Spirostanol saponins from Chinese onion (Allium chinense) exert pronounced anti-inflammatory and anti-proliferative activities

Journal of Functional Foods 25 (2016) 208–219

Available online at www.sciencedirect.com

ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Spirostanol saponins from Chinese onion (Allium chinense) exert pronounced antiinflammatory and anti-proliferative activities Yihai Wang, Chuan Li, Limin Xiang, Wenjie Huang, Xiangjiu He * School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

Chinese onion (Allium chinense) is widely cultivated as a vegetable, native to China and cul-

Received 17 March 2016

tivated in many other countries. Its bulbs are commonly processed into pickles and spices.

Received in revised form 31 May

In this study, seven new spirostanol saponins (5, 7, 9–11, 13 and 15) together with ten known

2016

congeners (1–4, 6, 8, 12, 14, 16 and 17) were isolated from the bulbs of Chinese onion and

Accepted 8 June 2016

their structures were elucidated by spectroscopic analysis and chemical degradation. The

Available online

isolated components were evaluated for anti-inflammatory and anti-proliferative activi-

Keywords:

lipopolysaccharides (LPS) induced nitric oxide (NO) production in murine macrophage

Chinese onion

RAW264.7. Compound 13 displayed potential inhibitory activity against five human cancer

Allium chinense

cell lines with IC50 values of 1.43–6.81 µM. Compound 14 exhibited significant selective in-

ties. As a result, compounds 4 and 8 showed potent inhibitory effects on the

Spirostanol saponins

hibition against these cancer cells (IC50 2.22–15.58 µM), in comparison with the normal cells

Anti-inflammatory

MRC-5 (IC50 > 100 µM).

Antiproliferative

1.

Introduction

Epidemiological studies have consistently shown that regular consumption of fruit and vegetables is associated with reduced risks of developing chronic diseases such as cancer, cardiovascular disease, stroke, Alzheimer disease, cataracts, and some of the functional declines associated with ageing (Block, Patterson, & Subar, 1992; Willett, 1994). Prevention is a more

© 2016 Elsevier Ltd. All rights reserved.

effective strategy than treatment for these diseases (Liu, 2003; Naczk & Shahidi, 2006). This suggests that a change in dietary behaviour, such as increasing the consumption of fruits, vegetables and whole grains rich in bioactive phytochemicals, may provide desirable health benefits for reducing the incidence of chronic diseases. The genus Allium belongs to family Liliaceae, more recently classified to the family Amaryllidaceae (subfamily Allioideae) (The Angiosperm Phylogeny Group, 2009). It

Chemical compounds: Laxogenin (PubChem CID: 10950057); Neogitogenin (PubChem CID: 44630243). * Corresponding author. School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. Tel.: +86 20 3935 2132; fax: +86 20 3935 2132. E-mail address: [email protected] (X. He). Abbreviations: A549, human lung cancer cells; CNE-1, human nasopharyngeal carcinoma; DEPT, distortionless enhancement by polarization transfer; FBS, foetal bovine serum; 1H–1H COSY, 1H–1H correlation spectroscopy; HepG2, human liver cancer cells; HMBC, heteronuclear multiple-bond correlation; HPLC, high-performance liquid chromatography; HR-ESI-Q-TOF-MS, high-resolution electrospray ionization quadrupole ion trap time-of-flight mass spectrometry; HSQC, heteronuclear single-quantum coherence; LPS, lipopolysaccharides; MGC803, human gastric cancer cells; MPLC, medium-pressure liquid chromatography; MRC-5, human foetal lung fibroblasts cells; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; NO, nitric oxide; iNOS, inducible nitric oxide synthase; RAW 264.7, murine macrophage; SPC-A-1, human lung cancer cells http://dx.doi.org/10.1016/j.jff.2016.06.005 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 25 (2016) 208–219

comprises more than six hundred different species found throughout North America, Europe, Asia and North Africa. Members of Allium family have been cultivated in the Middle and Far East for at least five thousand years. Many Allium species possess characteristic pungent flavour and are prized as foodstuffs in widespread areas of the world (Block, 1992; Fenwick, Hanley, & Whitaker, 1985). Because of their health benefits, extensive scientific investigations on the phytochemical and biological properties of Allium species have been conducted (Albishi, John, Al-Khalifa, & Shahidi, 2013). However, most of these researches have focused on particular species such as onion (A. cepa), garlic (A. sativum), leek (A. ampeloprasum) and chives (A. schoenoprasum), while chemical and biological properties of other species especially Chinese onion are still lacking. Chinese onion, commonly known as oriental onion, is a medicinal food native to China. It is widely cultivated in Southern China (where it derived the common name of Jiao Tou) and Japan, but also found spreading across a similar latitude to Southeast Asia and Northern America (Mann & Stearn, 1960; Wang & Tang, 1980). In China, the bulbs of Chinese onion have been widely used as sweet or sour pickles after steeped in salt. In Japan, they are consumed mainly in pickles as side dishes. Chinese onion is also used as a pickled meal during Vietnamese New Year–Tet celebrations. Raw flowers and young seed pods of Chinese onion are edible and used as garnishes on salads (Lim, 2015). The dried bulbs of Chinese onion are the major source of traditional Chinese medicine “Xiebai,” included in some classic prescription for the treatment of chest pain, stenocardia and heart asthma (Chinese Pharmacopeia Commission, 2010). During the past two decades, Chinese onion has attracted increasing attention in the field of food, nutraceuticals and phytomedicine due to its wide health beneficial effects. Previous phytochemical investigation has led to the identification of sulphur-containing compounds (Pino, Fuentes, & Correa, 2001), nitrogen-containing compounds (Okuyama et al., 1989) and steroidal saponins (Jiang, Wang, Yao, & Susumu, 1998; Matsuura, Ushiroguchi, Itakura, & Fuwa, 1989; Peng, Yao, Tezuka, & Kikuchi, 1996; Peng, Yao, Tezuka, Kikuchi, & Narui, 1996), among which steroidal saponins are believed to account for a variety of biological activities, such as antitumour (Baba et al., 2000; Yu et al., 2015), anticoagulation (Jiang et al., 1998), cardioprotective (Kuroda, Mimaki, Kameyama, Sashida, & Nikaido, 1995; Ren, Qiao, Yang, & Zhou, 2010), and cyclic AMP phosphodiesterase inhibitory activities (Kuroda et al., 1995). Inflammation is a biological response of the host to foreign challenge or tissue injury and can be classified as either an acute or a chronic response depending on the time of onset (Lawrence, Willoughby, & Gilroy, 2002). Chronic inflammation plays an important role in the initiation and progression of several chronic conditions including cancer, arteriosclerosis, diabetes, obesity, and even neurodegenerative diseases (Chung et al., 2009; Sun et al., 2016). Up-regulation of iNOS isoenzymes and the consequent overproduction of NO, an endogenous mediator of numerous physiological processes, has been implicated as a promoter of the severity of a series of disease such as cancer, diabetes, stroke and so on. Therefore, NO levels have been used as markers of inflammation and diseases pathogenesis (Miranda, Espey, & Wink, 2001).

209

In our continuing efforts to seek bioactive components from medicinal plants, fruits and vegetables (He & Liu, 2007; Qiao, Wang, Xiang, Zhang, & He, 2015; Wang, Xiang, Wang, Tang, & He, 2013), a detailed phytochemical characterization of steroidal saponins from the bulbs of Chinese onion was conducted, and seven new steroidal saponins along with ten known congeners were isolated and identified. The potent antiinflammatory and anti-proliferative activities of the isolated components were also evaluated in vitro.

2.

Materials and methods

2.1.

General experimental procedures

Optical rotations were measured on a JASCO P-1020 digital polarimeter (Tokyo, Japan). Infrared spectra (4000–450 cm−1) were acquired with a Perkin Elmer Spectrum 100 FT-IR spectrometer (Perkin Elmer Inc., Waltham, MA, USA). All NMR spectra were obtained on a Bruker Avance III 500 MHz digital NMR spectrometer (Bruker Inc., Fällanden, Switzerland). HR-ESI-MS data were obtained on an Acquity UPLC-Q-TOF Micro MS mass spectrometer (Waters Corp., Milford, MA, USA). Preparative and semipreparative HPLC were carried out on an HPLC system composed of a Dynamax model HPXL solvent delivery system (Rainin Instrument Co. Inc., Woburn, MA, USA) and a model 133 Gilson Refractive Index Detector (Gilson, Villier-le-Bel, France). Their chromatographic separation was performed on C 18 columns (Cosmosil 5C 18 -MS-II, 5 µm, 10ID × 250 mm; Cosmosil 5C18-AR-II, 5 µm, 20ID × 250 mm; Nacalai Tesque, Kyoto, Japan) using a gradient solvent system composed of MeOH and H2O as mobile phase at a flow rate of 8.0 mL/min and 4.0 mL/ min, respectively. N2000 software (Zhejiang University, Hangzhou, China) was connected to the detector for data acquisition and the retention time (tR) of the isolated peak was obtained from the corresponding chromatograms.

