furostanol saponins from the seeds of allium cepa l

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FITOTE-03019; No of Pages 8

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Fitoterapia

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FFurostanol saponins from the seeds of Allium cepa L.

Chuang-Jun Li 1, Ling Yuan 1, Teng-Fei Ji, Jian-Bo Yang, Ai-Guo Wang, Ya-Lun Su⁎State Key Laboratory of Bioactive Substance and Function of NaturalMedicines, Institute ofMateriaMedica, Chinese Academy ofMedical Sciences and PekingUnionMedical College,Beijing 100050, People's Republic of China

a r t i c l e i n f o

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⁎ Corresponding author at: Institute of Materia MedicMedical Sciences and Peking Union Medical College, 1Xicheng District, Beijing 100050, People's Republic o63165327; fax: +86 10 631017757.

E-mail addresses: [email protected] (C.-J. Li), s(Y.-L. Su).

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fitote.2014.08.0220367-326X/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Li C-J, et al, Furos10.1016/j.fitote.2014.08.022

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Article history:Received 17 June 2014Accepted in revised form 21 August 2014Available online xxxx

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Allium cepa L. is one of themost widely cultivated and used plants. In addition to its bulb (onion),which is used as food in many cultures, the seeds of A. cepa L. are used as a traditional herbalmedicine by the Uygur nationality in China to treat diarrhea and promote blood flow. In abioactivity-screening, the ethanol extract of seeds ofA. cepa L. showed inhibitory effects on proteintyrosine phosphatase 1B (PTP1B) enzyme, with 81.1% inhibition. Phytochemical investigation ofthe ethanol extract of red onion (Allium cepa L.) seeds led to the isolation of eight new furostanolsaponins, named ceparosides E–L (1–8). Their structures were established using 1D and 2D NMRspectroscopy, mass spectrometry and chemical methods. Compounds 1–8 were screened forinhibitory effects on the PTP1B enzyme and cytotoxic activity against five human cells, includingHCT-8, Bel-7402, BGC-823, A549 and A2780, but all were found to be inactive.

© 2014 Elsevier B.V. All rights reserved.

Keywords:LiliaceaeAllium cepa LFurostanol saponinsCeparosides E–L

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Allium cepa L. is one of the most widely cultivated and usedplants, and its bulb (onion) is used both as food and medicine. Ithas been shown that onion possesses various biological activities,including antibiotic, antidiabetic, antioxidant, antiatherogenic andanticancer activities [1]. Our co-researcher has found that onionextract can dose-dependently decrease plasma total cholesterol(TC) levels [2]. Furostanol saponins were recognized as majorconstituents of the Allium genus [1,3–5] and some of themexhibited cytotoxic activity in vitro against several human tumorcell lines [6]. The seeds of A. cepa L. were used as a traditionalherbal medicine by the Uygur nationality in China and canimprove the functions of internal organs, treat diarrhea,fervescence, calenture, and puffiness of the face and eyes, andpromote blood flow [7]. HPLC analysis of the n-butanol fraction of

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a, Chinese Academy ofXian Nong Tan Street,f China. Tel.: +86 10

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tanol saponins from the

the ethanol extract revealed the presence of saponins, whichwere considered the bioactive constituents of onion. Our previousstudy on an ethanol extract of the seeds of A. cepa L. reported foursaponins [8,9]. In a continuing search, we found that the ethanolextract of onion seeds had conspicuous activity in inhibiting thePTP1Benzyme invitro. To clarify thebioactivity of the compoundsof the ethanol extract, a detailed chemical investigation wasconducted to identify the chemical constituents of this extract. Asa result, eight new furostanol saponins were obtained (Fig. 1). Inthis work, we described the isolation, structure determination,and biological activity evaluation of compounds 1–8.

2. Experimental part

2.1. General experimental procedures

Optical rotations were measured on a P2000 automaticdigital polarimeter. IR spectra were recorded on a Nicolet 5700FT-IR spectrometer. NMR spectroscopic data were recorded onINOVA-500, and Bruker AV600 spectrometers in pyridine-d5.ESI-MS (LC–MS) spectra were measured on an Agilent 1100Series LC/MSDtrapmass spectrometer (USA).HRESI-MSdatawererecorded using an Agilent 6520 Accurate-Mass Q-TOF LC/MSspectrometer (USA). Preparative HPLC was performed on a

seeds of Allium cepa L., Fitoterapia (2014), http://dx.doi.org/

http://dx.doi.org/10.1016/j.fitote.2014.08.022

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mailto:[email protected]

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Fig. 1. Chemical structures of compounds 1–8.

2 C.-J. Li et al. / Fitoterapia xxx (2014) xxx–xxx

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RRShimadzu LC-6AD instrument with an SPD-20A detector

using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm).Sephadex LH-20 (GE Healthcare, Sweden), ODS (50 μm,YMC, Japan), macroporous resin D101 (20–60 mesh, TianjinHaiguang Chemistry Company, Tianjin, China) and silica gel(200–300 mesh, Qingdao Marine Chemical Inc., Qingdao,China) were used for column chromatography. TLC wascarried out with glass precoated silica gel GF254 glass plates.

2.2. Plant materials

Red onion (A. cepa L.) seeds were purchased in Xinjiang(Autonomous Region), China, in 2007 and identified byProfessor Yan-Fu Zhang of the Institute of Xinjiang MateriaMedica. A voucher specimen (No. 050219) has been depositedin the herbarium at the Department of Medicinal Plants,Institute of Materia Medica, Chinese Academy of MedicalSciences.

