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Discovery and LC-MS Characterization of New Crocins in Gardeniae Fructus and Their Neuroprotective Potential Yang Ni, Lin Li, Weiyang Zhang, Dan Lu, Caixia Zang, Dan Zhang, Yang Yu, and Xinsheng Yao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03866 • Publication Date (Web): 09 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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Journal of Agricultural and Food Chemistry

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Discovery and LC-MS Characterization of New Crocins in Gardeniae Fructus and

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Their Neuroprotective Potential

3 ⊥

4

Yang Ni,‡,

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Yao, *,†,‡

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†

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China

8

‡

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P. R. China

Lin Li,†,

⊥

Weiyang Zhang,# Dan Lu, † Caixia Zang,§ Dan Zhang,§ Yang Yu, *,†and Xinsheng

Institute of TCM & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P. R.

College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016,

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§

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Beijing, 100050, P. R. China

12

#

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Technology, Macau, P. R. China

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,

State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and

14

15

⊥

Both authors contributed equally to this work.

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*To whom correspondence should be addressed. Tel: +86-20-85225849. Fax: +86-20-85221559. Email:

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[email protected], [email protected].

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ACS Paragon Plus Environment


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Abstract

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Ten new crocins, neocrocins B-J (1-9), and 13-cis-crocetin-8′-O-β-D-gentiobioside (14), along with ten

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known crocins, were isolated from the fruits of Gardenia jasminoides Ellis (Gardeniae Fructus). The

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structures of these compounds were elucidated by analyzing HRESIMS, UV/Vis and 1D and 2D NMR

22

spectra, and their neuroprotective effects against hydrogen peroxide- and L-glutamic acid-induced

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SH-SY5Y cell injury were evaluated. The UPLC-Q/TOF-MS chromatogram of a crocin-rich fraction

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derived from gardenia fruit extracts was established using the obtained crocin compounds as references.

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Most of the peaks were identified (the total integral area of the identified peaks accounted for 95% of total

26

peak areas), and bioactive crocins were a large portion of this fraction (the areas of peaks from the

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neuroprotective compounds accounted for 70% of the total).

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Key words

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crocin, Gardeniae Fructus , neuroprotective, UPLC-Q/TOF-MS

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Introduction

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Gardenia jasminoides Ellis, an evergreen shrub, is commonly found in the tropical and subtropical

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regions of Asia. Gardeniae Fructus, the dried ripe fruit of Gardenia jasminoides Ellis, is officially listed

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in the Pharmacopoeia of the People’s Republic of China (Ch. P. 2015) and widely used in traditional

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Chinese medicine (TCM) for its cholagogue, diuretic, anti-inflammatory and analgesic effects.1 Previous

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chemical investigation of Gardeniae Fructus demonstrated the presence of iridoid glucosides,2 quinic

37

acids,3 and crocins.4, 5

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Crocins are a family of natural water-soluble carotenoids firstly discovered in saffron (Crocus sativus L.),

39

which is an important Chinese herbal medicine and one of the most expensive spices in the world. Saffron

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extracts, crocetin, and crocins exhibit antitumor properties,6, 7 cardiovascular protective effects,8, 9 and most

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attractively, neuroprotective activities. As reported, crocins are potent in protecting cerebral cells from

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ischemia

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These properties render crocins useful as medicines or food additives. Since Gardeniae Fructus is

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commonly used as food ingredient and textile colorant, the production of Gardeniae Fructus is

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flourishing. Comparing to saffron, Gardeniae Fructus is much more accessible in both price and resource.

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Therefore, as a crocin source, Gardeniae Fructus is an economical alternative to saffron.

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Thus, a comprehensive chemical examination of the crocin components in gardenia fruits was performed.

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Twenty crocins were obtained including ten new compounds, and the analysis of the NMR data and MS

49

fragmentation patterns enabled structural elucidation. The neuroprotective effects of each crocin obtained

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against hydrogen peroxide- and L-glutamic acid-induced cell injury were evaluated in the SH-SY5Y

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human neuroblastoma cell line. Moreover, using the crocins that we obtained as reference compounds, a

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UPLC-Q/TOF-MS chromatogram was established for the crocin-rich fraction derived from Gardeniae

53

Fructus extracts.

10

and oxidative damage 11, 12 and alleviating subsequent behavioral and recognitive impair. 13

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Materials and Methods

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Instrumentation and Reagents UV/Vis spectra were measured using a JASCO V-550 UV/Vis

57

spectrometer (JASCO International Co. Ltd., Hachioji, Tokyo, Japan). IR spectra were acquired using a

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JASCO FT/IR-480 plus spectrometer (JASCO International Co. Ltd., Tokyo, Japan) in KBr, and

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HRESIMS spectra were obtained using a Waters Synapt G2 mass spectrometer (Waters, Manchester, U.

60

K.). 1D and 2D NMR data were acquired with a Bruker AV 600 (Bruker Co. Ltd., Bremen, German) using

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solvent signals (DMSO-d6: δH 2.50/δC 39.5) as internal reference. HPLC analysis was performed on a

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Waters 2695 separations module (Waters, Manchester, U.K.) equipped with a 2998 photodiode array

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detector (Waters, Manchester, U.K.) and an Alltech 3300 evaporative light scattering detector (Alltech

64

Inc., Deerfield, Illinois, U.S.A.) using an RP-18 column (5 µm, ϕ 4.6 × 250 mm; Welch Ultimate AQ;

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Welch Tech., Shanghai, China), an RP-18 column (5 µm, ϕ 4.6 × 250 mm; COSMOSIL MS-II; Nacalai

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Tesque, Kyoto, Japan ) and a chiral column (5 µm, ϕ 4.6 × 300 mm; Marshal BIO-C18, Research &

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Creativity Biotech, Co., Ltd, Guangdong, China). The semi-preparative and preparative HPLC analyses

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were performed on a Waters 1515 isocratic HPLC pump (Waters, Manchester, U.K.) coupled to a 2489

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UV/Vis detector (Waters, Manchester, U.K.), RP-18 columns (5 µm, ϕ 10 × 250 mm; 5 µm, ϕ 20 × 250

70

mm; Welch Ultimate A, Welch Tech., Shanghai, China) and RP-18 columns (5 µm, ϕ 10 × 250 mm; 5 µm,

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ϕ 20 × 250 mm; COSMOSIL MS-II, Nacalai Tesque, Kyoto, Japan). The UPLC-Q/TOF-MS was operated

72

on a Waters Synapt G2 Q-TOF-MS (Waters, Manchester, U.K.) with an RP-18 column (1.7 µm, ϕ 3.0 ×

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150 mm; BEH).