2.2.

Chemicals and reagents

D101 macroporous resin (Xi′an Lanxiao Resin Corporation Ltd., Xi′an, China), silica gel (200–300 mesh, Anhui Liangchen Silicon Material Co. Ltd., Anhui, China) and ODS (40–60 µm, Merck KGaA, Darmstadt, Germany) were used for column chromatography. HPLC grade methanol was purchased from Oceanpak Alexative Chemical Co., Ltd. (Gothenburg, Sweden). The deuterated solvents for NMR experiment, standard sugars (Dglucose, D-galactose, D-xylose and L-arabinose) for GC analysis, LPS and MTT were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). FBS and cell culture medium were purchased from Gibco Life Technologies (Grand Island, NY, USA). Cell lines used for biological study were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All other analytical chemicals and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

2.3.

Plant material

Fresh bulbs of Chinese onion (122.9 kg) were collected in June 2012 from Shu’an Country of Jiangxia District (Wuhan, China)

210

Journal of Functional Foods 25 (2016) 208–219

and was identified to be A. chinense G. Don. The fresh materials were steamed for fifteen minutes over the boiled water and then air dried for use. A voucher specimen (No. GDPU-NPR2012-ACG) was deposited in the School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China.

2.4.

Extraction and isolation

Dried bulbs of Chinese onion were pulverized and then extracted three times with 60% aqueous methanol (3 × 40 L) for 2 h at reflux. The extracted solution was filtered through three layers of gauze, and the filtrate was then concentrated under reduce pressure in a rotary evaporator at 55 °C to remove methanol. The obtained extract was resuspended in distilled water and subjected to a D101 macroporous resin column (100 × 1200 mm), subsequently eluted with 100 L of H2O, 40 L of MeOH/H2O (30:70, v/v), and 50 L of MeOH. The MeOH elution was evaporated to dryness (152.1 g). The residue was redissolved in methanol and subjected to silica gel chromatography (200–300 mesh, 100 × 1100 mm) and eluted with a CHCl 3 / MeOH gradient elution (100:0 to 0:100, v/v) to afford 14 fractions (Frs. 1–14). Compound 1 (1.5 g) was obtained from Fr. 5. Fr. 7 (915.7 mg) was applied to a silica gel column (15 × 400 mm) which was subsequently eluted with 1000 mL of cyclohexane/EtOAc (5:1, v/v), 400 mL of cyclohexane/EtOAc (4:1, v/v) and 500 mL of cyclohexane/EtOAc (3:1, v/v). Compound 12 (56.2 mg) was obtained from cyclohexane/EtOAc (3:1, v/v) elution. Fr. 10 (4.19 g) was chromatographed on an ODS MPLC column (30 × 300 mm) eluted gradiently with 6000 mL of MeOH/H2O (20:80, v/v), 5500 mL of MeOH/H2O (50:50, v/v), 1200 mL of MeOH/H2O (70:30, v/v), and 2500 mL of MeOH/H2O (90:10, v/v) to give seven subfractions (Subfrs. 10.1–10.7). Subfr. 10.5 was further purified with the preparative HPLC C18 column (20 × 250 mm) which eluted with MeOH/H2O (65:35, v/v) and compound 10 (4.0 mg, tR = 23.2 min) was obtained. Subfr. 10.6 was also separated with the preparative HPLC C18 column and eluted with MeOH/H2O (75:25, v/v) to afford compounds 5 (42.6 mg, tR = 29.8 min), 3 (13.4 mg, tR = 32.1 min) and 2 (54.4 mg, tR = 34.2 min). Fr. 11 (4.40 g) was subjected to an ODS MPLC column (30 × 300 mm) chromatography and eluted gradiently with 3000 mL of MeOH/H2O (30:70, v/v), 2000 mL of MeOH/H2O (50:50, v/v), 2500 mL of MeOH/H2O (80:20, v/v), and 1500 mL of MeOH/H2O (90:10, v/v), to give six subfractions (Subfrs. 11.1–11.6). Compound 4 (719.0 mg) was obtained from Subfr. 11.5 (840.0 mg).The filtrate of Subfr. 11.5 was further purified with the semi-preparative HPLC C18 column (10 × 250 mm) which eluted with MeOH/H2O (50:50, v/v), to give compound 11 (3.3 mg, tR = 34.2 min). Fr.12 (9.18 g) was divided to ten subfrs. (Subfrs. 12.1–12.10) with an ODS MPLC column (30 × 300 mm) eluted gradiently with 1750 mL of MeOH/H2O (30:70, v/v), 2500 mL of MeOH/H2O (40:60, v/v), 3250 mL of MeOH/ H2O (50:50, v/v), 4250 mL of MeOH/H2O (60:40, v/v), 1750 mL of MeOH/H2O (70:30, v/v) and 3500 mL of MeOH/H2O (80:20, v/v). Compound 6 (1.60 g) was obtained from Subfr.12.8 (1.92 g). The filtrate of Subfr. 12.8 was chromatographed on the semipreparative HPLC C18 column (10 × 250 mm) eluted with MeOH/ H2O (70:30, v/v), to afford compound 7 (3.3 mg, tR = 34.1 min). The Subfr.12.9 was separated with the preparative HPLC C18 column (20 × 250 mm), which eluted with MeOH/H2O (75:25, v/v) to yield compounds 15 (10.3 mg, tR = 26.9 min) and 16 (46.0 mg, tR = 29.9 min). Compounds 13 (54.0 mg, tR = 48.9 min) and 14

(11.4 mg, tR = 52.3 min) were obtained from the Subfr. 12.10 (240.5 mg) by the preparative HPLC C18 column (20 × 250 mm) eluted with MeOH/H2O (80:20, v/v). Fr. 13 (8.50 g) was subjected to an ODS MPLC column (30 × 300 mm) eluted gradiently with 2000 mL of MeOH/H2O (30:70, v/v), 2000 mL of MeOH/H2O (40:60, v/v), 1750 mL of MeOH/H2O (50:50, v/v), 2250 mL of MeOH/ H2O (60:40, v/v), 2750 mL of MeOH/H2O (70:30, v/v), 2500 mL of MeOH/H2O (80:20, v/v) and 2000 mL of MeOH to yield ten subfrs. (Subfr. 13.1–13.10). Subfr. 13.6 was finally purified by the semipreparative HPLC C18 column (10 × 250 mm) eluted with MeOH/ H2O (70:30, v/v) to afford compounds 9 (4.5 mg, tR = 67.5 min) and 8 (12.7 mg, t R = 70.6 min). Compound 17 (13.0 mg, t R = 40.8 min) was isolated from Subfr. 13.9 by the semipreparative HPLC C18 column (10 × 250 mm) eluted with MeOH/ H2O (70:30, v/v).

2.4.1. (25R)-5α-spirostan 3-O-{O-(4-O-acetyl-α-Larabinopyranosyl)-(1→6)-β-D-glucopyranoside} (5) White amorphous powder (MeOH); [α]14 D–54.2 (c 0.50, MeOH); IR νmax (KBr) cm−1: 3405 (OH), 2950 (CH), 1735 (C=O), 1708 (C=O), 1453, 1430, 1376, 1242, 1174, 1019, 983, 920, 900, 866, 800, 782 (intensity 920 < 900); 1H and 13C NMR data see Tables 1–4; HRESI-Q-TOF-MS (positive) m/z 789.4011 [M+Na] + , calcd. for C40H62O14Na 789.4037.

2.4.2. (25R)-3β-hydroxy-5β-spirostan-6-one3-O-β-Dxylopyranosyl(1→4)-[α-L-arabinopyranosyl-(1→6)]-β-Dglucopyranoside (7) White amorphous powder (MeOH); [α]14 D–71.7 (c 0.23, MeOH); IR νmax (KBr) cm−1: 3322 (OH), 2931 (CH), 1701 (C=O), 1456, 1379, 1242, 1158, 1054, 1010, 983, 920, 900, 866, 781 (intensity 920 < 900); 1 H and 13C NMR data see Tables 1–4; HR-ESI-Q-TOF-MS m/z 879.4326 [M+Na]+, calcd. for C43H68O17Na 879.4354.

