New flavonoids, emarginin A-C from Vaccinium emarginatum Hayata

Journal Pre-proof New flavonoids, emarginin A-C from Vaccinium emarginatum Hayata

Ping-Chen Tu, Yu-Chia Liang, Guan-Jhong Huang, Hui-Chi Huang, Ming-Ching Kao, Te-Ling Lu, Yueh-Hsiung Kuo PII:

S0367-326X(19)31609-0

DOI:

https://doi.org/10.1016/j.fitote.2019.104446

Reference:

FITOTE 104446

To appear in:

Fitoterapia

Received date:

8 August 2019

Revised date:

28 November 2019

Accepted date:

29 November 2019

Please cite this article as: P.-C. Tu, Y.-C. Liang, G.-J. Huang, et al., New flavonoids, emarginin A-C from Vaccinium emarginatum Hayata, Fitoterapia (2019), https://doi.org/ 10.1016/j.fitote.2019.104446

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© 2019 Published by Elsevier.

Journal Pre-proof

New

flavonoids,

emarginin

A-C

from

Vaccinium

emarginatum Hayata Ping-Chen Tua, Yu-Chia Liangb, Guan-Jhong Huangb, Hui-Chi Huangb, Ming-Ching Kaoc, Te-Ling Lud, Yueh-Hsiung Kuob,e,f,* [email protected] a

The Ph.D. Program for Cancer Biology and Drug Discovery, China Medical University and

Academia Sinica, Taichung, Taiwan b

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Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China

Medical University, Taichung, Taiwan

Department of Biological Science and Technology, China Medical University, Taichung,

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c

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Taiwan d

e

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School of Pharmacy, China Medical University, Taichung, Taiwan

Department of Biotechnology, Asia University, Taichung, Taiwan

f

*

Abstract

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Corresponding author.

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Chinese Medicine Research Center, China Medical University, Taichung, Taiwan

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Phytochemical investigation of methanolic extract of the whole plants of Vaccinium emarginatum allowed for the characterization of one epicatechin derivative (1) that was isolated from a natural source for the first time and three new flavonoids, emarginin A (2), emarginin B (3), and emarginin C (4), together with 11 known compounds (5–15). The structures of compounds 1–4 were elucidated by combination of spectroscopic analysis (MS, IR, and NMR) and by comparison with that of literature analogues. Compounds 1–8 and 11–15 were evaluated for their preliminary in vitro anti-proliferative activity against Du145 and PC-3 prostate cancer cell lines. Among them, compound 15 exhibited most potent cytotoxicity against Du145 and PC-3 cells, with IC50 values of 8.46 and 10.98 μM, respectively. Furthermore,

Journal Pre-proof compounds 1–7 were assessed for their anti-inflammatory potential against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Compound 4 exhibited moderate anti-inflammatory activity, with an IC50 value of 27.99 μM. Keywords:

Vaccinium

emarginatum;

Flavonoids;

Anti-proliferation;

Anti-inflammation

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1. Introduction Vaccinium, an important source of food and pharmaceutical ingredients in the

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family Ericaceae, consists of approximately 450 species including blueberry, bilberry,

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cranberry, huckleberry, and lingonberry. Some of the Vaccinium species have been

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reported as one of the rich sources of naturally occurring bioactive flavonoids [1] with

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broad-spectrum activities, including antioxidative [2, 3], anti-inflammatory [4, 5], anti-bacterial [6], anti-diabetic [7], and anti-proliferative properties [3]. Flavonoids

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are divided into several subgroups, including chalcones, flavonols, flavanols, flavones,

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flavanones, isoflavones, and anthocyanins. They are widely found in foods and natural beverages, such as fruits, vegetables, cocoa, tea, wine, and coffee [8]. Today, many botanical extracts, containing mainly flavonoid components, are developed as herbal products and botanical drug. V. emarginatum Hayata, a medicinal endemic plant in Taiwan, is an epiphytic shrub distributed at elevation 1,500–3,000 m. The root nodules are used as an herbal Yang tonic, and its fruits and leaves are used as a traditional herbal medicine for treating inflammatory disease and infection. In this study, we report our characterization on the isolates together with their preliminary in vitro anti-proliferative activity against Du145 and PC-3 cells and anti-inflammatory activity against LPS-induced NO production in RAW 264.7 cells.

