1H 7.25, COSY, d (7.5) HSQC, ROESY 130.2 (Figure 2A), and 7.28, HMBC d (7.5) (Figure 2B) spectral 131.0 analyses.COMe-2 Thus, the structure203.9 of 1 was elucidated 203.7as Int. J. Mol. Sci. 2021, 22, 7448 1-(3-hydroxy-9-(2-hydroxy-4-methylphenyl)-6-methyldibenzo[b,d]furan-2-yl)ethenone,4 of 16 COMe-2 2.41, s 26.2 2.40, s 26.2 named eupatodibenzofuran A. OH-3 12.65, s 12.62, s Me-6 2.62, s 15.1 2.60, s 15.1 OH-2′ 4.97, s OMe-2′ 3.70, s 55.5 Me-4′ 2.44, s 21.4 2.49, s 21.7 a Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses; b Recorded in CDCl3 at 125 MHz.

Compound 2 was isolated as amorphous powder with molecular formula C23H20O4, as determined by positive-ion HRESIMS, showing an [M + Na]+ ion at m/z 383.12612

(calcd for C23H20O4Na, 383.12593). The presence of a conjugated carbonyl group was re- vealedFigure 2.2. byKey Key a ROESY ROESYband (Aat ()A and1645) and HMBC HMBCcm− (1B in) correlations(B the) correlations IR spectrum, of 1. of 1. and was confirmed by the resonance at δC 203.7 in the 13C NMR spectrum. The 1H NMR data of 2 were similar to eupatodiben- TableCompound 1. 1H and 132Cwas NMR isolated data of as 1 amorphousand 2. powder with molecular formula C23H20O4, zofuranas determined A (1 by), except positive-ion that HRESIMS, the 2′-methoxy showing group an [M + [ Na]δH +3.70ion at(3H,m/z 383.12612s), δC 55.5] (calcd of 2 replaced the

2for′-hydroxy C23H20O4 Na,group 383.12593). [δH 4.97 The (1H, presence1 s)] of a1 conjugated. This was carbonyl supported group wasby2 revealedthe ROESY by correlations −1 a band at 1645 cm in the IRa spectrum, andb was conﬁrmed by the resonancea at δ 203.7b betweenPosition OMe-2 δH′ (J( δinH Hz)3.70) and H-3δ′ C( δ H 6.91) andδH ( Jby in Hz)the HMBC correlationsC δC between in the 13C NMR spectrum. The 1H NMR data of 2 were similar to eupatodibenzofuran OMe-21′ (δH 3.70) and 7.65,0 C-2s ′ (δC 156.7) 125.4 (Table 1). The position 7.59, s of each substituent 125.10 was sup- A(1), except that the 2 -methoxy group [δH 3.70 (3H, s), δC 55.5] of 2 replaced the 2 - 2 116.6 H H 116.0 H portedhydroxy by group ROESY [δH 4.97 correlations (1H, s)] of 1between. This was H-1 supported (δ 7.59)/Ac-2 by the ROESY (δ 2.40), correlations Me-6 (δ 2.60)/H-7 0 0 (betweenδH 7.27),3 OMe-2 H-7 ((δH 3.70)7.27)/H-8 and H-3 (δ(Hδ H7.17),6.91) 163.2 andOMe-2 by the′ (δ HMBCH 3.70)/H-3 correlations ′ (δH between6.91), H-3 162.9 OMe-′ (δH 6.91)/Me-4′ 0 0 (2δH(δ 2.49),H 3.70)4 and and C-2Me-4 7.07,(δC′ (156.7) δsH 2.49)/H-5 (Table1).′ The ( 99.8δH position 6.95) and of each by7.05, substituentHMBC s correlation was supported 99.6 between ‘H-1 (δH by ROESY4a correlations between H-1 (δH 161.27.59)/Ac-2 (δH 2.40), Me-6 (δH 2.60)/H-7 161.3 (δH 7.59)/MeCO-2 (δC 203.7), C-3 (δC 162.9),0 C-4a (δC 161.3),0 C-9a (0δC 121.7)’, ‘Ac-20 (δH 2.40)/C-2 7.27), H-75a (δH 7.27)/H-8 (δH 7.17), OMe-2 155.9(δH 3.70)/H-3 (δH 6.91), H-3 (δH 6.91)/Me-4 155.4 ((δδC 116.0)’,2.49), and ‘OH-3 Me-40 ((δδH 12.62)/C-22.49)/H-50 ( δ(δC6.95) 116.0)’, and by‘H-4 HMBC (δH 7.05)/C-2 correlation ( betweenδC 116.0), ‘H-1 C-9b (δC 117.8)’, H 6 H H 121.8 120.5 ‘Me-6(δH 7.59)/Me (δH 2.60)/C-5aCO-2 (δC 203.7), (δC 155.4), C-3 (δC 162.9),C-7 (δ C-4aC 127.9)’, (δC 161.3), ‘H-7 C-9a (δH ( δ7.27)/C-5aC 121.7)’, ‘Ac-2 (δC ( δ155.4)’,H ‘H-8 (δH 7 7.33, br d (7.5) 128.6 7.27, br d (7.6) 127.9 7.17)/C-62.40)/C-2 ((δδCC116.0)’, 120.5), ‘OH-3 C-9a ( δ(HδC12.62)/C-2 121.7), C-1 (δC′ 116.0)’,(δC 125.4)’, ‘H-4 (‘H-6δH 7.05)/C-2′ (δH 7.28)/C-9 (δC 116.0), (δ C-C 130.6), C-2′ (δC 9b (δ 117.8)’,8 ‘Me-6 7.22, (δ d (7.5)2.60)/C-5a (δ 125.1155.4), C-7 (δ 127.9)’, 7.17, d ‘H-7 (7.6) (δ 7.27)/C-5a 125.0 (δ 156.7),C C-4′ (δC 139.8)’,H ‘OMe-2′ (δCH 3.70)/C-2′ C(δC 156.7)’, ‘Me-4H ′ (δH 2.49)/C-4C ′ (δC 139.8), δ δ δ 0 δ 0 δ 155.4)’,9 ‘H-8 ( H 7.17)/C-6 ( C 120.5), C-9a 127.7 ( C 121.7), C-1 (1 C 125.4)’, 13 ‘H-6 ( H 7.28)/C- 130.6 C-5′ (δC 121.4)’0 (Figure 3). The0 full assignment0 of H and 0 C NMR resonances0 was con- 9 (δC 130.6),9a C-2 (δC 156.7), C-4 (δC 139.8)’, 121.9 ‘OMe-2 (δH 3.70)/C-2 (δC 156.7)’, ‘Me-4 121.7 firmedδ by 1H–0 δ1H COSY, ROESY0 δ (Figure 3A), HSQC, and HMBC1 (Figure13 3B) techniques. ( H 2.49)/C-49b ( C 139.8), C-5 ( C 121.4)’ 116.3 (Figure 3). The full assignment of H and 117.8C AccordingNMR resonances to wasthe conﬁrmed evidence by 1H– 1above,H COSY, ROESYthe (Figurestructure3A), HSQC,of 2 and was HMBC elucidated as 1′ 122.4 125.4 1-(3-hydroxy-9-(2-methoxy-4-methylphenyl)-6-methyldibenzo[(Figure3B) techniques. According to the evidence above, the structure of 2 wasb,d elucidated]furan-2-yl)ethan-1-on e,as named 1-(3-hydroxy-9-(2-methoxy-4-methylphenyl)-6-methyldibenzo[ eupatodibenzofuran B. b,d]furan-2-yl)ethan-1- one, named eupatodibenzofuran B.