2.4.3. (25R)-3β-hydroxy-5α-spirostan-6-one3-O-{[O-β-Dglucopyranosyl-(1→3)-O-β-D-xylopyranosyl]-(1→4)-O-[α-Larabinopyranosyl-(1→6)]}-β-D- glucopyranoside (9) White amorphous powder (MeOH); IR νmax (KBr) cm−1: 3322 (OH), 2950 (CH), 1709 (C=O), 1455, 1380, 1242, 1167, 1059, 984, 920, 900, 866, 783 (intensity 920 < 900); 1H and 13C NMR data see Tables 1–4; HR-ESI-Q-TOF-MS m/z 1041.4944 [M+Na]+, calcd. for C49H78O22Na 1041.4882.

2.4.4. (25S)-3β,24β-dihydroxy-5α-spirostan-6-one3-O[α-L-arabinopyranosyl(1→6)]-β-D-glucopyranoside (10) White amorphous powder (MeOH); IR νmax (KBr) cm−1: 3347 (OH), 2955 (CH), 1705 (C=O), 1452, 1382, 1188, 1075, 994, 965, 900; 1H and 13 C NMR data see Tables 1–4; HR-ESI-Q-TOF-MS m/z 763.3859 [M+Na]+, calcd. for C38H60O14Na 763.3881.

2.4.5. (25S)-24-O-β-D-glucopyranosyl-3β,24β-dihydroxy5α-spirostan-6-one (11) White amorphous powder (MeOH); IR νmax (KBr) cm−1: 3420 (OH), 2955 (CH), 1700 (C=O), 1455, 1382, 1242, 1187, 1075, 993, 965, 900, 871; 1H and 13C NMR data see Tables 1–4; HR-ESI-Q-TOFMS m/z 631.3436 [M+Na]+, calcd. for C33H52O10Na 631.3458.

2.4.6. (25R)-5α-spirostan-3β-yl-3-O-acetyl-O-β-Dglucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-Dglucopyranosyl-(1→4)-β-D-galactopyranoside (13) White amorphous powder (MeOH); [α]14 D–43.6 (c 0.50, MeOH); IR νmax (KBr) cm−1: 3348 (OH), 2931 (CH), 1735, 1451, 1374, 1244,

211

Journal of Functional Foods 25 (2016) 208–219

Table 1 – 1H NMR (500 MHz, in Pyr-d5) data for aglycone moieties of compounds 5, 7, 9–11, 13, 15. No.

1 2 3 4 5 6 7 8 9 11 12 14 15 16 17 18 19 20 21 23 24 25 26 27 a

δH (J in Hz) 5

7

9

10

11

13

15

1.54 m 1.07 m 2.12 m 2.00 m 4.08a 2.34 m 1.57 m 2.09a

1.95a 1.56a 1.94a 1.67a 4.32 m 1.95a 1.50–1.66a 2.91 dd (13.0,4.1)

1.52 m 1.09 m 2.06a 1.58a 3.89a 2.36 m 1.73 m 2.15 dd (12.6, 2.3)

1.56a 1.14a 2.16a 1.63a 4.06a 2.41 d (12.5) 1.74 m 2.15a

1.62a 1.16a 2.12a 1.91a 3.85 m 2.32a 1.92a 2.26 dd (12.2, 3.0)

2.18 dd (12.8, 4.7) 1.11a 4.00a

2.33 dd (12.9, 4.4) 1.97a 1.86 m 1.08a 1.46 m 1.22 m 1.65a 1.05a 1.20 m 1.93a 1.29a 4.51 dd (14.6, 7.4) 1.80 dd (8.3, 6.6) 0.76 s 0.61 s 1.93a 1.13 d (6.9) 1.66a 1.66a 1.57 m 1.45 m 1.64 m 3.57 dd (11.4, 3.4) 3.47 t (10.7) 0.67 d (5.9)

2.24 dd (14.2, 4.1) 2.33 dd (14.0, 12.5) 1.88 m 1.23a 1.39a 1.21a 1.71a 1.19a 1.23a 1.97a 1.37a 4.60 dd (14.6, 7.6) 1.81 dd (8.2, 6.5) 0.77 s 0.69 s 1.94 m 1.15 d (6.9) 1.65a 1.65a 1.57a 1.16a 1.95a 3.58 m 3.49 t (10.8) 0.68 d (5.9)

2.36 dd (12.9, 4.4) 2.00 t (12.6) 1.88 m 1.13a 1.48 m 1.22 m 1.67a 1.09a 1.23a 1.93a 1.37a 4.52 dd (15.4, 6.5) 1.81 dd (8.3, 6.6) 0.77 s 0.62 s 1.93 m 1.14 d (6.9) 1.64a 1.64a 1.56a 1.25a 1.64 m 3.48 t (10.8) 3.58 dd (11.3, 4.2) 0.68 d (6.0)

2.35 dd (13.1, 4.3) 1.99 t (12.1) 1.86a 1.12a 1.45 m 1.22 m 1.65a 1.09a 1.22a 1.92a 1.36 m 4.55 dd (14.8, 7.4) 1.82 dd (8.4, 6.6) 0.77 s 0.63 s 2.02a 1.18 d (6.9) 2.33a 2.04a 4.03a

2.36 dd (12.9, 4.4) 2.00 t (13.6) 1.86a 1.14a 1.50 m 1.24a 1.65a 1.13a 1.23a 1.94a 1.32 m 4.50 dd (14.5, 7.5) 1.78 dd (8.4, 6.6) 0.71 s 0.75 s 1.92a 1.07 d (7.0) 2.69 dd (13.0, 4.9) 1.97 t (13.1) 4.03 dd (10.6, 4.8)

1.85a 3.72 dd (11.3, 4.8) 3.60 t (11.3) 1.10 d (6.5)

1.66a 3.65 dd (11.3, 5.1) 3.56 t (11.4) 1.15 d (6.5)

1.56a 0.80a 2.08a 1.70a 3.83a 1.79a 1.38a 0.90 m 1.53a 1.12a 1.53a 0.79a 1.34a 0.47 td (12.0, 3.9) 1.41a 1.22a 1.69a 1.05a 1.00 m 2.02a 1.42a 4.56a 1.81a 0.80 s 0.61 s 1.94 p (6.9) 1.13 d (7.0) 1.68a 1.68a 1.59a 1.10a 2.06a 3.58a 3.49 t (10.7) 0.67 d (5.7)

3.88a 1.75a 1.40a 1.12a 1.48a 1.10a 1.53a 0.74a 1.37a 0.55 td (12.2, 3.6) 1.49a 1.11a 1.58a 1.00a 1.01a 2.03a 1.41a 4.52a 1.79a 0.78 s 0.67 s 1.93 m 1.06 d (6.9) 1.76a 1.76a 2.69 m 2.25 m 4.45 d (12.1) 4.04a 4.81s; 4.77 s

Signals are overlapped.

1160, 1075, 984, 922, 900, 865 (intensity 922 < 900); 1H and 13C NMR data see Tables 1–4; HR-ESI-Q-TOF-MS m/z 1129.5430 [M+Na]+, calcd. for C38H60O14Na 1129.5407.

2.4.7. 5α-spirostane 25(27)-ene-2α,3β-diol-3-O-{O-β-Dglucopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-β-Dgalactopyranoside} (15) White amorphous powder (MeOH); [α]14 D–38.6 (c 0.35, MeOH); IR νmax (KBr) cm−1: 3357 (OH), 2929 (CH), 1648 (C=C), 1451, 1378, 1230, 1171, 1076, 1045, 955, 924, 900, 880, 792; 1H and 13C NMR data see Tables 1–4; HR-ESI-Q-TOF-MS m/z 939.4606 [M+Na]+, calcd. for C45H72O19Na 939.4566.

2.5.

Acid hydrolysis and GC analysis

The hydrolysis and GC analysis of the chiral derivatives of the sugars of new steroidal saponins were performed as previously described (Xiang, Wang, Yi, Feng, & He, 2016).

2.6. Bioassay for inhibitory activity on LPS-induced NO production Macrophage RAW264.7 cells were cultured in DMEM containing 10% FBS, penicillin (100 units/mL) and streptomycin (100 µg/ mL) at 37 °C in a humid atmosphere of 5% CO2/95% air. NO produced in the medium was measured by assaying the levels of NO2− via the Griess reaction (Miranda et al., 2001).

2.7.

Anti-proliferative assays

Cell culture and anti-proliferative activity assay were performed as previously described (Wang, Zhang, Wang, & He, 2015).

3.