2.3. Extraction and isolation

Red onion (A. cepa L.) seeds (14.75 kg) were powdered anddefatted with petroleum ether (3 × 3 L), and the residue wasextracted with 95% EtOH under reflux (3 × 120 L). The filtratewas evaporated under reduced pressure to yield a dark brown

Please cite this article as: Li C-J, et al, Furostanol saponins from the10.1016/j.fitote.2014.08.022

residue (2100 g). The residuewas suspended inwater (6 L) andthen partitioned with EtOAc (3 × 3 L), and n-BuOH (3 × 3 L),successively. After removing the solvent, the n-butanol portion(100 g) was subjected to column chromatography onmacroporous resin with an EtOH–H2O gradient (0, 30%, 50%,70%, and 95%) to give five fractions (Fractions I–V). Fraction III(eluted with 50% EtOH) (20 g) was separated on a C-18medium-pressure column eluted with a MeOH-H2O gradient(30–100%). The 70% MeOH eluted fraction was separated on aC-18 medium pressure column eluted with MeOH–H2O(50–100%) and then purified with preparative HPLC (elutedwith 70% MeOH–H2O) repeatedly to give compounds 1(20 mg), 2 (40 mg), 3 (8 mg), 4 (5 mg), 5 (50 mg), 6 (30 mg),7 (9 mg), and 8 (6 mg) successively.

2.3.1. Ceparocide E (1)White amorphous powder, [α]D20 −59 (c 0.01, MeOH); IR

νmax (cm−1): 3378, 2935, 2902, 1691, 1642, 1452, 1376,1258, 1161, 1045, 911 and 638; 1H NMR (pyridine-d5,500 MHz) and 13C NMR (pyridine-d5, 125 MHz) data aregiven in Tables 1 and 2; ESI-MS m/z: 1101 [M + Na]+, 1077[M − H]−; ESI-MSn m/z: 915 [M − H − 162]−, 769 [M −H − 162 − 146]−, 607 [M − H − 162 − 146 − 162]−. HR-FAB-MS (positive ionmode)m/z: 1101.5405 [M+Na]+ (calcd.for C52H86O23Na, 1101.5458).


image of Fig.�1



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2.3.2. Ceparocide F (2)White amorphous powder, [α]D20 −66 (c 0.01, MeOH); IR

νmax (cm−1): 3364, 2935, 2900, 1646, 1451, 1376, 1258, 1161,1044, 989 and 637; 1H NMR (pyridine-d5, 500 MHz) and 13CNMR (pyridine-d5, 125 MHz) data are given in Tables 1 and 2;ESI-MS m/z: 1087 [M + Na]+, 1063 [M − H]−; HR-FAB-MS(positive ion mode) m/z: 1087.5249 [M + Na]+ (calcd. forC51H84O23Na, 1087.5301).

2.3.3. Ceparocide G (3)White amorphous powder, [α]D20 −45 (c 0.01, MeOH); IR

νmax (cm−1): 3352, 2925, 1690, 1650, 1449, 1375, 1048, 912and 633; 1H NMR (pyridine-d5, 600 MHz) and 13C NMR(pyridine-d5, 150 MHz) data are given in Tables 1 and 2; ESI-MSm/z: 909 [M+Na]+, 885 [M − H]−; HR-FAB-MS (negativeion mode) m/z: 885.4411 [M − H]− (calcd. for C44H69O18,885.4484).

2.3.4. Ceparocide H (4)White amorphous powder, [α]D20 −43 (c 0.01, MeOH); IR

νmax (cm−1): 3367, 2927, 1692, 1649, 1450, 1375, 1050, 912and 636; 1H NMR (pyridine-d5, 600 MHz) and 13C NMR(pyridine-d5, 150 MHz) data are given in Tables 1 and 2; ESI-MSm/z: 909 [M+Na]+, 885 [M − H]−; HR-FAB-MS (negativeion mode) m/z: 885.4485 [M − H]− (calcd. for C44H69O18,885.4484).

2.3.5. Ceparocide I (5)White amorphous powder, [α]D20 −31 (c 0.01, MeOH); IR

νmax (cm−1): 3395, 2912, 1692, 1645, 1451, 1378, 1072, 1050,913 and 635; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR(pyridine-d5, 125MHz) data are given in Tables 1 and 2; ESI-MSm/z: 893 [M+ Na]+, 869 [M − H]−; HR-FAB-MS (positive ionmode) m/z: 893.4535 [M + Na]+ (calcd. for C44H70O17Na,893.4511).

2.3.6. Ceparocide J (6)White amorphous powder, [α]D20 −18 (c 0.01, MeOH); IR

νmax (cm−1): 3394, 2910, 1691, 1646, 1450, 1377, 1048, 913and 636; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR(pyridine-d5, 125 MHz) data are given in Tables 1 and 2; ESI-MS m/z: 893 [M+ Na]+, 869 [M − H]−. HR-FAB-MS (positiveion mode) m/z: 893.4576 [M + Na]+ (calcd. for C44H70O17Na,893.4511).

2.3.7. Ceparocide K (7)White amorphous powder, [α]D20 −64 (c 0.01, MeOH); IR

νmax (cm−1): 3361, 2910, 1689, 1649, 1450, 1376, 1044, 913and 634; 1H NMR (pyridine-d5, 600 MHz) and 13C NMR(pyridine-d5, 150 MHz) data are given in Tables 1 and 2; ESI-MS m/z: 923.5 [M+ Na]+, 899.5 [M − H]−; ESI-MSn m/z: 753[M − H − 146]−, 591 [M − H − 146 − 162]−, 429 [M −H − 146 − 162 − 162]−. HR-FAB-MS (negative ion mode)m/z: 899.4590 [M − H]− (calcd. for C45H71O18, 899.4640).