2. Materials and methods

Journal Pre-proof 2.1. General experimental procedures Chromatography was carried on Silica gel 60 (40–63 µm, Merck). Thin-Layer Chromatography (TLC) was performed on silica gel 60 F254 plates (200 µm, Merck). High Performance Liquid Chromatography (HPLC) was performed using a KNAUER HPLC system (Germany) and Phenomenex Luna Silica (2) (5 µm, 250 x 10 mm, USA). Specific rotation was recorded on a JASCO P-2000 polarimeter. UV spectra were measured with a PerkinElmer Lambda 265 UV-Visible spectrophotometer. IR

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spectra were acquired on a Shimadzu IRPrestige-21 fourier transform infrared (FTIR)

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spectrophotometer. 1D- and 2D- Nuclear Magnetic Resonance (NMR) spectra were

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obtained with a Bruker AVANCE 500 MHz FTNMR spectrometer. High resolution

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electrospray mass (HRESIMS) spectra were recorded on an ESI-Q-TOF mass

2.2. Chemicals

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spectrometer (Bruker Daltonics, maXis impact).

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ACS grade solvents (acetone, dichloromethane, ethyl acetate, methanol, and n-hexane), HPLC grade solvents (acetone, dichloromethane, ethyl acetate, and

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n-hexane), and deuterated solvent (acetone-d6 and methanol-d4) were purchased from the branch of Merck in Taiwan. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), glutamine, and antibiotics (penicillin-streptomycin) were purchased from GIBCO (Grand Island, NY, USA). LPS (endotoxin from Escherichia coli, serotype 055: B5), sodium nitrite, thiazolyl blue tetrazolium bromide (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), doxorubicin, and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.3. Plant Material The whole plants of V. emarginatum were collected in Taichung, Taiwan, in June

Journal Pre-proof 2013 and identified by Prof. Y.-C. Chen (Kaohsiung Medical University, Taiwan). A voucher specimen (CMU-VE-201307) was deposited in Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University. 2.4. Extraction and Isolation The methanolic extract of the whole plants of V. emarginatum (17.4 kg) was concentrated under vacuum and suspended in water and fractionated with ethyl acetate (EtOAc) and n-BuOH, sequentially. The EtOAc soluble fraction (500 g) was

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then subjected to silica gel column chromatography (CC, 5.0 kg, 70-230 mesh) using

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a gradient solvent system (n-hexane/EtOAc/MeOH). Collected fractions were

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checked by TLC and combined into 10 main subfractions (Fr.A–Fr.J). Fr.F (42.8 g)

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was separated by silica gel CC (0.5 kg, n-hexane/acetone, gradient) to obtain 10 fractions: Fr.F-1–Fr.F-10. Fr.F-1 (9.5 g) was isolated by silica gel CC (150 g,

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CH2Cl2/EtOAc, gradient) followed by normal phase semipreparative HPLC

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(CH2Cl2/EtOAc = 40/1) to provide compounds 4 (2.1 mg, tR = 16.5 min) and 3 (3.0 mg, tR = 21.3 min). Fr.F-4 (7.5 g) was re-separated by silica gel CC (0.5 kg, n-hexane

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and EtOAc, 33%) and followed by Sephadex LH-20 (20 g, CH2Cl2/MeOH = 1/1) to give compounds 12 (4.3 mg), 13 (5.2 mg), and 14 (4.8 mg). Fr.F-4-5 (2.0 g) was purified by normal phase semipreparative HPLC (n-hexane/acetone = 2/1) to provide compound 15 (23.6 mg, tR = 11.5 min). Fr.F-6 (5.1 g) was separated by silica gel CC (2 g, CH2Cl2 and acetone, gradient) followed by normal phase HPLC (CH2Cl2/acetone = 6/1) to give 8 (5.2 mg, tR = 10.9 min), 11 (3.4 mg, tR = 11.4 min), and 9 (2.1 mg, tR = 12.2 min). Fr.F-6-4 was purified by normal phase semipreparative HPLC (n-hexane/EtOAc = 1/1) to afford compound 7 (0.7 g, tR = 9.6 min). Fr.F-7 (1.5 g) was separated by silica gel CC (25 g, CH2Cl2 and acetone, gradient) followed by normal phase HPLC (n-hexane/EtOAc = 2/3) to give compound 10 (2.0 mg, tR = 8.8 min).