FigureFigure 3. 3.Key Key ROESY ROESY (A) ( andA) HMBCand HMBC (B) correlations (B) correlations of 2. of 2.

Compound 3 was isolated as light brown amorphous powder. Its molecular formula, Compound 3 was isolated as light brown amorphous powder. Its molecular+ formu- C14H16O4, was determined on the basis of the positive HRESIMS at m/z 249.1123 [M + H] la,(calcd C14 249.1121)H16O4, was and wasdetermined supported byon the the1H basis and 13 ofC NMRthe positive data. The HRESIMS IR absorption at bands m/z 249.1123 [M + H]of 3+ revealed(calcd 249.1121) the presence and of carbonylwas supported (1686 cm −by1) function.the 1H and Analyses 13C NMR of the 1data.H and The13C IR absorption bands of 3 revealed the presence of carbonyl (1686 cm−1) function. Analyses of the 1H and

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NMR data of 3 showed the signals for two methyl groups [δH 1.55 (6H, s, Me-2 × 2); δC 26.5 (Me-2 × 2)], an acetyl group [δH 2.60 (3H, s, Ac-6); δC 26.2 (COMe), 196.6 (COMe)], a methoxy group [δH 3.95 (3H, s, OMe-8); δC 56.4 (OMe-8)], two meta-coupled aromatic protons [δH 7.70, 8.08 (each 1H, each d, J = 2.0 Hz, H-7 and H-5); δC 114.3 (C-7), 120.0 (C-5)], and two methylene protons [δH 2.79 (2H, s, H-3); δC 48.5 (C-3)] (Table2). The position of each substituent was supported by the ROESY correlations between Me-2 (δH 1.55)/H-3 (δH 2.79), H-5 (δH 8.08)/Ac-6 (δH 2.60), Ac-6 (δH 2.60)/H-7 (δH 7.70), and H-7 (δH 7.70)/OMe-8 (δH 3.95) and HMBC correlations between ‘Me-2 (δH 1.55)/C-2 (δC 81.0), C-3 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW(δC 48.5)’, ‘H-3 (δH 2.79)/C-2 (δC 81.0), C-4 (δC 191.7), Me-2 (δC 26.5)’, ‘H-5 (δH 8.08)/C-4 5 of 17

(δC 191.7), MeCO-6 (δC 196.6), C-7 (δC 114.3), C-8a (δC 154.0)’, ‘H-7 (δH 7.70)/C-5 (δC 120.0), MeCO-6 (δC 196.6), C-8a (δC 154.0)’, and ‘OMe-8 (δH 3.95)/C-8 (δC 149.8)’ (Figure4). The full assignment of 1H and 13C NMR resonances was further conﬁrmed by the 1H-1H COSY, 13CHSQC, NMR 1D-selective data of 3 showed NOESY (Figure the signals4A), and for HMBC two (Figuremethyl4B) groups data. Conse [δH 1.55quently, (6H, the s, Me-2 × 2); δC structure of compound 3 was established as 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one. 26.5 (Me-2 × 2)], an acetyl group [δH 2.60 (3H, s, Ac-6); δC 26.2 (COMe), 196.6 (COMe)], a

methoxyTable 2. 1 Hgroup and 13C [ NMRδH 3.95 data of(3H,3. s, OMe-8); δC 56.4 (OMe-8)], two meta-coupled aromatic protons [δH 7.70, 8.08 (each 1H, each d, J = 2.0 Hz, H-7 and H-5); δC 114.3 (C-7), 120.0 a b Position δH J (Hz) δ (C-5)], and two methylene protons [δH 2.79 (2H, s, H-3); δC 48.5 C(C-3)] (Table 2). The posi- 2 81.0 tion of each substituent was supported by the ROESY correlations between Me-2 (δH 3 2.79, s 48.5 1.55)/H-3 (δH 2.79),4 H-5 (δH 8.08)/Ac-6 (δH 2.60), Ac-6 (δH 2.60)/H-7 191.7 (δH 7.70), and H-7 (δH 7.70)/OMe-8 (4aδH 3.95) and HMBC correlations between ‘Me-2 119.6 (δH 1.55)/C-2 (δC 81.0), C-3 5 8.08, d (2.0) 120.0 (δC 48.5)’, ‘H-36 (δH 2.79)/C-2 (δC 81.0), C-4 (δC 191.7), Me-2 (δC 26.5)’, 129.6 ‘H-5 (δH 8.08)/C-4 (δC 191.7), MeCO-67 (δC 196.6), C-7 (δC 7.70,114.3), d (2.0) C-8a (δC 154.0)’, ‘H-7 114.3 (δH 7.70)/C-5 (δC 120.0), 8 149.8 MeCO-6 (δC 196.6), C-8a (δC 154.0)’, and ‘OMe-8 (δH 3.95)/C-8 (δC 149.8)’ (Figure 4). The 8a 154.0 full assignmentMe-2 of 1H and 13C NMR 1.55, resonances s was further 26.5 confirmed by the 1H-1H COSY, HSQC,COMe-6 1D-selective NOESY (Figure 4A), and HMBC 196.6 (Figure 4B) data. Conse- COMe-6 2.60, s 26.2 quently, OMe-8the structure of 3.95, scompound 3 56.4was established as a b 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one.Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses. Recorded in CDCl3 at 125 MHz.

FigureFigure 4. 4. KeyKey 1D-selective NOESY NOESY (A) and(A) HMBCand HMBC (B) correlations (B) correlations of 3. of 3.

