α-Tetralonyl Glucosides from the Green Walnut Husks of Juglans mandshurica and Their Antiproliferative Effects

Abstract

Two new α-tetralonyl glucosides, (4S)-4,5,8-trihydroxy-α-tetralone-5-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (1) and (4S)-4,8-dihydroxy-α-tetralone-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), together with eight known compounds (3 – 10) were isolated from the green walnut husks of Juglans mandshurica. The structural characterization of all compounds was performed by spectroscopic analyses, including 1D and 2D NMR and HR-ESI-MS experiments. The isolated compounds were assayed for their cytotoxicity against two human cancer cell lines, A549 and HeLa. Four compounds (7 – 10) exhibited inhibitory effects against two human cancer cell lines with GI₅₀ values between 1.3 and 5.8 µM.

Key words

Juglans mandshurica - Juglandaceae - in vitro - α-tetralonyl glucosides - antiproliferative

Introduction

Juglans, a genus of the family Juglandaceae, comprises about 20 species mainly distributed in the temperate and subtropical regions of the northern hemisphere, extending into South America [1]. Juglans mandshurica Maxim. grows in the north of China in the Heilongjiang, Jilin, Liaoning, Hebei, and Shanxi Provinces [2]. Previous phytochemical studies of J. mandshurica led to the identification of naphthoquinones, diarylheptanoids, and flavonoids (as well as their glycosides) [3], [4], [5]. The biological screening of these compounds showed promising bioactivity, including cytotoxic and antimicrobial activities [5], [6], [7], [8], [9].

In the course of our studies on the genus Juglans, including J. mandshurica, we have analyzed the chemical content of the green walnut husks of J. mandshurica. In particular, our attention has been focused towards the n-butanol-soluble fraction of the 70% ethanol extract containing naphthoquinone and diarylheptanoid components. This study led us isolate ten compounds including two new α-tetralonyl glucosides, (4S)-4,5,8-trihydroxy-α-tetralone-5-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranosie (1) and (4S)-4,8-dihydroxy-α-tetralone-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), and a series of known compounds (3 – 10), some of which have not been reported before from J. mandshurica ([Fig. 1]). These compounds were screened for antiproliferative activity against two human cancer cell lines, A549 and HeLa. Herein, details of the isolation, structural elucidation, and antiproliferative activities of these compounds are described.

Fig. 1 Chemical structures of compounds 1 – 10 from the green walnut husks of J. mandshurica.

Results and Discussion

The 70% ethanol extract of the dried the green walnut husks of J. mandshurica was subjected to multiple chromatographic steps over silica gel, Sephadex LH-20, and recycling preparative HPLC. Ten compounds were obtained, including two new compounds, (4S)-4,5,8-trihydroxy-α-tetralone-5-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (1) (4S)-4,8-dihydroxy-α-tetralone-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), and eight known compounds comprising of four naphthoquinones (3 – 6) and four diarylheptanoids (7 – 10). The structures of 1 – 10 were identified by physical data analyses, including 1D and 2D NMR and HR-ESI-MS. The sugar residues were identified by GC analyses after hydrolysis.

Constituents 1 and 2 are new α-tetralonyl glucosides never described before in the literature. The full assignments of ¹H NMR and ¹³C NMR spectroscopic data of the new isolated compounds (1 and 2) are listed. Their key ¹H-¹H COSY and HMBC correlations are presented in [Fig. 2]. The respective NMR and HR-ESI-MS spectra are provided in the Supporting Information.

Fig. 2 The key ¹H-¹H COSY and HMBC of compounds 1 – 2.