Journal Pre-proof Fr.G (63.0 g) was separated by silica gel CC (0.7 kg, CH2Cl2 and acetone, gradient) to obtain 9 fractions (Fr.G-1–Fr.G-9) and compound 5 (27.1 g). Fr.G-2 (5.2 g) was re-separated by silica gel CC (250 g, n-hexane/EtOAc = 1/1) followed by normal phase HPLC (CH2Cl2/EtOAc = 1/1) to obtain compounds 2 (8.0 mg, tR = 7.3 min) and 1 (11.4 mg, tR = 8.3 min). Fr.G-2-7 (5.2 g) was separated by silica gel CC (250 g, CH2Cl2/MeOH = 9/1) and followed by silica gel CC (250 g, n-hexane/EtOAc = 1/1) to give compound 6 (0.8 g).

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(6aR,12aR)-5,5-dimethyl-7,12a-dihydro-6aH-isochromeno[4,3-b]chromene-2,3,8,10-t

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1 etrol. (1): Amorphous colorless powder; [α]22 𝐷 = +251.75 (0.1, MeOH); H and

13

C

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NMR data: see Table 1; HRESIMS m/z 353.0989 [M + Na]+ (calcd for C18H18O6Na).

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Emarginin A (2): Amorphous colorless powder; [α]22 𝐷 = +217.90 (0.1, MeOH); UV λmax (log ε, MeOH) 279.5 (3.35); IR (KBr) νmax 3335, 2974, 2932, 1624, 1611, 1497,

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1466, 1288, 1140, and 1065 cm-1; 1H and 13C NMR data: see Table 1; HRESIMS m/z

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353.1020 [M + Na]+ (calcd for C18H18O6Na). Emarginin B (3): Amorphous colorless powder; UV λmax (log ε, MeOH) 267.1 (3.79),

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302.1 (3.52); IR (KBr) νmax 2930, 2845, 1663, 1597, 1504, 1462, 1420, 1261, 1240, 1213, and 1126 cm-1; 1H and 13C NMR data: see Table 2; HRESIMS m/z 383.1493 [M + Na]+ (calcd for C20H24O6Na).

Emarginin C (4): Amorphous colorless powder; UV λmax (log ε, MeOH) 231.3 (4.24), 278.2 (3.80); IR (KBr) νmax 2928, 2851, 1670, 1614, 1591, 1506, 1472, 1340, 1275, 1263, 1126, and 1072 cm-1; 1H and

13

C NMR data: see Table 2; HRESIMS m/z

397.1313 [M + Na]+ (calcd for C20H22O7Na). 2.5. Preparation of acetone-conjugate from epicatechin A mixture of epicatechin (0.5 g, 1.7 mmol) and silica gel (50 g) was suspended in acetone (500 mL) and refluxed for 24 hr. The filtrate was evaporated under vacuum.

Journal Pre-proof Its 1H NMR spectrum was consistent with that of epicatechin (Fig. S1). 2.6. Cytotoxicity Assessment The cytotoxicity assessment of the compounds isolated from V. emarginatum against the cultured human cancer cell lines Du145 and PC-3 was evaluated using the MTT assay. Cell lines addressed in the study were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All cells were cultured in DMEM with 10% FBS at 37 °C in an incubator containing 5% CO2. Cells were seeded into 96-well

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plates (6,000 cells/well) and incubated overnight. After the cells were adhered to the

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plate, the cells were treated with different concentrations of the filtered isolates and

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incubated at 37°C for 72 h. After a 4 h incubation with MTT (0.5 mg/mL), the relative

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cell viability was calculated proportionally to the production of formazan crystals

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dissolved by DMSO. The absorbance of the final solution was read using a

reference.