Compound1 134 was obtained as colorless oil and the molecular formula was determined Table 2. H and C NMR data of 3. + to be C14H16O3 by HRESIMS [m/z 255.10030 [M + Na] (calcd for C14H16O3Na, 255.09971)]. The IRPosition spectrum showed the presence ofδ esterH J (Hz) and conjugated a carbonyl groups at 1736δ andC b 1684 cm−1, respectively. Analysis of the 1H and 13C NMR data of 4 revealed the signals for 2 81.0 an acetyl group [δH 2.52 (3H, s, Ac-2); δC 29.4 (COMe), 197.1 (COMe)], a methyl group [δH 2.40 (3H, s,3 Me-5); δC 21.4 (Me-5)], a (Z)-(2-methylbut-2-enoyl)oxy2.79, s group [δH 2.08 (3H, 48.5 dq, J = 7.3, 1.34 Hz, H-40), 2.08 (3H, qd, J = 1.8, 1.3 Hz, H-50), 6.30 (1H, qq, J = 7.3, 1.8 Hz, H-3 191.70); δ 0 0 0 0 0 C 16.0 (C-44a ), 20.6 (C-5 ), 127.0 (C-2 ), 141.4 (C-3 ), 166.1 (C-1 )], and three mutually coupled 119.6 aromatic protons [δH 6.94 (1H, br s, H-6), 7.12 (1H, br d, J = 8.0 Hz, H-4), and 7.74 (1H, d, J = 8.0 Hz5, H-3); δC 124.4 (C-6), 126.7 (C-4), 8.08, and d 130.4(2.0) (C-3)] (Table3). The position of each 120.0 6 129.6 7 7.70, d (2.0) 114.3 8 149.8 8a 154.0 Me-2 1.55, s 26.5 COMe-6 196.6 COMe-6 2.60, s 26.2 OMe-8 3.95, s 56.4 a Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses. b Recorded in CDCl3 at 125 MHz.

Compound 4 was obtained as colorless oil and the molecular formula was determined to be C14H16O3 by HRESIMS [m/z 255.10030 [M + Na]+ (calcd for C14H16O3Na, 255.09971)]. The IR spectrum showed the presence of ester and conjugated carbonyl groups at 1736 and 1684 cm−1, respectively. Analysis of the 1H and 13C NMR data of 4 revealed the signals for an acetyl group [δH 2.52 (3H, s, Ac-2); δC 29.4 (COMe), 197.1 (COMe)], a methyl group [δH 2.40 (3H, s, Me-5); δC 21.4 (Me-5)], a (Z)-(2-methylbut-2-enoyl)oxy group [δH 2.08 (3H, dq, J = 7.3, 1.3 Hz, H-4′), 2.08 (3H, qd, J = 1.8, 1.3 Hz, H-5′), 6.30 (1H, qq, J = 7.3, 1.8 Hz, H-3′); δC 16.0 (C-4′), 20.6 (C-5′), 127.0 (C-2′),

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substituent was supported by the ROESY correlations between Ac-2 (δH 2.52)/H-3 (δH 7.74), H-3 (δH 7.74)/H-4 (δH 7.12), H-4 (δH 7.12)/Me-5 (δH 2.40), Me-5 (δH 2.40)/H-6 (δH 6.94), 0 0 0 0 H-3 (δH 6.30)/H-4 (δH 2.08), and H-3 (δH 6.30)/H-5 (δH 2.08) and by HMBC correlations between ‘COMe-2 (δH 2.52)/C-2 (δC 128.3), COMe-2 (δC 197.1)’, ‘H-3 (δH 7.74)/C-1 (δC 149.3), COMe-2 (δC 197.1), C-5 (δC 144.7)’, ‘H-4 (δC 7.12)/C-2 (δC 128.3), C-6 (δC 124.4), Me-5 (δC 21.4)’, ‘Me-5 (δH 2.40)/C-4 (δC 126.7), C-5 (δC 144.7), C-6 (δC 124.4)’, ‘H-6 (δH 0 0 6.94)/C-1 (δC 149.3), C-2 (δC 128.3), C-4 (δC 126.7), Me-5 (δC 21.4)’, ‘H-4 (δH 2.08)/C-2 (δC 0 0 0 0 127.0)’, and ‘H-5 (δH 2.08)/C-1 (δC 166.1), C-2 (δC 127.0), C-3 (δC 141.4)’ (Figure5). The full assignment of 1H and 13C NMR resonances was supported by 1H–1H COSY, HSQC, ROESY (Figure5A), and HMBC (Figure5B) spectral analyses. According to the above data, Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWthe structure of 4 was elucidated as (Z)-2-acetyl-5-methylphenyl 2-methylbut-2-enoate, 6 of 17

named eupatofortunone.

Table 3. 1H and 13C NMR data of 4. 141.4 (C-3′), 166.1 (C-1′)], and three mutually coupled aromatic protons [δH 6.94 (1H, br s, a b H-6), 7.12 (1H,Position br d, J = 8.0 Hz, H-4), δandH J (Hz) 7.74 (1H, d, J = 8.0 Hz, H-3);δC δC 124.4 (C-6), 126.7 (C-4), and 130.41 (C-3)] (Table 3). The position of each substituent 149.3 was supported by the ROESY correlations2 between Ac-2 (δH 2.52)/H-3 (δH 7.74), H-3 (δH 128.37.74)/H-4 (δH 7.12), H-4 3 7.74, d (8.0) 130.4 (δH 7.12)/Me-5 (δH 2.40), Me-5 (δH 2.40)/H-6 (δH 6.94), H-3′ (δH 6.30)/H-4′ (δH 2.08), and H-3′ 4 7.12, br d (8.0) 126.7 (δH 6.30)/H-5′5 (δH 2.08) and by HMBC correlations between ‘COMe-2 144.7 (δH 2.52)/C-2 (δC 128.3), COMe-26 (δC 197.1)’, ‘H-3 (δH 7.74)/C-1 6.94, br s (δC 149.3), COMe-2 (δ 124.4C 197.1), C-5 (δC 144.7)’, 10 166.1 ‘H-4 (δC 7.12)/C-2 (δC 128.3), C-6 (δC 124.4), Me-5 (δC 21.4)’, ‘Me-5 (δH 2.40)/C-4 (δC 126.7), 20 127.0 C-5 (δC 144.7),30 C-6 (δC 124.4)’, ‘H-66.30, (δH qq 6.94)/C-1 (7.3, 1.8) (δC 149.3), C-2 (δ 141.4C 128.3), C-4 (δC 126.7), 0 Me-5 (δC 21.4)’,4 ‘H-4′ (δH 2.08)/C-22.08,′ (δC dq127.0)’, (7.3, 1.3) and ‘H-5′ (δH 2.08)/C-1 16.0′ (δC 166.1), C-2′ (δC 0 127.0), C-3′ (δ5C 141.4)’ (Figure 5). The2.08, full qd (1.8,assignment 1.3) of 1H and 13C 20.6 NMR resonances was COMe-21 1 197.1 supported COMeby H–-2H COSY, HSQC, ROESY 2.52, s (Figure 5A), and HMBC 29.4 (Figure 5B) spectral analyses. AccordingMe-5 to the above 2.40,data, s the structure of 4 21.4 was elucidated as a b (ZRecorded)-2-acetyl-5-methylphenyl in CDCl3 at 500 MHz. Values 2-methylbu in ppm (δ). J (int-2-enoate, Hz) in parentheses. namedRecorded eupatofortunone. in CDCl3 at 125 MHz.