2.3.8. Ceparocide L (8)White amorphous powder, [α]D20 −64 (c 0.01, MeOH); IR

νmax (cm−1): 3379, 2934, 1647, 1452, 1376, 1064, 912 and 636;1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5,125 MHz) data are given in Tables 1 and 2; ESI-MS m/z: 941[M + Na]+, 917 [M − H]−; ESI-MSn m/z: 771 [M − H −


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146]−, 609 [M − H − 146 − 162]−, 447 [M − H − 146 −162 − 162]−. HR-FAB-MS (positive ion mode) m/z: 941.4786[M+ Na]+ (calcd. for C45H74O19 Na, 941.4722).

2.4. Acid hydrolysis and sugar analysis

Saponin (2mg) was dissolved in 2 N HCl (2mL) and stirredat 80 °C for 4 h. The reactionmixture was extractedwith CHCl3,and the aqueous layer was evaporated to give a mixture ofmonosaccharides. The residue was further dissolved in anhy-drous pyridine (1 mL) followed by the addition of L-cysteinemethyl ester hydrochloride (2 mg). After heating at 60 °C for2 h, the solvent was eliminated under N2, and trimethyl-silylimidazole (0.2 mL) was added. Next, the mixture washeated at 60 °C for another 2 h and partitioned with n-hexaneand water. The organic layer was analyzed by gas chromatog-raphy (GC) under the following conditions: capillary column,HP-5 (30 m × 0.25 mm × 0.25 μm, Dikma); FID detector at280 °C; injection temperature, 250 °C; initial temperature of160 °C raised to 280 °C at 5 °C/min, final temperature main-tained for 10 min; carrier gas, N2. The standard sugars weresubjected to the same reaction and GC conditions. The retentiontimes of persilylated L-rhamnose, D-glucose, D-galactose andL-arabinose were found to be 20.76 min, 22.35 min, 22.55 minand 20.46 min, respectively.

2.5. PTP1B activity assay

Recombinant human GST-PTP1B proteinwas over expressedby hGST-PTP1B-BL21 Escherichia coli and purified by GST affinitychromatography. The reagent pNPP was used as a substrate forthe measurement of PTP1B activity. Compounds were pre-incubated with the enzyme at room temperature for 5 min.Assays were performed in a final volume of 100 μL in the activesystem containing 50 mM HEPES, 5 mM DTT, 150 mM NaCl,2 mM EDTA and 2 mM pNPP (pH 7.0), incubated at 30 °C for10 min and stopped by the addition of 50 μL of 3 M NaOH. Theabsorbance was then determined at a wavelength of 405 nm. Asimilar system without GST-PTP1B protein was used as a blank.The effects of the ethanol extract of A. cepa L. seeds andcompounds 1–8 on PTP1B activity were measured [10].

2.6. Cytotoxicity assay

The cytotoxic activity of compounds 1–8 were evaluatedusing lung adenocarcinoma (A549), stomach cancer (BGC-823), hepatoma (BEL-7402), colon cancer (HCT-8) and hela(A2780) cell lines. Paclitaxel was used as a positive control.Following 72 h of continuous treatment of the cells with thesamples, the supernatant was doffed off and 0.1 mL of MTT(0.5 mg/mL in RPM1640) was added after each well had beencarefully washed with RPM1640. The cell growth was mea-sured with an MTT assay procedure [11], and the IC50 valueswere calculated from a dose-dependent curve for A 549, BGC-823, BEL-7402, HCT-8 and A2780 cell lines.

3. Results and discussion

The n-butanol fraction of the ethanol extract of onion seedswas subjected to a succession of chromatographic procedures,




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t1:1 Table 1t1:2 aH NMR data for compounds 1–8 in pyridine-d5 (δ in ppm, J in Hz).

t1:3 No 1a 2a 3b 4b 5a 6a 7b 8a

t1:4 1 0.97 m1.72 m

0.98 m1.71 m

3.81 dd (12.4, 3.6) 3.81 brd (12.0) 3.82 dd (11.5, 3.0) 3.84 dd (11.5, 3.0) 3.86 dd (12.0, 3.6) 3.84 dd (12.0, 4.0)

t1:5 2 2.08 m2.72 m

2.14 m2.65 m

2.35 m2.72 m

2.36 m2.72 m

2.38 m2.76 m

2.36 m2.71 m

2.43 m2.70c

2.40 m2.68 m

t1:6 3 3.88 m 3.94 m 3.85 m 3.86 m 3.85 m 3.85 m 3.80c 3.80 mt1:7 4 1.94c

2.76c2.04c

2.73c2.55 m2.68 m

2.56 dd (10.5, 3.0)2.67 m

2.58 dd (10.5, 3.0)2.70 m

2.57 m2.67 m

2.55 dd (12.6, 3.6)2.66 m

2.53 m2.68 m

t1:8 6 5.31 d (4.0) 5.30 d (4.0) 5.57 d (4.8) 5.57 d (4.8) 5.58 d (4.5) 5.58 d (5.0) 5.56 d (5.4) 5.58 d (5.0)t1:9 7 1.49 m

2.01 m1.51 m2.11 m

1.48 m2.01 m

1.48 m2.04 m

1.45 m2.08 m

1.45 m2.08 m

1.50 m2.06 m

1.56 m2.07 m

t1:10 8 1.92 m 1.89 m 1.86 m 1.83 m 1.52 m 1.52 m 1.50 m 1.88 mt1:11 9 0.86 m 0.93 m 1.47 m 1.49 m 1.52 m 1.49 m 1.58 m 1.55 mt1:12 11 2.79c