74

Methanol for HPLC was purchased from BCR International Co. Ltd. (Shanghai, China), acetonitrile was

75

purchased from Merck (Darmstadt, Germany), and reference substances for sugar analysis were

76

purchased from Sigma Aldrich (Shanghai, China). Silica gel (200-300 mesh, Qingdao Marine Chemical

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Ltd., Shandong, China), Diaion HP20 (Mitsubishi Chemical Co., Tokyo, Japan), octadecylsilanized (ODS)

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silica gel (12 nm, S-50 µm, YMC Ltd., Tokyo, Japan) and Sephadex LH-20 (Amersham Pharmacia

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Biotech, Sweden) were used for column chromatography (CC). TLC was performed on pre-coated silica

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gel plates (SGF254, 0.2 mm, Yantai Chemical Industry Research Institute, Shandong, China).

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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum and L-glutamic acid were purchased

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from Solarbio Life Science (Beijing, China), DMSO for dissolving the compounds was purchased from

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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Hydrogen peroxide solution (30%, w/w) was

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purchased from Beijing Beihua Fine Chemicals Co. Ltd. (Beijing, China). The cell line (SHSY5Y

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neuroblastoma cell line) was obtained from Cell Center of Beijing Peking Union Medical College. Methyl

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thiazolyl tetrazolium (MTT) was purchased from Amresco LCC. (Ohio, U.S.). Micro-plate reader was

87

purchased from Bio-Tek Instruments (Vermont, U.S.).

88

Plant Materials The dried fruits of Gardenia jasminoides Ellis that were collected in Jiangxi Province on

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October 2014 were purchased from the Bozhou Kunyuan Pharm Corporation. The species was identified

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by Prof. G. X. Zhou of Jinan University. A voucher specimen (No. JNU-GJ-2014) was deposited in Jinan

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University, Guangzhou, China.

92

Extraction and Isolation Air-dried shredded pieces of gardenia fruits (40 kg) were heat-refluxed three

93

times with 60% EtOH (160 L, 2 h each time) to yield crude extract (6.2 kg). The crude extract was

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suspended in H2O (10 L) and subjected to column chromatography over an HP-20 macroporous resin (ϕ

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20 × 90 cm) eluted with EtOH-H2O (0:100 - 30:70 - 50:50 - 70:30 - 95:5). The 70% (v/v) ethanol elution

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portion (150.0 g) was identified as a crocin-rich fraction by HPLC-DAD online analysis with the

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characteristic visible light absorption peak at 440 nm. The crocin-rich fraction was separated into 11

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fractions using a silica gel column (CHCl3/MeOH/H2O, 98:2:0-6:4:0.8, v/v/v). Compound 10 (49.1 mg) in

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Fr. 2, compound 11 (136.5 mg) in Fr. 4, and compound 12 (7.0 g) in Fr. 6 were isolated by

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recrystallization in CH3OH. Fr. 6 (19.0 g) was further separated with octadecylsilane column

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chromatography (ODS CC) (MeOH/H2O, 50:50 to 90:10, v/v) to isolate compound 15 (315.7 mg), and

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the sub-fractions were subjected to preparative HPLC to yield compounds 5 [66.2 mg, tR = 13.3 min,

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MeOH/H2O (0.1% formic acid), 65/35, v/v], 7 [10.0 mg, tR = 21.5 min, MeOH/H2O (0.1% formic acid),

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70/30, v/v], 8 [2.0 mg, tR = 10.3 min, MeOH/H2O (0.1% formic acid), 65/35, v/v] and 9 [10.0 mg, tR =

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18.0 min, MeOH/H2O (0.1% formic acid), 60/40, v/v]. Compound 19 (545.1 mg) was obtained from Fr. 9

106

(13.5 g) by ODS CC eluted with MeOH/H2O (30:70 to 70:30, v/v) followed by recrystallization in

107

CH3OH, whereas compound 20 (265.7 mg, tR = 16.6 min) was obtained from Fr. 9 by preparative HPLC

108

with 60% MeOH. A mixture of compounds 3 and 4 (104.8 mg, 1:2, tR = 18.6 min) was obtained from Fr. 9

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by preparative HPLC with 68% MeOH (0.1% formic acid). Compound 16 (143.7 mg) was isolated by

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ODS CC and preparative HPLC followed by recrystallization in CH3OH from Fr. 8 (17.7 g). Compounds

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1 [400.9 mg, tR = 9.5 min, MeOH/H2O (0.1% formic acid), 68/32, v/v], 2 [21.9 mg, tR = 9.5 min,

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MeOH/H2O (0.1% formic acid), 55/45, v/v] and 6 [59.1 mg, tR = 9.0 min, MeOH/H2O (0.1% formic acid),

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55/45, v/v] were obtained from Fr. 8 with preparative HPLC, and compounds 17 (1.8 mg, tR = 10.2 min)

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and 18 (3.6 mg, tR = 10.2 min) were also isolated by HPLC [CH3CN/H2O, (0.1% formic acid), 32/68, v/v]

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from Fr. 8. Fr. 7 (18.3 g) was subjected to ODS CC (MeOH/H2O, 40:60 to 80:20, v/v), and compounds 13

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(8.0 mg, tR = 17.9 min) and 14 (16.0 mg, tR = 21.5 min) were further separated by preparative HPLC

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[CH3CN/H2O, (0.1% formic acid), 42/58, v/v] from Fr. 7.

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Neocrocin B (1). Red amorphous powder; UV (MeOH) λmax (log ε): 433 (5.32), 458 (5.28), 331 (4.68),

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253 (4.52) nm; IR (KBr) νmax 3401, 2920, 1694, 1610, 1576, 1268, 1224, 1061, 968 cm-1; 1H NMR (in

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DMSO-d6, 600 MHz) and 13C NMR (in DMSO-d6, 150 MHz) data, see Table 1; HRESIMS m/z 989.3642

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[M + H]+ (calcd for C48H61O22, 989.3654).

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Neocrocin C (2). Red amorphous powder; UV (MeOH) λmax (log ε): 431 (4.63), 457 (4.56), 331 (4.12),

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249 (3.85); IR (KBr) νmax 3417, 2921, 1698, 1602, 1230, 1279, 1064 cm-1; 1H NMR (in DMSO-d6, 600

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MHz) and

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(calcd for C48H61O22, 989.3654).