FigureFigure 5. Key ROESYROESY (A (A) and) and HMBC HMBC (B) ( correlationsB) correlations of 4. of 4.

TableCompound 3. 1H and 135Cwas NMR isolated data of as 4. light brown amorphous powder. Its molecular formula, C20H20O4S2, was determined on the basis of the positive HRESIMS at m/z 411.07026 + 1 13 [M + Na]Position(calcd 411.07007) and wasδH J supported (Hz) a by the H and C NMR data.δC b The IR absorption bands of 5 revealed the presence of alkynyl (2230 cm−1) and conjugated carbonyl 1 149.3 (1639 cm−1) functions. Analysis of the 1H and 13C NMR data of 5 revealed the signals for 2 128.3 an acetyl group [δH 2.50 (3H, s, Ac-2); δC 29.4 (COMe-2), 190.2 (COMe-2)], a prop-1-yn- 1-yl group3 [δH 2.09 (3H, s, C≡CMe); 7.74,δC d4.8 (8.0) (C ≡CMe), 73.2 (C≡CMe), 94.8 (C 130.4≡CMe)], a methoxy group4 [δH 3.94 (3H, s, OMe-5); 7.12, brδC d58.8 (8.0) (OMe-5)], and a singlet aromatic 126.7 proton [δH 6.84 (1H, s,5 H-4); δC 119.1 (C-4)] (Table4). The position of each substituent was 144.7 supported by the HMBC6 correlation between6.94, ‘COMe br (sδ H 2.50)/C-2 (δC 122.6), COMe-2 124.4 (δC 190.2)’, ‘C≡CMe (δ 2.09)/C≡CMe (δ 73.2), C≡CMe (δ 94.8)’, ‘H-4 (δ 6.84)/C-2 (δ 122.6), C-3 1′H C C H 166.1C (δC 130.6), C≡CMe (δC 73.2)’, ‘OMe-5 (δH 3.94)/C-5 (δC 159.3)’ and by the 1D selective 2′ 127.0 NOESY correlation between H-4 (δH 6.84) and OMe-5 (δH 3.94) (Figure6). According to 3′ 6.30, qq (7.3, 1.8) 141.4 4′ 2.08, dq (7.3, 1.3) 16.0 5′ 2.08, qd (1.8, 1.3) 20.6 COMe-2 197.1 COMe-2 2.52, s 29.4 Me-5 2.40, s 21.4

a Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses. b Recorded in CDCl3 at 125 MHz.

Compound 5 was isolated as light brown amorphous powder. Its molecular formula, C20H20O4S2, was determined on the basis of the positive HRESIMS at m/z 411.07026 [M + Na]+ (calcd 411.07007) and was supported by the 1H and 13C NMR data. The IR absorption bands of 5 revealed the presence of alkynyl (2230 cm−1) and conjugated carbonyl (1639 cm−1) functions. Analysis of the 1H and 13C NMR data of 5 revealed the signals for an acetyl group [δH 2.50 (3H, s, Ac-2); δC 29.4 (COMe-2), 190.2 (COMe-2)], a prop-1-yn-1-yl group [δH 2.09 (3H, s, C≡CMe); δC 4.8 (C≡CMe), 73.2 (C≡CMe), 94.8 (C≡CMe)], a methoxy group [δH 3.94 (3H, s, OMe-5); δC 58.8 (OMe-5)], and a singlet aro-

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the 1H, 13C NMR, and HR-ESI-MS data, the number of resonances observed was half that expected, suggesting that 5 had a symmetrical structure. The full assignment of 1H and 13C NMR resonances was further conﬁrmed by 1H-1H COSY, 1D-selective NOESY (Figure6A ), HSQC, and HMBC (Figure6B) data. Consequently, the structure of compound 5 was established as 1,10-((2E,4Z,7Z,9E)-5,7-dimethoxy-3,9-di(prop-1-yn-1-yl)-1,6-dithiecine-2,10- diyl)diethanone, named eupatodithiecine.

Table 4. 1H and 13C NMR data of 5.

a b Position δH J (Hz) δC 2 122.6 3 130.6 4 6.84, s 119.1 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 17 5 159.3 7 159.3 8 6.84, s 119.1 9 130.6 matic proton 10[δH 6.84 (1H, s, H-4); δC 119.1 (C-4)] (Table 4). The position 122.6 of each substituent was supportedCOMe-2 by the HMBC correlation between ‘COMe (δH 190.22.50)/C-2 (δC 122.6), COMe-2 (δCOMeC 190.2)’,-2 ‘C≡CMe (δH 2.09)/C 2.50,≡CMe s (δC 73.2), C≡CMe 29.4(δC 94.8)’, ‘H-4 (δH C≡CMe-3 73.2 6.84)/C-2 (δC 122.6), C-3 (δC 130.6), C≡CMe (δC 73.2)’, ‘OMe-5 (δH 3.94)/C-5 (δC 159.3)’ and C≡CMe-3 94.8 by the 1D selective NOESY correlation between H-4 (δH 6.84) and OMe-5 (δH 3.94) (Figure C≡CMe-3 2.09, s 4.8 1 13 6). AccordingOMe-5 to the H, C NMR, and 3.94,HR-ESI-MS s data, the number 58.8 of resonances observed wasOMe-7 half that expected, suggesting 3.94, that s 5 had a symmetrical 58.8 structure. The full assignmentC≡ ofCMe-9 1H and 13C NMR resonances was further confirmed 73.2 by 1H-1H COSY, ≡ 1D-selectiveC CNOESYMe-9 (Figure 6A), HSQC, and HMBC (Figure 6B) data. 94.8 Consequently, the C≡CMe-9 2.09, s 4.8 structure COMe-10of compound 5 was established 190.2 as 1,1′-((2E,4ZCOMe,7Z,9E-10)-5,7-dimethoxy-3,9-di(prop-1-yn-1-yl)-1,6-dithiecine-2,10-diyl)diethan 2.50, s 29.4 a b one,Recorded named in CDCl eupatodithiecine.3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses. Recorded in CDCl3 at 125 MHz.

Figure 6.6. KeyKey 1D-selective1D-selective NOESY NOESY (A ()A and) and HMBC HMBC (B )(B correlations) correlations of 5 of. 5.