1.43 m2.75c

1.43 m2.95c

1.48 m2.95c

1.43 m2.97 d (13.5)1.45 m

2.97 d (13.5)1.47 m

3.03 brd (11.4)1.46 m

3.02 brd (11.5),1.45 m

t1:13 12 1.37 m1.72 m

1.13 m1.71 m

1.37 m1.77 m

1.37 m,1.60 m

1.30 m1.62 m

1.30 m1.61 m

1.40 m1.63 m

1.40 m1.62 m

t1:14 14 1.05 m 0.88 m 1.02 m 1.04 m 0.90 m 0.90 m 0.97 m 1.14 mt1:15 15 1.88 (2H, m) 1.91 (2H, m) 2.04 (2H, m) 1.88 (2H, m) 1.86 (2H, m) 1.86 (2H, m) 1.90 (2H, m) 1.89 (2H, m)t1:16 16 4.44 m 4.40 m 5.16 m 5.17 m 4.74 m 4.74 m 4.72 m 4.84 mt1:17 17 1.73 m 1.74 m 2.17 d (5.4) 2.32 d (12.0) 2.35 m 2.35 m 2.38 d (10.2) 1.88 mt1:18 18 0.80 s 0.88 s 0.92 s 0.92 s 0.75 s 0.73 s 0.74 s 0.92 st1:19 19 1.04 s 1.06 s 1.44 s 1.43 s 1.43 s 1.43 s 1.45 s 1.42 st1:20 21 1.18 d (6.5) 1.33 d (7.0) 1.65 s 1.64 s 1.58 s 1.56 s 1.55 s 1.21 d (6.5)t1:21 23 2.10 m

2.20 m1.98 m2.22 m

4.49 (t, 7.2) 4.47 (t, 7.2) 2.08 m2.18 m

2.06 m2.17 m

2.14 m2.21 m

2.14 m2.21 m

t1:22 24 1.43 m1.84 m

1.43 m1.84 m

2.40 m2.10 m

2.38 m2.07 m

1.46c

1.84c1.46c

1.84 c1.50c

1.86c1.52 m1.92 m

t1:23 25 1.84c 1.94 m 2.26 m 2.24 m 1.94 m 1.96 m 1.94 m 1.96 mt1:24 26 3.59 m

3.94 m3.61 m3.94 m

3.50 dd (6.0, 9.0) 4.12 m 3.60 dd (6.0, 9.0)3.92 m

3.59 dd (6.0, 9.0), 3.92 m 3.45 dd (7.0, 9.0)4.03 m

3.45 dd (6.6, 9.0)4.02 m

3.44 dd (7.5, 9.0) 4.02 m

t1:25 27 0.99 d (7.0) 0.97 d (6.5) 1.05 d (6.0) 1.05 d (6.0) 1.00 d (6.0) 1.00 d (6.5) 1.00 d (7.2) 0.99 d (6.0)

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t1:26 -OCH3 3.25 st1:27 Gal-1 4.89 d (8.0) 4.87 d (8.0) 4.79 d (7.8) 4.78 d (7.8)t1:28 2 4.54 m 4.53 m 4.65 m 4.65 mt1:29 3 4.26 m 4.24 m 4.20 m 4.20 mt1:30 4 4.58 m 4.58 m 4.50 m 4.59 mt1:31 5 4.23 m 4.20 m 3.92 m 3.92 mt1:32 6 4.17 m, 4.54 m 4.16 m, 4.52 m 4.39 m, 4.53 m 4.39 m, 4.53 mt1:33 Ara-1 4.72 d (7.2) 4.72 d (6.6) 4.73 d (7.5) 4.73 d (7 )t1:34 2 4.61 m 4.61 m 4.59 m 4.60 mt1:35 3 4.17 m 4.17 m 4.16 m 4.16 mt1:36 4 4.17 m 4.17 m 4.16 m 4.16 mt1:37 5 3.65 d (11.4)

4.25 m3.65 d (10.2)4.25 m

3.66 d (12.0)4.27 m

3.66 d (1 5)4.27 m

t1:38 Rha-1 6.25 s 6.26 s 6.35 s 6.35 s 6.34 s 6.33 s 6.41 s 6.33 st1:39 2 4.77 brs 4.76 brs 4.74 brs 4.74 brs 4.72 brs 4.73 brs 4.76 brs 4.73 brst1:40 3 4.58 m 4.58 m 4.64 m 4.63 m 4.63 m 4.63 m 4.66 m 4.63 mt1:41 4 4.31 t (9.5) 4.31 t (9.5) 4.32 t (9.0) 4.32 t (9.0) 4.29 m 4.30 m 4.32 m 4.30 mt1:42 5 4.94 m 4.94 m 4.87 m 4.87 m 4.82 m 4.83 m 4.90 m 4.83 mt1:43 6 1.70 d (6.0) 1.71 d (6.0) 1.75 d (6.6) 1.75 d (6.0) 1.73 d (6.0) 1.73 d (5 ) 1.76 d (6.6) 1.73 d (5.0)t1:44 Glu-1 5.17 d (7.5) 5.17 d (7.5)t1:45 2 4.06 m 4.06 mt1:46 3 4.18 m 4.18 mt1:47 4 4.02 m 4.02 mt1:48 5 3.95 m 3.95 mt1:49 6 4.39c, 4.60c 4.39c, 4.60c