Results and discussion

In this study, bulbs of Chinese onion were collected, dried and extracted with 60% methanol. The obtained extract was then

212

Journal of Functional Foods 25 (2016) 208–219

Table 2 – 13C NMR (125 MHz, in Pyr-d5) data for aglycone moieties of compounds 5, 7, 9–11, 13, 15 (δC in ppm). No.

5

7

9

10

11

13

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

36.9 29.7 77.1 27.2 56.6 210.0 46.9 37.6 53.8 41.1 21.7 39.8 41.3 56.6 32.0a 81.1 63.0 16.7 13.3 42.2 15.2 109.5 31.9a 29.4 30.8 67.1 17.5

30.2 26.8 73.0 29.9 55.4 213.9 43.5 36.8 40.0 38.3 21.5 40.0 41.5 56.8 32.1a 81.3 63.2 16.7 24.0 42.3 15.4 109.6 32.2a 29.5 30.9 67.2 17.7

37.0 29.8 77.3 27.3 56.8 209.9 47.1 37.7 54.0 41.2 21.9 40.0 41.4 56.8 32.2a 81.2 63.2 16.8 13.4 42.3 15.3 109.6 32.1a 29.6 30.9 67.2 17.7

37.1 29.9 77.1 27.4 56.8 210.0 47.1 37.7 54.0 41.4a 21.9 39.9 41.2a 56.8 32.2 81.6 62.8 16.8 13.5 42.6 15.3 112.2 42.2 70.9 40.4 65.7 14.0

37.3 32.2a 70.3 31.6 57.3 210.4 47.1 37.7 54.1 41.2 21.9 40.0 41.2 56.9 32.1a 81.6 62.7 16.8 13.6 42.5 15.2 112.0 41.4 81.8 38.6 65.5 13.8

37.4 30.1 77.6 35.0 44.8 29.1 32.6 35.5 54.6 36.0 21.5 40.4 41.0 56.7 32.4 81.4 63.2 16.9 12.5 42.2 15.3 109.5 32.0 29.5 30.8 67.1 17.6

45.8 70.6 84.7 34.2 44.8 28.3 32.2 34.7 54.5 37.1 21.6 40.2 41.0 56.5 32.4 81.7 63.1 16.8 13.6 42.0 15.2 109.6 33.4 29.1 144.6 65.2 109.0

a

Assignments may be interchanged in each column.

successively chromatographed on macroporous resin D101, silica gel, ODS MPLC and finally purified by RP-HPLC to give seven new steroidal saponins (5, 7, 9–11, 13 and 15), as well as ten known congeners (Fig. 1).

3.1.

Structural elucidation

Compound 1 was obtained as colourless needle crystal (CHCl3/ MeOH) and reacted positive to Anisaldehyde but negative to Ehrlich reaction. The IR spectrum showed strong absorption bands for hydroxyl (3496 cm −1 ) and carbonyl (1713 cm −1 ) functionalities. Four characteristic absorption bands of the spirostanol rings were observed at 982, 920, 901 and 867 cm−1, with the 901 cm−1 band stronger than the 920 cm−1 band, which suggested the R-configuration of C-25 (Eddy, Wall, & Scott, 1953). The 1H NMR spectrum displayed two angular methyl signals at δH 0.76 (s, 6H), two secondary methyl signals at δH 0.96 (d, J = 7.0 Hz, 3H) and 0.77 (d, overlapped), indicative of the methyl groups of the spirostanol skeleton. The signals in the 13C NMR spectrum showed twenty-seven carbons in the molecule, including four methyl groups at δC 17.1, 16.4, 14.4, 13.2 and a typical quaternary carbon signal at δC 109.5 (C-22), respectively, ascribable to the spirostanol skeleton. These data were identical to the reported sapogenin laxogenin (Woo, Do, & Son, 1992). Compounds 2–4, 6 and 8 possessed the same aglycone which exhibited similar spectroscopic features as that of laxogenin in 1H and 13C NMR spectra as well as IR spectrum. The sugar moieties of these steroidal saponins were further determined by the NMR data and acid hydrolysis. By comparing

the NMR data with those reported in the literature, they were identified as laxogenin 3-O-β-D-glucopyranoside (2) (Matsuura et al., 1989). laxogenin 3-O-{β-D-xylopyranosyl(1→4)-β-Dglucopyranoside} (3) (Matsuura et al., 1989), laxogenin 3-O-{αL-arabinopyranosyl(1→6)-β-D-glucopyranoside} (4) (Matsuura et al., 1989), laxogenin 3-O-{β-D-xylopyranosyl-(1→4)-O-[α-Larabinopyranosyl-(1→6)]-β-D-glucopyranoside} (6) (Kuroda et al., 1995), and laxogenin 3-O-β-D-glucopyranosyl (1→4)-[α-Larabinopyranosyl(1→6)]-β-D-glucopyranoside (8) (Woo et al., 1992). Compound 12 was a 2,3-dihydroxy spirostanol and was indentified to be Neogitogenin (Achenbach, Hübner, Brandt, & Reiter, 1994). Compound 14 was identified as (25S)-5αspirostane-3β-ol-3-O-{O-β-D-glucopyranosyl-(1→2)-O-β-Dglucopyranosyg-(1→4)-β-D-galactopyranoside} (Kuroda et al., 1995). Compounds 16 and 17 were identified as (25R,S)-5αspirostane-2α,3β-diol-3-O-{O-β-D-glucopyranosyl-(1→2)-O-βD-glucopyranosyl-(1→4)-β-D-galactopyranoside} (16) (Kuroda et al., 1995) and (25R,S)-5α-spirostane-3β-ol-3-O-{O-β-Dglucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-O-β-Dglucopyranosyl-(1→4)-β-D-galactopyranoside} (17) (Matsuura et al., 1989), respectively. Data for these known compounds were used to aid in structural elucidation of the newly described saponins. Compound 5 was positive to Anisaldehyde but negative to Ehrlich reaction. The HR-ESI-MS spectrum showed a molecular formula of C40H62O14 with a pseudo molecular ion at m/z 789.4011 [M+Na]+ (calcd. 789.4037). The R-configuration of C-25 was drawn from the IR spectrum (Eddy et al., 1953). The 1H NMR spectrum displayed two angular methyl signals at δH 0.76 and

213

Journal of Functional Foods 25 (2016) 208–219

Table 3 – 1H NMR (500 MHz, in Pyr-d5) data for sugar moieties of compounds 5, 7, 9–11, 13, 15. No.

δH (J in Hz) 5

7

9

10

C-3: 1 2 3 4 5 6

Glc 4.97 d (7.7) 3.99 m 4.19a 4.20–4.31a 4.08a 4.84 dd (11.2, 1.5) 4.33 dd (11.3, 6.0)

Glc 4.79 d (7.9) 3.88a 4.34a 4.48a 4.04a 4.77 d (11.7) 4.70 dd (10.8, 3.0) Xyl (1→4) 5.52 d (7.9) 4.03a 4.19 t (7.0) 4.20a 4.20a 3.96 d (12.4)

Glc 4.93 d (7.8) 4.14a 4.70a 4.44a 3.91a 4.80 d (9.4) 4.67 dd (10.8, 3.2) Xyl (1→4) 5.50 d (7.8) 4.07 m 4.35 t (8.8) 4.10a 4.18a 3.90a

Glc 5.01 d (7.7) 4.04a 4.25 t (8.6) 4.19a 4.10a 4.87 dd (11.3, 1.8) 4.35 dd (11.5, 6.3)

1 2 3 4 5

11

6 Ara (1→6) 4.94 d (7.3) 4.20–4.31a 4.20–4.31a 5.56 m 4.22a 3.74 d (11.9)

1 2 3 4 5

Ara (1→6) 5.06 d (7.5) 4.47 t (7.4) 3.83–4.07a 4.23a 4.25a 3.69 d (11.2)

Ara (1→6) 5.04 d (7.5) 4.47a 4.91a 4.22a 4.20a 3.69 d (11.0)

Ara (1→6) 5.00 d (6.8) 4.49 t (7.4) 4.04a 4.36a 4.32a 3.76 d (10.4)

6 Glc (1→3) 5.32 d (7.8) 4.10a 4.20a 4.18a 3.95a 4.52 dd (15.4, 6.5) 4.28 m

1 2 3 4 5 6 Ac a

C-24: Glc 4.93 d (7.8) 4.08 t (8.2) 3.90 m 4.28 t (9.1) 4.24 t (8.8) 4.55 d (12.1) 4.28 t (9.1)

1.95 s

13

15

Gal 4.87 d (7.6) 4.42 dd (9.3, 8.0) 4.14a 4.60 d (3.1) 4.04a 4.69 d (9.4) 4.25 dt (10.7, 5.4) Glc (1→4) 5.14 d (7.9) 4.30a 4.15a 3.84a 3.95a

Gal 4.94 d (7.8) 4.58a 4.13a 4.59a 3.97a 4.87 dd (9.9, 9.5) 4.25a Glc (1→4) 5.13 d (7.8) 4.16a 4.18a 4.19a 3.86a

4.50a 4.20a Glc (1→2) 5.59 d (7.9) 3.98a 5.70 t (9.6) 4.27a 4.00a

4.63a 4.36a Glc (1→2) 5.27 d (7.6) 4.15a 4.27a 3.97a 3.96a

4.44a 4.00a Glc (1→3) 5.21 d (8.0) 4.04a 3.75-4.18a 4.14a 3.75-4.18a 4.55a 4.35a 1.80 s

4.64a 4.14a

Signals are overlapped.