Table2.3. Structure 4. 1H and Identiﬁcation 13C NMR data of the of 5 Known. Isolated Compounds The known compounds were readily identified by a comparison of their physical and spectroscopicPosition data (UV, IR, 1H NMR,δH J and (Hz) MS) a with those of authentic samplesδC b or literature values. They2 include four thymol derivatives, thymyl angelate (6)[10], 8,9-dehydrothymol122.6 3-O-tiglate3 (7)[4], 9-angeloyloxythymol (8)[5], and 9-O-angeloyl-8,10-dehydrothymol 130.6 (9)[11], five4 phenol derivatives, 2-hydroxy-4-methylacetophenone6.84, s (10)[12], trans 119.1-o -coumaric acid (11)[13], 6-hydroxy-7-methoxy-2-isopropenyl-5-acetylcumaran (12)[14], 2,4-di-tert- 5 159.3 butylphenol (13)[15], and 1-(2-hydroxy-5-methoxy-4-methylphenyl)ethanone (14)[16], a coumarin7 (15 )[17], and a triterpenoid, taraxasterol (16)[18]. 159.3 8 6.84, s 119.1 9 130.6 10 122.6 COMe-2 190.2 COMe-2 2.50, s 29.4 C≡CMe-3 73.2 C≡CMe-3 94.8 C≡CMe-3 2.09, s 4.8 OMe-5 3.94, s 58.8 OMe-7 3.94, s 58.8 C≡CMe-9 73.2 C≡CMe-9 94.8 C≡CMe-9 2.09, s 4.8 COMe-10 190.2 COMe-10 2.50, s 29.4

a Recorded in CDCl3 at 500 MHz. Values in ppm (δ). J (in Hz) in parentheses. b Recorded in CDCl3 at 125 MHz.

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2.4. Biological Studies The cytotoxic effects of the isolated compounds from E. fortunei were evaluated by their activities to suppress A549 and MCF-7 cells. The cytotoxic activity data are shown in Table5. Among the isolated compounds, compound 1 exhibited potent inhibitory activities with IC50 values of 5.95 ± 0.89 and 5.32 ± 0.31 µM, respectively, against A549 and MCF-7 cells. In addition, colony-formation assay was performed to estimate the effects of compound 1 on the proliferation of A549 and MCF-7 cells. As shown in Figure7, compound 1 reduced colony formation in a dose-dependent manner in both cell lines. In addition, we generated physicochemical properties (Table S1) of compound 1 using Pipeline Pilot [19]. The ALogP98 value, molecular polar surface area, and ADMET absorption level suggest that compound 1 is hydrophobic and may have good permeability to cross the cell membrane, which may account for the cytotoxic effects of compound 1.

Table 5. In vitro inhibitory effects of compounds 1-16 isolated from the aerial part of E. fortunei against A549 and MCF-7 cells.

a IC50 (µM) Compound A549 MCF-7 Eupatodibenzofuran A (1) 5.95 ± 0.89 ** 5.55 ± 0.23 ** Eupatodibenzofuran B (2) 93.37 ± 1.14 85.91 ± 3.94 6-Acetyl-8-methoxy-2,2-dimethylchroman-4-one (3) >100 >100 Eupatofortunone (4) 86.63 ± 10.89 * 82.15 ± 8.26 ** Eupatodithiecine (5) 39.44 ± 2.81 * 31.20 ± 4.23 * Thymyl angelate (6) >100 >100 8,9-Dehydrothymol 3-O-tiglate (7) >100 >100 9-Angeloyloxythymol (8) 60.08 ± 3.39 * 52.11 ± 2.16 * 9-O-Angeloyl-8,10-dehydrothymol (9) 55.36 ± 0.80 * 51.70 ± 0.48 * 2-Hydroxy-4-methylacetophenone (10) >100 >100 trans-o-Coumaric acid (11) >100 >100 6-Hydroxy-7-methoxy-2-isopropenyl-5- acetylcumaran 73.97 ± 2.88 * 72.67 ± 3.51 * (12) 2,4-Di-tert-butylphenol (13) >100 >100 1-(2-Hydroxy-5-methoxy-4-methylphenyl)ethanone (14) >100 >100 Coumarin (15) >100 >100 Taraxasterol (16) 94.79 ± 10.23 * 93.59 ± 6.11 * 5-Fluorouracil (5FU) b 10.57 ± 1.89 ** 8.59 ± 1.03 ** a The IC50 values were calculated from the slope of the dose-response curves (SigmaPlot). Values are expressed as average ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared with the control. b 5-Fluorouracil (5-FU) was used as a positive control against A549 and MCF-7 cells.

To further confirm whether apoptosis was triggered, annexin V/propidium iodide (PI) assay was performed and the expression levels of apoptosis-associated proteins, Bcl-2, Bax, and caspase-3 were analyzed by Western blot analysis after A549 and MCF-7 cells were treated with compound 1. As shown in Figure8A,B, compound 1 significantly induced the cell apoptosis in A549 and MCF-7 cells, respectively. Furthermore, compound 1 increased the expression of Bax and cleaved-caspase-3, and decreased Bcl-2 and pro-caspase-3 levels in a dose-dependent manner in both A549 and MCF-7 cells (Figure9A,B). The above results confirm that compound 1 markedly induces apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways (Scheme1). Int. J. Mol. Sci. 2021, 22, 7448 9 of 16 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 17

FigureFigure 7. 7.CompoundCompound 1 1inhibitedinhibited the the growth ofof human human non-small-cell non-small-cell lung lung cancer cancer cell cell (A549) (A549) and and human human breast breast cancer cancer cell cell(MCF-7) (MCF-7) in in a dose-dependenta dose-dependent manner manner (1.25–10 (1.25–10µM) byμM) the by colony-formation the colony-formation assay. Theassay. data The were data expressed were expressed as mean ± asSEM mean ± SEM(n = 3).(n = Asterisks 3). Asterisks indicate indicate signiﬁcant significant differences differences (* p < 0.05(* p and< 0.05 *** andp < 0.001)*** p < compared0.001) compared with the with control the group.control group.

ToTo further further confirm understand whether the mechanism apoptosis was of compound triggered,1 annexinin this study, V/propidium we predicted iodide (PI)potential assay targetswas performed using the similarity and the ensembleexpression approach levels serverof apoptosis-associated [20]. This approach predictsproteins, Bcl-2,possible Bax, target and caspase-3 proteins of were a compound analyzed by by comparing Western blot chemical analysis similarities. after A549 Compound and MCF-71 was predicted to target four proteins (Supplementary Materials Table S2), including PON1, cells were treated with compound 1. As shown in Figure 8A,B, compound 1 significantly CELA1, CBR1, and NQO1. The Tanimoto coefﬁcients (Tc) of chemical similarity were induced the cell apoptosis in A549 and MCF-7 cells, respectively. Furthermore, com- generated for the predicted targets. The Tc is a pairwise score between the compound and pound 1 increased the expression of Bax and cleaved-caspase-3, and decreased Bcl-2 and the predicted target. The Tc score ranges from 0.0 (no similarity) to 1.0 (total similarity). pro-caspase-3The P-value indicateslevels in thea dose-dependent prediction reliability, manner and in aboth value A549 approaching and MCF-7 zero cells means (Figure a 9A,B).reliable The prediction. above results The confirm possible that targets compound may account 1 markedly for the inhibitioninduces apoptosis mechanisms of A549 of andcompound MCF-7 cells1. through mitochondrial- and caspase-3-dependent pathways (Scheme 1). To further understand the mechanism of compound 1 in this study, we predicted potential targets using the similarity ensemble approach server [20]. This approach pre- dicts possible target proteins of a compound by comparing chemical similarities. Com- pound 1 was predicted to target four proteins (Supplementary Materials Table S2), including PON1, CELA1, CBR1, and NQO1. The Tanimoto coefficients (Tc) of chemical similarity were generated for the predicted targets. The Tc is a pairwise score between the compound and the predicted target. The Tc score ranges from 0.0 (no similarity) to 1.0 (total similarity). The P-value indicates the prediction reliability, and a value approaching zero means a reliable prediction. The possible targets may account for the inhibition mechanisms of compound 1.