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spectrophotometer at a wavelength of 545 nm against a wavelength of 690 nm as

cells.

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2.7. NO Production and Viability in LPS-induced murine macrophage RAW 264.7

The murine macrophage cell line RAW 264.7 (BCRC No. 60001) was obtained from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). RAW 264.7 cells were maintained in DMEM with 10% FBS at 37 °C in an incubator containing 5% CO2. RAW 264.7 cells were seeded into 96-well plate (5×104 cells/well) and incubated overnight, then the cells were treated with various doses of compounds in the presence of LPS (100 ng/mL) for 24 h. For cell viability analysis, the cells were washed twice with PBS and incubated in 100 μL DMEM with MTT (0.5 mg/mL) for 3 h at 37 °C in the incubator. The medium was removed and 100 μL

Journal Pre-proof DMSO was added to dissolve the MTT formazan, then the absorbance was read at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The NO production was assessed by measuring the concentration of nitrite in the culture medium, which was calculated using a standard curve and sodium nitrite was being used as a standard sample. Each 100 μL of supernatant was reacted with the same volume

of

Griess

reagent

(containing

1%

sulphanilamide,

0.1%

naphthylethylenediamine dihydrochloride, and 6% phosphoric acid) and incubated at

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room temperature for 5 min, then the mixtures were detected at 540 nm using a

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microplate reader (Molecular Devices, Sunnyvale, CA, USA).

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3. Results and Discussion

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The structures of known compounds were determined by comparison with the

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literature spectroscopic data. Their structures were consequently identified as epicatechin (5) [9], proanthocyanidin A2 (6) [10], davidigenin (7) [11],

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1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-propan-1-one (9)

[13],

phloretin

(10)

[12], [14]

,

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α,4,2’,4’-tetrahydroxydihydrochalcone

(8)

dihydrokaempferol (11) [15], 7-hydroxy-4H-chromen-4-one (12) [16], isoscopoletin (13) [17], isofraxidin (14) [18], integracin A (15) [19] (Fig. 1). Compound 1 was obtained as a colorless amorphous solid with [α]D +251.75 (c 0.1, MeOH) and assigned as a molecular formula of C18H18O6 with 10 degrees of unsaturation based on its [M+Na]+ ion peak at m/z 353.0989 (calcd for C18H18O6Na) in the HRESIMS data. The 1H NMR spectrum (Table 1) showed the presence of two aromatic proton singlets (δH 6.83 and 6.70, each 1H), a pair of meta-aromatic proton signals (δH 5.99 and 5.79, each 1H, d, J = 2.2 Hz) assigned to an 1,3,4,5-tetrasubstitued aromatic ring, two oxymethine proton signals [δH 4.49 (1H, brs) and 4.27 (1H, brd, J = 5.2 Hz)], a

Journal Pre-proof pair of methylene proton signals [δH 2.88 (1H, dd, J = 17.3, 5.2 Hz) and 2.79 (1H, d, J = 17.3 Hz)], and two methyl singlets (δH 1.47 and 1.38). The characteristic flavan-3-ol pattern was suggested by the presence of 12 sp2 carbons, two oxymethine groups (δC/δH 71.3/4.49 and 64.3/4.27), and one methylene group (δC/δH 26.4/2.88 and 2.79). The suggestion was confirmed by the HMBC correlations (Fig. 2 and Table 1) from H-1 to C-12a; H-6a to C-7a; H-7 to C-7a and C-12a; and H-12a to C-4a. The structure of 1 was found to be closely comparable to that of epicatechin (5), with the difference

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being the presence of two additional methyl groups (δH 1.47 and 1.38) and an

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additional oxygenated quaternary carbon (δC 75.9). Furthermore, the HMBC

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correlations from the methyl protons (δH 1.47 and 1.38) to C-4a and C-5; H-4 to C-5;

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and H-6a to C-5 suggested that the hydroxyl group at C-6a was cyclized with the substituent group on C-4a. The NOESY correlation (Fig. 3) together with the

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negligible coupling of H-6a and H-12a indicated the cis configuration between them.