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Neocrocin D and Neocrocin E (3 and 4). Red amorphous powder; UV (MeOH) λmax (log ε): 429 (5.04),

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453 (4.99), 324 (4.68), 251 (4.04) nm; IR (KBr) νmax 3368, 2920, 1693, 1607, 1277, 1229, 1062, 969 cm-1;

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1

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m/z 1011.3471 [M + Na]+ (calcd for C48H60O22Na, 1011.3474).

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Neocrocin F (5). Red amorphous powder; UV (MeOH) λmax (log ε): 430 (5.33), 454 (5.28), 326 (4.80),

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242 (4.78); IR (KBr) νmax 3391, 2922, 1697, 1610, 1284, 1227, 1179, 1069, 972 cm-1; 1H NMR (in

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DMSO-d6, 600 MHz) and 13C NMR (in DMSO-d6, 150 MHz) data, see Table 2; HRESIMS m/z 881.3188

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[M + Na]+ (calcd for C43H54O18Na, 881.3208).

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Neocrocin G (6). Red amorphous powder; UV (MeOH) λmax (log ε): 434 (5.22), 459 (5.17), 330 (4.78),

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242(4.65); IR (KBr) νmax 3385, 2920, 1701, 1610, 1273, 1225, 1119, 1059 cm-1; 1H NMR (in DMSO-d6,

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600 MHz) and 13C NMR (in DMSO-d6, 150 MHz) data, see Table 2; HRESIMS m/z 1183.4479 [M + H]+

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(calcd for C55H75O28, 1183.4445).

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Neocrocin H (7). Red amorphous powder; UV (MeOH) λmax (log ε): 430 (4.64), 456 (4.59), 322 (3.84),

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257 (3.95); IR (KBr) νmax 3400, 2925, 1697, 1229, 1074 cm-1; 1H NMR (in DMSO-d6, 600 MHz) and 13C

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NMR (in DMSO-d6, 150 MHz) data, see Table 3.; HRESIMS m/z 703.2904 [M + Na]+ (calcd for

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C34H48O14Na, 703.2942).

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Neocrocin I (8). Red amorphous powder; UV (MeOH) λmax (log ε): 438 (4.63), 462 (4.60), 328 (3.90),

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261 (3.94); IR (KBr) νmax 3277, 2921, 1694, 1515, 1071 cm-1; 1H NMR (in DMSO-d6, 600 MHz) and 13C

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NMR (in DMSO-d6, 150 MHz) data, see Table 3; HRESIMS m/z 659.2657 [M + Na]+ (calcd for

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C32H44O13Na, 659.2680).

13

C NMR (in DMSO-d6, 150 MHz) data, see Table 1;. HRESIMS m/z 989.3646 [M + H]+

H NMR (in DMSO-d6, 600 MHz) and 13C NMR (in DMSO-d6, 150 MHz) data, see Table 1; HRESIMS

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Neocrocin J (9). Red amorphous powder; UV (MeOH) λmax (log ε): 428 (4.56), 453 (4.50), 320 (4.11),

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257(4.10); IR (KBr) νmax 3416, 2924, 1700, 1230, 1072 cm-1; 1H NMR (in DMSO-d6, 600 MHz) and 13C

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NMR (in DMSO-d6, 150 MHz) data, see Table 3; HRESIMS m/z 645.2519 [M + Na]+ (calcd for

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C31H42O13Na, 645.2523).

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13-cis-crocetin-8′-O-β-D-gentiobioside (14). Red amorphous powder; UV (MeOH) λmax (log ε): 424

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(4.92), 447 (4.86), 318 (4.43), 256 (4.13); IR (KBr) νmax 3399, 2924, 1690, 1607, 1232, 1069, 971 cm-1.

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1

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m/z 675.2617 [M + Na]+ (calcd for C32H44O14Na, 675.2629).

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Acid Hydrolysis and HPLC Analysis of Sugars

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The absolute configurations of the sugar moieties in the new crocetin esters were determined using a

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reported method.14 Compounds 1-9 (1.5 mg) were hydrolyzed with 2 mL 2 M HCl for 2 h at 90°C. The

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solvent was then removed under vacuum, then the residue was dissolved in 1 mL H2O and extracted with

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CHCl3. The aqueous layer was evaporated to dryness, and pyridine (1 mL)-containing L-cysteine methyl

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ester (2.5 mg) was added and heated at 60°C for 1 h before adding o-tolyl isothiocyanate (5 µL). This

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mixture was heated at 60°C for one additional hour and subjected to HPLC analysis. The standard

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monosaccharides, namely

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procedure. HPLC analysis was conducted on an RP-18 column (5 µm, ϕ 4.6 × 250 mm, COSMOSIL

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MS-II) at 35°C, and the detector wavelength was 250 nm. The mobile phase was 25% CH3CN containing

164

0.1% formic acid at a flow rate of 0.8 mL/min. The absolute configurations of the sugar moieties in the

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new crocetin esters were determined by comparing their retention time with those of the standard

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monosaccharides.

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SH-SY5Y Cell Culture

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SH-SY5Y cells

H NMR (in DMSO-d6, 600 MHz) and 13C NMR (in DMSO-d6, 150 MHz) data, see Table 3; HRESIMS

15

D-glucose, L-glucose, D-xylose

and L-xylose, were treated using the same

were cultured in DMEM with 5% fetal bovine serum and maintained at 37°C in a

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saturated humidity atmosphere containing 95% air and 5% CO2. The cells were passaged once every 3-4

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days, and cells in the logarithmic phase were selected for the experiment.

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Protection Activity against Hydrogen Peroxide (H2O2)-Induced SH-SY5Y Cell Injury

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SH-SY5Y cells were seeded at a density of 5×103 cells/well in 96-well plates. After a 24-hour incubation

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period, each well of cells was treated with 100 µL of a medium containing 400 µM H2O2 and various

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concentrations (10 µM, 1 µM, 0.1 µM) of the compounds (1-20). After 24 h incubation, the medium was

175

removed, and 100 µL methyl thiazolyl tetrazolium (MTT) (0.5 mg/mL) was added to each well for an

176

additional 4-hour incubation. The supernatant was discarded, and 150 µL DMSO was added for formazan

177

solubilization. The optical density was measured at 570 nm using a microplate reader. All of the tests were

178

performed in triplicate to ensure precision.

