Chemical Constituents and Their Activities From the Twigs of Euscaphis konishii Hayata

Abstract

A new ellagic acid derivative 3,3′-di-O-methylellagic acid 4′-α-l-arabinopyranoside (1), with 9 known compounds identified as 3,3′-di-O-methylellagic acid (2), 3,3′-di-O-methylellagic acid 4′-α-d-arabinofuranoside (3), 3,3′-di-O-methylellagic acid 4′-β-d-glucopyranoside (4), 3,3′-di-O-methylellagic acid 4′-β-d-xylopyranoglucoside (5), 3,3′,4-tri-O-methylellagic acid 4′-β-d-glucopyranoside (6), tormentic acid (7), ursolic acid (8), euscaphic acid (9), and betulinic acid (10), was isolated from the twigs of Euscaphis konishii Hayata. Compounds 1, 3, and 5 to 7 were isolated from this plant for the first time, and compounds 1 and 5 were obtained from the plant genus for the first time. The structure of the new compound was confirmed by HRESIMS, NMR, and compared with data from the literature . The cytotoxicities of 10 isolated compounds were tested, with compounds 1 to 6 showing moderately inhibited activity against the Human Hepatocarcinoma cell line (HepG2 cells) with an IC₅₀ value ranging from 69.7 to 181.8 μM.

Euscaphis konishii Hayata (E. konishii), belonging to the Staphyleaceae family and being widely distributed in Fujian, Jiangxi, Guangdong, and Hunan provinces of China, has been used in traditional Chinese medicine for the treatment of stomach pain, testicular pain, and prolapse of the anus. ^1,2 Previous chemical studies on the Euscaphis genus have led to the isolation of triterpenes, ³ flavonoids, ⁴ and lignans. ⁵ However, E. konishii, which is the most abundant species of the Euscaphis genus with more than 300 hectares of man-made forest in Fujian Province of China, has been underemphasized and overlooked for its chemical constituents and activities. ^6,7 Significant bioactive constituents of E. konishii, as well as anti-inflammatory and fungistatic compounds, have been reported in the previous literature. ^8,9 This paper reports continuing research, which resulted in finding a tumor-inhibiting extract, which led to the isolation of 10 constituents described in this study, among them a new ellagic acid with 9 known compounds (Figure 1). This study describes the isolation, and the identification of the structures and the biological activities of these compounds from the twigs of E. konishi

Figure 1

The structure of compounds 1-10. * indicates new compound.

Results and Discussion

3,3′-Di-O-methylellagic acid 4′-α-l-arabinopyranoside (1) was obtained as yellow powder. Its molecular formula was determined to be C₂₁H₁₈O₁₂ based on HRESIMS m/z: 463.08670 [M+H]⁺ (calculated for C₂₁H₁₈O₁₂, 463.08710). The IR spectrum clearly exhibited the absorption bands of a hydroxy group (3489 cm⁻¹), a carbonyl group (1730 cm⁻¹), and an aromatic group (1609 cm⁻¹). The ¹³C-NMR and DEPT spectra showed 12 aromatic quaternary carbons, 2 methine carbons at (δ _C 111.6) and (δ _C 111.7), Supplemental Material 2 ester carbons at (δ _C 158.5) and (δ _C 158.6), 2 methoxyl at (δ _C 61.0) and (δ _C 61.7), and the characteristic resonances of a pyranose unit at [δ _C 101.2 (C-1″), 72.0 (C-2″), 70.3 (C-3″), 66.9 (C-4″), 65.1 (C-5″)] ¹⁰ (Table 1). ¹H-NMR, ¹H-¹H COSY, and HSQC spectra allowed the identification of the sugar protons, 2 methoxy groups at [δ _H 4.10 (3H, s, 3-OCH₃); 4.05 (3H, s, 3′-OCH₃)], and 2 aromatic protons at [δ _H 7.53 (1H, s, H-5); 7.75 (1H, s, H-5′)], as well as a terminal proton of arabinose at [δ _H 5.17 (1H, d, J = 6.0 Hz, H-1″)].

Table 1

¹H- and ¹³C-NMR Spectroscopic Data for Compound 1.

Position	δ C a	δ H b	Position	δ C a	δ H b
1	111.9		5′	111.7	7.75 (s, 1H)
2	140.9		6′	112.8
3	140.3		7′	158.6
4	151.2		1″	101.2	5.16, d (1H, d, J = 6.0)
5	111.6	7.52 (s, 1H)	2″	72.0
6	111.8		3″	70.3
7	158.5		4″	66.9
1′	114.2		5″	65.1
2′	141.7		3-OCH3	61.0	4.05 (s, 3H)
3′	141.9		3′-OCH3	61.7	4.10 (s, 3H)
4′	152.8

^aIn DMSO-d₆ (100 MHz).

^bIn DMSO-d₆ (400 MHz).

