Two new cytotoxic glycosides isolated from the green walnut husks of Juglans mandshurica Maxim.

Abstract Two new glycosides including an alcohol glycoside and a phenolic glycoside: hexyl-1-O-α-d-arabinofuranosyl-(1 → 6)-β-d-glucopyranoside (1), 4-hydroxypropiophenone-4-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside(2), along with six known naphthalenyl glucosides (3–8) were isolated from green walnut husks of Juglans mandshurica, and their structures were elucidated on the basis of spectroscopic studies. All compounds were evaluated for their inhibitory effects on tumour cells (BGC-823, HepG-2, MCF-7). The results showed that new compounds 1 and 2 had superior inhibitory activity in comparison with other naphthalenyl glucosides.


Introduction
Juglans mandshurica Maxim. (Juglandaceae) is one of the rare species of trees for pharmacy resources and distributed throughout urban and rural areas in north-east of Asia (Wu & Raven 1999). Its leaves, roots, fruit and barks have been used as medicinal parts in use or development (Li et al. 2003;Liu et al. 2004;Lin et al. 2014;Yao et al. 2015a). Recent pharmacological studies of medicinal parts from J. mandshurica have reported they had many biological properties, including antitumour, of green husks, roots, barks and leaves Li et al. 2008;Liu et al. 2010;Guo et al. 2015;Zhou et al. 2015a;Gao et al. 2016), anti-inflammatory, of barks (Ju et al. 2009), anticomplement activity, of stem-barks (Min et al. 2003), antioxidant, of stem-barks (Ngoc et al. 2008), as well as protection of skin fibroblasts from damage by regulating the oxidative defence system of leaves (Park et al. 2012), anti-human immunodeficiency virus-type 1 of stem-barks (Min et al. 2002). In addition, the types of components isolated from different parts of J. mandshurica were multiple and different. Naphthoquinones and their derivatives widely existed in the roots, stem barks, leaves and green husks (Lee et al. 2000;Min et al. 2002;Li et al. 2008;Lin et al. 2014;Wang et al. 2014;Yao et al. 2015b;Zhou et al. 2015a). Diarylheptanoids were distributed among leaves, green husks and roots (Li et al. 2003;Chen et al. 2015;Jin et al. 2015;Yao et al. 2015b), also including flavonoids from stem-barks (Min et al. 2003), phenolic glycoside from barks and stem-barks (Machida et al. 2009;Yao et al. 2014), fatty acid from walnuts, barks and stem-barks (Bouabdallah et al. 2014;Yao et al. 2015a;Gao et al. 2016) and so on. According to the reports, the types of components isolated from green walnut husks of this plant mainly focused on naphthoquinones, α-tetralones and their glycosides, triterpenes and diarylheptanoids (Liu et al. 2004(Liu et al. , 2010Chen et al. 2015;Zhou et al. 2015b). As part of our continuing search for biologically active compounds from green walnut husks of J. mandshurica Maxim, we investigated the BuoH fraction of the etoH extract which was barely reported, since this polarity fraction exhibited potential cytotoxic activity in our screening procedures. Indeed, we isolated two new glycosides including an alcohol glycoside (1) and a phenolic glycoside (2), together with six known naphthalenyl glucosides (3)(4)(5)(6)(7)(8). The isolated compounds were tested for their cytotoxic activities against three human cancer cell lines in vitro. The results showed that new compounds 1, 2 showed superior inhibitory activity and the other naphthalenyl glucosides showed almost no activity.

