Studies on cytotoxic activity against HepG-2 cells of naphthoquinones from green walnut husks of Juglans mandshurica maxim

Molecules 2015, 20, 15572-15588; doi:10.3390/molecules200915572

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molecules

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Article

Studies on Cytotoxic Activity against HepG-2 Cells of

Naphthoquinones from Green Walnut Husks of

Juglans mandshurica Maxim

Yuanyuan Zhou ^1,†, Bingyou Yang ^1,†, Yanqiu Jiang ¹, Zhaoxi Liu ¹, Yuxin Liu ¹, Xiaoli Wang ²

and Haixue Kuang ^1,*

1

College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China;

E-Mails: zhouyuanyuan1998@163.com (Y.Z.); ybywater@163.com (B.Y.);

jiangyanqiu219@163.com (Y.J.); liu_zhao_xi@163.com (Z.L.); ZYC19901014@126.com (Y.L.)

2

College of Adult Education, Heilongjiang University of Chinese Medicine, Harbin 150040, China;

E-Mail: wangxiaolicando2000@126.com

†

These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: hxkuang@hotmail.com;

Tel.: +86-451-8219-3001; Fax: +86-451-8211-0803.

Academic Editor: Isabel C. F. R. Ferreira

Received: 20 July 2015 / Accepted: 19 August 2015 / Published: 26 August 2015

Abstract: Twenty-seven naphthoquinones and their derivatives, including four new

naphthalenyl glucosides and twenty-three known compounds, were isolated from green

walnut husks, which came from Juglans mandshurica Maxim. The structures of four new

naphthalenyl glucosides were elucidated based on extensive spectroscopic analyses. All of

these compounds were evaluated for their cytotoxic activities against the growth of human

cancer cells lines HepG-2 by MTT [3-(4,5-dimethylthiazo l-2-yl)-2,5 diphenyl tetrazolium

bromide] assay. The results were shown that most naphthoquinones in an aglycone form

exhibited better cytotoxicity in vitro than naphthalenyl glucosides with IC₅₀values in the

range of 7.33–88.23 μM. Meanwhile, preliminary structure-activity relationships for these

compounds were discussed.

Keywords: Juglans mandshurica Maxim; green walnut husks; naphthoquinones; cytotoxic

activity; structure-activity relationships

Molecules 2015, 20

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1. Introduction

With the increased use of natural product-based cancer chemotherapy, exploring the cytotoxic

activity of phytochemicals for anticancer drug design has gained extensive attention worldwide [1].

Juglans mandshurica Maxim is a well-known member of the Juglandaceae family which is widely

distributed throughout urban and rural areas in northeast China [2–4]. A few distrubute in Russia,

Korea and Japan. It is one of the most important medical plants of which the green husks, leaf, root and

bark all can be medically used [5–8]. Its green husks have been used as a folk medicine for treatment of

gastric ulcers, uterine prolapse, leukopenia, diarrhea and dysentery for many years in China [9]. In recent

years, many studies showed that green walnut husks have obvious advantages in tumor treatment like

liver cancer [9–11].

Based on our interest in natural antitumor sources prompted us to continue investigating the

phytochemicals and cytotoxicity of this plant [12–16]. A number of extracts and compounds obtained

from medical materials have been identified as in vitro tumor inhibitors [7,9]. These beneficial effects

have largely been ascribed to the presence of naphthoquinones. Naphthoquinones are the most important

and widely distributed chemical class in the quinone family. Their derivatives have exhibited a variety of

biological responses which include antiallergic, antibacterial, antifungal, anti-inflammatory, antithrombotic,

antiplatelet, antiviral, apoptosis, lipoxygenase, radical scavenging, and anti-ringworm activities. Many

studies have shown that naphthoquinones have biological activities specifically against pathogenic

protozoa and cancer cells owing to their privileged structures in medicinal chemistry [17]. In this

study, we obtained a series of naphthoquinone aglycones and glucosides, and then presented the isolation

and structural elucidation of four new naphthalenyl glucosides (compounds 18, 25–27), together with

23 known compounds from green walnut husks of J. mandshurica Maxim. These included juglone (1),

5-methoxy-1,4-naphthoquinone (2), 5,8-dihydroxy-1,4-naphthoquinone (3), 2-hydroxy-1,4-naphthoquinone

(4), 2,5-dihydroxy-1,4-naphthoquinone (5), 3,5-dihydroxy-1,4-naphthoquinone (6), 3-methoxy juglone

(7), 2-methoxy juglone (8), 3-ethoxy juglone (9), 2-ethoxy juglone (10), Engelharquinone (11),

(S)-regiolone (12), (4S)-4-hydroxy-α-tetralone (13), (4S)-5-hydroxy-4-methoxy-α-tetralone (14), 1,4,5-

trihydroxynaphthalene-1,4-di-O-β-D-glucopyrano side (15), 1,4,5-trihydroxynaphthalene-1,5-di-O-β-

D-glucopyranoside (16), 1,4,8-trihydroxynaphthalene-1-O-β-D-glucopyranoside (17), 1,4,8-trihydroxy-

3-naphthalenecarboxylic acid -1-O-β-D-glucopyranoside ethyl ester (18), 1,4,8-trihydroxynaphthalene-

1-O-β-D-[6′-O-(3′′,4′′,5′′-trihydroxybenzoyl)]glucopyranoside (19), (4S)-4-hydroxy-α-tetralone-4-O-β-

D-glucopyranoside (20), (4S)-4,5-dihydroxy-α-tetralone 4-O-β-D-glucopyranoside (21), (4S)-4,6-dihydroxy-

α-tetralone 4-O-β-D-glucopyranoside (22), (4S)-4,5,8-trihydroxy-α-tetralone 4-O-β-D-glucopyranoside

(23), (4S)-4,5,8-trihydroxy-α-tetralone 5-O-β-D-[6′-O-(3′′,4′′,5′′-trihydroxybenzoyl)] glucopyranoside (24),

(4S)-4-hydroxy-α-tetralone-4-O-β-D-(6′-O-4′′-hydroxylbenzoyl)glucopyranoside (25), (4S)-4,5-dihydroxy-

α-tetralone-4-O-β-D-(6′-O-4′′-hydroxylbenzoyl)glucopyranoside (26), (4S)-4,5,8-thihydroxy-α-tetralone-

5-O-β-D-(6′-O-4′′-hydroxylbenzoyl)glucopyranoside (27). In this study, we explored the antitumor

structure-activity relationships using above compounds against the growth of liver cancer cell HepG-2

by MTT assays. The aim of this work was to define the key naphthoquinone structural elements that

were required for cytotoxic activity through the determination of the ability of 27 naphthoquinones

belonging to main structural subtypes such as naphthoquinone, tetralone, and naphthols.

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2. Results and Discussion

2.1. Isolation and Characterization of Compounds 18, 25–27

The compounds were isolated using silica gel columns and semi-preparative HPLC chromatography

from 30% ethanol extract of fresh green husks of Juglans mandshurica Maxim. The structures of four

new naphthalenyl glucosides were elucidated based on extensive mass and spectroscopic analyses

1

13

including HR-ESI-MS, IR, H-NMR, C-NMR, DEPT, HSQC, HMBC, and CD. Their structures,

¹H- and ¹³C-NMR data, and HMBC correlations are shown in Figures 1 and 2 and Table 1.

Figure 1. The chemical structures of compounds 1–27.