0.61, two secondary methyl signals at δH 1.13 (d, J = 6.9 Hz, 3H) and 0.67 (d, J = 5.9 Hz, 3H), indicative of the methyl groups of the spirostanol skeleton. Two anomeric protons at δH 4.97 (d, J = 7.7 Hz, 1H), 4.94 (d, J = 7.3 Hz, 1H) and some overlapping signals from δH 3.48 to 5.56 indicated the presence of two sugar moieties in the molecule. Meanwhile, there was an additional singlet methyl signal that appeared at δH 1.96 (s, 3H), which might be due to an acetyl group. The 13C NMR spectrum displayed five methyl carbon signals in the higher field, including four methyl groups at δC 17.6, 16.7, 15.2, 13.3 ascribable to the spirostanol skeleton and a methyl group at δC 21.3 due to acetyl group. In the lower field of 13C NMR spectrum, a typical quaternary carbon signal of spirostanol and two carbonyl signals was observed at δC 109.5 (C-22), 210.0 and 171.1, respectively. Additionally, two anomeric carbon signals were observed at δC 102.5 and 106.0, and this was consistent with the presence of two sugar moieties. The sugars obtained from the hydrolysates were identified as D-glucose and L-arabinose at a ratio of 1:1 by GC analysis of their chiral derivatives. Their anomeric configurations were determined to be β-configuration for glucose (3J1,2 = 7.7 Hz) and α-configuration for arabinose

(3J1,2 = 7.3 Hz) by their coupling constants. From the results of H and 13C NMR, it was suggested that 5 was a 3β-hydroxy5α-spirostan-6-one saponin with two sugars and an acetyl group. Comparing the NMR data with those of laxogenin 3-O{α-L-arabinopyranosyl (1→6)-β-D-glucopyranoside} (Kuroda et al., 1995), 5 seemed to be the laxogenin with a arabinopyranosyl (1→6) glucopyranosyl residue linked to C-3 of aglycone and an acetyl group linked to C-4″ of arabinose. These could be drawn from the downfield shifts of C-3 (+6.5 ppm) and C-4″ (+3.1 ppm) and further evidenced by the long-range correlations of H-1′ (δH 4.97) of glucose with C-3 (δC 77.1) of aglycone, H-1″ (δH 4.92) of arabinose with C-6′ (δC 70.1) of glucose, and −CH3 (δH 1.95) of acetyl group with C-4″ (δC 72.5) of arabinose (Fig. 2). From these results, the structure of 5 was established as (25R)5α-spirostan-3-O-{O-(4-O-acetyl-α-L-arabinopyranosyl)-(1→6) -β-D-glucopyranoside}. Compound 7 was positive to Anisaldehyde but negative to Ehrlich agent. On acid hydrolysis, 7 liberated D-glucose, D-xylose and L-arabinose. These suggested 7 was a spirostanol saponin. The IR spectrum revealed the R-configuration of C-25. Its molecular formula was deduced as C43H68O17 by HR-ESI-MS. In 1

214

Journal of Functional Foods 25 (2016) 208–219

Table 4 – 13C NMR (125 MHz, in Pyr-d5) data for sugar moieties of compounds 5, 7, 9–11, 13, 15 (δC in ppm). No.

5

7

9

10

C-3: 1 2 3 4 5 6

Glc 102.5 75.4 78.7 71.7 77.3 70.1

Glc 103.5 75.3a 78.8 80.1 75.2a 68.4 Xyl (1→4) 105.4 75.0a 76.6 71.4 67.6a

Glc 102.4 74.9 76.6 80.0 75.2 68.3 Xyl (1→4) 104.8 74.1 88.9 69.8 66.8

Glc 102.5 75.6 79.0 72.3 77.5 70.2

Ara (1→6) 106.0 72.6 72.8 72.5 64.6

Ara (1→6) 106.0 72.9 74.9 70.2 67.7a

Ara (1→6) 106.0 72.9 75.2 70.2 67.7

Ara (1→6) 106.0 72.7 74.9 69.6 67.0

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 —COCH3 —COCH3 a

Glc (1→3) 106.2 76.0 78.9 72.0 78.5 62.9

11

C-24:Glc 106.8 76.0 78.4 72.1 79.0 63.2

171.1 21.3

13

15

Gal 102.6 73.4 75.7 80.4 75.5 60.9 Glc (1→4) 105.0 81.7 88.9 70.9 77.8 62.5 Glc (1→2) 104.9 74.3 79.2 68.7 78.5a 63.2 Glc (1→3) 104.6 75.5 78.8a 71.7 78.7a 61.9 171.0 21.4

Gal 105.3 73.0 75.7 81.1 75.7 60.7 Glc (1→4) 103.4 86.1 77.9 70.7 79.2 61.9 Glc (1→2) 107.0 76.8 78.6 71.9 78.4 63.3

Assignments with the same signs may be interchanged in each column.

higher field of the 1H NMR spectrum, four typical steroidal methyl were observed at δH 1.15 (d, J = 6.9 Hz, 3H), 0.77 (s, 3H), 0.69 (s, 3H) and 0.68 (d, J = 5.9 Hz, 3H), respectively. In the lower field, three anomeric protons were presented at δH 5.52 (d, J = 7.9 Hz, 1H), 5.06 (d, J = 7.5 Hz, 1H) and 4.79 (d, J = 7.9 Hz, 1H), which was consistent with the acid hydrolysis results, and revealed the presence of three sugar moieties. In the 13C and DEPT spectra, forty-three carbon signals were observed. In the higher field, four methyl groups presented at δC 15.4, 16.7, 17.7, and 24.0. Three oxygen-bearing carbons at δC 106.0, 105.4, and 103.5 were assignable to anomeric carbons of sugars. In the lower field, a carbonyl signal was observed at δC 213.9, and a typical quaternary carbon signal of spirostanol was measured at δC 109.6. The sugar linkages were determined by comparison with the known compound laxogenin 3-O-{β-D-xylopyranosyl-(1→4)O-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranoside}. These were further evidenced by the long-range correlations between H-1′ (δH 4.79) of glucose with C-3 (δC 73.0) of aglycone, H-1″ (δH 5.52) of xylose with C-4′ (δC 80.1) of glucose, and H-1‴ (δH 5.06) of arabinose with C-6′ (δC 68.4) of glucose. The location of the carbonyl group was determined to be at C-6 of the aglycone by the long-range correlations between H-5 (δH 2.91) and H-7 (δH 2.24) of aglycone with the carbonyl group (δC 213.9) (Fig. 2). These NMR spectra largely resembled those of the above mentioned known compound, with changes focused on those signals originating from the A/B ring. The downfield chemical

shift of C-19 by approximately +10.6 ppm and the higher field chemical shift of the A/B ring revealed the β-orientation of H-5. Consequently, compound 7 was identified as (25R)-3βhydroxy-5β-spirostan-6-one 3-O-β-D-xylopyranosyl(1→4)[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranoside. Acid hydrolysis of compound 9 gave D-glucose, D-xylose and L-arabinose. Its molecular formula was determined as C43H68O17 by HR-ESI-MS. The IR and NMR spectral data, especially the 13CNMR signals, were in good agreement with those of laxogenin 3-O-{β-D-xylopyranosyl-(1→4)-O-[α-L-arabinopyranosyl-(1→6)]β-D-glucopyranoside} (6), with exceptions of an additional glucose. The downfield chemical shift of C-3″ (δC 88.9) of xylose by +12.3 ppm suggested the remaining glucose was linked to the C-3″ (δC 88.9) of xylose. This was further supported by the long-range correlations between H-1‴ (δH 5.32) of the terminal glucose with the C-3″ (δC 88.9) of xylose (Fig. 2). Therefore, the structure of 9 was identified as (25R)-3β-hydroxy-5αspirostan-6-one3-O-{[O-β-D-glucopyranosyl-(1→3)-O-β-Dxylopyranosyl]-(1→4)-O-[α-L-arabinopyranosyl- (1→6)]}-β-Dglucopyranoside. Compound 10 reacted positive to Anisaldehyde but negative to Ehrlich agent. Acid hydrolysis of 10 gave D-glucose and L-arabinose. These suggested 10 was a spirostanol saponin. Its molecular formula C38H60O14 was determined by HR-ESI-MS at m/z 763.3859 [M+Na]+ (calcd. 763.3.881). The NMR features of 10 were similar to those of laxogenin 3-O-{α-L-arabinopyranosyl

Journal of Functional Foods 25 (2016) 208–219

215

Fig. 1 – Chemical structures of compounds 1–17 isolated from the bulbs of Chinese onion.