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Figure 8.FigureCompound 8. Compound1 induced cell1 induced apoptosis cell in A549apoptosis (A) and in MCF-7A549 ( (AB) cells.and MCF-7 Cells were (B) treatedcells. Cells with compoundwere treated1 (0, 5, 10 µM) forwith 24 h.compound Apoptotic 1 cells (0, 5, with 10 AnnexinμM) for 24 V-FITC h. Apoptotic and PI staining cells with were A analyzednnexin V-FITC by ﬂow and cytometer. PI staining The data were were expressedanalyzed as means by± SEMflow ( ncytometer.= 3). Asterisks The indicate data were signiﬁcant expressed differences as means (* p < ± 0.05) SEM compared (n = 3). Asterisks to the control indicate group. significant differences (* p < 0.05) compared to the control group.

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Figure 9. Compound 1 inhibited the anti-apoptotic protein Bcl-2, increased pro-apoptotic protein Bax, and activated Figure 9. Compound 1 inhibited the anti-apoptotic protein Bcl-2, increased pro-apoptotic protein Bax, and activated caspase-3 in A549 (A) and MCF-7 (B) cells. Cells were treated with 1 (1.25–10 μM) for 72 h. Bcl-2, Bax, pro-caspase-3, and µ caspase-3cleaved-caspase-3 in A549 ( Awere) and analyzed MCF-7 (byB) immunoblotting. cells. Cells were Densitometric treated with 1 analysis(1.25–10 ofM) tested for 72compounds h. Bcl-2, Bax, was pro-caspase-3,normalized to the and cleaved-caspase-3corresponding expression were analyzed of β-actin. by immunoblotting.The data were expressed Densitometric as means analysis ± SEM of(n tested= 3). Asterisks compounds indicate was significant normalized dif- to theferences corresponding (* p < 0.05, expression ** p < 0.01, of andβ-actin. *** p < The0.001) data compared were expressed to the control as means group.± SEM (n = 3). Asterisks indicate signiﬁcant differences (* p < 0.05, ** p < 0.01, and *** p < 0.001) compared to the control group.

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Scheme 1. The mechanism of apoptosis for compound 1 in A549 and MCF-7 cells. Scheme 1. The mechanism of apoptosis for compound 1 in A549 and MCF-7 cells.

3.3. MaterialsMaterials andand Methods Methods 3.1.3.1. GeneralGeneral ExperimentalExperimental Procedures Procedures UltravioletUltraviolet (UV) (UV) spectra spectra were were obtained obtained on aon Jasco a Jasco UV-240 UV-240 spectrophotometer. spectrophotometer. Infrared In- (IR)frared spectra (IR) (neatspectra or (neat KBr) wereor KBr) recorded were recorded on a Perkin on Elmera Perkin 2000 Elmer FT-IR 2000 spectrometer. FT-IR spectrometer.Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), rotatingNuclear frame magnetic nuclear resonance Overhauser (NMR) effect spectra, spectroscopy including (ROESY), correlation nuclear spectroscopy Overhauser (COSY), effect spectroscopyrotating frame (NOESY), nuclear heteronuclear Overhauser effect multiple-bond spectroscopy correlation (ROESY), (HMBC), nuclear and Overhauser heteronuclear ef- single-quantumfect spectroscopy coherence (NOESY), (HSQC) heteronuclear experiments, multiple-bond were acquired correlation using a BRUKER (HMBC), AVIII-500 and het- 1 13 spectrometereronuclear single-quantum (Bruker, Bremen, coherence Germany), (HSQ operatingC) experiments, at 500 MHz (wereH) and acquired 125 MHz using ( C), a δ respectively,BRUKER AVIII-500 with chemical spectrometer shifts given (Bruker, in the Bremen, ppm ( )Germany) using tetramethylsilane, operating at 500 (TMS) MHz as ( an1H) internaland 125 standard. MHz (13C), Electrospray respectively, ionization with chemical (ESI) and shifts high-resolution given in the electrospray ppm (δ) using ionization tetra- (HRESI)-massmethylsilane spectra(TMS) wereas an recorded internal on standard. a Bruker APEXElectrospray II Mass Spectrometerionization (ESI) (Bruker, and µ µ Bremen,high-resolution Germany). electrospray Silica gel ionization [70–230 mesh (HRESI)-mass (63–200 m) spectra and 230–400were recorded mesh (40–63 on a Brukerm), Merck] was used for column chromatography (CC). Silica gel 60 F-254 (Merck, Darm- APEX II Mass Spectrometer (Bruker, Bremen, Germany). Silica gel [70–230 mesh (63–200 stadt, Germany) was used for thin-layer chromatography (TLC) and preparative thin-layer μm) and 230–400 mesh (40–63 μm), Merck] was used for column chromatography (CC). chromatography (PTLC). Silica gel 60 F-254 (Merck, Darmstadt, Germany) was used for thin-layer chromatog- 3.2.raphy Plant (TLC) Material and preparative thin-layer chromatography (PTLC). The aerial part of E. fortunei collected from Dihua St., Datong Dist., Taipei City, Taiwan, 3.2. Plant Material in May 2019 and identiﬁed by Prof. J.-J. Chen. A voucher specimen was deposited in the DepartmentThe aerial of Pharmacy, part of E. National fortunei Yangcollected Ming from Chiao Dihua Tung University,St., Datong Taipei,Dist., Taiwan.Taipei City, Taiwan, in May 2019 and identified by Prof. J.-J. Chen. A voucher specimen was depos- 3.3.ited Extraction in the Department and Isolation of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan.The aerial part of E. fortunei (5.0 kg) was pulverized and extracted three times with MeOH (30 L each) for 3 days. The MeOH extract was concentrated under reduced pres- 3.3. Extraction◦ and Isolation sure at 35 C, and the residue (123.7 g) was partitioned between EtOAc and H2O (1:1) to provideThe aerial the EtOAc-soluble part of E. fortunei fraction (5.0 kg) (fraction was pulverized A, 25.6 g). and Fraction extracted A (25.6 three g) times was chro- with matographedMeOH (30 L oneach) silica for gel 3 days. (70–230 The mesh, MeOH 1.3 extract kg), eluting was concentrated with n-hexane, under gradually reduced increas- pres- ingsure the at polarity35 °C, and with the EtOAc residue to (123.7 give teng) was fractions: partitioned A1 (1 between L, n-hexane/EtOAc, EtOAc and H 100:1),2O (1:1) A2 to