179

Protection rate % = (ODsample – ODmodel)/(ODcontrol – ODmodel) × 100%

180

Protection Activity against L-Glutamic Acid-Induced SH-SY5Y Cell Injury

181

SH-SY5Y cells were seeded at a density of 5×103 cells/well in 96-well plates. After a 24-hour incubation

182

period, each well of cells was treated with 100 µL of a medium containing 160 mM L-glutamic acid and

183

various concentrations (10 µM, 1 µM, 0.1 µM) of the compounds (1-20). After 24 h incubation, the

184

medium was removed, and 100 µL MTT (0.5 mg/mL) was added to each well for an additional 4-hour

185

incubation. The supernatant was discarded, and 150 µL DMSO was added for formazan solubilization.

186

The optical density was measured at 570 nm using a microplate reader. All of the tests were performed in

187

triplicate to ensure precision.

188

Protection rate % = (ODsample – ODmodel)/(ODcontrol – ODmodel) × 100%

189

UPLC-Q/TOF-MS and HPLC Analysis

190

Pretreatment of the samples for LC-MS analysis was as follows: 5.0 mg of the crocin-rich fraction was

191

dissolved in 1.0 mL of methanol and centrifuged for 10.0 min at 13000 rpm. The supernatant was loaded

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onto a Waters Oasis HLB SPE column (6 cc, 200 mg) and eluted with methanol. The methanol-eluted

193

portion was filtered through a 0.22-µm syringe filter; 1.0 mg of each compound (1 - 20) was pretreated

194

following the procedure described above.

195

UPLC-Q/TOF-MS analysis was performed on a Waters Synapt G2 UPLC-Q-TOF-MS system. The

196

capillary voltage was 2.0 kV, and the source temperature was 100°C. The desolvation gas was N2 at 600

197

L/h, and the drying gas temperature was 300°C. In full scan positive mode, the mass range was m/z 50 -

198

2000.

199

The mobile phases were composed of A (water containing 0.1% formic acid) and B (acetonitrile

200

containing 0.1% formic acid). The flow rate was 0.6 mL/min, and the program was set as follows:

201

•0 to 0.5 min – isocratic at 80% A, 20% B;

202

•0.5 to 19 min – linear gradient to 50% A, 50% B;

203

•19 to 20 min – linear gradient to 0% A, 100% B;

204

•20 to 23 min – isocratic at 0% A, 100% B.

205

An RP-18 column (5 µm, ϕ 4.6 × 250 mm; COSMOSIL) was used in the HPLC analysis and the column

206

oven was set at 30°C. The mobile phases were A (water containing 0.1% acetic acid) and B (methanol) at

207

1.0 mL/min, and the gradient program was as follows:

208

•0 to 40 min – linear gradient from 50% A, 50% B to 10% A, 90% B;

209

•40 to 45 min – linear gradient to 0% A, 100% B.

210 211 212

Results and Discussion

213

Structural Elucidation of New Crocins

214

Compound 1 was obtained as red amorphous powder. The UV/Vis spectrum of 1 showed the typical

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absorption of crocins at 433 and 458 nm. HRESIMS gave a quasi-molecular ion peak at m/z 989.3642 [M

216

+ H]+ (calcd for C48H61O22, 989.3654), and the molecular formula C48H60O22 was inferred. After acid

217

hydrolysis and derivatization, HPLC analysis revealed the presence of

218

fragment at m/z 687 [M + Na – 324]+ given by the ESI-MS2 spectrum of the ion at m/z 1011 [M + Na]+

219

indicated the presence of a sugar chain consisting of two glucosyl residues.

220

The 1H and 13C NMR data of 1 showed characteristic signals of crocin-ester, which include a conjugated

221

polyene moiety (δC 125.4-144.7; δH 6.49-7.35), four methyl groups (δC 12.6-12.8; δH 1.92-2.00), two

222

conjugated carbonyl carbons (δC 166.2 and 167.0) and glucosyl residues. In the HMBC spectrum,

223

correlations observed at Glc-H-6 (δH 3.58, 3.99)/Glc-C-1′ (δC 103.1), Glc-H-1′ (δ 4.17)/Glc-C-6 (δC 67.9)

224

and Glc-H-1 (δ 5.42)/C-8 (δC 166.2) established the main structure as a crocetin mono-gentiobioside.

225

Furthermore, the 1H and 13C NMR spectra of 1 showed a set of trans double-bond signals [δ 7.44 (1H, d,

226

J = 16.2 Hz, H-3′′′), 6.16 (1H, d, J = 16.2 Hz, H-2′′′), 145.6 (C-3′′′), 113.6 (C-2′′′)] and a set of

227

1,3,4-trisubstituted benzene signals [δ 7.03 (1H, d, J = 1.8 Hz, H-5′′′), 6.98 (1H, dd, J = 8.4, 1.8 Hz,

228

H-9′′′), 6.74 (1H, d, J = 8.4 Hz, H-8′′′), 148.5 (C-7′′′), 145.6 (C-6′′′), 125.2 (C-4′′′), 121.6 (C-9′′′), 115.7

229

(C-8′′′), 114.9 (C-5′′′)]. The HMBC correlations of H-3′′′/C-4′′′, C-5′′′, C-9′′′ and C-1′′′, H-2′′′/C-1′′′ and

230

C-4′′′ revealed the presence of a trans-caffeoyl moiety.

231

Based on the molecular formula, the remaining 7 carbon signals could be assigned to a carbonyl carbon,

232

two methylenes, three methines and a quaternary carbon. The sequential 1H-1H COSY correlations from

233

H-2′′ to H-6′′, together with the HMBC correlations of H-2′′b, H-6′′b/C-1′′ and H-6′′a/C-1′′, C-7′′, led to the

234

construction of a substituted quinic acid moiety. The typical signal of δ 4.88 (1H, dd, J = 8.4, 3.0 Hz,

235

H-4′′) and the correlations of H-4′′/C-8′ in the HMBC spectrum revealed that the quinic acid was

236

connected with a crocetin mono β-D-gentiobioside at C-4′′. Simultaneously, a down-field H-3′′ shift to δ

237

5.42 indicated that the trans-caffeoyl is positioned at C-3′′ of the quinic acid, which could be verified by

11


D-glucose

in 1. The positive


Page 12 of 33

238

the HMBC H-3′′/C-1′′′ correlation. Thus, compound 1 was elucidated as shown in Fig. 1. With the aid of

239

1D and 2D NMR experiments, all of the 1H and 13C NMR signals for 1 were assigned as shown in Table 1,

240

and the key correlations are shown in Fig. 2.