Compound 18 was a red amorphous powder. The molecular formula C₁₉H₂₂O₁₀was determined from

HR-ESI-MS and ¹³C-NMR data. There were two major differences between 18 and 25–27: two methylene

groups located at C-2 and C-3, respectively, at δ_C33.0–35.0 and 30.0–31.5 in compounds 25–27 were

replaced by methenyl groups at δ_C109.9 and 105.8 in compound 18, indicating no presence of a

hydrogenated position. Furthermore, the independent existence of the glucopyranosyl moiety was not

13

together with p-hydroxybenzoly on the basis of 1D-, 2D-NMR data. Noise-decoupled C-NMR and

the distortionless enhancement by polarization transfer (DEPT) spectrum of 18 showed 19 carbon

peaks, including one methyl, two methylenes, nine methynes, and seven quaternary carbons. There

were 10 carbons due to the naphthalene ring, six carbons due to the glucose, and a carbonyl ketone at

Molecules 2015, 20

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δ_C171.8 correlated with one ethyl group, which was assigned to acetyl group. In the ¹H-NMR spectrum,

there were ABC-spin aromatic proton signals at δ_H6.99 (dd, J = 1.0, 7.8 Hz, H-5), 7.40 (t, J = 7.8 Hz,

H-6), and 7.86 (dd, J = 1.0, 7.8 Hz, H-7), which couple among themselves. Moreover, one isolated

proton signal due to H-2 at δ_H7.72 and one double-peak signal due to an anomeric proton at δ_H4.99

were distinct. In the HMBC spectrum of 18 (Figure 2), the correlation peak between the anomeric

proton and C-1 at δ_C148.0 was observed. The results implied that the glucopyranosyl was linked to C-1

of the aglycone (Table 1, Figure 2). Thus, the structure of 18 was elucidated as 1,4,8-trihydroxy-3-

naphthalenecarboxylic acid 1-O-β-D-glucopyranoside ethyl ester.

Figure 2. Key HMBC correlations of compounds 18, 25–27.

Compound 25 was obtained as a yellow amorphous powder and the molecular formula was assigned

13

1

13

as C₂₃H₂₄O₉from its HR-ESI-MS and C-NMR data. H-NMR and C-NMR spectra revealed that 25

contained a typical β-D-glucopyranosyl (δ_H4.42 (d, J = 7.6 Hz, H-1′); δ_C103.7, 75.2, 78.1, 72.2, 75.5,

65.0), which was confirmed by acid hydrolysis and co-chromatography in comparison with an authentic

sample. Moreover, the remaining 17 carbon signals, which respectively belong to the tetralone moiety

and a p-hydroxybenzoly group, were attributable to two methylenes, nine methines, four olefinic

quaternary carbons, and two quaternary carbonyl groups. To ascertain the structure of the aglycone and

the glycosidic connection, a complete ¹H- and ¹³C-NMR spectral assignment was carried out utilizing a

combination of DEPT, HSQC, HMBC, and CD experiments. To be specific, the ¹H-NMR spectrum of

25 showed two methylenes of tetralone at δ_H2.87 (ddd, J = 4.5, 8.9, 17.5 Hz, H_ax-2) and δ_H2.41 (ddd,

J = 4.5, 6.5, 17.5 Hz, H_eq-2); 2.34 (dddd, J = 2.2, 4.5, 8.9, 13.4 Hz, H_ax-3) and 2.28 (dddd, J = 3.8, 4.5,

6.5, 13.4 Hz, H_eq-3), corresponding to C-atom signals at δ_C35.5 and 31.5 in the HSQC spectrum.

In the ¹H-NMR spectrum, there was a set of correlation signals at δ_H7.65 (br.d, J = 7.6 Hz, H-5), 7.52

(dt, J = 1.2, 7.6 Hz, H-6), 7.43 (dt, J = 1.2, 7.6 Hz, H-7), and 7.93 (dd, J = 1.2, 7.6 Hz, H-8) due to an

Molecules 2015, 20

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ortho-disubstituted aromatic ring. All above data implied that 25 was an α-tetralone derivative.

Hydrolysis of 25 yielded glucose, which was identified on a thin layer chromatography (TLC) plate

by comparison with a reference sample. Moreover, a suggestive correlation was observed between the

anomeric proton signal of glucose and a methane carbon signal at δ_C75.9 (C-4) in the HMBC spectrum

(Figure 2), indicating that the sugar moiety was linked at the C-4 position. The β-anomeric configuration

for glucopyranose was determined from the J_H1,H2value (7.6 Hz). At the same time, it was also observed

that the δ_H4.66 (dd, J = 2.2, 11.8 Hz, H-6′a) and 4.47 (dd, J = 7.2 Hz, 11.8 Hz, H-6′b) had a linkage

with formyl group. There were two sets of high peaks at δ_H7.95 (d, J = 8.8 Hz, H-2′′, 6′′), 6.84 (d,

1

13

J = 8.8 Hz, H-3′′, 5′′), and δ_C132.9 (C-2′′, 6′′), 116.3 (C-3′′, 5′′) in the H- and C-NMR spectrum,

indicating the presence of p-hydroxybenzoly. To determine the absolute configuration of the chiral center

at the C-4 position, 25 was hydrolyzed to give the aglycone, which was identified to be S configuration

by comparing its NMR data with those of the reference [18,19] and the circular dichrosim CD spectrum,

where a negative Cotton effect at 236 nm was observed. On the basis of the above evidence, the structure

of 25 was established as (4S)-4-hydroxy-α-tetralone 4-O-β-D-(6′-O-4′′-hydroxybenzoyl) glucopyranoside.

Compound 26, a yellow amorphous powder, was assigned as C₂₃H₂₄O₁₀on the basis of its HR-ESI-MS

and ¹³C-NMR data. The 1D- and 2D-NMR spectrographic data were similar as compound 25 except for

1

the aryl ring moiety of the tetralone. The H-NMR spectrum of 26 showed a set of proton signals that

was in accordance with the ABC-type aromatic proton signals, indicating the presence of a hydroxyl

group at the C-5 position on the aromatic ring. The position of the hydroxyl group was also deduced to

the C-5 position by observation of the correlations between δ_H5.37 (H-4) and δ_C157.0 (C-5) in the

HMBC spectrum (Figure 2). The C-6, C-8, and C-10 located in the ortho- and para-position of C-5

were different from compound 25 due to the influence of the hydroxyl group. Moreover, the absolute

configuration of 26 was determined as 4S from the CD spectrum of its aglycon [18], which had a

negative Cotton effect. Thus, the structure of 26 was established as (4S)-4,5-dihydroxy-α-tetralone-4-

O-β-D-(6′-O-4′′-hydroxybenzoyl) glucopyranoside.

Compound 27 was isolated as a yellow powder, which had the molecular formula C₂₃H₂₄O₁₁,

established in HR-ESI-MS. Hydrolysis of 27 was similar to 25 and 26. Glucose was further confirmed

1

13

by H-, C-NMR, and the DEPT spectrum (δ_H4.81 (d, J = 7.5 Hz, H-1′); δ_C104.4, 75.2, 78.0, 72.0,

75.8, 64.8). The correlation position between the aglycone and glucose was different from compounds

25 and 26, which was deduced to transfer to δ_C148.3 (C-5), implying the connection at the aryl ring of

the tetralone by the HMBC spectrum. The relative configuration of the glucopyranose moiety was

determined as β by the coupling constant (J = 7.5 Hz) of the anomeric proton. Furthermore, the

¹H-NMR spectrum showed the AB-type aromatic proton signals at δ_H7.40 (d, J = 9.1 Hz, H-6) and

6.67 (d, J = 9.1 Hz, H-7) in this aryl ring. It was also observed that a new quaternary carbon signal

appeared at δ_C159.3 due to the C-8 position in DEPT spectrum. It was also worth noting that the carbon

signal at δ_C116.2 (C-3′′, 5′′) not only had connections with H-3′′, 5′′ and H-2′′, 6′′, but also related with

H-7 and H-4 (Figure 2) in HMBC. So we deduced that C-9 and C-3′′, 5′′ occurred in the same position.

The absolute configuration of the chiral center at the C-4 position was deduced to be S by CD spectrum

analysis of its aglycon [18]. Thus, the structure of 27 was determined to be (4S)-4,5,8-thihydroxy-α-

tetralone 5-O-β-D-(6′-O-4′′-hydroxybenzoyl) glucopyranoside.

Molecules 2015, 20

Table 1. ¹H-(400 MHz) and ¹³C-(100 MHz) NMR data of 18, 25–27 in CD₃OD.

15577

18

δ_H(J in Hz)

—

25

δ_H(J in Hz)

—

26

δ_H(J in Hz)

—

27

δ_H(J in Hz)

—

No.