Compound 1 was obtained as a white amorphous powder. The molecular formula of C₂₂H₃₀O₁₄ was determined on the basis of the HR-ESI-MS (m/z 541.1524, [M + Na]⁺). Acidic hydrolysis of 1 gave D-glucose as the mono sugar residue. The ¹H NMR and ¹³C NMR data of 1 exhibited signals arising from a CO group at δ _C 206.5, a benzene ring at δ _H 7.60 (1H, d, J = 9.0 Hz, H-6), 6.93 (1H, d, J = 9.0 Hz, H-7), two β-glucopyranosyl moieties [anomeric H-atom at δ _H 4.83 (1H, d, J = 7.7 Hz, H-1′), 4.40 (1H, d, J = 7.7 Hz, H-1″) and C-atom signals at δ _C 104.8 (C-1″), 104.2 (C-1′), 78.0 (C-3″), 77.9 (C-3′), 77.8 (C-5″), 77.4 (C-5′), 75.2 (C-2′), 75.1 (C-2″), 71.6 (C-4″), 71.3 (C-4′), 70.0 (C-6′), and 62.7 (C-6″)]. These NMR characteristics resembled those observed for 3 [10]. However, instead of a β-glucopyranosyl moiety in 3, compound 1 had two β-glucopyranosyl moieties in view of the anomeric H-atom at δ _H 4.83 (1H, d, J = 7.7 Hz, H-1′), 4.40 (1H, d, J = 7.7 Hz, H-1″). The linkage of the second sugar moiety at OH-C(6′) of the first sugar moiety was established from the HMBC correlations between δ _H 4.40 (1H, d, J = 7.7 Hz, H-1″) and δ _C 70.0 (C-6′). The absolute configuration of the saccharides was determined to be D-glucose by GC analysis of chiral derivatives in the hydrolysate of this compound. As for the absolute configuration of the chiral carbon, (4S)-1 was determined on the basis of a negative Cotton effect at ca. 220 – 240 nm in the CD spectrum of 1 (Fig. 2S, Supporting Information) and comparison of its optical rotation ([α]_D ²⁰: − 45 in MeOH) with that of literature [11].

After further comprehensive analysis of its NMR spectra (Figs. 3S – 8S, Supporting Information), compound 1 was identified as (4S)-4,5,8-trihydroxy-α-tetralone-5-O-β-D-glucopyranosyl(1 → 6)- β-D-glucopyranoside.

Compound 2 was obtained as a white amorphous powder. The molecular formula of C₂₂H₃₀O₁₃ was determined on the basis of the HR-ESI-MS (m/z 525.1563, [M + Na]⁺). Acidic hydrolysis of 2 gave D-glucose as the mono sugar residue. The ¹H NMR and ¹³C NMR data of 2 exhibited the signals arising from a CO group at δ _C 206.6, a benzene ring at δ _H 7.53 (1H, t, J = 8.4 Hz, H-6), 7.21 (1H, d, J = 7.4 Hz, H-5), 6.92 (1H, dd, J = 8.4, 1.0 Hz, H-7), two β-glucopyranosyl moieties [anomeric H-atom at δ _H 4.47 (1H, d, J = 7.8 Hz, H-1″), 4.39 (1H, d, J = 7.7 Hz, H-1′)] and C-atom signals at δ _C 105.1 (C-1″), 102.7 (C-1′), 78.1 (C-5″), 78.0 (C-3′), 78.0 (C-3″), 77.1 (C-5′), 75.2 (C-2′), 75.1 (C-2″), 71.7 (C-4″), 71.6 (C-4′), 70.1 (C-6′), and 62.8 (C-6″). These NMR characteristics resembled those observed for 4 [12]. However, instead of a β-glucopyranosyl moiety in 4, compound 2 had two β-glucopyranosyl moieties in view of the anomeric H-atom at δ _H 4.47 (1H, d, J = 7.8 Hz, H-1″), 4.39 (1H, d, J = 7.7 Hz, H-1′). The linkage of the second sugar moiety at OH-C(6′) of the first sugar moiety was established from the HMBC correlations between δ _H 4.47 (1H, d, J = 7.8 Hz, H-1″) and δ _C 70.1 (C-6′). The absolute configuration of the saccharides was determined to be D-glucose by GC analysis of the chiral derivatives in the hydrolysate of this compound. As for the absolute configuration of the chiral carbon, (4S)-2 was determined on the basis of a negative Cotton effect at ca. 210 – 230 nm in the CD spectrum of 2 (Fig. 10S, Supporting Information) and comparison of its optical rotation ([α]_D ²⁰: − 51 in MeOH) with that of the literature [11].