(1→6)-β-D-glucopyranoside}, with changes focused on those signals originating from the F ring. The downfield chemical shift of C-24 (δC 70.9) revealed the presence of 24-OH. This was consistent with those reported in the literature (Hong, Luo, Guo, & Kong, 2012) and further verified by the HMBC correlations (Fig. 2) of H-27 (δH 1.10) with C-24 (δC 70.9), C-25 (δC 40.4), and C-26 (δC 65.7), and H-23 (δH 2.33, 2.03) with C-22 (δC 112.2) and C-24 (δC 70.9). The 24S and 25S configurations were revealed by the large J values between the H-23ax proton and H-24 proton

(13.0 Hz), and between the H-26ax proton and H-25 proton (11.3 Hz). These were further supported by ROESY correlations of H-27 (δH 1.10) with H-26ax (δH 3.60) and H-24 (δH 4.02). Thus, 10 was identified as (25S)-3β,24β-dihydroxy-5α-spirostan6-one3-O-[α-L-arabinopyranosyl(1→6)]-β-D-glucopyranoside. Compound 11 reacted positive to Anisaldehyde but negative to Ehrlich agent. Its molecular formula was determined as C33H52O10 by HR-ESI-MS with the ion of m/z 631.3436 [M+Na]+ (calcd. 631.3458). Acid hydrolysis of 11 gave D-glucose. The 1H

216

Journal of Functional Foods 25 (2016) 208–219

Fig. 2 – Selected key HMBC and 1H–1H COSY correlations of compounds 5, 7, 9 and 10.

and 13C NMR signals of the aglycone moiety of 11 were similar to those of 10 except for the glycosidation shift at C-24. These suggested 11 possessed the same aglycone as 10. The downfield chemical shift of C-24 revealed the glucose linked to C-24 of the aglycone. This was further evidenced by the HMBC correlations from H-1′ of glucose at δH 4.93 (d, J = 7.8 Hz, 1H) to C-24 (δC 81.8) of the aglycone. Therefore, 11 was identified as (25S)-24-O-β-D-glucopyranosyl-3β,24β-dihydroxy5α-spirostan-6-one. Compound 13 reacted positive to Anisaldehyde but negative to Ehrlich agent. On acid hydrolysis, 13 liberated D-glucose and D-galactose at a ratio of 3:1. Four characteristic absorptions observed in the IR spectrum revealed the R-configuration of C-25. Its molecular formula was deduced as C53H86O24 by HR-ESI-MS. The 1H and 13C NMR signals of 13 were almost identical to those of macrostemonoside A, (Kuroda et al., 1995; Wu, Peng, Yao, & Okuyama, 1992) except for the presence of acetyl group and some changes focused on those signals originating from the terminal glucose that attached to C-2″ of the inner glucose. The downfield chemical shift at δC 79.2 (C-3‴) and up field chemical shifts at 74.3 (C-2‴) and 68.7 (C-4‴) in the 13C NMR spectra suggested the acetyl group attached to C-3‴ position. This was further confirmed by HMBC correlations between H-3‴ (δH 5.70) and C-2‴ (δC 74.3), C-4‴ (δC 68.7), and C=O (δC 171.0 ppm), as well as the H–H COSY correlations between H-2‴ (δH 3.98) and H-3‴ (δH 5.70), H-1‴ (δH 5.59). Consequently, 13 was elucidated as (25R)-5α-

spirostan-3β-yl-3-O-acetyl-O-β-D-glucopyranosyl-(1→2)-O-[βD-glucopyranosyl-(1→3)]-O-β-D- glucopyranosyl-(1→4)-β-Dgalactopyranoside. The HR-ESI-MS and NMR data for compound 15 suggested a molecular formula of C45H72O19. Acid hydrolysis of 15 afforded D-glucose and D-galactose. Two signals at δC 144.6 and 109.0 were a pair of olefinic carbons, which were assigned to the C-25 and C-27 positions respectively (He, Qiu, Shoyama, Tanaka, & Yao, 2002). The 1H and 13C NMR of 15 were almost identical to those of (25R,S)-5α-spirostane-2α,3β-diol-3-O-{Oβ-D-glucopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-β-Dgalactopyranoside} (Hong et al., 2012) except for some signals originating from the F ring. These indicated that 15 was a 25(27)ene steroidal saponin. The location of Δ25(27) was further verified by HMBC correlations between H2-27 (δH 4.81 and 4.77) and C-24 (δC 29.1) and C-26 (δC 65.2). The linkages of the sugar residues were also unambiguously confirmed by the HMBC experiment. The HMBC correlations between H-1‴ (δH 5.27) of the terminal glucose and C-2″ (δC 86.1) of the inner glucose, H-1″(δH 5.13) of the inner glucose and C-4′(δC 81.1) of galactose, H-1′(δH 4.94) of galactose and C-3 (δC 84.7) of aglycone revealed a saccharide chain of β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl(1→4)-β-D-galactopyranosyl attached to C-3 of the aglycone. Based on the above evidence, the structure of 15 was identified to be 5α-spirostane 25(27)-ene-2α,3β-diol-3-O-{O-β-Dglucopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-β-Dgalactopyranoside}.

217

Journal of Functional Foods 25 (2016) 208–219

Table 5 – Inhibitory activities of the isolated compounds 1–10, 12–17 from the bulbs of Chinese onion on LPSinduced NO production in murine macrophage RAW264.7.a Compd.

IC50 (µM)

Compd.

IC50 (µM)

1 2 3 4 5 6 7 8 9 10

>100 88.12 ± 4.54 >100 32.20 ± 0.65 59.56 ± 5.11 65.94 ± 1.95 >100 34.33 ± 5.04 >100 >100

12 13 14 15 16 17

>100 >100 >100 >100 >100 >100

Indomethacinb

47.40 ± 4.50

a b

Values are the mean ± SD; n = 3. Indomethacin was used as positive control.

3.2.

Anti-inflammatory activity

Except for compound 11 which was too few and not available for screening, all the isolated compounds were evaluated for their inhibitory effects on the LPS induced NO production in murine macrophage RAW264.7 cells. As shown in Table 5, compounds 4 and 8 showed potential inhibitory effects against NO production with IC 50 values of 32.20 ± 0.65 and 34.33 ± 5.04 µM respectively. Compounds 2, 5 and 6 exhibited moderate inhibitory effects with IC50 values of 59.56 ± 5.11, 88.12 ± 4.54, and 65.94 ± 1.95 µM respectively.

3.3.