t1:50 Glu-(26)-1 4.84 d (7.5) 4.81 d (7.5) 4.83 d (8.4) 4.81 d (7.8) 4.82 d (8.0) 4.81 d (8 ) 4.82 d (7.8) 4.81 d (8.0)t1:51 2 4.02 m 4.02 m 4.03 m 4.03 m 4.02 m 4.02 m 3.60 m 4.02 mt1:52 3 4.22 m 4.21 m 4.24 m 4.24 m 4.23 m 4.23 m 4.24 m 4.23 mt1:53 4 4.22 m 4.21 m 4.24 m 4.24 m 4.23 m 4.23 m 4.24 m 4.23 mt1:54 5 3.95 m 3.95 m 3.93 m 3.92 m 3.96 m 3.94 m 3.94 m 3.94 mt1:55 6 4.37 c, 4.52c 4.37c, 4.50c 4.39c, 4.53c 4.39c, 4.53c 4.38 c, 4.55c 4.39 c, 4.5 c 4.40 c, 4.54 mc 4.39c, 4.53c

t1:56 a Measured in 500 MHz,t1:57 b Measured in 600 MHz.t1:58 c Overlap with other signals

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(2014),http://dx.doi.org/10.1016/j.fitote.2014.08.022

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t2:1 Table 2t2:2 13C NMR data for compounds 1–8 in pyridine-d5 (δ in ppm).

t2:3 No. 1a 2a 3b 4b 5a 6a 7b 8a

t2:4 1 37.5 37.5 83.7 83.7 83.5 83.5 84.0 84.0t2:5 2 30.2 30.2 37.5 37.5 37.4 37.4 38.0 37.9t2:6 3 77.9 77.9 68.2 68.3 68.2 68.2 68.2 68.1t2:7 4 38.9 39.0 43.9 43.9 43.9 43.9 43.9 43.9t2:8 5 140.9 140.9 139.7 139.6 139.6 139.6 139.7 139.6t2:9 6 121.7 121.8 124.6 124.6 124.7 124.7 124.7 124.7t2:10 7 32.3 32.5 32.6 32.6 34.7 34.7 32.9 32.0t2:11 8 31.6 31.7 31.8 31.8 32.9 32.8 34.8 33.1t2:12 9 50.3 50.4 50.2 50.2 50.4 50.3 50.6 50.6t2:13 10 37.1 37.1 42.8 42.8 42.9 42.8 42.9 42.8t2:14 11 21.0 21.1 23.5 23.5 24.2 24.2 24.2 24.1t2:15 12 39.7 39.9 39.9 39.9 40.2 40.2 40.3 40.5t2:16 13 40.8 40.8 40.1 40.1 43.1 43.1 43.2 40.5t2:17 14 56.5 56.6 57.1 57.2 55.1 55.1 55.4 57.0t2:18 15 32.2 32.4 33.8 33.8 32.1 32.1 32.2 32.7t2:19 16 81.3 81.2 84.3 84.3 84.5 84.5 84.5 81.1t2:20 17 64.1 64.1 68.0 67.9 64.6 64.6 64.7 63.9t2:21 18 16.4 16.4 14.2 14.0 14.4 14.4 14.5 16.9t2:22 19 19.4 19.4 15.0 15.1 15.1 15.1 15.1 15.0t2:23 20 40.5 40.8 76.8 76.8 103.8 103.8 103.8 40.8t2:24 21 16.4 16.4 21.9 21.9 11.8 11.8 11.8 16.3t2:25 22 112.6 110.7 164.0 163.9 152.1 152.2 152.1 110.6t2:26 23 30.8 37.5 91.2 91.6 23.7 23.6 23.7 37.1t2:27 24 28.1 28.4 29.6 29.9 31.5 31.4 31.4 28.2t2:28 25 34.2 34.3 34.8 35.1 33.5 33.7 33.8 34.4t2:29 26 75.2 75.2 75.3 75.4 75.0 75.2 75.3 75.2t2:30 27 17.1 17.5 17.4 17.6 17.3 17.1 17.2 17.4t2:31 OCH3 47.3t2:32 Gal-1 100.4 100.4 100.5 100.5t2:33 2 76.7 76.7 75.1 75.0t2:34 3 76.4 76.4 76.8 76.8t2:35 4 81.2 81.2 70.5 70.4t2:36 5 78.5 78.5 76.4 76.3t2:37 6 62.9 63.1 62.0 61.9t2:38 Ara-1 100.5 100.5 100.3 100.3t2:39 2 75.2 75.3 75.2 75.2t2:40 3 75.9 75.9 75.9 75.9t2:41 4 70.2 70.2 70.1 70.1t2:42 5 67.4 67.4 67.3 67.3t2:43 Rha-1 102.2 102.2 101.8 101.8 101.8 101.7 101.8 101.7t2:44 2 72.4 72.5 72.6 72.6 72.6 72.6 72.7 72.6t2:45 3 72.7 72.8 72.7 72.7 72.7 72.7 72.6 72.6t2:46 4 74.1 74.1 74.3 74.3 74.2 74.2 74.3 74.3t2:47 5 69.5 69.5 69.5 69.5 69.5 69.4 69.4 69.3t2:48 6 18.6 18.6 19.1 19.1 19.0 19.0 19.1 19.0t2:49 Glu-1 107.2 107.2t2:50 2 75.6 75.6t2:51 3 78.8 78.9t2:52 4 72.2 72.2t2:53 5 78.6 78.6t2:54 6 60.9 60.9t2:55 Glu-(26)-1 105.0 104.9 105.2 105.2 104.9 105.2 105.3 105.1t2:56 2 75.2 75.2 75.3 75.3 75.2 75.2 75.3 75.2t2:57 3 78.6 78.6 78.6 78.6 78.6 78.6 78.6 78.6t2:58 4 71.7 71.7 71.7 71.7 71.7 71.7 71.7 71.7t2:59 5 78.5 78.5 78.5 78.5 78.5 78.5 78.6 78.5t2:60 6 62.9 62.8 62.8 62.9 62.9 62.8 62.9 62.8

t2:61 a Measured in 125 MHz.t2:62 b Measured in 150 MHz.


including silica gel column chromatography,MPLC andHPLC toafford eight new furostanol saponins (1–8) (Fig. 1).