After further comprehensive analysis of its NMR spectra (Figs. 11S – 16S, Supporting Information), compound 2 was identified as (4S)-4,8-dihydroxy-α-tetralone-4-O-β-D-glucopyranosyl(1 → 6)- β-D-glucopyranoside.

α-Tetralonyl glucosides with one β-glucopyranosyl moiety have been reported from the green walnut husks of J. mandshurica before. But the naphthoquinone with two β-glucopyranosyl moieties has not been described in the literature so far. The putative biosynthetic pathway begins from erythrose-4-phosphate, which was derived through a series of reactions, including intermolecular cyclization, oxidation, hydroxylation, and glycosylation ([Fig. 3]). The eight known compounds identified by comparing their spectroscopic and spectrometric data (NMR and HRESIMS) with those in the literature are juglanoside E (3) [10], berchemiaside A (4) [12], regiolone (5) [13], berchemiaside B (6), 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7-(20),15,17,18-hexaen-10-one (7), juglanin A (8), 2-oxatrycyclo[13.2.2.13,7]eicosa-3,5,7(20),15,17,18-hexaen-10 – 16-diol (9), and (11S)-11,17-dihydroxy-3,4-dimethoxy-[7, 0]-metacyclophane (10) [3], [14], [15]. Copies of the original spectra for the known compounds are obtainable from the corresponding author.

Fig. 3 Plausible biosynthetic pathway of new compounds.

All J. mandshurica components isolated were tested for their cytotoxic activity against A549 and HeLa cell lines, with etoposide as the positive control ([Table 1]). Compounds 1 – 6 showed no or weak cytotoxic activity, whereas compounds 7 – 10 exerted strong cytotoxicity with GI₅₀ values in the range of 1.3 – 5.8 µM. Based on the available data, it seems that diarylheptanoids (compounds 7 – 10) are favorable for the observed cytotoxic activity.

Table 1 Cytotoxicity data of isolated compounds 1 – 10.^a

Compound	GI50 (µM)
	A549	HeLa
A549: human lung cancer cell lines. HeLa: human cervical carcinoma cancer cell lines. aData expressed as GI50 values (µM); bpositive control
1	51.1	73.6
2	67.6	51.9
3	> 100	70.2
4	> 100	> 100
5	36.4	> 100
6	61.6	81.4
7	1.6	2.1
8	5.8	3.3
9	2.4	1.9
10	1.3	2.7
Etoposideb	4.4	0.7

Diarylheptanoid is a group of compounds that bears the 1,7-dihenylheptane skeleton as a special characteristic in the natural product estate [16]. Since the first diarylheptanoid isolated in 1815, approximately 400 such materials have been obtained from natural resources [17]. Hirsutanone and oregonin, from Alnus japonica Steud, exhibited potent antiproliferative activity against two colon cancer cell lines (HCT-15 and Colo205) [18]. Tian et al. had suggested that a diarylheptanoid, 1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxyphenyl)-4E-en-3-heptanone, was isolated from Alpinia officinarum and provided new insight into neuroblastoma chemotherapy [19]. Furthermore, diarylheptanoids have been reported by numerous studies to possess diverse bioactivities, including anti-inflammatory [20] and antiemetic [21].

In summary, two new α-tetralonyl glucosides (1 and 2) and eight known compounds (3 – 10) were isolated from the green walnut husks of J. mandshurica, and the structures were determined by MS, 1D NMR, and 2D NMR. The results obtained in this study show that diarylheptanoids (7 – 10) exhibited inhibitory effects against the two human cancer cell lines (A549 and HeLa) with GI₅₀ values below 6 µM, while naphthoquinones (1 – 6) were inactive.