Anti-proliferative activity

The pure compounds were also evaluated for their antiproliferative activity against five human cancer cell lines (HepG2,

A549, SPC-A-1, CNE-1, MGC80-3) and a human lung fibroblast cell line MRC-5 (used as normal cell). The results were summarized in Table 6. Among the tested compounds, 13–17 showed inhibitory activities against all the five tumour cell lines, with IC50 values below 25 µM. The new saponin 13 exhibited the strongest inhibition against these cancer cells with IC50 values of 1.43–6.81 µM. However, when tested against the normal cells MRC-5, compound 13 also showed significant cytotoxicity (IC50 = 2.15 ± 0.63 µM). Compound 14 exhibited a significant selective inhibition against cancer cells with IC50 values ranging from 2.22 to 15.58 µM, in comparison with the normal cells MRC-5 (IC50 > 100 µM). Steroidal saponins are considered as an important class of secondary metabolites in many food and medicinal plants, and were found to have a variety of pharmacological qualities, such as cytotoxic, haemolytic, anti-platelet aggregation, antiinflammatory, and anti-bacterial activities (Sparg, Light, & Staden, 2004). In the present study, spirostanol saponins isolated from the medicinal food Chinese onion were found to exert pronounced anti-inflammatory and anti-proliferative activities, and the inhibition effects were sensitive to the change of their structures. In the NO production assay, some observation could be concluded. Firstly, it seems that the introduction of a carbonyl group in the B-ring is in generally favourable to the inhibitory activity. However, hydroxylation at C-24 position led to the disappearance of inhibitory activity in glycoside 10 (IC50 > 100 µM). Secondly, the spatial conformation of the A and B-rings significantly affects the inhibitory activity. For example, compound 6 (5α-H) showed moderate inhibition (IC50 = 65.94 ± 1.95 µM) while compound 7 (5β-H) was inactive (IC50 > 100 µM). Moreover, the sugar moiety is also important for NO inhibitory activity. Laxogenin 3-O-β-D-glucopyranoside (2) exhibited moderated inhibition. The presence of an α-Larabinose moiety at the C-6 position of the glucose significantly

Table 6 – Anti-proliferative activities of the isolated compounds 1–10, 12–17 from the bulbs of Chinese onion against HepG2, SPC-A-1, A549, CNE-1, MGC80-3 and MRC-5 cells.a Compd.

1 2 3 4 5 6 7 8 9 10 12 13 14 15 16 17 Cisplatinb a b

IC50 (µM) HepG2

SPC-A-1

A549

CNE-1

MGC80-3

MRC-5

39.21 ± 1.25 85.18 ± 4.65 72.38 ± 1.67 81.64 ± 7.52 76.57 ± 1.77 25.29 ± 3.09 79.15 ± 18.46 36.79 ± 5.86 46.16 ± 8.12 80.11 ± 10.24 46.09 ± 1.52 2.35 ± 0.08 2.22 ± 0.17 5.52 ± 0.04 15.51 ± 1.27 3.87 ± 0.22 3.48 ± 0.31

>100 >100 >100 83.91 ± 0.95 >100 69.08 ± 2.63 >100 74.59 ± 1.77 >100 >100 45.03 ± 3.14 5.09 ± 1.07 10.80 ± 1.19 22.32 ± 0.60 18.63 ± 1.03 15.38 ± 1.06 7.22 ± 0.71

>100 >100 >100 92.90 ± 0.86 90.43 ± 7.16 56.14 ± 2.70 >100 >100 >100 >100 84.18 ± 9.23 1.43 ± 0.16 15.58 ± 6.34 15.42 ± 1.31 19.36 ± 5.78 10.63 ± 0.96 4.07 ± 0.40

60.27 ± 1.61 >100 >100 46.93 ± 1.36 >100 46.25 ± 0.73 >100 68.52 ± 0.80 >100 >100 89.45 ± 6.80 1.8 ± 1.11 3.50 ± 0.35 6.63 ± 1.06 9.68 ± 2.01 6.80 ± 0.56 7.14 ± 0.77

>100 >100 79.30 ± 7.60 66.00 ± 0.46 >100 72.39 ± 0.75 >100 66.16 ± 4.75 >100 >100 50.79 ± 0.30 6.81 ± 1.81 12.14 ± 0.92 11.44 ± 1.16 12.64 ± 0.53 4.76 ± 0.29 10.22 ± 1.43

>100 >100 >100 >100 >100 >100 >100 80.40 ± 9.29 >100 >100 >100 2.15 ± 0.63 >100 11.97 ± 1.24 3.62 ± 0.38 25.17 ± 4.32

Values are the mean ± SD; n = 3. Cisplatin was used as positive control.

218

Journal of Functional Foods 25 (2016) 208–219

increased the activity (compound 4: IC50 = 32.20 ± 0.65 µM), while the introduction of a β-D-xylose to the C-4 position of the glucose led to the disappearance of the activity (compound 3: IC50 > 100 µM). When incorporated an α-L-arabinose to the C-6 position and a β-D-xylose to the C-4 position of glucose at the same time, compound 6 showed moderate activity with IC50 = 65.94 ± 1.95 µM. Intriguingly, when a β-Dglucose instead of β-D-xylose was introduced to the C-4 position of glucose, compound 8 displayed strong inhibitory activity (IC 50 = 34.33 ± 5.04 µM). However, further attachment of a β-D-glucose to C-3 of the β-D-xylose caused significant reduction of the activity (compound 9: IC50 > 100 µM). It is also interesting to note that acetylation of C-4 OH of the α-Larabinose slightly decreased the activity (compound 9: IC50 = 59.56 ± 5.11 µM). In the cytotoxic bioassay, analysis of Table 5 provides important information regarding the effect of aglycone functionalization as well as the variation of the sugar moiety on the cytotoxicity of spirostanol saponin. Firstly, regardless the difference between the sugar chains, the incorporation of polar oxygenated groups (C=O) into C-6 of B-ring of compounds 1–10 led to significant reduction of the inhibitory activity, when compared to compounds 12–17. This is in good agreement with literature report that 5α-hydroxy-laxogenylβ-D-glucoside and hecogenyl-β-D-glucoside showed poor cytotoxicity (PerezLabrada et al., 2012). Besides, in the case of compounds 4 and 10, which possess the same sugar chain, hydroxylation of F-ring at C-24 position significantly reduced the activity. Secondly, the new compound 7 (5β-H) showed weak inhibition against all the five selected cancer cell lines (IC50 > 79 µM), while its stereoisomer compound 6 (5α-H) showed moderate inhibition against these cells. This suggests that the spatial conformation of the A- and B-rings considerably affects the cytotoxicity. In contrast to the literature reported that a 5β-H steroidal sapogenin smilagenin exhibited strong inhibition while its stereoisomer tigogenin induced moderate inhibition (Trouillas, Corbière, Liagre, Duroux, & Beneytout, 2005). This difference may be due to the presence of carbonyl group at the C-6 position and the sugar chain at the C-3 position. Thirdly, among the analogous of laxogenyl β-D-glycoside (2–10), laxogenin 3-O-β-D-glucopyranoside (2) was not active (IC50 > 80 µM); an additional β-D-xylose moiety at C-4 (3) or α-Larabinose moiety at the C-6 (4) of the glucose slightly increased the activity. When the β-D-xylose and α-L-arabinose presented at the same time in compound 6, considerable increase of the inhibition was observed (IC50 values ranging from 25.29 to 72.39 µM). However, when another β-D-glucose was attached to the C3-OH position of D-xylose in compound 9, the inhibitory activity generally disappeared (IC50 > 100 µM), except against HepG2 cells (IC50 = 46.16 ± 8.12 µM). It is also interesting to note that acetylation of C4-OH of the L-arabinose in compound 5 caused only a slight increase of inhibition against HepG2 and A549 cells (IC50 values decreased 2.47 and 5.07 µM respectively), but led to significant reduction of the inhibition against SPC-A-1, CNE-1 and MGC80-3 cells. This information suggests that selective acetylation of the hydroxyl group of sugar moiety could considerably influence the cytotoxicity of steroidal saponins. Although further SAR study that focuses on functionalization and stereoisomer in the spirostanol aglycone, as well as the

variation of the sugar moiety, must be carried out, our results along with previous studies confirm that anti-inflammatory and anti-proliferative activities of spirostanol saponins are influenced both by the aglycone and the sugar moiety.

4.

Conclusions

Seventeen steroidal saponins derivatives, including seven new compounds, were isolated from Chinese onion. Compounds 4 and 8 showed potential inhibitory effects on the LPS induced NO production in RAW264.7 cells. Compound 13 displayed potential inhibitory activity against five human cancer cell lines (HepG2, A549, SPC-A-1, CNE-1, MGC80-3), while 14 exhibited significant selective inhibition against these cancer cells in comparison with the normal cells MRC-5. Anti-inflammatory and anti-proliferative activities of spirostanol saponins are influenced both by the aglycone and the sugar moiety. This study provides a basis for further development and utilization of this medicinal food as natural nutraceuticals and functional food ingredients, or as source of new potential anti-inflammatory or antitumour chemotherapy agent.

Conflict of interest The authors declare that there was no competing financial interest.

Acknowledgements This research was supported by 2013 Special Scientific Research Fund for the Doctoral Program of Higher Education, Ministry of Education of the People’s Republic of China (No. 20130141110050) and the National Natural Science Foundation of China (No. 81573303). The authors also greatly acknowledge Prof. Dr. Hao Gao, Jinan University, for his generous help in the measurement of optical rotation.

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2016.06.005.