1

δ_C

148.0

200.0

200.9

206.4

H_ax: 2.87, ddd

(4.5, 8.9, 17.5)

H_eq: 2.41, ddd

(4.5, 6.5, 17.5)

H_ax: 2.34, dddd

(2.2, 4.5, 8.9, 13.4)

H_eq: 2.28, dddd

(3.8, 4.5, 6.5, 13.4)

4.97, dd (3.6, 6.5)

7.65, brd

H_ax: 3.03, ddd

(5.0, 13.4, 17.0)

H_eq: 2.37, dt

(3.6, 17.0)

H_ax: 3.01, ddd

(5.9,12.9, 17.6)

H_eq: 2.44, dt

(3.6, 17.6)

7.72, s

109.9

35.5

33.9

33.5

2

3

H_ax: 2.48, dddd

(1.3, 3.2, 4.7, 12.6)

H_eq: 2.10, tt

(4.2, 12.6)

—

105.8

31.5

30.0

2.16, m

30.3

4

5

6

7

—

6.99, dd

(1.0, 7.8)

7.40, t (7.8)

7.86, dd

(1.0, 7.8)

—

155.1

116.0

128.6

116.1

75.9

130.0

134.8

129.4

5.37, t (2.9)

69.9

157.0

122.3

130.6

5.32, t (3.1)

—

61.3

148.3

128.9

118.9

—

(7.6)

7.52, dt (1.2, 7.6)

7.43, dt

7.08, dd (0.8, 8.0)

7.40, d (9.1)

6.67, d

7.27, t

(1.2, 7.6)

(8.0)

7.46, dd (0.8, 8.0)

—

(9.1)

8

9

158.0

120.2

131.1

105.5

7.93, dd (1.2, 7.6)

—

127.9

132.9

143.9

103.7

118.9

134.4

129.6

103.8

—

159.3

116.2

135.5

104.4

—

10

1′

—

4.99, d (7.6)

4.42, d (7.6)

4.60, d (7.8)

4.81, d (7.5)

3.53, dd

2′

3′

3.50, m

3.53, m

3.43, m

78.9

75.0

71.3

78.2

62.5

3.34, m

75.2

78.1

72.2

75.5

65.0

3.2, m

3.38, m

75.3

78.0

72.1

75.7

65.0

75.2

78.0

72.0

75.8

64.8

(8.8, 16.5)

3.49, t (8.6)

3.43, dd

3.36, dd (2.5, 7.0)

4′

(10.6, 16.2)

3.68, dt (2.2, 8.4)

4.61, dd

5′

3.48, m

3.96, dd

(2.1, 12.0)

3.77, dd

(5.5, 12.0)

—

3.62, m

4.66, dd

3.65, m

4.64, dd

6′a

(2.2, 11.8)

4.40, dd

6′b

1′′

2′′

4.47, dd (7.2, 11.8)

4.43, dd (6.8, 11.8)

(7.4, 11.8)

—

171.8

62.8

—

7.95, d

122.2

132.9

—

7.95, dt

121.9

133.0

122.1

132.9

4.44, dq

(3.6, 17.7)

1.43, t (7.2)

—

7.82, dt

(8.8)

(2.6, 8.8)

(2.7, 8.8)

6.81, dt (2.7, 8.8)

—

3′′

4′′

5′′

6′′

7′′

14.5

—

6.84, d (8.8)

—

116.3

163.7

116.3

132.9

167.9

6.83, dt (2.6, 8.8)

—

116.2

163.6

116.2

133.0

168.1

116.2

163.7

116.2

132.9

167.8

—

6.84, d (8.8)

7.95, d (8.8)

—

6.83, dt (2.6, 8.8)

7.95, dt (2.6, 8.8)

—

6.81, dt (2.7, 8.8)

7.82, dt (2.7, 8.8)

—

2.2. Cytotoxic Activity

It was reported that green husks of Juglans mandshurica Maxim had an obvious effect on liver

cancer. HepG-2 is a kind of human liver cancer cells which are often applied to evaluate cytotoxic

activity in vitro [20,21].Therefore, we tested the cytotoxicity of compounds 1–27 against HepG-2 by

the MTT method and compared with references for some compounds [22–24].

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The results were shown that most naphthoquinones in an aglycone form exhibited better cytotoxicity

in vitro than naphthalenyl glucosides with IC₅₀values in the range of 7.33–88.23 μM. None of them

had better IC₅₀values than cisplatin itself, but some naphthoquinone aglycones including juglone (1)

and 3,5-dihydroxy-1,4-naphthoquinone (6) had obvious inhibition effects similar with cisplatin. The

IC₅₀value of juglone was 8.14 ± 1.95, and that of 3,5-dihydroxy-1,4-naphthoquinone was 7.33 ± 0.52

at 24 h of MTT assay, respectively (Table 2). Furthermore, these naphthoquinone aglycones with the

structural features of 2,3-unsaturated moieties showed better and stronger cytotoxicity effects compared

to other tetralones with a partial saturated aryl ring.

Table 2. Cytotoxicities of compound 1–27 from J. mandshurica Maxim on HepG-2 cells lines.

Structural

Features

Structural

Features

Compd.

IC₅₀(μM) ^a

SD ^bCompd.

IC₅₀(μM) ^aSD ^b

1

2

3

4

5

6

7

8

9

10

11

12

13

8.14

68.72

16.11

18.83

15.37

7.33

43.54

22.38

30.42

32.51

34.80

56.87

67.95

1.95

1.50

3.54

2.98

1.63

0.52

0.15

0.66

2.48

0.46

0.33

4.27

3.22

15

16

17

18

19

20

21

22

23

24

25

26

27

NA

83.32

NA

78.61

NA

3.21

-

4.54

-

2.38

-

NA

14

88.23

1.90

PC ^c

metal complex

4.51

0.38

a

IC₅₀, concentration required for inhibiting growth of HepG-2 by 50% (in μM). These results are average

results of three experiments; ^bSD, standard deviation; ^cPC, positive control (cisplatin); NA = not active.

Above results were merely obtained from the distinction of the mother nucleus structure. The different

nature of the substituent in the naphthoquinone also seemed to influence the cytotoxicity activity.

One or two phenolic hydroxyl groups without other substituents, which were introduced to a set of

analogues (compounds 1, 3, 4, 5 and 6), were responsible for the lower IC₅₀value and better inhibition

effect. However, it was worth noting that the position and number of the hydroxyl group had a limited or

negligible effect on HepG-2 inhibitory activities. For example, the IC₅₀value of compound 1 with one

hydroxyl group was similar with compound 6 with two hydroxyl groups; the IC₅₀value of compound 3

which was substituted at the 5, 8-position was similar to compound 5 which was substituted at the

2, 5-position. In addition, the introduction of the methoxy or ethyoxyl group to some naphthoquinones

with 2, 3-unsaturated moieties resulted in a slight decrease in the inhibition effect, including compounds

7, 8, 9 and 10. Among of them, compound 2 had the worst effect on the inhibition of HepG-2 cells.

A majority of naphthoquinone glycosides exhibited no activity against HepG-2 cells. These results

were in accordance with previous reports [24] that the most active compounds were those without the

linkage of saccharide. Some glycosides like compound 17 and 19, of which aglycone was the integrated

Molecules 2015, 20

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conjugation structure assigned to the naphthols, possessed slight cytotoxicity in vitro with IC₅₀values

of 83.3 ± 4.54 and 78.61 ± 2.38, respectively. However, some naphthols substituted with more than

one saccharide or other groups, except for phenolic hydroxyl groups, had no cytotoxic activity against

HepG-2 cells. The results indicated that there were differences in cytotoxic activity between these

naphthoquinone glycosides according to the way of substitution and the type of aglycone.