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Furthermore, the 6aR,12aR absolute configuration of 1 was determined by comparison of its specific optical rotation ([α]D +251.8) with that of a reference compound with

Thus,

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known absolute configuration ([α]D +90.9 in 7-O-galloylplumbocatechin A) [20]. compound

1

was

identified

as

(6aR,12aR)-5,5-dimethyl-7,12a-dihydro-6aH-isochromeno[4,3-b]chromene-2,3,8,10-t etrol, which belongs to unusual bent epicatechin analogues. The bent geometry of that is due to a cis junction between C ring and the newly generated D ring. The acetone-conjugated epicatechin derivative (1) has been synthesized previously by acetone-conjugation from epicatechin [21]. In the preparation of the acetone-conjugated epicatechin derivative (1) reported by Meng [22], epicatechin was suspended in acetone with H2SO4 and stirred for 24 h at room temperature (yield 17.5 %). In order to confirm whether compound 1 was yielded artificially during extraction

Journal Pre-proof or chromatography procedures, a mixture of epicatechin and silica gel was suspended in acetone and refluxed for 24 hr. As shown in Fig. S1, the acetone-conjugation reaction would not occur during extraction or chromatography. Consequently, compound 1 is a naturally occurring compound isolated from a natural source for the first time. Compound 2 was obtained as a colorless amorphous solid with [α]D +217.90 (c 0.1, MeOH) and assigned as a molecular formula of C18H18O6 with 10 degrees of

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unsaturation based on its [M+Na]+ ion peak at m/z 353.1020 (calcd for C18H18O6Na)

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and 1624, 1611, and 1497 cm-1 (aromatic ring).

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in the HRESIMS data. Its IR spectrum showed absorption bands at 3335 cm-1 (OH)

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C NMR data (Table 1) revealed the

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The combined analysis of the 1H and

presence of an oxygenated quaternary carbon, two methyls, a methylene, two

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oxymethines, and two aromatic rings. The overall 1D NMR suggested that it shares

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close structural similarities with 1 except for the 3,4-dihydroxy substitution of B-ring in 2, which was determined by a pair of ortho-aromatic protons (δH 6.73 and 6.75)

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with a characteristic coupling constant of 8.2 Hz. The 3,4-dihydroxy substitution of B-ring was further supported by the absence of the NOESY correlations (Fig. 3) between H-4 and the methyl groups on C-5. In addition, the dimethyl substitution on C-5 at downfield (δH 1.64 and 1.66) was deshielded by the lone pair electrons from the O atom of OH-4. Due to the consistent specific optical rotation, the absolute configuration of 2 seems to be identical to 1. Thus, compound 2 was identified as a new flavonoid, named emarginin A. Compound 3 was afforded as an amorphous solid. It was assigned to a molecular formula of C20H24O6 with 9 degrees of unsaturation on the basis of its [M+Na]+ ion peak at m/z 383.1493 (calcd for C20H24O6Na) in the HRESIMS data. The IR spectrum

Journal Pre-proof showed absorption bands at 1670 cm-1 (conjugated carbonyl) and 1614, 1591, and 1506 (aromatic ring) cm-1. The 13C NMR data (Table 2) showed the presence of 15 carbon signals assigned to two methylenes, one carbonyl carbon, and 12 aromatic carbons, indicative of a tetrahydrochalcone skeleton. Additionally, the 1H NMR spectrum (Table 2) displayed the presence of a set of ABX coupling aromatic proton signals [δH 7.69 (1H, d, J = 8.7 Hz), 6.60 (1H, d, J = 2.3 Hz), and 6.58 (1H, dd, J = 8.7, 2.3 Hz)] assigned to the

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A-ring, a pair of meta-aromatic proton singlets [δH 6.49 (2H, s)] assigned to the