Material and Methods

General experimental procedures

The NMR spectra were measured in methanol-d ₄ on a Bruker ARX-400 or AV600 instrument with TMS as an internal standard. The CD spectrum was tested using a JASCO pu-2080 spectrometer. IR spectra were taken on a Bruker IFS-55 infrared spectrophotometer with a KBr disk. Optical rotations were measured on a Peking-Elmer 241 MC Spectropolarimeter at 20 °C. ESI-MS spectra were recorded on a Waters Quattro micro API LC/MS/MS spectrometer (Waters). HR-TOF-MS spectra were performed on an Agilent LC/MS spectrometer (Agilent). HPLC was performed on JAI LC9103 Recycling preparative HPLC (Japan Analytical Industries) equipped with JAIGEL-ODS-AP-P column and JAIGEL-GS310 column using a JAI refractive index detector and a JAI UV-3702 detector with MultiChro 2000 workstation. TLC was performed on precoated GF₂₅₄ plates (Merck) and detected by spraying with 10% H₂SO₄ followed by heating.

Plant material

The green walnut husks of J. mandshurica were collected in October 2014 at Yichun, Heilongjiang, China, and authenticated by Professor Jin-cai Lu (The School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University). A voucher specimen (No. 1014023) is stored at the key laboratory of Modern Analysis of TCM, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China.

Extraction and isolation

The green walnut husks of J. mandshurica (20 kg) were extracted with 70% (v/v) aqueous ethanol three times (3 × 80 L) to give 1 kg of crude extract. The crude extract was suspended in 3 L of water. The suspension was successively partitioned with EtOAc (3 × 3 L) and n-BuOH (3 × 3 L). The n-BuOH-soluble fraction (120 g) was subjected to silica gel chromatography, eluting with gradients of CH₂Cl₂ : MeOH (100 : 13 000 mL, 100 : 33 000 mL, 100 : 73 000 mL, 100 : 153 000 mL, 100 : 303 000 mL, 100 : 703 000 mL) on a silica gel column (column dimension: 6.0 × 80 cm) to give six fractions, Fr. A – F (4.7 g, 6.3 g, 12.2 g, 8.4 g, 8.1 g, and 3.0 g). Fraction A was purified by silica gel CC (column dimension: 2.5 × 40 cm) with CH₂Cl₂ : MeOH (300 : 11 000 mL, 150 : 3800 mL, 100 : 11 000 mL) to afford 230 mg of Fr. A-1 and 130 mg of Fr. A-2. Fraction A-1 was purified by recycling preparative HPLC with MeOH-H₂O (85 : 15) as the eluent to obtain pure compound 7 (23.1 mg, t _R 43 min). Fraction A-2 was purified by recycling preparative HPLC with MeOH-H₂O (85 : 15) as the eluent to obtain pure compounds 8 (15.3 mg, t _R 34 min) and 9 (30.1 mg, t _R 38 min). Fraction B was purified by silica gel CC (column dimension: 2.5 × 40 cm) with CH₂Cl₂ : MeOH (100 : 11 000 mL, 100 : 21 000 mL, 100 : 31 000 mL) to afford 400 mg of Fr. B-1. Fraction B-1 was purified by recycling preparative HPLC with MeOH-H₂O (70 : 30) as the eluent to obtain pure compounds 5 (9.1 mg, t _R 29 min) and 6 (6.2 mg, t _R 35 min). Fraction C was purified by silica gel CC (column dimension: 2.5 × 40 cm) with CH₂Cl₂: MeOH (100 : 31 000 mL, 100 : 51 000 mL, 100 : 81 000 mL) to afford 210 mg of Fr. C-1 and 310 mg of Fr. C-2. Fraction C-1 was purified by recycling preparative HPLC with MeOH-H₂O (55 : 45) as the eluent to obtain pure compounds 1 (10.5 mg, t _R 21 min) and 3 (9.0 mg, t _R 25 min). Fraction C-2 was purified by recycling preparative HPLC with MeOH-H₂O (55 : 45) as the eluent to obtain pure compounds 2 (17.7 mg, t _R 24 min), 4 (8.7 mg, t _R 31 min), and 10 (7.9 mg, t _R 35 min).

