Studies on Cytotoxic Activity against HepG-2 Cells of Naphthoquinones from Green Walnut Husks of Juglans mandshurica Maxim

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 IC50 values in the range of 7.33–88.23 μM. Meanwhile, preliminary structure-activity relationships for these compounds were discussed.


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][3][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][6][7][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][10][11].

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 including HR-ESI-MS, IR, 1 H-NMR, 13 C-NMR, DEPT, HSQC, HMBC, and CD. Their structures, 1 H-and 13 C-NMR data, and HMBC correlations are shown in Figures 1 and 2   Compound 18 was a red amorphous powder. The molecular formula C19H22O10 was determined from HR-ESI-MS and 13 C-NMR data. There were two major differences between 18 and 25-27: two methylene groups located at C-2 and C-3, respectively, at δC 33.0-35.0 and 30.0-31.5 in compounds 25-27 were replaced by methenyl groups at δC 109.9 and 105.8 in compound 18, indicating no presence of a hydrogenated position. Furthermore, the independent existence of the glucopyranosyl moiety was not together with p-hydroxybenzoly on the basis of 1D-, 2D-NMR data. Noise-decoupled 13 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 δC 171.8 correlated with one ethyl group, which was assigned to acetyl group. In the 1 H-NMR spectrum, there were ABC-spin aromatic proton signals at δH 6.99 (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 δH 7.72 and one double-peak signal due to an anomeric proton at δH 4.99 were distinct. In the HMBC spectrum of 18 (Figure 2), the correlation peak between the anomeric proton and C-1 at δC 148.0 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-3naphthalenecarboxylic acid 1-O-β-D-glucopyranoside ethyl ester. Compound 25 was obtained as a yellow amorphous powder and the molecular formula was assigned as C23H24O9 from its HR-ESI-MS and 13 C-NMR data. 1 H-NMR and 13 C-NMR spectra revealed that 25 contained a typical β-D-glucopyranosyl (δH 4.42 (d, J = 7. 6 Hz, H-1′); δC 103.7, 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 1 H-and 13 C-NMR spectral assignment was carried out utilizing a combination of DEPT, HSQC, HMBC, and CD experiments. To be specific, the 1 H-NMR spectrum of 25 showed two methylenes of tetralone at 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 δC 75.9 (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 JH1,H2 value (7. 6 Hz). At the same time, it was also observed that the δH 4.66 (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 δH 7.95 (d, J = 8. 8 Hz, H-2′′, 6′′), 6.84 (d, J = 8. 8 Hz, H-3′′, 5′′), and δC132.9 (C-2′′, 6′′), 116.3 (C-3′′, 5′′) in the 1 H-and 13 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 Compound 26, a yellow amorphous powder, was assigned as C23H24O10 on the basis of its HR-ESI-MS and 13 C-NMR data. The 1D-and 2D-NMR spectrographic data were similar as compound 25 except for the aryl ring moiety of the tetralone. The 1 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 δH 5.37 (H-4) and δC 157.0 (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- 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 by 1 H-, 13 C-NMR, and the DEPT spectrum (δH 4.81 (d, J = 7.5 Hz, H-1′); δC 104.4, 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 δC 148.3 (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 1 H-NMR spectrum showed the AB-type aromatic proton signals at δH 7.40 (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 δC 159.3 due to the C-8 position in DEPT spectrum. It was also worth noting that the carbon signal at δC 116.2 (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-

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][23][24].
The results were shown that most naphthoquinones in an aglycone form exhibited better cytotoxicity in vitro than naphthalenyl glucosides with IC50 values in the range of 7.33-88.23 μM. None of them had better IC50 values 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 IC50 value 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. 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 IC50 value 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 IC50 value of compound 1 with one hydroxyl group was similar with compound 6 with two hydroxyl groups; the IC50 value 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 conjugation structure assigned to the naphthols, possessed slight cytotoxicity in vitro with IC50 values 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.

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.
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          (18  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).

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.