Results and discussion
Compound 1 was obtained as white amorphous powder. The positive-mode-HR-eSI-MS data showed a quasi-molecular ion peak [M + Na] + at m/z 419.4137, suggesting a molecular mass of 396.2011 and thus a molecular formula of 1 as C 17 H 32 o 10 . The 1 H NMR data of 1 (Table S1 and Figure S2) showed the methyl group at δ H 0.90 (3H, t, J = 6.8 Hz), and four methylene proton signals at δ H 1.61 (2H, dt, J = 14.9, 6.8 Hz) and 1.28-1.35 (6H, m) indicating the presence of saturated straight-chain paraffin. Further, the 1 H NMR data revealed two anomeric proton signals [δ H 4.24 (d, J = 7.8 Hz, Glc H-1), δ H 4.95 (d, J = 1.1 Hz, Ara H-1)] implying the occurrence of two sugar units: one β-glucopyranose and one α-arabinofuranose which were determined by the coupling constant of the anomeric proton. The proton signals for the disaccharide sugar unit of 1 were identical to the published compound Bumaldoside A (Hideaki et al. 2010) The 13 C NMR data (Table S1 and Figure S3) and distortionless enhanced polarisation transfer spectra (DePT, Table S1 and Figure S4) of 1 showed 17 carbons including seven methylene carbons, nine methine carbons and one methyl group. The 13 C NMR data also revealed two anomeric carbon signals at δ C 104.4 (Glc C-1) and 110.0 (Ara C-1) in accordance with the two anomeric signals in the 1 H NMR spectrum. In the HMBC spectrum ( Figure  S6), a suggestive correlation was observed between the anomeric proton signal of glucopyranose and an oxymethylene carbon signal at δ C 71.1 (C-1) of the aglycone, indicating that the sugar moieties were linked at the C-1 position. Moreover, the connectivity of the arabinofuranose with the glucopyranose was indicted by the cross-peak between the anomeric proton H-1 of arabinofuranose and C-6 of glucopyranose. The sugar moieties of 1 linked at 1-O-were assigned as 1-O-α-l-arabinofuranosyl (1 → 6)-β-d-glucopyranoside, combined with the result of acid hydrolysis of 1. The aqueous layer was separated by HPLC to give d-glucose and l-arabinose. on the basis of the above evidence, the structure of 1 was established as the new compound hexyl-1-O-α-l-arabinofuranosyl (1 → 6)-β-d-glucopyranoside ( Figure 1).
The isolated compounds (1-8) were assessed for cytotoxic activity against three human tumour cell lines, BGC-823 (gastric carcinoma), HepG-2 (liver carcinoma), MCF-7 (breast carcinoma) and cisplatin as positive control using the MTT method. The in vitro cytotoxic activity of these compounds and cisplatin was evaluated at different concentrations against above-mentioned tumour cells, and the IC 50 values are shown (Table S3). Among all the compounds tested, only the new compounds 1 and 2 showed superior cytotoxic activity against tumour cell, but the remaining compounds of naphthalenyl glucosides 3-8 had less or no activity. These results were in accordance with previous reports (Liu et al. 2010).

Plant material
The green husks of J. mandshurica were collected in Changbai Mountains (Jilin, China), in late July 2014 and identified by one of us (Z.Y.Y.). The dried samples were grounded into fine powder (60 mesh), dried thoroughly in an oven at 40 °C for 3 days. A voucher specimen (QLY 20140112) has been stored at the College of Pharmacy, Heilongjiang university of Chinese Medicine.

Acid hydrolysis
Compounds 1 and 2 (each about 1.5 mg) were refluxed with 1.0 mol/L HCl (5 mL, dioxane-H 2 o, v/v) for 7 h under 80 °C water bath. After cooling down to room temperature, the reaction mixture was extracted three times with CH 2 Cl 2 :H 2 o (each 1 : 1, v/v, 1 mL). The aqueous layer was neutralised with 5% NaoH and desalted with Sephadex LH-20 to obtain the sugar residue (0.8 mg). The monosaccharides in 1 were first determined as l-arabinose and d-glucose by co-TLC with authentic sugar, eluting with chloroform/n-BuoH/MeoH/acetic acid/ water 17 : 10 : 6 : 2 : 3 [Rf ( l -Ara) = 0.41, Rf ( d -Glc) = 0.38). In the same way, the monosaccharide in 2 was identified as d-glucose. Then, the PMP derivative of the monosaccharide mixture was prepared as the past report (Honda et al. 1989). The resulted PMP-monosaccharide was dissolved in etoH/n-hexane solution (1 : 4, v/v) and analysed by HPLC using CHRIALPAK AD-H column, with an elution solvent system of n-hexane/2-propanol (87 : 13), at 25 °C, and detected by DAD detector. The standard sugars were operated with the same methods. The monosaccharides in 1 were determined to be l-arabinose (t R = 11.3 min) and d-glucose (t R = 9.6 min). The monosaccharide in 2 was determined to be d-glucose (t R = 9.6 min).

Cytotoxicity assay
Tumour cells in logarithmic growth phase were seeded in a 96-well microtiter plates and kept overnight for attachment. eight compounds and positive control (cisplatin), dissolved in dimethyl sulfoxide and PB (phosphate buffer), were added at various concentrations from 200 to 0.5 μM for 24 h. The optical density was measured at 570 nm using a multiscan microplate reader. All experiments were performed in triplicate. Data were expressed as the concentration required for inhibiting growth of tumour cells by 50% (IC 50 ).