(4S)-4,5,8-Trihydroxy-α-tetralone-5-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (1)

White amorphous power, [α]_D ²⁰ = − 45 (c 0.6, MeOH); HR-ESI-MS: m/z 541.1524, [M + Na]⁺ (calcd. for C₂₂H₃₀O₁₄Na, 541.1523). ¹H NMR (600 MHz, methanol-d ₄) δ 7.60 (1H, d, J = 9.0 Hz, H-6), 6.93 (1H, d, J = 9.0 Hz, H-7), 5.37 (1H, d, J = 2.7 Hz, H-4), 4.83 (1H, d, J = 7.7 Hz, H-1′), 4.40 (1H, d, J = 7.7 Hz, H-1″), 4.21 (1H, dd, J = 11.7, 1.9 Hz, H-6′_a), 3.88 (1H, dd, J = 11.9, 2.1 Hz, H-6″_a), 3.83 (1H, dd, J = 11.7, 6.2 Hz, H-6′_b), 3.66 (2H, m, H-5′/H-6″_b), 3.56 (1H, t, J = 9.0 Hz, H-2′), 3.49 (1H, t, J = 9.0 Hz, H-5″), 3.44 (1H, t, J = 9.0 Hz, H-4′), 3.37 (1H, t, J = 9.0 Hz, H-3′), 3.31 (1H, d, J = 9.0 Hz, H-4″), 3.26 (2H, m, H-2″/H-3″), 3.08 (1H, ddd, J = 17.4, 12.7, 6.0 Hz, H-2a), 2.56 (1H, d, J = 17.4 Hz, H-2b), 2.26 (1H, m, H-3a), 2.24 (1H, m, H-3b). ¹³C NMR (100 MHz, methanol-d ₄) δ 206.5 (C-1), 159.0 (C-8), 148.7 (C-5), 134.5 (C-4a), 128.5 (C-6), 119.2 (C-7), 116.1 (C-8a), 104.8 (C-1″), 104.2 (C-1′), 78.0 (C-3″), 77.9 (C-3′), 77.8 (C-5″), 77.4 (C-5′), 75.2 (C-2′), 75.1 (C-2″), 71.6 (C-4″), 71.3 (C-4′), 70.0 (C-6′), 62.7 (C-6″), 61.3 (C-4), 33.6 (C-2), 30.2 (C-3).

(4S)-4,8-Dihydroxy-α-tetralone-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2)

White amorphous power, [α]_D ²⁰ = − 51 (c 0.6, MeOH); HR-ESI-MS: m/z 525.1563, [M + Na]⁺ (calcd. for C₂₂H₃₀O₁₃Na, 525.1570). ¹H NMR (600 MHz, methanol-d ₄) δ 7.53 (1H, t, J = 8.4 Hz, H-6), 7.21 (1H, d, J = 7.4 Hz, H-5), 6.92 (1H, dd, J = 8.4, 1.0 Hz, H-7), 5.07 (1H, t, J = 6.3 Hz, H-4), 4.47 (1H, d, J = 7.8 Hz, H-1″), 4.39 (1H, d, J = 7.7 Hz, H-1′), 4.23 (1H, dd, J = 11.7, 2.0 Hz, H-6′_a), 3.92 (1H, dd, J = 11.8, 1.8 Hz, H-6″_a), 3.83 (1H, dd, J = 11.7, 6.5 Hz, H-6′_b), 3.71 (1H, dd, J = 11.8, 5.1 Hz, H-6″_b), 3.51 (1H, ddt, J = 8.4, 4.2, 1.9 Hz, H-5′), 3.39 (1H, m, H-3″), 3.34 (4H, m, H-3′/H-4′/H-4″/H-5″), 3.28 (2H, t, J = 9.0 Hz, H-2′/H-2″), 3.11 (1H, ddd, J = 17.9, 9.6, 5.0 Hz, H-2a), 2.69 (1H, ddd, J = 17.9, 6.3, 4.8 Hz, H-3b), 2.40 (1H, m, H-3a), 2.34 (1H, m, H-3b). ¹³C NMR (100 MHz, methanol-d ₄) δ 206.6 (C-1), 163.8 (C-8), 144.1 (C-4a), 137.5 (C-6), 120.7 (C-5), 118.6 (C-7), 116.8 (C-8a), 105.1 (C-1″), 102.7 (C-1′), 78.1 (C-5″), 78.0 (C-3′), 78.0 (C-3″), 77.1 (C-5′), 75.2 (C-2′), 75.1 (C-2″), 74.7 (C-4), 71.7 (C-4″), 71.6 (C-4′), 70.1 (C-6′), 62.8 (C-6″), 35.2 (C-2), 31.1 (C-3).