3. Experimental Section

3.1. General Information

High resolution-electron spray ionization (HR-ESI) mass spectra were obtained on a micromass

1

13

LCT spectrometer. H-, C-NMR, DEPT, HSQC, HMBC were obtained on Bruker DPX 400 NMR

instrument (Bruker, Rheinstetten, Germany). Chemical shifts (δ) are expressed in parts per million

(ppm) using tetramethyl-silane (TMS) as an internal standard. Spin multiplicities are given as s

(singlet), d (doublet), t (triplet), dd (double doublet), and m (multiplet). The UV spectra were recorded on

a Thermo Scientific Evolution 300 UV-visible spectrophotometer (Thermo Fisher Scientific, Waltham,

MA, USA). Optical rotations were recorded using an Anton Paar-MCP 600 polarimeter r (Anton Paar,

Graz, Austria). GC was run on Agilent 7890A Gas Chromatograph System (Agilent Technologies,

Santa Clara, CA, USA). CD spectra were obtained on a Bio-Logic MOS-450 CD spectrometer. The IR

spectra were obtained on a Shimadzu FTIR-8400S spectrometer (Shimadzu, Kyoto, Japan). Melting

points are uncorrected and were obtained on a Hoover capillary melting point. HPLC chromatograms

were obtained with an Agilent Technologies 1260 infinity HPLC system (Agilent Technologies,

Germany) and semi-preparative HPLC (Waters, 515-2414, Milford, MA, USA) was performed using a

Hypersil-ODS II column (300 mm × 20 mm i.d., 10 μm, Ylite, Dalian, China). De-ionized water was

prepared with a Milli-Q system (Milford, MA, USA). HepG-2 cell line obtained from Institute of

Biochemistry and Cell Biology (Shanghai, China) were grown in Dulbecco’s Modified Eagle’s Medium

(DMEM) (Hyclone, NRH0020), supplemented with 5% fetal bovine serum and 1% antibiotic mixture

comprising penicillin-streptomycin, in a humidified atmosphere at 37 °C with 5% CO₂. A multiscan

microplate reader (Thermo Labsystems, Helsinki, Finland) was used for the MTT assays. The solvents

used for open column isolation, such as ethyl acetate, methanol, acetonitrile, and chloroform were

purchased from Merck (Darmstadt, Germany). MTT and Dulbecco’s Modified Dagle’s Medium

(DMEM) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

3.2. Plant Material

The green husks of J. mandshurica were collected in late July from the Changbai Mountains

(Jilin, China), and identified by the professor Zhen-Yue Wang. The dried samples were grounded into

fine powder (60 mesh), and dried thoroughly in an oven at 40 °C for 3 days.

3.3. Extraction and Isolation

The air-dried parts of materials (10.0 kg) were powdered and soaked in 80 L of CHCl₃for 7 days. The

extraction was repeated three times and then concentrated under reduced pressure to afford the CHCl₃

extract (350 g). CHCl₃extract was subjected to silica gel (200–300 mesh) column chromatography

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(CC), eluted with PE:EtOAc (40:1→1:1, v/v), to create twelve fractions (Fr1-Fr12). Fraction 3

(15.50 g) was subjected to silica gel (200–300 mesh) CC, eluted with PE:EtOAc (40:1→1:1, v/v), to

give fractions 3a–3c. Compounds 1 (245.4 mg), 3 (12.1 mg), 4 (33.8 mg), and 8 (11.8 mg), were

isolated from fraction 3a by repeated column chromatography over silica gel, eluted with PE:EtOAc

(40:1→10:1, v/v). Fraction 6 (12.80 g) was subjected to silica gel (200–300 mesh) CC, eluted with

PE:EtOAc (20:1→5:1, v/v) to obtain compounds 5 (15.5 mg), 7 (22.0 mg), 9 (25.3 mg), 10 (15.2 mg),

12 (75.0 mg), and 13 (23.4 mg). Fraction 8 (8.92 g) was subjected to silica gel (200–300 mesh) CC,

eluted with PE:EtOAc (5:1→1:1, v/v) to obtain 2 (5.8 mg), 6 (7.7 mg), 11 (6.8 mg), and 14 (12.8 mg).

The residue of materials were reflux extracted three times with 60 L EtOH (95% v/v), then concentrated

under reduced pressure to afford the EtOH extract (750 g). The EtOH extract was subjected to

Macroporous Resin AB-8 CC, sequentially eluted with H₂O, 30% EtOH, and 95% EtOH. Compounds

15–27 were isolated from 30% EtOH fraction. Next, the isolation procedure of these compounds was

explained. The 30% EtOH elution fraction was evaporated and concentrated to yield a crude residue

(98 g). The residue was further purified by octadecyl silane (ODS) CC with MeOH/H₂O (2:8→1:0) to

give eleven fractions (Fr1-Fr11). Fraction 2 (6.50 g) was fractionated by ODS CC with MeOH/H₂O

(2:8→1:0) to afford a number of subfractions: 2a, 2b, 2c. Subfraction 2c (1.0 g) was subjected to

semi-preparative HPLC chromatography (MeOH/H₂O 35:65, v/v, flow rate 3 mL/min) to yield compounds

15 (5.3 mg, t_R= 21 min), 16 (6.1 mg, t_R= 23 min), and then subjected to semi-preparative HPLC

chromatography (MeOH/H₂O 45:55, v/v, flow rate 3 mL/min) to yield compound 19 (3.2 mg, t_R=

22 min). Fraction 5 (9.40 g) was fractionated twice by ODS CC with MeOH/H₂O (2:8→1:0) to afford

a number of subfractions: 5a, 5b, 5c, 5d and 5e. Subfraction 5b (0.84 g) was purified by semi-preparative

HPLC chromatography (MeOH/H₂O 45:55, v/v, flow rate 3 mL/min) to yield compound 24 (4.7 mg,

t_R= 32 min) and purified by semi-preparative HPLC chromatography (MeOH/H₂O 55:45, v/v, flow

rate 3 mL/min) to obtain compound 17 (4.3 mg, t_R= 25 min). Similarly, Subfraction 5c was purified

by semi-preparative HPLC chromatography (MeOH/H₂O 70:30, v/v, flow rate 3 mL/min, t_R= 25 min)

to yield compounds 18 (4.5 mg, t_R= 13 min), 26 (2.8 mg, t_R= 38 min), 25 (3.1 mg, t_R= 40 min), and 27

(5.9 mg, t_R= 42 min). Fraction 8 (5.70 g) was subjected to silica gel (200–300 mesh) CC, eluted with

CH₂Cl₂:MeOH (5:1→0:1, v/v) to afford compounds 20 (7.5 mg), 21 (5.3 mg), 22 (4.6 mg), 23 (4.4 mg).

3.4. Spectral Data

Juglone (1). Orange needle crystal, ¹H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.99 (2H, d, J = 12.2 Hz, H-2, 3),

7.27 (1H, dd, J = 1.9, 7.7 Hz, H-6), 7.65 (1H, t, J = 7.6 Hz, H-7), 7.63 (1H, dd, J = 7.6, 7.7 Hz, H-8),

13

11.90 (1H, s, 5-OH). C-NMR (CDCl₃, 100 MHz) δ (ppm): 190.3 (C-1), 138.7 (C-2), 139.6 (C-3),

184.3 (C-4), 161.5 (C-5), 124.5 (C-6), 136.6 (C-7), 119.2 (C-8), 115.1 (C-9), 131.8 (C-10).

5-Methoxy-1,4-naphthoquinone (2). Light yellow powder, ¹H-NMR (CDCl₃, 400 MHz) δ: 7.58 (1H, d,

J = 7.6 Hz, H-2), 7.48 (1H, d, J = 7.6 Hz, H-3), 6.73 (1H, br.d, J = 7.7 Hz, H-6), 7.36 (1H, t,

J = 7.7 Hz, H-7), 7.42 (1H, br.d, J = 7.7 Hz, H-8), 3.83 (3H, s, 5-OCH₃), 11.82 (1H, s, 5-OH).

¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 190.4 (C-1), 138.3 (C-2), 139.3 (C-3), 186.3 (C-4), 161.2 (C-5),

119.2 (C-6), 124.3 (C-7), 136.1 (C-8), 131.4 (C-9), 114.0 (C-10), 55.4 (5-OCH₃).

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1

5,8-Dihydroxy-1,4-naphthoquinone (3). Light yellow powder, H-NMR (CDCl₃, 400 MHz) δ (ppm):

7.13 (4H, s, H-2, 3, 6, 7). ¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 173.1 (C-1, 4, 5, 8), 134. 4 (C-2, 3, 6, 7),

112. 0 (C-9, 10).