Its structure was further confirmed by 2D NMR, which indicated the HMBC correlations from H-5 to C-1 (δ _C 111.9), C-7 (δ _C 158.5), and H-5′ to C-1′ (δ _C 114.2), C-7′ (δ _C 158.6), and H-1″ to C-4′ (δ _C 152.8). These data fully demonstrate that the compound is a glycoside derivative of ellagic acid. ¹¹ The 2 methoxyl groups were located at C-3 and C-3′ on the basis of their ¹³C-NMR chemical shifts at (δ _C 61.0) and (δ _C 61.7). ¹²

The sugar was hydrolyzed and compared with α-l-arabinose, with the spot position being consistent after color development by TLC. Thus, compound 1 is the new ellagic acid derivative 3,3′-di-O-methylellagic acid 4′-α-l-arabinopyranoside, with a structure as shown in Figure 2 and the ¹H- and ¹³C-NMR data shown in Table 1.

Figure 2

The key HMBC correlations of compound 1.

Except for the 3,3′-di-O-methylellagic acid 4′-α-l-arabinopyranoside, there are 5 known ellagic acid derivatives, 3,3′-di-O-methylellagic acid (2), ¹³ 3,3′-di-O-methylellagic acid 4′-α-d-arabinofuranoside (3), ¹⁴ 3,3′-di-O-methylellagic acid 4′-β-d-glucopyranoside (4), ¹⁵ 3,3′-di-O-methylellagic acid 4′-β-d-xylopyranoglucoside (5), ¹⁶ 3,3′,4-tri-O-methylellagic acid 4′-β-d-glucopyranoside (6), ¹⁷ and 4 triterpenic compounds, tormentic acid (7), ¹⁸ ursolic acid (8), ¹⁹ euscaphic acid (9), ²⁰ and betulinic acid (10). ²¹ Their structure was confirmed by comparing their data with ESIMS, ¹H- and ¹³C-NMR data in the literature. The cytotoxicities of compounds 1 to 10 and different fractions of the extractions were evaluated by the Tetramethylazoazole salt trace enzyme reaction colorimetry (MTT) method, with 5-fluorouracil (5-FU) being used as a positive control.

Their cytotoxicities were determined against the Human Hepatocarcinoma cell line (HepG2 cells). The results in Table 2 indicate that ellagic derivative compounds showed moderate cytotoxic activities against HepG2 cells lines. Compounds 1 to 6 showed moderately inhibited activity against HepG2 cells with an IC₅₀ value ranging from 69.7 to 181.8 μM, suggesting that ellagic acid derivatives may be important constituents relating to the tumor-inhibiting properties of E. konishii.

Table 2

Cytotoxicity Values of Compounds 1-6.

Compounds	IC50 (μM)
3,3′-Di-O-methylellagic acid 4′-α-l-arabinopyranoside (1)	69.7
3,3′-Di-O-methylellagic acid (2)	108.4
3,3′-Di-O-methylellagic acid 4′-α-d-arabinofuranoside (3)	180.8
3,3′-Di-O-methylellagic acid 4′-β-d-glucopyranoside (4)	181.8
3,3′-Di-O-methylellagic acid 4′-β-d-xylopyranoglucoside (5)	72.0
3,3′,4-tri-O-methylellagic acid 4′-β-d-glucopyranoside (6)	101.9
EtOAc extract of Euscaphis konishii	203.5
Fr.12 extract of Euscaphis konishii	126.8
5-Fluorouracil	11.4

Experimental

General Experimental Procedures

Waters W2695-QDA High Performance Liquid Chromatograph-Mass Spectrometer was purchased from Waters Corporation, United States; CPA225D electronic analytical balance from Sedolis Scientific Instrument Beijing Co., Ltd.; high-speed multifunction pulverizer from Yongkang Tianhao Shengshi Industry and Trade Co., Ltd.; BS-100A automatic partial collector from Shanghai Huxi Analytical Instrument Factory Co., Ltd.; WRR melting point instrument from Zhengzhou Nanbei Instrument Equipment Co., Ltd.; BS-214D electronic balance from Sedolis Scientific Instrument Beijing Co., Ltd.; LC-20AP preparative high performance liquid chromatography (HPLC) from Shimadzu Corporation, Japan; CO₂ constant temperature cell incubator from USA Shellan company; Bruker AV400 (400MHz) nuclear magnetic resonance instrument from German Bruker; Agilent 6520 high-resolution mass spectrometer from USA Agilent Technologies; Nicolet impact 5700 Fourier transform infrared spectrometer from USA Thermoelectric Corporation; silic agel from Qingdao Ocean Chemical Co., Ltd.; D101 macroporous adsorption resin, Sephadex LH-20 from General Electric Company, United States; PRP-512A resin from Beijing Jufu Resin Factory; YMC-Pack ODS-A reversed phase chromatography column (20 × 250 mm, 5 µm and 10 × 250 mm, 5 µm) from YMC, Japan; Diamonsil C18 analytical reversed-phase chromatography column (C18 250 × 4.6 mm, 5 µm) from Beijing Dima Technology Co., Ltd.; methanol, dichloromethane, petroleum ether, and ethyl acetate from Xiyu Chemical Co., Ltd.; methanol and acetonitrile from Merck Company; 5-FU and MTT from Sigma company.