Acid hydrolysis of compounds 1 and 2

Each compound (2 mg) was treated with 1 M HCl (4 mL) at 90 °C for 2 h. Then the reaction mixture was extracted with CHCl₃ (3 × 5 mL). The aqueous layer was collected and the water was evaporated under vacuum with the repeated addition of MeOH to remove the solvent completely. The residue was redissolved in anhydrous pyridine (2 mL) and mixed with a pyridine solution of L-cysteine methyl ester hydrochloride (2 mL). After the mixed solution was heated at 60 °C for 1 h, trimethylchlorosilane (0.5 mL) was added and the resulting mixture was stirred at 60 °C for another 30 min. Then, the solution was concentrated to dryness and taken up in water (3 × 1 mL), followed by extraction with n-hexane (3 × 1 mL). The supernatant was analyzed by GC. Separations were carried out on HP-5 columns (320 µm × 30 cm, 0.25 µm). Highly pure N₂ was employed as a carrier gas (1.0 mL/min), and the FID detector operated at 280 °C (column temperature 160 – 200 °C). The retention time of the monosaccharide derivative was as follows: D-glucose (14.43 min).

Cell culture

Human lung cancer cells (A549) and human cervical carcinoma cancer cells (HeLa) was provided by the ATCC. The cells were cultured in medium (RPMI 1640 for A549 and DMEM for HeLa) supplemented with 10% heat-inactivated FBS and antibiotics/antimycotics (PSF; 100 units/mL penicillin G sodium, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B). The cells were incubated at 37 °C and 5% CO₂ in a humidified atmosphere. Etoposide (purity > 98%; Sigma) was used as a positive control.

In vitro cell proliferation assay

Cell viability was determined by the sulforhodamine B (SRB) protein staining method. Cells were seeded in 96-well plates and incubated for 24 h, and then fixed (for zero day controls) or treated with the test compounds for 72 h. All compounds were solved in DMSO (final concentration of 0.1% [v/v]), stored at − 20 °C, and diluted to the desired concentration (0.01, 0.1, 1, 10, 100 µM) in normal saline immediately prior to each experiment. Each concentration was tested thrice. At least three experiments were performed. Data leading to the determination of GI₅₀ values are provided as dose-response curves in Fig. 17S, Supporting Information. After incubation, cells were fixed with 10% trichloroacetic acid (TCA), dried, and stained in 0.4% SRB in 1% acetic acid solution. Unbound dye was washed, and stained cells were dried and dissolved in 10 mM Tris (pH 10.0). Absorbance was measured at 515 nm and cell proliferation was determined as follows: cell proliferation (%) = (average absorbance_compound – average absorbance_{zero day})/(average absorbance_control – average absorbance_{zero day}) × 100%. GI₅₀ values were calculated by nonlinear regression analysis using Table Curve 2D software (Version 5.01, Systat Software Inc.).

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the General Project of the Department of Education of Liaoning Province (grant no. 201610163L13) and the Natural Science Foundation of Liaoning Province (grant no. 201602712).