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B-ring, five methoxy singlets [δH 3.91 (3H), 3.86 (3H), 3.80 (6H), and 3.72 (3H)], and

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two methylene triplets [δH 3.25 (2H, t, J = 7.6 Hz) and 2.90 (2H, t, J = 7.6 Hz)]

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attributed to -CO-CH2-CH2-Ar. The assignments were supported by the HMBC correlations (Fig. 2 and Table 2) from H-6, H-α, and H-β to the carbonyl carbon (δC

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202.2); H-α to C-1’; and H-2’ and H-6’ to C-β. Furthermore, the HMBC correlations

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from OMe-2 (δH 3.91) to C-2 (δC 166.5); OMe-4 (δH 3.86) to C-4 (δC 162.5); OMe-3’ and OMe-5’ (δH 3.80) to C-3’ and C-5’ (δC 138.5); and from OMe-4’ (δH 3.72) to C-4’

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(δC 154.5) further assigned the positions of these methoxy groups. Their positions were supported by the NOESY correlations (Fig. 3). Therefore, compound 3 was identified as a new chalcone, named emarginin B. Compound 4 was isolated as a colorless powder. It was assigned to a molecular formula of C20H22O7 with 10 degrees of unsaturation on the basis of its [M+Na]+ ion peak at m/z 397.1313 (calcd for C20H22O7Na) in the HRESIMS data. The IR spectrum showed absorption bands at 1663 cm-1 (conjugated carbonyl) and 1663, 1597, and 1504 cm-1 (aromatic ring). The 1H and 13C NMR data (Table 2) of 4 were similar to those of 3 except for the different signals assigned to ring A: a pair of ortho-aromatic proton signals (δH 7.23

Journal Pre-proof and 6.60, each 1H, d, J = 8.3 Hz), an additional methylenedioxy signal (δH 6.03, 2H, s), and one less methoxy signal. Furthermore, the HMBC correlations (Fig. 2 and Table 1) from the methylenedioxy protons (δH 6.03) to C-3 (δC 138.5) and C-4 (δC 154.5); from OMe-2 (δH 4.05) to C-2 (δC 144.8) indicated the assignment of the methylenedioxy group between C-3 and C-4 and the methoxy group at C-2. The position of OMe-2 was supported by the absence of the NOESY correlations between

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OMe-2 and H-5 or H-6. On the basis of the above, compound 4 was identified as a new chalcone, named emarginin C.

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Besides, compounds 1–8 and 11–15 were evaluated for their anti-proliferative

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activity against Du145 and PC-3 prostate cancer cell lines using MTT cell viability

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assay (Table 3). Doxorubicin was used as the positive control. Among the compounds tested, compound 15 exhibited most potent cytotoxicity against Du145 and PC-3 cells,

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with IC50 values of 8.46 and 10.98 μM, respectively. Compounds 3 and 4 showed

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moderate cytotoxicity against Du145 and PC-3 cells, with IC50 values ranging from 21.20–27.06 μM, while other flavonoids (1, 5, and 6) and coumarins (12–14)

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displayed poor anti-proliferative activity. In previous study, the presence of 3,4,5-trimethoxy moiety on either ring of chalcone is essential to enhance cytotoxic activity [23, 24]. This may provide the rationale for more potent cytotoxicity of compounds 3 and 4 than other chalcones. Additionally, compounds 1-7 were evaluated for their anti-inflammatory activity through inhibiting LPS-induced NO production in RAW 264.7 cells (Table 4). Compound 4 exhibited more potent anti-inflammatory activity than the positive control indomethacin. However, other compounds showed poor anti-inflammatory activity. The acetone-conjugated epicatechin derivative (1) has been reported to show good radical scavenging activity and exhibit 10-fold enhanced activity to protect H2O2-induced erythrocyte hemolysis

Journal Pre-proof when compared with epicatechin [22]. Dickerhof [25] reported that oxidation of the catechol moiety of epicatechins to an o-quinone by myeloperoxidase led to the enhanced inhibitory activity against macrophage migration inhibitory factor, which is an important therapeutic target for treating inflammatory diseases. However, compound 1 together with epicatechin (5) exhibited poor inhibitory activity against LPS-induced NO production. Accordingly, epicatechin derivatives might act as potential precursors of bioactive products. The oxidation of the catechol moiety of

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epicatechins might be essential.