Ceparocide E (1) was obtained as a white amorphouspowder. The compound was positive to the Ehrlich reagent,which indicated the presence of a furostanol skeleton [12]. ItsIR absorption spectrum revealed the presence of hydroxy(3378 cm−1) and olefinic (1642 cm−1) groups. The positive


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HRFABMS revealed a quasi-molecular peak at m/z 1101.5405[M + Na]+ (calcd for m/z 1101.5458), consistent with themolecular formula of C52H86O23. The 1H NMR spectrum of 1showed twomethyl singlets at δH 0.80 (H3-18) and 1.04 (H3-19);twomethyl doublets at δH 1.18 (d, J=6.5Hz, H3-21) and 0.99 (d,J = 7.0 Hz, H3-27); a methoxy signal at δH 3.25 (OCH3-22); anolefinic proton at δH 5.31 (d, J = 4.0 Hz, H-6); and fourdeshielded methines (anomeric) at δH 4.84 (1H, d, J = 7.5 Hz,Glu-(26)-1), 4.89 (1H, d, J = 8.0 Hz, Gal-1), 5.17 (1H, d, J =7.5 Hz, Glu-1) and 6.25 (1H, s, Rha-1). The 13C NMR spectrumshowed 52 carbon resonances, of which fourwere attributed toanomeric carbons (δC 100.4, 102.2, 107.2 and 105.0), two wereattributed to a trisubstituted double bond (δC 140.9 and 121.7),and one was attributed to a hemiacetal carbon at δC 112.6. Theabove information coupled with literature references [13]indicated the presence of a furostanol saponin skeleton.

The structure of 1 was finalized by analysis of the 2D NMRdata. The HSQC experiment allowed for the assignments of theproton and protonated carbon resonances in the NMR spectraof 1. In the HMBC spectrum (Fig. 2), two- and three-bondcorrelations of H-6/C-5, C-4 and C-10; H3-19/C-1, C-5, C-9 andC-10; H3-18/C-12, C-13, C-14 and C-17; H3-21/C-20, C-17 andC-22; H-26/C-25, C-26 and C-27; H3-27/C-24, C-25 and C-26;CH3O/C-22; and H-16/C-13, C-14 and C-17, together with thepresence of a hemiacetal carbon singlet at δH 112.6, suggested aΔ5,6 furostan nucleus of 1 [14]. ROESY correlations (Fig. 2) ofH-11/H3-19, H-11/H3-18, H-9/H-14, H-14/H-16, H-16/H-17,and H-17/H3-21 completed the relative stereochemistry of 1,indicating the usual furostanol ring junctions with rings B/Ctrans, C/D trans, D/E cis, and α-orientation of CH3-21. Theα-orientation of 22-OCH3 of 1 was determined by a NOEexperiment; when irradiating the proton signal at δH 3.25 (s, 22-OCH3), the signal at δH 4.44 (m, H-16) was enhanced, revealingthat the H-16 and 22-OCH3 were on same side of the five-membered ring. Thus the orientation of 22-OCH3 should be anαin 1. The small difference between the chemical shifts of the twoprotons of C-26 (δH-1, 3.94; δH-2, 3.59) suggested that C-25 of 1had an R configuration [15]. The 3-β-hydroxyl group of 1 wasdetermined by the chemical shift and literature references[14,15]. Based on the above spectral data, the aglycone of com-pound 1was identified as (25R)-22α-O-methyl-furost-5(6)-en-3β,26-diol [14,15].

The monosaccharides in 1 were confirmed to be D-(+)-glucose, L-(−)-rhamnose and D-(+)-galactose after the acidhydrolysis of 1 with 2 N HCl and the GC analysis of theirtrimethylsilyl L-cysteine derivatives. Relatively large 3JH1–H2coupling constants (6.5–8.0 Hz) of these monosaccharides intheir pyranose form indicated the β-anomeric orientation ofGlu and Gal. Furthermore, the positions of the glycosidiclinkages were clarified by the HMBC spectrum. The HMBCcorrelation between H-1 of Glu-(26) (δH 4.84) and C-26 (δC75.2) indicated that the anomeric carbon of the glucose (Glu-1)was connected to C-26 of the aglycone. Moreover, the trisaccha-ride was established as 3-O-β-D-glucopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside due to thelong-range correlations between H-1 of Gal (δH 4.89) and C-3(δC 77.9); H-1 of Rha (δH 6.25) and C-2 of Gal (δC 76.7); andH-1 of Glu-2 (δH 5.17) and C-4 of Gal (δC 81.2). Accordingly,the structure of 1 was determined to be 26-O-β-D-glucopyranosyl-(25R)-furost-5-en-3β,22α,26-triol,3-O-β-D- glucopyranosyl-(1→ 4)-[α-L-rhamnopyranosyl-(1→ 2)]-β-D-galactopyranoside.




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Fig. 2. Key HMBC correlations (H→ C) and selected ROESY correlations (H↔H) for compound 1.