2-Hydroxy-1, 4-naphthoquinone (4). Light yellow powder, ¹H-NMR (CDCl₃, 400 MHz) δ (ppm): 7.38

(1H, s, H-3), 8.13 (2H, d, J = 7.3 Hz, H-5, 8), 7.82 (1H, m, H-6), 7.74 (1H, m, H-7). ¹³C-NMR (CDCl₃,

100 MHz) δ (ppm): 182.2 (C-1), 156.5 (C-2), 110.7 (C-3), 185.0 (C-4), 135.3 (C-5), 126.8 (C-6), 126.7

(C-7), 133.4 (C-8), 130.0 (C-9), 129.5 (C-10).

2,5-Dihydroxy-1, 4-naphthoquinone (5). Light yellow powder, ¹H-NMR (CDCl_3,400 MHz) δ (ppm): 6.12

(1H, s, H-3), 7.23 (1H, d, J = 8.2 Hz, H-6), 7.57 (1H, m, H-7), 7.40 (1H, d, J = 7.6 Hz, H-8), 12.10 (1H,

13

s, 5-OH). C-NMR (CDCl₃, 100 MHz) δ (ppm): 180.3 (C-1), 160.2 (C-2), 110.4 (C-3), 191.7 (C-4),

159.3 (C-5), 124.2 (C-6), 135.3 (C-7), 118.2 (C-8), 130.3 (C-9), 113.7 (C-10).

1

3,5-Dihydroxy-1, 4-naphthoquinone (6). Light yellow powder, H-NMR (CDCl₃, 400 MHz) δ (ppm):

6.14 (1H, s, H-2), 7.26 (1H, d, J = 7.8 Hz, H-6), 7.66 (1H, t, J = 7.8 Hz, H-7), 7. 43 (1H, d, J = 7.8 Hz,

13

H-8), 12.02 (1H, s, 5-OH). C-NMR (CDCl₃, 100 MHz) δ (ppm): 183.0 (C-1), 160.0 (C-2), 111.4

(C-3), 185.3 (C-4), 160.3 (C-5), 122.2 (C-6), 136.0 (C-7), 117.0 (C-8), 132.5 (C-9), 114.2 (C-10).

3-Methoxy juglone (7). Orange powder, ¹H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.12 (1H, s, H-2), 7.27

(1H, dd, J = 7.5, 1.2 Hz, H-6), 7.62 (1H, t, J = 7.5 Hz, H-7), 7.67 (1H, d, J = 7.5, 1.2 Hz, H-8), 3.90

(3H, s, OCH₃), 11.75 (1H, s, OH-5). ¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 184.9 (C-1), 110.5 (C-2),

160.6 (C-3), 183.9 (C-4), 162.0 (C-5), 123.9 (C-6), 138.2 (C-7), 118.9 (C-8), 132.1 (C-9), 114.3 (C-10),

56.6 (3-OCH₃).

1

2-Methoxy juglone (8). Orange-red needle crystal, H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.11 (1H, s,

H-3), 7.28 (1H, dd, J = 8, 1.2 Hz, H-6), 7.58 (1H, t, J = 8 Hz, H-7), 7.67 (1H, dd, J = 8, 1.2 Hz, H-8),

3.93 (3H, s, 2-OCH3), 12.22 (1H, s, 5-OH). ¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 179.3 (C-1), 161.1

(C-2), 109.5 (C-3), 190.8 (C-4), 161.1 (C-5), 125.2 (C-6), 135.4 (C-7), 119.5 (C-8), 131.1 (C-9), 114.2

(C-10), 56.6 (2-OCH₃).

3-Ethoxy juglone (9). Orange powder, ¹H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.13 (H, s, H-2), 7.24 (1H,

dd, J = 7.5, 1.2 Hz, H-6), 7.62 (1H, t, J = 7.5 Hz, H-7), 7.62 (1H, dd, J = 7.5, 1.2 Hz, H-8), 4.11 (3H, q,

J = 6.9 Hz, H-1a), 1.60 (2H, t, J = 6.9 Hz, H-2a), 11.78 (1H, s, OH-5). ¹³C-NMR (CDCl₃, 100 MHz) δ

(ppm): 185.1 (C-1), 110.8 (C-2), 159.3 (C-3), 184.1 (C-4), 161.9 (C-5), 123.8 (C-6), 118.8 (C-7),

137.1 (C-8), 132.0 (C-9), 114.3 (C-10), 65.6 (C-1a), 13.9 (C-2a).

1

2-Ethoxy juglone (10). Light yellow flaky crystal, H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.08 (1H, s,

H-3), 7.67 (1H, dd, J = 7.5, 1.2 Hz, H-6), 7.59 (1H, t, J = 7.5 Hz, H-7), 7.26 (1H, dd, J = 7.5, 1.2 Hz,

13

H-8), 4.12 (2H, q, J = 7.2 Hz, -OCH₂-), 1.53 (3H, t, J = 7.2 Hz, -CH₃), C-NMR (CDCl₃, 100 MHz)

δ (ppm): 190.0 (C-1), 160.1 (C-2), 109.3 (C-3), 179.2 (C-4), 161.0 (C-5), 114.6 (C-6), 135.3 (C-7),

125.0 (C-8), 131.1 (C-9), 119.0 (C-10), 65.5 (C-1′), 13.8 (C-2′).

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1

Engelharquinone (11). Yellow needle crystal, H-NMR (CDCl₃, 400 MHz) δ (ppm): 6.92 (1H, d,

J = 8.4 Hz, H-2), 7.48 (1H, dd, J = 7.4, 8.4 Hz, H-3), 7.14 (1H, d, J = 7.4 Hz, H-4), 7.25 (1H, dd,

J = 7.6, 1.9 Hz, H-8), 7.59 (1H, dd, J = 7.6, 7.4 Hz, H-9), 7.65 (1H, dd, J = 7.6, 1.9 Hz, H-10), 4.23

(1H, d, J = 3.1 Hz, H-12), 3.08 (1H, dd, J = 3.8, 10.6 Hz, H-14a), 3.04 (1H, brd, J = 3.8, 10.6 Hz,

H-14b), 11.50 (1H, s, 1-OH), 4.80 (1H, s, 5-OH), 11.52 (1H, s, 7-OH). ¹³C-NMR (CDCl₃, 100 MHz) δ

(ppm): 198.1 (C-13), 188.8 (C-6), 180.5 (C-11), 163.5 (C-1), 162.1 (C-7), 154.4 (C-5a), 148.7 (C-11a),

146.3 (C-4a), 137.3 (C-9), 136.8 (C-3), 132.6 (C-11a), 125.2 (C-8), 120.4 (C-10), 119.4 (C-2), 115.3

(C-6a), 110.5 (C-13a), 82.0 (C-5), 54.1 (C-14), 52.6 (C-12).

1

(S)-Regiolone (12). White powder, H-NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.74 (2H, m, H-2), 2.20

(1H, m, H-3a), 2.00 (1H, m, H-3b), 4.76 (1H, m, H-4), 7.06 (1H, d, J = 8.0 Hz, H-5), 7.53 (1H, t,

J = 8.0, 8.2 Hz, H-6), 6.83 (1H, d, J = 8.2 Hz, H-7), 5.60 (H, s, OH-4), 12.42 (1H, s, OH-8).

¹³C-NMR (DMSO-d₆, 100 MHz) δ (ppm): 205.5 (C-1), 35.5 (C-2), 31.8 (C-3), 66.6 (C-4), 117.8 (C-5),

137.0 (C-6), 116.1 (C-7), 162.0 (C-8), 149.4 (C-9), 115.3 (C-10).

1

(4S)-4-Hydroxy-α-tetralone (13). Claybank oil substance, H-NMR (CDCl₃, 400 MHz) δ (ppm): 2.53

(1H, ddd, J = 17.8, 9.6, 4.8 Hz, H-2a), 2.86 (1H, ddd, J = 17.8, 7.5, 4.6 Hz, H-2), 2.15 (1H, m, H-3a),

2.37 (1H, m, H-3b), 4.95 (1H, dd, J = 8.1, 3.9 Hz, H-4), 7.52 (1H, m, H-5, 6), 7.38 (1H, m, H-7), 7.98

(1H, brd, J = 7.8 Hz, H-8). ¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 197.8 (C-1), 35.2 (C-2), 32.3 (C-3),

67.7 (C-4), 126.9 (C-5), 134.2 (C-6), 127.3 (C-7), 128.5 (C-8), 130.8 (C-9), 145.5 (C-10).