241

Compound 2 was an isomer of 1, and its molecular formula was C48H60O22, as revealed by HRESIMS

242

(m/z 989.3645 [M + H]+). The 1H and

243

β-D-gentiobioside with trans-caffeoyl and quinic acid moieties. The chemical shift values of protons [H-3′′

244

(δ 5.21), H-4′′ (δ 3.80), H-5′′ (δ 5.18)] on the quinic acid moiety indicated that the hydroxyls at C-3′′ and

245

C-5′′ were esterified. The entire structure was further confirmed by the correlations of H-5′′/C-8′ and

246

H-1/C-8 in the HMBC spectrum. The full assignment of 2 was established by 2D NMR analyses, and the

247

key correlations are shown in Fig. 2. Therefore, the structure of compound 2 was elucidated as a new

248

crocin and named neocrocin C.

249

Compounds 3 and 4 were the 13-cis and 13′-cis isomers of 1, respectively, both of which showed very

250

close retention times in HPLC. Similar to the description of cis isomers in the literature,

251

absorption peak at approximately 330 nm was observed in the UV spectrum. Light exposure can convert 3

252

and 4 into 1, though the conversion was a very slow process. The conversion can be largely prevented by

253

shielding the compounds from light, so these compounds should be handled with care to reduce their

254

exposure on light. Compared with the all trans isomer, a down-field shift of a methyl group (C-20) to δ

255

20.0 in

256

observed. Furthermore, as 1 is asymmetric, the cis/trans isomerization of the C-C double-bond could

257

occur at either 13-cis or 13′-cis under phytochemical conditions; thus, 1 has a 13-cis isomer and a 13′-cis

258

isomer, that is, 3 and 4. Although we failed to obtain pure 13-cis (3) and 13′-cis (4) isomers from a limited

259

amount of the mixture, their ratio in the mixture was 1:2, as determined by HPLC on a chiral column

260

(Marshal Bio-C18, 5 µm, ϕ 4.6 × 300 mm) [CH3CN/H2O, (0.3% TEAA), 45/55, v/v] (see Fig. S63 in

13

C NMR signals demonstrated that it is a crocetin mono-

16,17

a strong

13

C NMR, down-field shifts of H-12 and H-15, and an up-field shift of H-14 in 1H-NMR were

12


Page 13 of 33


261

Supporting Information).

262

Meanwhile, we obtained three groups of cis/trans isomers, 12 and its 13-cis isomer (13) and 13′-cis

263

isomer (14); 16 and its 13′-cis isomer (17) and 13-cis isomer (18); 19 and its 13-cis isomer (20). Based on

264

the NMR data comparison of these isomers under the same experimental conditions, we found that in the

265

13-cis structures, β-D-gentiobiosyl substitution on C-8 can shift H-1′ slightly from δ 5.42 to δ 5.44,

266

whereas β-D-gentiobiosyl substitution on C-8′ had little effect on the H-1′ chemical shift. In the 1H-NMR

267

spectrum, of the mixture of 3 and 4, the integral peak area of peak at δ 5.42 was larger than the peak at δ

268

5.44. As compound 4 was the major component as shown by HPLC (Fig. S63), β-D-gentiobiosyl was

269

determined to be at C-8 in compound 3 and at C-8′ in compound 4. The NMR data of compounds 3 and 4

270

were assigned as shown in Table 1, and the compounds were named neocrocin D and neocrocin E,

271

respectively.

272

Compound 5, a red amorphous powder, had a molecular formula of C43H54O18 as determined by

273

HRESIMS (m/z 881.3188 [M + Na]+, calcd for C43H54O18Na, 881.3208). The 1H and

274

indicated that compound 5 was the sinapoyl derivative of crocetin mono β-D-gentiobioside. A typical

275

group of signals including a trans-double bond [δ 7.55 (1H, d, J = 15.6 Hz, H-3′′), 6.57 (1H, d, J=15.6 Hz,

276

H-4′′), 145.5 (C-3′′), 114.8 (C-2′′)], a symmetrical 1,3,4,5-tetrasubstituted benzene ring [δH 7.03 (2H, s,

277

H-5′′ 9′′), δC 106.3 (C-5′′ 9′′), 124.4 (C-4′′), 138.3 (C-7′′), 148.0 (C-6′′ 8′′)], and a methoxyl group [δ 3.80

278

(6H, s, OCH3-6′′ 8′′), 56.1 (OCH3-C-6′′ 8′′)], was assigned to the trans-sinapoyl moiety. The sinapoyl

279

substitution caused the H-1′ of the outer glucosyl of gentiobioside to shift down-field from δ 4.17 to δ

280

4.25, and caused H-6′ to shift down-field from δ 3.65/3.44 to 4.36/4.18. The sinapoyl moiety was located

281

at C-6′ of the gentiobioside, according to the HMBC correlation peak at H-6′ (δH 4.36, 4.18)/C-1′′ (δC

282

166.7). Thus, compound 5 was a new crocin and named neocrocin F.

283

Compound 6 was obtained as red amorphous powder, and the molecular formula was established as

13


13

C NMR data


Page 14 of 33

284

C55H74O28 by HRESIMS (m/z 1183.4452 [M + H]+, calcd for C55H75O28, 1183.4445), indicating the

285

presence of an extra gentiobiosyl moiety comparing with 5. Characteristic signals from the trans-sinapoyl

286

moiety were observed by NMR (Table 2), and compound 6 was identified as a sinapoyl derivative of

287

crocetin di-β-D-gentiobioside (Comp. 19), a major crocin found in saffron stigmas (Croci Stigma) and

288

Gardeniae Fructus. The highly symmetric structure caused overlap of the signals from two inner glucose

289

residues. The sinapoyl group was attached to an outer glucosyl, as demonstrated by an HMBC correlation

290

[H-6′ (δH 4.36, 4.18)/C-1′′ (δC 166.7)], and the chemical shifts of the other protons in the glucose unit

291

changed in a similar manner to compound 5. Compound 6 was named neocrocin G.

292

Compound 7 had the molecular formula C34H48O14 based on HRESIMS at m/z 703.2885 [M + Na]+. The

293

NMR data (Table 3) were almost the same as that of crocetin mono β-D-gentiobioside, except for two

294

additional signals [δH 4.15 (2H, H-1′′), δC 1.24 (3H, t, H-2′′)] in the 1H spectrum and two carbon signals

295

[δC 60.1 (C-1′′), 14.2 (C-2′′)] in the 13C spectrum. Moreover, H-1′′/H-2′′ correlations in 1H-1H COSY and

296

HMBC correlations, including H-1′′ (δH 4.15)/C-8′(δC 167.4)/C-2′′ (δC 12.4), and H-2′′(δH 1.24)/C-1′′ (δC

297

60.1), indicated that there was an ethyl ester at the C-8′ carboxyl. Compound 7 was named neocrocin H.