7C.-J. Li et al. / Fitoterapia xxx (2014) xxx–xxx

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Compound 2 exhibited a molecular formula of C51H84O23,as deduced from its [M + Na]+ion at m/z 1087.5249 (calcd.for 1087.5301) in the positive HR-FAB-MS, which indicatedone CH2 unit less than that of 1. The IR, 1H and 13C NMRspectral data of the 2 were quite similar to those of 1 exceptfor the disappearance of the 22-OCH3 signal. The monosac-charides in 2 were confirmed to be D-(+)-glucose, L-(−)-rhamnose and D-(+)-galactose after the acid hydrolysis of 2with 2 NHCl and the GC analysis of their trimethylsilyl L-cysteinederivatives. Accordingly, the structure of2was identified as 26-O-β-D-glucopyranosyl-22-O-methyl-(25R)-furost-5-en-3β,22α,26-triol, 3-O-β-D-glucopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-galactopyranoside.

Ceparocide G (3) displayed an [M − H]− ion peak at m/z885.4411 in the negative HRFABMS, indicating a molecularformula of C44H70O18. The 1DNMR data also featured signals ofthree singlet methyls at δH 0.92, 1.44 and 1.65, a doublet signalat δH 1.05 (d, J=6.0 Hz, CH3-27), two olefinic protons (δH 5.57and 5.16) with four olefinic carbons (δC 164.0, 139.7, 124.6 and91.2) and two oxygen bearing methine protons at δH 3.81 (1H,dd, J = 12.4, 3.6 Hz, H-1) and 3.85 (1H, m, H-3). A pair ofsignals at δH 3.50 and 4.12 (δC 75.3) attributed to C-26.

The aglycone structure of 3was constructed by the detailedanalysis of 1D and 2D NMR (TOCSY, HSQC, and HMBC) data,especially the HMBC spectrum. The HMBC correlations of H-6/C-5, C-4 and C-10, H3-21/C-20, C-17 and C-22, H-23/C-22 andH-24/C-23 indicated unsaturation between C-5 and C-6, C-22and C-23, respectively. In particular, H-1 resonated as a doubledoublet (J= 12.5, 3.6 Hz),whereasH-3 appeared as amultipletwith two large (axial axial) and two small (axial equatorial)coupling constants. These data indicated an axial position forboth H-1 and H-3.6 The upfield shift of C-20 from δC 40.5 (in 1)to 76.8 (in 3) suggested an OH substitute in C-20. The absoluteconfiguration of C-20 was determined by the NOE experiment,when the signals at δH 1.65 (H3-21)were irradiated, NOEswereobserved at the signals of H3-18 and H-23. Thus the CH3-21of 3 should be a β-orientation and that the configuration ofC-20 is S. The large difference between the chemical shifts ofthe two protons of C-26 (δH1, 4.12; δH2, 3.50) provided evidenceof a C-25S configuration in 3 [14]. Thus, the aglycone of 3 wasdeduced to be (25S, 20S)-20-O-methyl-5-furost- 22(23)-dien-1β, 3β,22α,26-tetrol.

The linkage and sequence of the saccharidic chain of 3were confirmed by the HMBC correlations of H-1 of Glu/C-26,H-1 of Arab/C-1, and H-1 of Rha/C-2 of Gal. Accordingly, thestructure of 3was determined to be 26-O-β-D-glucopyranosyl-


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RO(25S,20S)-furost-5,22(23)-dien-1β,3β,20α,26-tetrol 1-O-α-L-

rhamnopyranosyl-(1→ 2)-α-L-arabinopyranoside.Ceparocide H (4) gives amolecular formula of C44H70O18 by

HR FAB-MS (negative ion mode) at m/z 885.4485 [M − H]−

(calculated for C44H69O18, 885.4484), which was consistentwith compound 3. Comparing of the NMR spectra of 4 withthose of 3 (Tables 1 and 2), all signals appeared at almost thesame positions except for the two protons of C-26, the smalldifference between which indicated that C-25 had an Rconfiguration in 4. Accordingly, the structure of 4was deducedto be 26-O-β-D-glucopyranosyl-(25R,20S)-furost-5,22(23)-dien-1β,3β,20α,26-tetrol, 1-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside.

Compounds 5 and 6 were determined to possess thesame molecular formula of C44H70O17 from the HR-FAB-MS.The NMR spectrum of each indicated a 5-furost aglyconesuch as ascalonicoside B,6 and three sugar residues. The Δ5

and Δ20 double bond was assignable based on the HMBCcorrelations of H-6/C-5, C-4, C-10; H-21/C-20, C-22, C-17;and H-23/C-20, C-22. The HMBC correlation between H-1 ofGlu and C-26 indicated that the glucose was attached at C-26 ofthe furostanol skeleton. The linkage of the disaccharidic chainwas also confirmed by the HMBC correlations between H-1 ofArab and C-1, H-1 of Rham and C-2 of Gal. Comparison of theNMR signals of 5 and 6 indicated many similarities but adifference in the proton signals of C-26. The small differencebetween the chemical shifts of those proton signals (δH1,3.92; δH2, 3.59) in 5 indicated the C-25R configuration, and thelarge difference in 6 (δH1, 4.03; δH2, 3.45) indicated the C-25Sconfiguration. Thus, compound 5 was determined to be 26-O-β-D-glucopyranosyl-(25R)-furost-5,20(22)-dien-1β,3β,26-triol,1-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside (5),while compound 6 was determined to be 26-O-β-D-glucopyranosyl-(25S)-furost-5,20(22)-dien-1β,3β,26-triol 1-O-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside (6).