Int. J. Mol. Sci. 2021, 22, 7448 13 of 16

(1 L, n-hexane/EtOAc, 80:1), A3 (1.5 L, n-hexane/EtOAc, 70:1), A4 (2 L, n-hexane/EtOAc, 50:1), A5 (2 L, n-hexane/EtOAc, 30:1), A6 (2 L, n-hexane/EtOAc, 10:1), A7 (3.5 L, n- hexane/EtOAc, 5:1), A8 (2.5 L, n-hexane/EtOAc, 3:1), A9 (3 L, n-hexane/EtOAc, 1:1), and A10 (2.5 L, EtOAc). Fraction A2 (1.8 g) was subjected to column chromatography (CC) (85 g of silica gel, 230–400 mesh, n-hexane/acetone, 50:1–0:1, 250 mL–fractions) to give 11 subfractions: A2-1–A2-11. Part (125 mg) of fraction A2-2 was further purified by preparative TLC (silica gel, n-hexane/EtOAc, 30:1) to yield thymyl angelate (6)(5.2 mg) and 8,9-dehydrothymol 3-O-tiglate (7) (4.1 mg). Part (95 mg) of fraction A2-5 was further purified by preparative TLC (silica gel, n-hexane/CH2Cl2, 9:1) to obtain 2-hydroxy-4- methylacetophenone (10) (3.2 mg). Part (76 mg) of fraction A2-8 was further purified by preparative TLC (silica gel, n-hexane/EtOAc, 19:1) to afford eupatofortunone (4) (4.6 mg). Fraction A3 (1.6 g) was subjected to CC (72 g of silica gel, 230–400 mesh, n-hexane/EtOAc, 20:1–0:1, 250 mL–fractions) to give ten subfractions: A3-1–A3-10. Part (135 mg) of fraction A3-9 was further purified by preparative TLC (silica gel, n-hexane/acetone, 5:1) to obtain coumarin (15) (7.8 mg). Fraction A4 (2.3 g) was purified by medium pressure liquid chromatography (MPLC) (105 g of silica gel, 230–400 mesh, n-hexane/acetone, 19:1–0:1, 250 mL–fractions) to give eight subfractions: A4-1–A4-8. Fraction A4-5 (265 mg) was purified by MPLC (11.9 g of silica gel, n-hexane/EtOAc, 7:1) to afford four subfractions (each 150 mL, A4-5-1–A4-5-4). Fraction A4-5-3 (48 mg) was further purified by preparative TLC (silica gel, CH2Cl2/EtOAc, 9:1) to obtain trans-O-coumaric acid (11) (8.9 mg). Frac- tion A4-7 (53 mg) was further purified by preparative TLC (silica gel, CH2Cl2/acetone, 8:1) to obtain 2,4-di-tert-butylphenol (13) (3.2 mg). Faction A6 (2.2 g) was subjected to MPLC (100 g of silica gel, 230–400 mesh; CH2Cl2/EtOAc, 15:1–0:1, 250 mL–fractions) to give 13 subfractions: A6-1–A6-13. Part (92 mg) of fraction A6-6 was further purified by preparative TLC (silica gel, CH2Cl2/acetone, 7:1) to afford eupatodibenzofuran A (1) (4.3 mg) and eupatodibenzofuran B (2) (3.2 mg). Fraction A6-7 (226 mg) was purified by MPLC (silica gel, CH2Cl2/acetone, 4:1) to afford 5 subfractions (A6-7-1–A6-7-5, each 150 mL). Fraction A6-7-2 (49 mg) was further purified by preparative TLC (silica gel, CH2Cl2/EtOAc, 5:1) to obtain taraxasterol (16) (4.7 mg). Part (92 mg) of fraction A6-10 was further purified by preparative TLC (silica gel, CH2Cl2/EtOAc, 4:1) to yield 9-angeloyloxythymol (8) (5.1 mg) and 9-O-angeloyl-8,10-dehydrothymol (9) (4.3 mg). Part (111 mg) of fraction A6-11 was purified by preparative TLC (silica gel, CH2Cl2/EtOAc, 2:1) to obtain 6-hydroxy-7-methoxy-2-isopropenyl-5-acetylcumaran (12) (5.3 mg). Part (75 mg) of fraction A6-13 was further purified by semipreparative normal-phase high- −1 performance liquid chromatography (HPLC) (silica gel, CH2Cl2/EtOAc, 4:1, 2.0 mL min ) to afford 1-(2-hydroxy-5-methoxy-4-methylphenyl)ethanone (14) (4.2 mg). Faction A8 (1.6 g) was subjected to CC (72 g of silica gel, 230–400 mesh, CH2Cl2/MeOH 9:1–0:1, 250 mL–fractions) to give ten subfractions: A8-1–A8-10. Fraction A8-4 (88 mg) was further purified by semipreparative normal-phase HPLC (silica gel, CH2Cl2/MeOH, 9:1, 2.0 mL min−1) to afford eupatodithiecine (5) (3.7 mg). Part (93 mg) of fraction A8-7 was further purified by preparative TLC (silica gel, CH2Cl2/MeOH, 4:1) to yield 6-acetyl-8-methoxy- 2,2-dimethylchroman-4-one (3) (4.4 mg). ◦ Eupatodibenzofuran A (1): Colorless needles; mp 153–155 C (CH2Cl2); UV (MeOH) −1 λmax (log ε) 238 (4.09), 272 (3.97), 352 (1.43) nm; IR (KBr) υmax 3439 (OH), 1645 (C=O) cm ; 1 13 + H and C NMR data, see Table1; HRESIMS m/z 369.11041 [M + Na] (calcd for C22H18O4Na, 369.11028); HRESIMS, 1D-, and 2D-NMR spectra, see Supplementary Materials Figures S1–S7. Eupatodibenzofuran B (2): Amorphous powder; UV (MeOH) λmax (log ε) 238 (4.29), −1 1 13 271 (4.12), 348 (3.24) nm; IR (neat) υmax 3429 (OH), 1645 (C=O) cm ; H and C NMR + data, see Table1; HRESIMS m/z 383.12612 [M + Na] (calcd for C23H20O4Na, 383.12593); HRESIMS, 1D-, and 2D-NMR spectra, see Supplementary Materials Figures S8–S14. 6-Acetyl-8-methoxy-2,2-dimethylchroman-4-one (3): Light brown amorphous powder; UV (MeOH) λmax (log ε) 247 (4.08), 280 (sh, 3.64), 329 (3.24) nm; IR (neat) υmax 1686 (C=O) cm−1; 1H and 13C NMR data, see Table2; HRESIMS m/z 249.1123 [M + H]+ Int. J. Mol. Sci. 2021, 22, 7448 14 of 16