298

Compound 8 was assigned the molecular formula C32H44O13 by HRESIMS at m/z 659.2657 [M + Na]+. A

299

comparison between the NMR data for 5 and 8 indicated that both compounds shared a similar skeleton

300

with the difference in the chemical shift of C-8′ (δC 169.2 in 5 and δC 194.5 in 8). In addition, an aldehyde

301

proton signal was observed at δH 9.44 (1H, s, H-8′) in the 1H-NMR spectrum of 8 (Table 3). The structural

302

elucidation was further verified by the HMBC correlations of H-19′/C-8′ and H-8′/C-10′, 19′. Compound

303

8 was named neocrocin I.

304

Compound 9 showed a quasi-molecular peak in HRESIMS ion at m/z 645.2532 [M + Na]+ (calcd for

305

C31H42O13Na, 645.2523), and the molecular formula was C31H42O13. Judging from the 1H and

306

data (Table 3), compound 9 was a crocin with two sugar moieties, glucose and xylose. The acid hydrolysis

14


13

C NMR

Page 15 of 33


307

and derivatization of 9 further demonstrated the presence of

308

configurations were established as β according to the coupling constants of the anomeric protons [δ 5.41

309

(1H, d, J = 7.8 Hz, H-1) and 4.14 (1H, d, J = 7.8 Hz, H-1′)]. Compound 9 was elucidated as crocetin

310

mono-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside and named neocrocin J.

311

Compounds 13 and 14 were the 13-cis and 13′-cis isomers of compound 12 (crocetin mono

312

β-D-gentiobioside)

313

13-cis-crocetin-8′′-O-β-D-gentiobioside (13′cis-crocetin-8-O-β-D-gentiobioside), respectively. Compound

314

14 was a new compound. These compounds were isolated on a COSMOSIL MS-II column with 42%

315

CH3CN (0.1% formic acid) as the mobile phase in the dark. The asymmetry of the structure made NMR

316

data assignments possible (see the Supporting Information, Fig. S57-S62). This is the first report of the

317

successful isolation of 13-cis and 13′-cis crocin isomers. The NMR data for each of the 13-cis isomers of

318

crocetin mono β-D-gentiobioside were assigned as shown in Table 3.

319

Compounds 17 and 18 were the 13-cis and 13′-cis isomers of compound 16 (crocetin-β-D-

320

glucopyranosyl-β-D-gentiobioside), and HPLC separation was performed using 32% CH3CN (0.1%

321

formic acid) in the dark. As the NMR signals from 16 were almost symmetrical despite the difference

322

between gentiobiose and glucose, there were also few differences between 17 and 18 in terms of their 1H

323

and

324

8′-O-β-D-glucopyranoside

325

(13′-cis-crocetin-8-O-β-D-gentiobiosyl-8′-O-β-D-glucopyranoside), respectively, although they were

326

almost indistinguishable.

327

Compound 20 was the 13-cis isomer of compound 19 (crocetin-di-β-D-gentiobioside). Compound 20

328

(13-cis crocetin di-β-D-gentiobioside) is one of the most common cis-crocins found in botanical sources.

329

Crocetin-esters have been reported as characteristic compounds in both saffron stigmas and gardenia

13

and

identified

as

D-glucose

and

D-xylose,

13-cis-crocetin-8-O-β-D-gentiobioside

and the

and

C spectra. Compounds 17 and 18 were identified as 13-cis-crocetin-8-O-β-D-gentiobiosyland

13-cis-crocetin-8-O-β-D-glucopyranosyl-8′-O-β-D-gentiobioside

15



Page 16 of 33

330

fruits. Based on our findings, compounds 1-3 might be biosynthetically derived from a crocin molecule

331

(crocetin mono β-D-gentiobioside) with quinic acid in gardenia fruits. To our knowledge, these

332

compounds are the first examples of crocin-acylated quinic acid derivatives from a natural source. In

333

addition, we successfully isolated 13-cis and 13′-cis crocin isomers for the first time. The contribution of

334

the new neocrocins (1-9 and 14) to the overall effect of crocins is worth investigating.

335

Neuroprotective Activity The efficacy of crocins on Alzheimer’s disease has been demonstrated at the

336

clinical bedside, as shown by a pair of clinical trials on saffron extracts containing crocins as the major

337

constituents.

338

peptides to prevent amyloid formation. 20 In the meantime, linkage between antioxidative effect of crocins

339

and memory enhancement effect of saffron has been demonstrated.

340

Alzheimer’s disease is now full of uncertainty, what is known for sure is that oxidative stress

341

excitatory damage 23 are among the primary pathological factors. The protective potency of the crocins we

342

obtained against H2O2 and

343

neuroblastoma cell line SH-SY5Y is a typical in vitro neurodegenerative disease model. In a model of

344

H2O2-induced SH-SY5Y cell injury, compounds 6, 7, 12 and 19 improved the viability of cells, and 6 and

345

19 were the most effective (Table 4). In addition, compounds 1, 5-9, and 12 exhibited good

346

neuroprotective effects against the L-glutamic acid-induced injury in a dose-dependent manner (Table 4).

347

UPLC-Q/TOF-MS Chromatogram of the Crocin-rich Fraction The obtained compounds (1-20) were

348

used as references to establish a UPLC-Q/TOF-MS chromatogram, and 18 peaks were unambiguously

349

identified by referring to the compounds (Fig. 3, Table 5). The identified peak areas accounted for 95% of

350

the total peak areas both in the UPLC-UV/Vis chromatogram at 440 nm and in the HPLC-ELSD

351

chromatogram (Fig. 4), whereas the peak areas of the active constituents accounted for approximately 70%

352

of the total peak area.

13, 18

Crocins are potential acetylcholinesterase inhibitors,

L-glutamic

19

21

they can also interact with Aβ

Although the pathogenesis of 22

and

acid-induced SH-SY5Y cell injury was assayed. Human

16


Page 17 of 33


353

Compounds 1-4 (Peak 8, 9, 12, 14) with a molecular formula of C48H60O22 were a group of isomers

354

substituted with caffeoyl quinic acid. Compound 1 (Peak 8), for example, showed a [M + H]+ ion at m/z

355

989.3597, and the base peak at m/z 665.2532 was formed by losing a neutral gentiobioside residue (324

356

Da). The quinic acid moiety could also be detached to produce a weak daughter ion at m/z 827.3133 (Fig.