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4. Conclusion

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Phytochemical investigation of methanolic extract of the whole plants of V.

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emarginatum allowed for the characterization of four new naturally occurring

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flavonoids (1–4), including an epicatechin derivative (1) isolated from a natural source for the first time, together with 11 known compounds (5–15). Among them,

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compound 15 exhibited most potent cytotoxicity against Du145 and PC-3 cells, with IC50 values of 8.46 and 10.98 μM, respectively. When compared with the positive

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control indomethacin, compound 4 exhibited more potent anti-inflammatory activity, with an IC50 value of 27.99 μM. Our study may provide the basis for further phytochemical investigations on the medicinal plant V. emarginatum. Supplementary data Supplementary data related to this article can be found at XXX. Acknowledgements Mass spectrometric analyses were performed by the Proteomics Research Core Laboratory, Office of Research and Development at China Medical University, Taichung, Taiwan. This work was financially supported by Taiwan Ministry of Health and Welfare Clinical Trial Center (MOHW108-TDU-B-212-133004) and “Chinese

Journal Pre-proof Medicine Research Center, China Medical University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (CMRC-CHM-4). This work

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was supported by China Medical University in Taiwan (CMU108-Z-08).

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Fig. 1. The structures of compounds 1-15 from V. emarginatum.

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Fig. 2. Selected HMBC correlations of the compounds 1-4.

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Fig. 3. Selected NOESY correlations of the compounds 1-4.

Journal Pre-proof Table 1 H (500 MHz) and 13C NMR (125 MHz) spectral data of compounds 1 and 2 (δ in ppm, J in Hz). 1a

Position

2b

HMBC

δH, mult (J, Hz)

δC

δH, mult (J, Hz)

1

117.3

6.83 brs

122.6

6.75 d (8.2)

3, 4a, 12a

2

144.4

114.1

6.73 d (8.2)

4, 4b

3

146.9

147.1

4

112.8

4a

136.2

130.5

4b

124.8

126.0

5

75.9

77.4

6a

64.3

4.27 brd (5.2)

7a, 12a

64.9

7

26.4

2.88 dd (17.3, 5.2)

6a, 7a, 12a

26.7

2, 3, 4a, 12a

6.70 brs

2, 3, 4b, 5

2.79 d (17.3) 99.4

8

157.4

9

96.0

10

157.3

11

95.7

11a

156.8

12a

71.3

4.49 brs

5-Me

28.1

1.47 s

5-Me

32.0

1.38 s

4.26 brd (5.4)

5, 7a, 12a

2.87 dd (17.5, 5.4)

6a, 7a, 11a, 12a

2.77 dd (17.5)

100.0

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7a

142.8

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δC

HMBC

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1

157.9

7a, 10, 11

96.4

5.92 d (2.3)

1a, 10, 11

5.78 d (2.3)

7a, 9, 10, 11a

72.7

4.50 brs

1, 4a, 4b, 6a

4a, 5

24.4

1.66 s

4a, 5

4a, 5

28.4

1.64 s

4a, 5

re

5.99 d (2.2)

157.6

Measured in acetone-d6.

b

Measured in methanol-d4.

lP

Jo ur

a

7a, 9, 11a

1, 4a, 4b, 6a

na

5.79 d (2.2)

95.9

156.8

Journal Pre-proof Table 2 1

H (500 MHz) and 13C NMR (125 MHz) spectral data of compounds 3 and 4 in methanol-d4 (δ in ppm, J in Hz). Position

3 δC

4 δH, mult

δC

HMBC

δH, mult

(J in Hz)

HMBC

(J in Hz)

1

121.9

127.0

2

162.5

144.8

3

99.3

4

166.5

5

106.9

6.58 dd (8.7, 2.3)