(4S)-5-Hydroxy-4-methoxy-α-tetralone (14). White amorphous powder, ¹H-NMR (CDCl₃, 400 MHz) δ

(ppm): 2.63 (1H, m, H-2), 2.87 (1H, m, H-2), 2.11 (1H, m, 3a), 2.24 (1H, m, H-3b), 4.98 (1H, m, H-4),

7.07 (1H, dd, J = 8.0, 1.0 Hz, H-6), 7.58 (1H, dd, J = 8.0, 1.0 Hz, H-7), 7.33 (1H, t, J = 8.0 Hz, H-8),

13

8.55 (1H, s, 5-OH), 3.57 (3H, s, 4-OCH₃). C-NMR (CDCl₃, 100 MHz) δ (ppm): 196.2 (C-1), 35.6

(C-2), 27.3 (C-3), 80.2 (C-4), 156.3 (C-5), 122.32 (C-6), 129.6 (C-7), 119.3 (C-8), 132.5 (C-9), 126.7

(C-10), 55.3 (C-11).

1

1,4,5-Trihydroxynaphthalene-1,4-di-O-β-D-glucopyranoside (15). Yellow amorphous powder, H-NMR

(CD₃OD, 400 MHz) δ (ppm): 7.16 (1H, d, J = 8.8 Hz, H-2), 7.30 (1H, d, J = 8.8 Hz, H-3), 6.85 (1H, dd,

J = 7.6, 1.2 Hz, H-6), 7.34 (1H, dd, J = 8.4, 7.6 Hz, H-7), 7.89 (1H, dd, J = 8.4, 1.2 Hz, H-8), 5.04 (1H,

d, J = 7.7 Hz, H-1′), 3.63 (1H, dd, J = 9.0, 7.7 Hz, H-2′), 3.53 (1H, dd, J = 9.0, 8.8 Hz, H-3′), 3.45 (1H,

m, H-4′), 3.44 (1H, m, H-5′), 3.73 (1H, dd, J = 11.8, 5.8 Hz, H-6′a), 3.89 (1H, br.d, J = 11.8 Hz, H-6′b),

5.06 (1H, d, J = 7.8 Hz, H-1″), 3.58 (1H, dd, J = 9.0, 7.8 Hz, H-2″), 3.50 (1H, dd, J = 9.0, 8.5 Hz,

H-3″), 3.43 (1H, m, H-4″), 3.46 (1H, m, H-5″), 3.74 (1H, dd, J = 12.4, 5.2 Hz, H-6″a), 3.94 (1H, dd,

J = 12.4, 1.8 Hz, H-6″b). ¹³C-NMR (CDCl₃, 100 MHz) δ (ppm): 150.9 (C-1), 111.3 (C-2), 112.2 (C-3),

151.0 (C-4), 155.1 (C-5), 112.5 (C-6), 128.3 (C-7), 114.8 (C-8), 130.4 (C-9), 117.5 (C-10), 103.0 (C-1′),

75.0 (C-2′), 78.5 (C-3′), 71.4 (C-4′), 78.5 (C-5′), 62.4 (C-6′), 105.4 (C-1″), 75.0 (C-2″), 78.4 (C-3″),

71.4 (C-4″), 78.9 (C-5″), 62.5 (C-6″).

1

1,4,5-Trihydroxynaphthalene-1,5-di-O-β-D-glucopyranoside (16). Yellow amorphous powder, H-NMR

(CD₃OD, 400 MHz) δ (ppm): 7.20 (1H, d, J = 8.2 Hz, H-2), 6.72 (1H, d, J = 8.2 Hz, H-3), 7.41 (1H, dd,

J = 7.8, 1.6 Hz, H-6), 7.36 (1H, dd, J = 8.0, 7.8 Hz, H-7), 8.13 (1H, dd, J = 8.0, 1.6 Hz, H-8), 4.92 (1H,

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d, J = 7.8 Hz, H-1′), 3.60 (1H, dd, J = 8.7, 7.9 Hz, H-2′), 3.49 (1H, m, H-3′), 3.45 (1H, m, H-4′), 3.42

(1H, m, H-5′), 3.70 (1H, dd, J = 12.2, 5.1 Hz, H-6′a), 3.88 (1H, dd, J = 12.2, 2.0 Hz, H-6′b), 5.11 (1H,

d, J = 7.9 Hz, H-1″), 3.58 (1H, dd, J = 9.2, 7.9 Hz, 2″), 3.50 (1H, m, H-3″), 3.43 (1H, m H-4″), 3.52

(1H, m, H-5″), 3.76 (1H, dd, J = 12.2, 5.8 Hz, H-6″a), 3.97 (1H, dd, J = 12.2, 2.1 Hz, H-6″b). ¹³C-NMR

(CDCl₃, 100 MHz) δ (ppm): 147.4 (C-1), 114.0 (C-2), 110.9 (C-3), 150.6 (C-4), 155.8 (C-5), 112.7 (C-6),

126.6 (C-7), 119.4 (C-8), 130.2 (C-9), 117.3 (C-10), 104.0 (C-1′), 75.2 (C-2′), 78.2 (C-3′), 71.5 (C-4′),

78.3 (C-5′), 62.4 (C-6′), 104.6 (C-1″), 75.2 (C-2″), 78.3 (C-3″), 71.5 (C-4″), 78.8 (C-5″), 62.4 (C-6″).

1

1,4,8-Trihydroxynaphthalene-1-O-β-D-glucopyranoside (17). Yellow amorphous powder, H-NMR

(CD₃OD, 400 MHz) δ (ppm): 6.70 (1H, d, J = 8.6 Hz, H-2), 7.22 (1H, d, J = 8.6 Hz, H-3), 6.81 (1H, dd,

J = 7.7, 1.1 Hz, H-6), 7.26 (1H, dd, J = 8.8, 7.7 Hz, H-7), 7.64 (1H, dd, J = 8.8, 1.1 Hz, H-8), 4.98 (1H,

d, J = 7.8 Hz, H-1′), 3.53 (1H, dd, J = 9.0, 7.8 Hz, H-2′), 3.49 (1H, m, H-3′), 3.42 (1H, m, H-4′), 3.49

13

(1H, m, H-5′), 3.74 (1H, dd, J = 12.0, 5.8 Hz, H-6′), 3.94 (1H, dd, J = 12.0, 2.1 Hz, H-6′). C-NMR

(CDCl₃, 100 MHz) δ (ppm): 150.8 (C-1), 108.5 (C-2), 113.2 (C-3), 148.8 (C-4), 154.9 (C-5), 112.6 (C-6),

127.1 (C-7), 114.7 (C-8), 129.2 (C-9), 117.9 (C-10), 105.2 (C-1′), 75.2 (C-2′), 78.3 (C-3′), 71.6 (C-4′),

78.8 (C-5′), 62.8 (C-6′).

1,4,8-Trihydroxy-3-naphthalenecarboxylic acid-1-O-β-D-glucopyranoside ethyl ester (18). red powder,

mp 136-138 °C;[α]²_D⁵−12.3 (c 0.50, MeOH); UVλ_max= 243 nm; IR (KBr)ν_max3401, 2956, 1646, 1506 cm⁻¹;

13

¹H-NMR and C-NMR data see Table 1; HR-ESI-MS (positive): m/z 433.1375 [M + Na]⁺(calcd for

C₁₉H₂₂NaO₁₀, 433.1381).