357

5). Similar fragments can be found in the other three compounds.

358

Compound 6 (Peak 4), the sinapoyl derivative of crocetin di-β-D-gentiobioside, showed an [M+Na]+ ion

359

at m/z 1205.4243 with an elemental composition of C55H74O28. The cleavage of the saccharide moiety

360

produced ions at m/z 881.3331 [M + Na – Gen]+, 675.2614 [M + Na – Gen – sinapoyl]+ and the

361

sinapoyl-gentiobioside fragment at m/z 531.1690 [M + H - 652]+. The sinapoyl moiety promoted cleavage

362

of the internal gentiobioside linkage to generate the fragments at m/z 369.1180 [M + H – 652 – Glc]+ and

363

m/z 207.0654 [M + H – 652 – Glc – Glc]+ (Fig. 5). The MS fragmentation pattern was same as that in

364

compound 5 (Peak 17).

365 366

Acknowledgement

367

We hereby present our sincere gratitude to the financial support from National Natural Science

368

Foundation of China (NSFC key program No. 81630097, “The investigation of mechanism and

369

drugability of anti-AD bioactive components in Chinese natural medicines”) and “the Fundamental

370

Research Funds for the Central Universities (21616104)”.

17



Page 18 of 33

References (1) Tang, W. C.; Eisenbrand, G. Chapter 70: Gardenia jasminoides Ellis. In Chinese Drugs of Plant Origin. Springer-Verlag: Berlin, Germany, 1992, 539. (2) Yu, Y.; Xie, Z. L.; Gao, H.; Ma, W. W.; Dai, Y.; Wang, Y.; Zhong, Y.; Yao, X. S. Bioactive iridoid glucosides from the fruit of Gardenia jasminoides. J. Nat. Prod. 2009, 72, 1459−1464. (3) Kim, H. J.; Kim, E. J.; Seo, S. H.; Shin, C. G.; Jin, C.; Lee, Y. S. Vanillic acid glycoside and quinic acid derivatives from Gardeniae Fructus. J. Nat. Prod. 2006, 69, 600−603. (4) Van Calsteren, M. R.; Bissonnette, M. C.; Cormier, F.; Dufresne, C.; Ichi, T.; LeBlanc, J. Y.; Perreault, D.; Roewer, I. Spectroscopic characterization of crocetin derivatives from Crocus sativus and Gardenia jasminoides. J. Agric. Food Chem. 1997, 45, 1055−1061. (5) Carmona, M.; Zalacain, A.; Sánchez, A. M.; Novella, J. L.; Alonso, G. L. Crocetin esters, picrocrocin and its related compounds present in Crocus sativus stigmas and Gardenia jasminoides fruits. Tentative identification of seven new compounds by LC-ESI-MS. J. Agric. Food Chem. 2006, 54, 973−979. (6) Mousavi, S. H.; Moallem, S. A.; Mehri, S.; Shahsavand, S.; Nassirli, H.; Malaekeh-Nikouei, B. Improvement of cytotoxic and apoptogenic properties of crocin in cancer cell lines by its nanoliposomal form. Pharm. Biol. 2011, 49, 1039−1045. (7) Zarei Jaliani, H.; Riazi, G. H.; Ghaffari, S. M.; Karima, O.; Rahmani, A. The effect of the Crocus sativus L. carotenoid, crocin, on the polymerization of microtubules, in vitro. Iran J. Basic Med. Sci. 2013, 16, 101−107. (8) Higashino, S.; Sasaki, Y.; Giddings, J. C.; Hyodo, K.; Fujimoto Sakata, S.; Matsuda, K.; Horikawa, Y.; Yamamoto, J. Crocetin, a carotenoid from Gardenia jasminoides Ellis, protects against hypertension and cerebral thrombogenesis in stroke-prone spontaneously hypertensive rats. Phytother. Res. 2014, 28, 1315−1319. (9) Zheng, Y. Q.; Liu, J. X.; Wang, J. N.; Xu, L. Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Res. 2007, 1138, 86−94. (10) Papandreou, M. A.; Kanakis, C. D.; Polissiou, M. G.; Efthimiopoulos, S.; Cordopatis, P.; Margarity, M.; Lamari, F. N. Inhibitory activity on amyloid-β aggregation and antioxidant

18


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properties of Crocus sativus stigmas extract and its crocin constituents. J. Agric. Food Chem. 2006, 54, 8762−8768. (11) Ochiai, T.; Ohno, S.; Soeda, S.; Tanaka, H.; Shoyama, Y.; Shimeno, H. Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of α-tocopherol. Neurosci. Lett. 2004, 362, 61−64. (12) Pitsikas, N.; Sakellaridis, N. Crocus sativus L. extracts antagonize memory impairments in different behavioural tasks in the rat. Behav. Brain Res. 2006, 173, 112−115. (13) Akhondzadeh, S.; Sabet, M. S.; Harirchian, M. H.; Togha, M.; Cheraghmakani, H.; Razeghi, S.; Hejazi, S. S.; Yousefi, M. H.; Alimardani, R.; Jamshidi, A. A 22-week, multicenter, randomized, double-blind controlled trial of Crocus sativus in the treatment of mild-to-moderate Alzheimer’s disease. Psychopharmacology 2010, 207, 637−643. (14) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 2007, 55, 899−901. (15) Zhu, X.; Wang, K.; Zhang, K.; Lin, X.; Zhu, L.; Zhou, F. Puerarin protects human neuroblastoma SH-SY5Y cells against glutamate-induced oxidative stress and mitochondrial dysfunction. J. Biochem. Mol. Toxic. 2016, 30, 22−28. (16) Tarantilis, P. A.; Tsoupras, G.; Polissiou, M. Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass spectrometry. J. Chromatogr. A 1995, 699, 107-118. (17) Assimiadis, M. K.; Tarantilis, P. A.; Polissiou, M. G. UV-vis, FT-raman, and 1H NMR spectroscopies of cis-trans carotenoids from saffron (Crocus sativus L.). Appl. Spectrosc. 1998, 52, 519−522. (18) Akhondzadeh, S.; Sabet, M. S.; Harirchian, M.; Togha, M.; Cheraghmakani, H.; Razeghi, S.; Hejazi, S. S.; Yousefi, M.; Alimardani, R.; Jamshidi, A. Saffron in the treatment of patients with mild to moderate Alzheimer’s disease: a 16-week, randomized and placebo-controlled trial. J. Clin. Pharm. Ther. 2010, 35, 581−588. (19) Geromichalos, G. D.; Lamari, F. N.; Papandreou, M. A.; Trafalis, D. T.; Margarity, M.; Papageorgiou, A.; Sinakos, Z. Saffron as a source of novel acetylcholinesterase inhibitors: molecular docking and in vitro enzymatic studies. J. Agric. Food Chem. 2012, 60, 6131−6138. (20) Ghahghaei, A.; Bathaie, S. Z.; Bahraminejad, E. Mechanisms of the effects of crocin on 19