Plant Material

The twigs of E. konishii were collected from San’ming District, Fuzhou, Fujian Province, the People’s Republic of China (PRC) with 26°34′39″N and 117°41′11″E. The plant was identified by Professor Shuangquan Zou from Fujian Agriculture and Forestry University (FAFU), PRC. A voucher specimen (No. 20161105) was preserved in the Department of Pharmaceutical Engineering, College of Plant Protection, FAFU (PRC). The twigs were oven-dried at 65 °C for 24 hours and crushed before extraction.

Extraction and Isolation

The air-dried and powdered twigs of E. konishii (10 kg) were extracted with 95% EtOH (3 × 80 L) by reflux. The crude extract (670 g) was mixed with the diatomite (670 g) and successively extracted with petroleum ether (PE), ethyl acetate (EtOAc), and ethanol (EtOH). The solvents were evaporated to obtain PE (106 g), EtOAc (150 g), and EtOH (150 g). The EtOAc fraction subjected to chromatography on silica gel (200-300 mesh) and eluted with a CH₂Cl₂-MeOH gradient (100:0-0:100) to yield 15 fractions (Fr.1-Fr.15). Fraction 12 (4.46 g) was subjected to a Sephadex LH-20 column and eluted with methanol to obtain 5 fractions (Fr.12.1-Fr.12.5). Fraction 12.2 (214 mg) by preparative HPLC [CH₃CN-H₂O (55:45), v/v, 8.0 mL/min, 210 nm] resulted in the isolation of compounds 8 (15 mg) and 9 (24.8 mg). Fraction 12.3 (109 mg) by preparative HPLC [CH₃CN-H₂O (25:75), v/v, 8.0 mL/min, 254 nm] resulted in the isolation of compounds 2 (15 mg), 3 (4.5 mg), and 4 (4.8 mg). Fraction 12.4 (210 mg) by preparative HPLC [CH₃CN-H₂O (25 : 75), v/v, 8.0 mL/min, 254 nm] resulted in the isolation of compounds 1 (4 mg), 5 (4.5 mg), and 6 (9.9 mg). Fraction 12.5 (2.1 g) was subjected to a silica gel column and eluted with CH₂Cl₂/MeOH (10:1 to 1:1) to obtain compounds 7 (45 mg) and 10 (12 mg) 3,3′-di-O-methylellagic acid 4′-α-l-arabinopyranoside (1) yellow powder; [α] ${}_{\mathrm{D}}^{25}$ – 112°(c 0.1 CH₃OH); HRESIMS m/z: 463.08670 [M+H]⁺(calcd for C₂₁H₁₈O₁₂, 463.08710). UV (CH₃OH) λ _max (log ε): 246 (4.68), 364 (4.1) nm; IR (KBr) ν_max: 3489, 1730, 1609, 1575, 1491, 1365, 1072 cm^{−1; 1}H-NMR (400 MHz, Dimethyl sulfoxide [DMSO]-d ₆) and ¹³C-NMR (100 MHz, DMSO-d ₆) (Table 1).

Acid hydrolysis of compound 1: Compound 1 (4 mg) was dissolved in 10 mL MeOH:H₂O (1:1) and refluxed at 90 °C for 3 hours with 5 mL HCl (2 M). After evaporation of the organic solvent in vacuum and extraction of the aqueous phase with EtOAc, ²² the aqueous residue was concentrated to dryness by adding MeOH to remove the acid. l-Arabinose was identified by comparative TLC with a standard sample.

Cytotoxicity Assay

The compounds and different fractions of the extractions were tested for their cytotoxicity against HepG2 cells by the MTT method, ²³ with 5-FU being used as a positive control. The cells were plated in a 96-well plate at a density of 5 × 10³ cells per well in 180 µL and incubated for 24 hours. After different concentrations of the compounds were added to the plates, the cells were cultivated for another 48 hours, and subsequently 20 µL of MTT (5 mg/mL) were added to each well. After 4 hours, the culture medium was removed, and the cells were dissolved with 150 µL DMSO in each well by vigorously shaking the plate. Finally, the absorbance was assessed by a microplate reader at 570 nm. The IC₅₀ values were calculated for the inhibition rateas follows: (OD value of control group – experiment group)/(OD value of control group – OD value of blank group) × 100%

Supplemental Material

Supplementary Material 1 - Supplemental material for Chemical Constituents and Their Activities From the Twigs of Euscaphis konishii Hayata

Supplemental material, Supplementary Material 1, for Chemical Constituents and Their Activities From the Twigs of Euscaphis konishii Hayata by Jingxin Chen, Lin Ni, Yao Zhang, Yingsa Zhu, Wei Huang and Shuangquan Zou in Natural Product Communications