1,4,8-Trihydroxynaphthalene-1-O-β-D-[6'-O-(3'',4'',5''-trihydroxybenzoyl)] glucopyranoside (19). Yellow

amorphous powder, ¹H-NMR (CD₃OD, 400 MHz) δ (ppm): 6.50 (1H, d, J = 8.4 Hz, H-2), 7.18 (1H, d,

J = 8.4 Hz, H-3), 7.68 (1H, dd, J = 8.0, 3.1 Hz, H-5), 7.23 (1H, dd, J = 8.0, 7.8 Hz, H-6), 6.80 (1H, dd,

J = 8.2, 3.1 Hz, H-7), 4.96 (1H, d, J = 7.8 Hz, H-1′), 3.58 (1H, m, H-2′), 3.53 (1H, m, H-3′), 3.29 (1H,

m, H-4′), 3.84 (1H, m, H-5′), 4.53 (1H, dd, J = 12.0, 6.9 Hz, H-6′a), 4.66 (1H, dd, J = 12.0, 2.1 Hz,

13

H-6′b), 7.18 (2H, s, H-2″, H-6″). C-NMR (CDCl₃, 100 MHz) δ (ppm): 148.8 (C-1), 108.6 (C-2),

113.3 (C-3), 150.7 (C-4), 114.7 (C-5), 127.1 (C-6), 112.2 (C-7), 154.8 (C-8), 117.9 (C-9), 128.9 (C-10),

105.4 (C-1′), 78.2 (C-2′), 75.1 (C-3′), 72.0 (C-4′), 76.2 (C-5′), 65.1 (C-6′), 168.6 (C-7″), 121.6 (C-1″),

110.4 (C-2″, C-6″), 146.7 (C-3″, C-5″), 140.3 (C-4″).

(4S)-4-Hydroxy-α-tetralone-4-O-β-D-glucopyranoside (20). White amorphous powder, ¹H-NMR (CD₃OD,

400 MHz) δ (ppm): 3.05 (1H, ddd, J = 17.8, 9.6, 4.8 Hz, H-2a), 2.62 (1H, ddd, J = 17.8, 6.8, 4.8 Hz,

H-2b), 2.45 (1H, dddd, J = 13.2, 9.6, 4.8, 3.4 Hz, H-3a), 2.38 (1H, dddd, J = 13.2, 6.8, 6.2, 4.8 Hz,

H-3b), 5.10 (1H, dd, J = 6.2, 3.4 Hz, H-4), 7.71 (1H, dd, J = 7.8, 1.6 Hz, H-5), 7.63 (1H, td, J = 7.8,

1.6 Hz, H-6), 7.47 (1H, td, J = 7.8, 1.6 Hz, H-7), 7.97 (1H, dd, J = 7.8, 1.6 Hz, H-8), 4.38 (1H, d,

J = 7.8 Hz, H-1′), 3.24 (1H, dd, J = 8.5, 7.8 Hz, H-2′), 3.32 (1H, m, H-3′), 3.31 (1H, m, H-4′), 3.32 (1H,

13

m, H-5′), 3.71 (1H, dd, J = 11.8, 5.3 Hz, H-6′a), 3.95 (1H, dd, J = 11.8, 1.0 Hz, H-6′b). C-NMR

(CD₃OD, 100 MHz) δ (ppm): 200.2 (C-1), 35.6 (C-2), 31.6 (C-3), 75.1 (C-4), 130.4 (C-5), 135.1 (C-6),

129.9 (C-7), 128.2 (C-8), 133.1 (C-9), 144.1 (C-10), 103.3 (C-1′), 75.4 (C-2′), 78.2 (C-3′), 71.9 (C-4′),

78.1 (C-5′), 63.0 (C-6′).

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1

(4S)-4,5-Dihydroxy-α-tetralone-4-O-β-D-glucopyranoside (21). White amorphous powder, H-NMR

(CD₃OD, 400 MHz) δ (ppm): 3.12 (1H, ddd, J = 17.6, 14.0, 5.1 Hz, H-2a), 2.49 (1H, dt, J = 17.6,

3.3 Hz, H-2b), 2.19 (1H, tt, J = 14.0, 3.3 Hz, H-3a), 2.56 (1H, ddt, J = 14.0, 5.1, 3.3 Hz, H-3b), 5.41

(1H, t, J = 3.3 Hz, H-4), 7.12 (1H, dd, J = 7.9, 1.1 Hz, H-6), 7.30 (1H, t, J = 7.9 Hz, H-7), 7.48 (1H,

dd, J = 7.9, 1.1 Hz, H-8), 4.62 (1H, d, J = 7.8 Hz, H-1′), 3.19 (1H, dd, J = 8.7, 7.8 Hz, H-2′), 3.35 (1H,

m, H-3′), 3.36 (1H, m, H-4′), 3.36 (1H, m, H-5′), 3.75 (1H, dd, J = 12.2, 4.8 Hz, H-6′a), 3.90 (1H, dd,

J = 12.2, 1.8 Hz, H-6′b). ¹³C-NMR (CD₃OD, 100 MHz) δ (ppm): 201.2 (C-1), 34.0 (C-2), 30.3 (C-3),

70.1 (C-4), 156.8 (C-5), 122.3 (C-6), 130.7 (C-7), 119.2 (C-8), 134.7 (C-9), 130.0 (C-10), 104.1 (C-1′),

75. 6 (C-2′), 78.1 (C-3′), 71.6 (C-4′), 78.1 (C-5′), 62.9 (C-6′).

1

(4S)-4,6-Dihydroxy-α-tetralone-4-O-β-D-glucopyranoside (22). white amorphous powder, H-NMR

(CD₃OD, 400 MHz) δ (ppm): 2.97 (1H, ddd, J = 17.7, 9.7, 5.1 Hz, H-2a), 2.52 (1H, ddd, J = 17.7, 6.4,

4.8 Hz, H-2b), 2.38 (1H, dddd, J = 13.6, 9.6, 4.8, 3.5 Hz, H-3a), 2.30 (1H, dddd, J = 13.6, 6.4, 6.2,

5.1 Hz, H-3b), 5.02 (1H, dd, J = 6.2, 3.5 Hz, H-4), 7.04 (1H, d, J = 2.2 Hz, H-5), 6.85 (1H, dd, J = 8.4,

2.2 Hz, H-7), 7.88 (1H, d, J = 8.4 Hz, H-8), 4.38 (1H, d, J = 7.8 Hz, H-1′), 3.26 (1H, dd, J = 8.7,

7.8 Hz, H-2′), 3.33 (1H, t, J = 8.7 Hz, H-3′), 3.30 (1H, m, H-4′), 3.28 (1H, m, H-5′), 3.70 (1H, dd,

J = 11.9, 5.8 Hz, H-6′a), 3.93 (1H, dd, J = 11.9, 1.6 Hz, H-6′b). ¹³C-NMR (CD₃OD, 100 MHz) δ (ppm):

199.6 (C-1), 35.3 (C-2), 31.5 (C-3), 75.1 (C-4), 116.0 (C-5), 164.4 (C-6), 117.0 (C-7), 131.1 (C-8),

125.5 (C-9), 146.4 (C-10), 103.0 (C-1′), 75.3 (C-2′), 78.2 (C-3′), 71.9 (C-4′), 78.1 (C-5′), 63.2 (C-6′).

(4S)-4,5,8-Trihydroxy-α-tetralone-4-O-β-D-glucopyranoside (23). White amorphous powder, ¹H-NMR

(CD₃OD, 400 MHz) δ (ppm): 3.22 (1H, ddd, J = 18.0, 13.8, 4.8 Hz, H-2a), 2.52 (1H, dt, J = 18.0,

3.8 Hz, H-2b), 2.17 (1H, tdd, J = 13.8, 3.8, 3.0 Hz, H-3a), 2.48 (1H, m, H-3b), 5.40 (1H, t, J = 3.0 Hz,

H-4), 7.11 (1H, d, J = 9.1 Hz, H-6), 6.81 (1H, d, J = 9.1 Hz, H-7), 4.52 (1H, d, J = 7.8 Hz, H-1′), 3.17

(1H, dd, J = 8.9, 7.8 Hz, H-2′), 3.32 (1H, m, H-3′), 3.32 (1H, m, H-4′), 3.32 (1H, m, H-5′), 3.72 (1H,

13

dd, J = 12.1, 4.8 Hz, H-6′a), 3.91 (1H, dd, J = 12.1, 1.8 Hz, H-6′b). C-NMR (CD₃OD, 100 MHz) δ

(ppm): 207.2 (C-1), 34.3 (C-2), 29.9 (C-3), 69.3 (C-4), 148.6 (C-5), 127.0 (C-6), 119.6 (C-7), 157.3 (C-8),

116.9 (C-9), 127.5 (C-10), 103.4 (C-1′), 75.4 (C-2′), 78.2 (C-3′), 71.7 (C-4′), 78.1 (C-5′), 62.8 (C-6′).