1, 3

104.1

6.60 d (8.3)

3, 6

6

133.5

7.69 d (8.7)

2, 4, C=O

126.1

7.23 d (8.3)

2, 4, C=O

1’

139.3

2’

106.8

3’

154.4

4’

137.3

5’

154.4

6’

106.8

6.49 s

1’, 2’, 3’, 4’, β

α

46.3

3.25 t (7.6)

β

32.3

2.90 t (7.6)

C=O

202.2

2-OMe

56.2

3.91 s

4-OMe

56.1

3.86 s

4

3’,5’-OMe

56.5

3.80 s

4’-OMe

61.1

3.72 s

1, 2, 4, 5,

138.5

of

154.5

139.1

6.50 s

3’, 4’, 5’, 6’, β

106.8

6.50 s

2’, 3’, 4’, 5’, β

1’, β, C=O

46.0

3.23 t (7.6)

1’, β, C=O

1’, 6’, α, C=O

32.1

2.91 t (7.6)

1’, 2’, 6’, α, C=O

106.8

ro

1’, 3’, 4’, 6’, β

6.49 s

154.4

-p

137.3

na

lP

re

154.4

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O-CH2-O

6.60 d (2.3)

2

202.3 60.5

4.05 s

3’, 5’

56.5

3.80 s

4’

61.1

3.72 s

103.3

6.03 s

3’, 5’

3, 4

Journal Pre-proof Table 3 Anti-proliferative activity of compounds 1–8 and 11–15 against PC-3 and Du145 prostate cancer cell lines. IC50 (μM) a

Compounds

Du145

1

>200

>200

2

106.43±33.82

159.63±6.73

3

21.20±0.94

27.06±9.17

4

22.15±1.29

26.03±5.92

5

>200

>200

6

>200

>200

7

58.15±17.68

8

60.91±4.67

11

162.93±28.03

195.49±30.12

12

>200

>200

13

>200

>200

14

>200

>200

10.98±0.37

8.46±0.47

1.81±0.36

0.85±0.08

Doxorubicin

162.79±23.89 167.99±3.71

The IC50 values were calculated from the slope equation of the dose-response curves. Values are

na

expressed as mean SD of three independent experiments. Doxorubicin was used as a positive control.

Jo ur

b

ro

-p

re

b

lP

15

a

of

PC-3

Journal Pre-proof Table 4 Anti-inflammatory activity of compounds 1–7 against NO production in LPS-induced RAW 264.7 cells

1

>200

2

113.41±7.66

3

64.04±0.69

4

27.99±1.36

5

>200

6

159.39±6.89

7

122.96±5.46

Indomethacin

45.79±2.89

The IC50 values were calculated from the slope equation of the dose-response curves. Values are

expressed as mean SD of three independent experiments.

na

lP

re

-p

Indomethacin was used as a positive control.

Jo ur

b

ro

a

IC50 (μM) a

of

b

Compounds

Journal Pre-proof References

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3'R,4'R-Di-O-(−)-camphanoyl-2',2'-dimethyldihydropyrano[2,3-f]chromone (DCP) analogues as potent anti-HIV agents, J. Med. Chem. 47 (2004) 4072-4082. [17] X. Bai, R. Pan, M. Li, X. Li, H. Zhang, HPLC profile of longan (cv. Shixia)

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Journal Pre-proof inhibition

of

macrophage

migration

inhibitory

factor

(MIF)

by

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myeloperoxidase-dependent oxidation of epicatechins, Biochem. J. 462 (2014) 303-14.

Journal Pre-proof

CRediT author statement Kuo Yueh-Hsiung: Funding acquisition, Writing- Reviewing and Editing Tu Ping-Chen: Investigation, Writing- Original draft preparation. Liang Yu-Chia: Data curation, Validation. Huang Guan-Jhong: Resources. Huang Hui-Chi: Visualization, Software. Kao Ming-Ching: Supervision.

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Lu Te-Ling: Conceptualization, Methodology.

Journal Pre-proof

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Conflicts of Interest The authors declare no conflict of interest.