REFERENCES

Achenbach, H., Hübner, H., Brandt, W., & Reiter, M. (1994). Cardioactive steroid saponins and other constituents from the aerial parts of Tribulus cistoides. Phytochemistry, 35, 1527– 1543. Albishi, T., John, J. A., Al-Khalifa, A. S., & Shahidi, F. (2013). Antioxidative phenolic constituents of skins of onion varieties and their activities. Journal of Functional Foods, 5, 1191–1203.

Journal of Functional Foods 25 (2016) 208–219

Baba, M., Ohmura, M., Kishi, N., Okada, Y., Shibata, S., Peng, J., Yao, S. S., Nishino, H., & Okuyama, T. (2000). Saponins isolated from Allium chinense G. Don and antitumor-promoting activities of isoliquiritigenin and laxogenin from the same drug. Biological and Pharmaceutical Bulletin, 23, 660–662. Block, E. (1992). The organosulfur chemistry of the genus Allium– Implications for the organic chemistry of sulfur. Angewandte Chemie, 31, 1135–1178. Block, G., Patterson, B., & Subar, A. (1992). Fruit, vegetables, and cancer prevention – A review of the epidemiologic evidence. Nutrition and Cancer, 18, 1–29. Chinese Pharmacopeia Commission. (2010). Pharmacopoeia of the People’s Republic of China 2010 (Vol. 1). Beijing: Chinese Medical Science and Technology Press. Chung, H. Y., Cesari, M., Anton, S., Marzetti, E., Giovannini, S., Seo, A. Y., Carter, C., Yu, B. P., & Leeuwenburgh, C. (2009). Molecular inflammation: Underpinnings of aging and age-related diseases. Ageing Research Reviews, 8, 18–30. Eddy, C., Wall, M., & Scott, M. (1953). Catalog of infrared absorption spectra of steroidal sapogenin acetates. Analytical Chemistry, 25, 266–271. Fenwick, G. R., Hanley, A. B., & Whitaker, J. R. (1985). The genus Allium—Part 1. CRC Critical Reviews in Food Science and Nutrition, 22, 199–271. He, X. J., & Liu, R. H. (2007). Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple’s anticancer activity. Journal of Agricultural and Food Chemistry, 55, 4366–4370. He, X. J., Qiu, F., Shoyama, Y., Tanaka, H., & Yao, X. S. (2002). Two new steroidal saponins from “Gualou-xiebai-baijiu-tang” consisting of Fructus trichosanthis and Bulbus allii macrostemi. Chemical & Pharmaceutical Bulletin, 50, 653–655. Hong, X. X., Luo, J. G., Guo, C., & Kong, L. Y. (2012). New steroidal saponins from the bulbs of Lilium brownii var. viridulum. Carbohydrate Research, 361, 19–26. Jiang, Y., Wang, N. L., Yao, X. S., & Susumu, K. (1998). Structural elucidation of the anticoagulation and anticancer constituents from Allium chinense. Acta Pharmaceutica Sinica, 33, 355–361. Kuroda, M., Mimaki, Y., Kameyama, A., Sashida, Y., & Nikaido, T. (1995). Steroidal saponins from Allium chinense and their inhibitory activities on cyclic AMP phosphodiesterase and Na+K+ ATPase. Phytochemistry, 40, 1071–1076. Lawrence, T., Willoughby, D. A., & Gilroy, D. W. (2002). Antiinflammatory lipid mediators and insights into the resolution of inflammation. Nature Reviews. Immunology, 2, 787–795. Lim, T. K. (2015). Edible medicinal and non medicinal plants: Modified stems, roots, bulbs (Vol. 9). Dordrecht: Springer Science+Business Media. Liu, R. H. (2003). Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. The American Journal of Clinical Nutrition, 78, 517S–520S. Mann, L. K., & Stearn, W. T. (1960). Rakkyo or Ch’iao T’ou (Allium chinense G. Don, Syn. A. Bakeri Regel) a little known vegetable crop. Economic Botany, 14, 69–83. Matsuura, H., Ushiroguchi, T., Itakura, Y., & Fuwa, T. (1989). A furostanol glycoside from Allium chinense G. Don. Chemical & Pharmaceutical Bulletin, 37, 1390–1391. Miranda, K., Espey, M., & Wink, D. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide: Biology and Chemistry/Official Journal of the Nitric Oxide, 5, 62–71. Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41, 1523–1542. Okuyama, T., Fujita, K., Shibata, S., Hoson, M., Kawada, T., Masaki, M., & Yamate, N. (1989). Effect of Oriental plant drugs on platelet aggregation. V. Effects of Chinese drugs “Xiebai” and

219

“Dasuan” on human platelet aggregation (Allium bakeri, A. sativum). Planta Medica, 55, 242–244. Peng, J. P., Yao, X. S., Tezuka, Y., & Kikuchi, T. (1996). Furostanol glycosides from bulbs of Allium chinense. Phytochemistry, 41, 283–285. Peng, J. P., Yao, X. S., Tezuka, Y., Kikuchi, T., & Narui, T. (1996). New furostanol glycosides, chinenoside IV and V, from Allium chinense. Planta Medica, 62, 465–468. PerezLabrada, K., Brouard, I., Estevez, S., Marrero, M., Estevez, F., Bermejo, J., & Rivera, D. (2012). New insights into the structure-cytotoxicity relationship of spirostan saponins and related glycosides. Bioorganic & Medicinal Chemistry, 20, 2690– 2700. Pino, J., Fuentes, V., & Correa, M. (2001). Volatile constituents of Chinese chive (Allium tuberosum Rottl. ex Sprengel) and Rakkyo (Allium chinense G. Don). Journal of Agricultural and Food Chemistry, 49, 1328–1330. Qiao, A. M., Wang, Y. H., Xiang, L. M., Zhang, Z. X., & He, X. J. (2015). Novel triterpenoids isolated from hawthorn berries functioned as antioxidant and antiproliferative activities. Journal of Functional Foods, 13, 308–313. Ren, G., Qiao, H. X., Yang, J., & Zhou, C. X. (2010). Protective effects of steroids from Allium chinense against H2O2-induced oxidative stress in rat cardiac H9C2 cells. Phytotherapy Research, 24, 404–409. Sparg, S. G., Light, M. E., & Staden, J. V. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219–243. Sun, Y. N., Li, W., Yang, S. Y., Kang, J. S., Ma, J. Y., & Kim, Y. H. (2016). Isolation and identification of chromone and pyrone constituents from Aloe and their anti-inflammatory activities. Journal of Functional Foods, 21, 232–239. The Angiosperm Phylogeny Group. (2009). An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society, 161, 105–121. Trouillas, P., Corbière, C., Liagre, B., Duroux, J., & Beneytout, J. (2005). Structure–function relationship for saponin effects on cell cycle arrest and apoptosis in the human 1547 osteosarcoma cells: A molecular modelling approach of natural molecules structurally close to diosgenin. Bioorganic & Medicinal Chemistry, 13, 1141–1149. Wang, C. H., Zhang, Z. X., Wang, Y. H., & He, X. J. (2015). Cytotoxic indole alkaloids against human leukemia cell lines from the toxic plant Peganum harmala. Toxins, 7, 4507. Wang, F. Z., & Tang, J. (1980). Flora of China (Vol. 14). Beijing: Science Publishing House. Wang, Y. H., Xiang, L. M., Wang, C. H., Tang, C., & He, X. J. (2013). Antidiabetic and antioxidant effects and phytochemicals of mulberry fruit (Morus alba L.) polyphenol enhanced extract. PLoS ONE, 8, e71144. Willett, W. C. (1994). Diet and health – What should we eat. Science, 264, 532–537. Woo, M., Do, J., & Son, K. (1992). Five new spirostanol glycosides from the subterranean parts of Smilax sieboldii. Journal of Natural Products, 55, 1129–1135. Wu, Y., Peng, J. P., Yao, X. S., & Okuyama, T. (1992). Macrostemonoside A: A new spirostane saponin from Allium macrostemon Bge. Shenyang Yao Ke Da Xue Xue Bao, 9, 69–70. Xiang, L. M., Wang, Y. H., Yi, X. M., Feng, J. Y., & He, X. J. (2016). Furospirostanol and spirostanol saponins from the rhizome of Tupistra chinensis and their cytotoxic and anti-inflammatory activities. Tetrahedron, 72, 134–141. Yu, Z., Zhang, T., Zhou, F., Xiao, X., Ding, X., He, H., Rang, J., Quan, M., Wang, T., Zuo, M., & Xia, L. (2015). Anticancer activity of saponins from Allium chinense against the B16 melanoma and 4T1 breast carcinoma cell. Evidence-based Complementary and Alternative Medicine, 2015, 725023.