(4S)-4,5,8-Trihydroxy-α-tetralone-5-O-β-D-[6'-O-(3",4",5"-trihydroxybenzoyl)] glucopyranoside (24).

Light yellow amorphous powder, ¹H-NMR (CD₃OD, 400 MHz) δ (ppm): 2.49 (1H, dd, J = 17.6, 3.5 Hz,

H-2a), 3.03 (1H, ddd, J = 17.6, 11.8, 6.5 Hz, H-2b), 2.15 (1H, m, H-3), 5.34 (1H, t, J = 3.2 Hz, H-4),

7.42 (1H, d, J = 9.2 Hz, H-6), 6.76 (1H, d, J = 9.2 Hz, H-7), 4.78 (1H, d, J = 7.7 Hz, H-1′), 3.55 (1H, t,

J = 8.0 Hz, H-2′), 3.51 (1H, m, H-3′), 3.45 (1H, m, H-4′), 3.68 (1H, td, J = 7.8, 1.9 Hz, H-5′), 4.45 (1H,

dd, J = 11.8, 6.8 Hz, H-6′a), 4.55 (1H, dd, J = 11.8, 2.1 Hz, H-6′b), 7.07 (2H, s, H-2″, H-6″). ¹³C-NMR

(CD₃OD, 100 MHz) δ (ppm): 206.2 (C-1), 33.5 (C-2), 30.3 (C-3), 61.1 (C-4), 148.6 (C-5), 129.1 (C-6),

119.4 (C-7), 159.5 (C-8), 116.1 (C-9), 135.1 (C-10), 104.5 (C-1′), 75.3 (C-2′), 77.9 (C-3′), 71.7 (C-4′),

75.8 (C-5′), 64.6 (C-6′), 121.0 (C-1″), 110.4 (C-2″), 146.9 (C-3″), 140.4 (C-4″), 146.9 (C-5″), 110.3

(C-6″), 168.3 (C-7″).

(4S)-4-Hydroxy-α-tetralone-4-O-β-D-(6'-O-4''-hydroxybenzoyl) glucopyranoside (25). yellow powder,

mp 128–132 °C; [α]²_D⁵−28.2 (c 0.50, MeOH); UVλ_max= 262 nm; CD (MeOH) λ_max(∆ε): 236 nm (−12.6),

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258 nm (+1.23). IR (KBr)ν_max3396, 1728, 1260, 1180 cm⁻¹; ¹H-NMR and ¹³C-NMR data see Table 1;

HR-ESI-MS (positive): m/z 445.0262 [M + H]⁺(calcd for C₂₃H₂₅O₉, 445.0267).

(4S)-4,5-Dihydroxy-α-tetralone-4-O-β-D-(6'-O-4''-hydroxybenzoyl) glucopyranoside (26). yellow powder,

mp 132–135 °C; [α]²_D⁵−30.2 (c 0.52, MeOH); UVλ_max= 274 nm; CD (MeOH) λ_max(∆ε): 234 nm (−8.60),

1

13

261 nm (+2.21). IR (KBr)ν_max3420, 2960, 1726, 1255, 1135 cm⁻¹; H-NMR and C-NMR data see

Table 1; HR-ESI-MS (positive): m/z 483.4276 [M + Na]⁺(calcd for C₂₃H₂₄NaO₁₀, 483.4279).

(4S)-4,5,8-Thihydroxy-α-tetralone-5-O-β-D-(6'-O-4''-hydroxybenzoyl) glucopyranoside (27). yellow

powder, mp 145–148 °C; [α]_D²⁵−31.4 (c 0.47, MeOH); UVλ_max= 264 nm; CD (MeOH) λ_max(∆ε):

241 nm (−9.46), 266 nm (+0.93), 271 nm (−0.55), 292 nm (+0.35). IR (KBr)ν_max3400, 2994, 1725,

1056 cm⁻¹; HR-ESI-MS (positive): m/z 477.1058 [M + H]⁺(calcd for C₂₃H₂₅O₁₁, 477.1054).

3.5. Acid Hydrolysis and Sugar Analysis

Compounds 18, 25–27 (1.5 mg) were refluxed with 1.0 mol/L HCl (5 mL, dioxane/H₂O, v/v) for 7 h.

After filtration, the acid aqueous layer was neutralized with 5% NaOH and desalted with Sephadex

LH-20 to obtain the sugar residue (0.8 mg). The residues were dissolved in pyridine (5 mL) and

1-(trimetrylsilyl)-imidazole (0.5 mL) at 60 °C for 10 min. The reaction mixtures were dried with a

stream of N₂, the residues were partitioned between CHCl₂and H₂O. The organic layers were analyzed

by GC using an L-Chirasil-Val column (0.32 mm × 25 m). Temperatures of injector and detector were

maintained at 200 °C. A temperature gradient system was used for the oven; the initial temperature

remained at 100 °C for 1 min and then was raised to 180 °C at the rate of 5 °C/min. Peaks of the

hydrolysate of 18 and 25–27 were respectively detected at 14.72 min, 14.72 min, 14.73 min, 14.72 min.

The final result was to compare the retention time of authentic sample of D-glucose (SigmaAldrich,

St. Louis, MO, USA) treated in the same manner with 1-(trimetrylsilyl)-imidazole in pyridine which

was detected at 14.72 min. Thus, it was concluded that all the sugar moieties of 18 and 25–27 are D-glucose.

3.6. Cytotoxicity Assays

3.6.1. Cell Culture

The cytotoxicity of compounds 1–27 was performed against human liver carcinoma cells (HepG-2)

by MTT assay [25,26]. HepG-2 cell line was maintained in DMEM supplemented with 10% fetal

bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco-BRL). The cells

were incubated in 5% CO₂humidified at 37 °C for growth.

3.6.2. Measurement of Cell Proliferation by MTT Assay

HepG-2 cells in logarithmic growth phase were seeded in a 96-well microtiter plates and kept

overnight for attachment. Twenty-seven compounds and positive control (cisplatin), dissolved in

dimethyl sulfoxide, were diluted to various concentrations with Dulbecco’s Modified Eagle Medium

(DMEM) from 200 to 0.5 μM for 24 h. The optical density (OD) was measured at 570 nm using a

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multiscan microplate reader. All experiments were performed in triplicate. Data were expressed as the

concentration required for inhibiting growth of HepG-2 by 50% (IC₅₀).

4. Conclusions

Twenty-seven naphthoquinones and their derivatives, including four new naphthalenyl glucosides

and twenty-three known compounds, have been isolated with the aim of exploring the relationship

between cytotoxicity and structures. The results indicated that in naphthoquinones with 2,3-unsaturated

moieties, the position of the substituents was at the aryl ring portion or the quinone ring portion of

naphthoquinone played an important role in the cytotoxic activity. Moreover, the type of substituents

also had an effect on the activity. And in general, when these compounds were substituted with the

phenolic hydroxyl group, they had stronger activity against the HepG-2 cells. Napthoquinone

glycosides had no activity or weaker activity. So far, we are not able to definitely confirm that the type

of saccharide is an essential factor for cytotoxic activity, since compounds obtained are all substituted

with glucose. These results will provide experimental bases for further structural modifications to yield

better active derivatives.

Acknowledgments

This work was kindly supported by the National Science Foundation (grant NO.81202890), China

Postdoctoral Science Foundation funded project (2013M530164/2014T70374), Heilongjiang Postdoctoral

Fund (LBH-Z13195), Harbin city youth science and technology innovation talents (2013RFQXJ052).

Author Contributions

Yuanyuan Zhou, Bingyou Yang and Haixue Kuang came up with the conception of this experiment.

Yuanyuan Zhou and Zhaoxi Liu wrote the manuscript. Yuxin Liu and Yanqiu Jiang carried out the

experiments and analyzed the spectral data. Xiaoli Wang conducted the cytotoxicity assays against

HepG-2 and deduced the preliminary structure-activity relationships for these naphthoquinones from

green walnut husks of Juglans mandshurica Maxim. All authors had read and approved the final

manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–27 are available from the authors.

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/4.0/).

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Studies on cytotoxic activity against HepG-2 cells of naphthoquinones from green walnut husks of Juglans mandshurica maxim

DOI: 10.3390/molecules200915572

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