(calcd for C14H17O4, 249.1121); HRESIMS, 1D-, and 2D-NMR spectra, see Supplementary Materials Figures S15–S23. Eupatofortunone (4): Colorless oil; UV (MeOH) λmax (log ε) 211 (4.18), 246 (sh, 3.97), −1 1 13 284 (sh, 3.06) nm; IR (neat) υmax 1736 (C=O), 1684 (C=O) cm ; H and C NMR data, see + Table3; HRESIMS m/z 255.10030 [M + Na] (calcd for C14H16O3Na, 255.09971); HRESIMS, 1D-, and 2D-NMR spectra, see Supplementary Materials Figures S24–S30. Eupatodithiecine (5): Light brown amorphous powder; UV (MeOH) λmax (log ε) 227 −1 1 13 (4.03), 323 (4.26) nm; IR (neat) υmax 2230 (C≡C), 1639 (C=O) cm ; H and C NMR + data, see Table4; HRESIMS m/z 411.07026 [M + Na] (calcd for C20H20O4S2Na, 411.07007); HRESIMS, 1D-, and 2D-NMR spectra, see Supplementary Materials Figures S31–S37.

3.4. Biological Assay 3.4.1. Cell Culture ◦ All cell lines were cultured at 37 C under a humidiﬁed atmosphere with 5% CO2. Human non-small-cell lung cancer cell (A549) and human breast cancer cell (MCF-7) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and culti- vated in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Himedia, India) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Avantor Seradigm/VWR, Radnor, PA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Himedia, India). Cells were washed by warm phosphate buffered saline (PBS) every day and changed medium every 2–3 days [21,22].

3.4.2. Cell Viability Assay The cell viability was conducted by the MTT assay as previously described method [23]. Brieﬂy, 5 × 103 cells in 200 µL per well were plated in 96-well culture plates and cultured in complete medium overnight. After 24 h, cells were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 µM) of compounds 1–16. Fluorouracil (5-FU) (Sigma- Aldrich, St. Louis, MO, USA) was used as a positive control against A549 and MCF- 7 cells with IC50 values of 10.57 ± 1.89 and 8.59 ± 1.03 µM, respectively. The optical density at 570 nm was measured by ELISA plate reader (µ Quant) and the IC50 value was calculated. The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

3.4.3. Colony-Formation Assay The colony-formation assay was determined by the reference method with a slight modiﬁcation [24]. In this assay, A549 and MCF-7 cells were seeded in 6-well plates with 1 × 103 cells per well and incubated for 12 h. The cells were then treated with the indicated concentrations of compound 1, and cultured for 10 days. The cells were washed three times using PBS and ﬁxed using 95% methanol for 30 min. After washing three times with distilled water, the cells were stained using 0.2% crystal violet dye for 20 min and rinsed with distilled water to wash away the excess dye. The visible colonies were compared with the control samples and photographed using a standard camera under natural light.

3.4.4. Flow Cytometry Annexin V/PI assay was used to determine the apoptotic and necrotic cells. The A549 and MCF-7 cells were seeded on 6-well microplates at a density of 106 cells/mL respectively. After 24 h incubation, the cells were treated with following concentrations of 0, 5, and 10 µM for compound 1. After 24 h, they were washed and re-suspended in PBS solution (500 µL). Then, Annexin V-FITC (5 µL) and PI staining solution (5 µL) were introduced to the mixture, and the incubation process was followed under the dark condition for 5 min at 25 ◦C. Finally, ﬂow cytometer analysis (Beckman Coulter®, Miami, FL, USA) was performed using an AnnexinV-FITC Apoptosis Detection Kit (Strong Biotech Corporation, Taipei, Taiwan) and Flowjo version 7.6.1. Software. Int. J. Mol. Sci. 2021, 22, 7448 15 of 16

3.4.5. Western Blot Western blot analysis was performed according to the method previously reported [25,26]. Briefly, A549 and MCF-7 (1 × 105 cells) were seeded into 6-wells plate and grown until 85–90% confluent. Then different concentrations (1.25, 2.5, 5, and 10 µM) of compound 1 were added. Cells were collected and lysed by radioimmunoprecipitation assay (RIPA) buffer. Lysates of total protein were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were incubated with anti-Bax, anti-Bcl-2 (Cell Signaling Inc., Danvers, MA, USA), anti-caspase-3, and anti-β-actin (GeneTex Inc., Irvine, CA, USA) primary antibodies at 4 ◦C overnight. Then each membrane was washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h while shaking. Finally, each membrane was developed using enhanced chemiluminescence (ECL) detection kit, and the images were visualized by ImageQuant LAS 4000 Mini biomolecular imager (GE Healthcare, Woburn, MA, USA). Band densities were quantiﬁed using ImageJ software (NIH, Bethesda, MD, USA).

3.4.6. Statistical Analysis Results were expressed as mean ± SEM, and comparisons were made applying Student’s t-test. A probability of 0.05 or less was deemed signiﬁcant. The software Microsoft Excel 2016 was used for the statistical analysis.

4. Conclusions Five new (including two with new carbon skeleton) and eleven known compounds have been isolated and identiﬁed from the aerial part of E. fortunei. Among these isolated compounds, compound 1 markedly induces apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways. The compound 1 belongs to hydrophobic molecule, so it can cross the cell membrane by passive diffusion, a nonselective process. During passive diffusion, compound 1 simply dissolves in the phospholipid bilayer, dif- fuses across it, and then dissolves in the aqueous solution at the other side of the membrane. During this process, no membrane proteins are involved and the direction of transport is determined simply by the relative concentrations of the molecule inside and outside of the cell. This suggests that eupatodibenzofuran A (1) is worth further investigation and may be expectantly developed as a candidate for the treatment or prevention of non-small-cell lung cancer and breast cancer.

Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/ article/10.3390/ijms22147448/s1. Author Contributions: C.-H.C. and J.-J.C. performed the isolation and structure elucidation of the constituents and prepared the manuscript. C.-H.C., J.-J.C., and S.W. conducted the bioassay and analyzed the data. K.-C.H. and W.-J.H. analyzed physicochemical properties and possible targets. J.-J.C. planned, designed, and organized all of the research of this study and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from the Ministry of Science and Technology (MOST), Taiwan (No. MOST 109-2320-B-010-029-MY3 and MOST 106-2320-B-010-033-MY3), awarded to J.-J.C. Data Availability Statement: The data presented in this study are available in the main text and the Supplementary Materials of this article. Acknowledgments: We gratefully thank Shou-Ling Huang and Iren Wang for the assistance in NMR experiments of the Instrumentation Center at NTU which is supported by the Ministry of Science and Technology, Taiwan. Conﬂicts of Interest: The authors declare no conﬂict of interest. Int. J. Mol. Sci. 2021, 22, 7448 16 of 16

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