TheHR FAB-MS (negative ionmode) spectrumof compound7 gives a molecular formula of C45H72O18 at m/z 899.4590[M − H]−. The NMR data of 7were similar to those of 5 exceptfor in the sugar residues. The α-L-arabonocose unit in 5 wasreplaced by a β-D-galactose unit in 7. This was confirmed byESI-MSn, which showed fragmentation signals at m/z 753[M − H − 146]−, 591 [M − H − 146 − 162]− and 429[M − H − 146 − 162 − 162]−. Acid hydrolysis also con-firmed the presence of galactose in compound 7. The linkagepoints of sugar residues were established from the followingHMBC correlations: δH 4.82 (H-1 of Glu) with δC 75.3 (C-26); δH


image of Fig.�2



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4.79 (H-1 of Gal) with δC 84.0 (C-1); and δH 6.41 (H-1 of Rha)with δC 75.1 (C-2 of Gal). Accordingly, the structure of 7 wasdetermined to be 26-O-β-D-glucopyranosyl-(25S)-furost-5,20(22)-dien-1β,3β,26-triol,1-O-α-L-rhamnopyranosyl-(1 → 2)-β-D galactopyranoside.

The NMR spectral data of 8 were generally similar tothose of 2 except for the disappearance of a rhamnosemoiety and the presence of one oxygen-bearing methineproton at δH 3.84 (H-1). Considering the HMBC correlationsof H-1/C-2, C-3, C-10, H-19/C-1 and H-3/C-1, C-5, compound8was assigned to have two oxygen-bearing methanes at C-1and C-3. On the basis of the literature and J values (12.0, 4.0)of H-1, the 1-OH and 3-OH should have a β-orientation [6].The keyHMBC correlations of H-1 of Gal/C-1 of aglycone, H-1 ofRha/C-2 of Gal and H-1 of Glu/C-26 of aglycone located the α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside and theglucose on C-1 and C-26, respectively. The large differencebetween the chemical shifts of the two protons of C-26 (δH1,4.02; δH2, 3.44) provided evidence for a C-25S configuration in8. Thus, the structure of 8 was deduced to be 26-O-β-D-glucopyranosyl-(25S)-furost-5-en-1β,3β,22α,26-tetrol, 1-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside.

The in vitro inhibition of protein tyrosine phosphatase 1B(PTP1B) activity by ethanol extract and compounds 1–8 weretested at 10 μM,with the known effective compoundCCCF06240as the positive control [10]. As shown in Table S1, the ethanolextract shows activity with an inhibition of 81.1%, whilecompounds 1–8were inactive. Compounds 1–8were tested forcytotoxicity against lung adenocarcinoma (A549), stomachcancer (BGC-823), hepatoma (BEL-7402), colon cancer (HCT-8)and hela cell lines, but all of them were inactive (Table S2).

4. Conclusions

In conclusion, the ethanol extract of seeds of A. cepa L.showed inhibitory effect on protein tyrosine phosphatase 1B(PTP1B) enzyme, with 81.1% inhibition. Eight new saponinswere obtained from this extract, whose structures wereelucidated by 1D and 2D NMR spectroscopy, mass spectrom-etry and chemical methods. Compounds 1–8were screened forinhibitory effects toward PTP1B enzymes and cytotoxic activityagainst five human cancer cell lines, including HCT-8, Bel-7402,BGC-823, A549 and A2780, but all were found to be inactive.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgments

We thank the staff of the analytical group at the Institute ofMateria Medica for recording the spectral data. We also thankprofessor Fei Ye and Xiao Guang Chen for performing thebioactivity tests. This research programwas supported by grantsfrom the National Science and Technology Project of China (No.2011ZX09307-002-01 and No. 2012ZX09301002-002).

Appendix A. Supplementary data

NMR,MS, and IR spectra of compounds 1−8 are available asSupporting Information. Supplementary data to this article canbe found online at http://dx.doi.org/10.1016/j.jglr.2014.08.001

References

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[4] Fattorusso E, Lanzotti V, Taglialatela-Scafati O, Rosa MD, Ianaro A.Cytotoxic saponins from bulbs of Allium porrum L. J Agric Food Chem2000;48:3455–62.

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[6] Fattorusso E, Iorizzi M, Lanzotti V, Taglialatela-Scafati O. Chemical composi-tion of shallot (Allium ascalonicum Hort.). J Agric Food Chem 2002;50:5686–90.

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[9] Yuan L, Ji TF, Li CJ, Wang AG, Yang JB, Su YL. Two new steroidal saponinsfrom the seeds of Allium cepa L. J Asian Nat Prod Res 2009;11:213–8.

[10] Ma YM, Liu Q, Li J, Tian JY, Zhang XL, Xiao ZY, et al. PTP1B inhibitorimproves both insulin resistance and lipid abnormalities in vivo andin vitro. Mol Cell Biochem 2011;357:65–72.

[11] Carmichael J, De Graff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation ofa Tetrazolium-based semiautomated colorimetric assay: assessment ofchemosensitivity testing. Cancer Res 1987;47:936–42.

[12] Kiyosawa S, Hutoh M, Komori T, Nohara T, Hosokawa I, Kawasaki T.Detection of proto-type compounds of diosgenin and other spirostanol-glycosides. Chem Pharm Bull 1968;16:1162–4.

[13] Dini I, Tenore GC, Trimarco E, Dini A. Furostanol saponins inAllium caepa L.Var. tropeana seeds. Food Chem 2005;93:205–14.

[14] Mimaki Y, Satou T, Kuroda M, Sashida Y. Steroidal glycosides from thebulbs of Allium jesdianum. Phytochemistry 1999;51:567–73.

[15] Agrawal PK. Assigning stereodiversity of the 27-Me group of furostane-type steroidal via NMR chemical shifts. Steroids 2005;70:715–24.


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furostanol saponins from the seeds of allium cepa l

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