• References

• 1 Lin H, Zhang YW, Bao YL, Wu Y, Sun LG, Yu CL, Huang YX, Wang EB, Li YX. Secondary metabolites from the stem bark of Juglans mandshurica . Biochem Syst Ecol 2013; 51: 184-188
• 2 Editorial Committee of Flora of China, Chinese Academy of Sciences. Flora of China, Vol. 21. Beijing: Science Press; 1979: 31-33
• 3 Liu JX, Di DL, Wei XN, Han Y. Cytotoxic diarylheptanoids from the pericarps of walnuts (Juglans regia). Planta Med 2008; 74: 754-759
• 4 Zhao H, Bai H, Jing Y, Li W, Yin S, Zhou H. A pair of taxifolin-3-O-arabinofuranoside isomers from Juglans regia L. Nat Prod Res 2016; 31: 945-950
• 5 Yu HY, Zhang XQ, Li X, Zeng FB, Ruan HL. 2-methoxyjuglone induces apoptosis in HepG2 human hepatocellular carcinoma cells and exhibits in vivo antitumor activity in a H22 mouse hepatocellular carcinoma model. J Nat Prod 2013; 76: 889-895
• 6 Guo LN, Zhang R, Guo XY, Cui T, Dong W, Huo JH, Wang WM. Identification of new naphthalenones from Juglans mandshurica and evalution of their anticancer activities. Chin J Nat Medicines 2015; 13: 707-710
• 7 Yu HY, Li X, Meng FY, Pi HF, Zhang P, Ruan HL. Naphthoquinones from the root barks of Juglans cathayensis dode. J Asian Nat Prod Res 2011; 13: 581-587
• 8 Jin M, Sun J, Li R, Diao S, Zhang C, Cui J, Son JK, Zhou W, Li G. Two new quinones from the roots of Juglans mandshurica . Arch Pharm Res 2016; 39: 1237-1241
• 9 Li J, Xu KP, Zou ZX, Zou H, Long HP, Tan LH, Liu RH, Wang YK, Xu PS, Tan GS. Two new compounds from the green peel of Juglans mandshurica . J Asian Nat Prod Res 2017; 19: 1087-1092
• 10 Liu L, Li W, Koile K, Zhang S, Nikado T. New α-tetralonyl glucosides from the fruit of Juglans mandshurica . Chem Pharm Bull 2004; 52: 566-569
• 11 Machida K, Matsuoka E, Kasahara T, Kikuchi M. Studies on the constituents of Juglans species. I. Structural determination of (4S)- and (4R)-4-hydroxy-a-tetralone derivatives from the fruit of Juglans mandshurica MAXIM. var. sieboldiana MAKINO. Chem Pharm Bull 2005; 53: 934-937
• 12 Fujimoto H, Nagano J, Yamaguchi K, Yamazaki M. Immunosuppressive components from an Ascomycete, Diplogelasinospora grovesii . Chem Pharm Bull 1998; 46: 423-429
• 13 Talapatra S, Karmacharya B, Talapatra B. (−)-Regilone, an α-tetralone from Juglans Regia: structure, stereochemistry and conformation. Phytochemistry 1988; 27: 3929-3932
• 14 Jin W, Cai X, Na M, Lee J, Bae K. Triterpenoids and diarylheptanoids from Alnus hirsuta inhibit HIF-1 in AGS cells. Arch Pharm Res 2007; 30: 412-418
• 15 Yao D, Jin M, Zhang C, Luo J, Li R, Zheng M, Cui J, Li G. A new phenolic glycoside from Juglans mandshurica . Nat Prod Res 2014; 28: 998-1002
• 16 Lv H, She G. Naturally occurring diarylheptanoids – a supplementary version. Rec Nat Prod 2012; 6: 321-333
• 17 Alberti A, Riethmuller E, Beni S. Characterization of diarylheptanoids: an emerging class of bioactive natural products. J Pharmaceut Biomed 2018; 147: 13-34
• 18 Uto T, Tung NH, Appiah OR, Aning A, Morinaga O, Edoh D, Nyarko AK, Shoyama Y. Antiproliferative and pro-apoptotic activity of diarylheptanoids isolated from the bark of Alnus japonica in human leukemia cell lines. Am J Chinese Med 2015; 43: 757-767
• 19 Tian Z, An N, Zhou B, Xiao P, Kohane IS, Wu E. Cytotoxic diarylheptanoid induces cell cycle arrest and apoptosis via increasing ATF3 and stabilizing p53 in SH-SY5Y cells. Cancer Chemother Pharmacol 2009; 63: 1131-1139
• 20 Lai YC, Chen CK, Lin WW, Lee SS. A comprehensive investigation of anti-inflammatory diarylheptanoids from the leaves of Alnus formosana . Phytochemistry 2012; 73: 84-94
• 21 Yang Y, Kinoshita K, Koyama K. Structure-antiemetic-activity of some diarylheptanoids and their analogues. Phytomedicine 2002; 9: 146-152