aggregation and deposition of Aβ1–40 fibrils in Alzheimer’s disease. Int. J. Pept. Res. Ther. 2012, 18, 347−351. (21) Papandreou, M. A.; Tsachaki, M.; Efthimiopoulos, S.; Cordopatis, P.; Lamari, F. N.; Margarity, M. Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection. Behav. Brain Res. 2011, 219, 197−204. (22) Gella, A.; Durany, N. Oxidative stress in Alzheimer disease. Cell Adhes. & Migr. 2009, 3, 88−93. (23) Paula-Lima, A. C.; Brito-Moreira, J.; Ferreira, S. T. Deregulation of excitatory neurotransmission underlying synapse failure in Alzheimer’s disease. J. Neurochem. 2013, 126, 191−202.

20


Page 20 of 33

Page 21 of 33


Table 1. NMR Spectroscopic Data (600 MHz for 1H and 150 MHz for 13C, in DMSO-d6) of Compounds 1-4 1

2

3

4

No. δC 8, 8′

9, 9′

10, 10′

11, 11′

12, 12′

13, 13′

14, 14′

15, 15′

19, 19′

20, 20′

δH, J (HZ)

δC

δH, J (HZ)

δC

δH, J (HZ)

δC

166.2

166.2

166.2

166.9

167.0

167.1

166.9

166.2

125.4

125.2

125.4

126.8

126.1

127.1

125.9

125.1

δH, J (HZ)

140.0

7.35, d (10.8)

139.9

7.35, d (10.8)

140.0

7.44

139.0

7.35

139.0

7.23, d (10.2)

138.3

7.30, d (10.2)

139.0

7.23

140.0

7.37

6.67

123.8

6.67

125.2

6.64

125.2

6.63

123.9

a

124.0

a

6.63

124.2

6.65

123.7

6.61

123.6

6.61

144.7

6.81, d (15.0)

144.7

6.82, d (15.0)

136.8

7.46, br. d (8.4)

135.6

7.36

144.0

6.67

143.4

6.73, d (15.0)

144.1

6.68, d (12.6)

144.8

6.83, d (14.4)

a

137.0

135.1

135.0

136.8a

136.7

136.4

136.4

136.9

136.1

6.53, d (10.2)

136.0

6.53, br. d (9.6)

134.2

6.39, d (11.4)

134.2

6.37, d (11.4)

135.7

6.49, d (9.6)

135.3

6.53, br. d (9.6)

135.9

6.45, d (12.0)

136.0

6.52, d (12.0)

132.1

6.84, dd (10.2, 1.8)

132.1

6.86

130.9

7.13

130.9

7.13

131.9

6.84, dd (8.4, 1.8)

131.7

6.86

130.7

6.75

130.7

6.75

12.7

1.97, s

12.7

1.97, s

12.7

1.97, s

12.7

1.95, s

12.8

1.92, s

12.8

1.98, s

12.8

1.93, s

12.8

1.97, s

12.6

2.00, s

12.5

1.99, s

20.0

2.00, s

20.1

2.00, s

12.6

1.96, s

12.6

2.00, s

12.5

1.98, s

12.5

1.98, s

1′′

73.6

72.8

2′′

37.4

3′′

67.5

5.42

70.9

5.18

67.7

5.38

67.7

5.38

4′′

74.2

4.88, dd (8.4, 3.0)

68.5

3.80, br. s

73.4

4.95, dd (7.8, 3.0)

73.4

4.95, dd (7.8, 3.0)

5′′

66.3

4.19

70.9

5.18

66.1

4.19

66.1

4.19

1.99

35.1

2.15

73.6 1.92

37.0

2.13

73.6 2.01

37.0

2.17

21


2.01 2.17


1.87

35.1

6′′

37.6

7′′

174.8

172.1

174.7

174.7

1′′′

165.6

165.7

165.5

165.5

2.15

37.6

2.13

1.89

37.6

2.17

1.89 2.17

2′′′

113.6

6.16, d (16.2)

114.3

6.18, d (16.2)

113.6

6.14, d (16.2)

113.6

6.14, d (16.2)

3′′′

145.6

7.44, d (16.2)

145.0

7.45, d (16.2)

145.6

7.42, d (16.2)

145.6

7.42, d (16.2)

4′′′

125.2

125.6 7.03, d (1.8)

114.8

125.4 7.05, d (1.8)

114.9

125.4 7.02, d (1.8)

114.9

5′′′

114.9

6′′′

145.6

145.6

145.6

145.6

7′′′

148.5

148.4

148.5

148.5

7.02, d (1.8)

8′′′

115.7

6.74, d (8.4)

115.8

6.77, d (8.4)

115.7

6.75, d (8.4)

115.7

6.75, d (8.4)

9′′′

121.6

6.98, dd (8.4,1.8)

121.3

6.99, dd (8.4,1.8)

121.5

6.97, dd (8.4, 1.8)

121.5

6.97, dd (8.4, 1.8)

8-Gen

8-Gen

8-Gen

8′-Gen

1

94.5

5.42, d (7.8)

94.5

5.42, d (7.8)

94.5

5.44, d (7.8)

94.5

5.42, d (7.8)

2

72.5

3.23

72.4

3.23

72.5

3.22

72.5

3.22

3

76.3

3.26

76.2

3.26

76.3

3.25

76.3

3.25

4

69.2

3.25

69.2

3.24

69.2

3.24

69.2

3.24

5

76.3

3.42

76.3

3.42

76.3

3.42

76.3

3.42

6

67.9

3.99, br. d (10.2)

67.9

3.99, br. d (10.2)

67.9

3.99, br. d (9.6)

67.9

3.98, br. d (10.2)

1′

103.1

4.17, d (7.8)

103.1

4.17 d (7.8)

103.1

4.17 d (7.8)

103.1

4.17 d (7.8)

2′

73.5

2.96, t (7.8)

73.4

2.96, t (7.8)

73.5

2.95

73.5

2.95

3.58, dd (10.8, 4.8)

3.59, dd (10.8, 4.8)

3.58, dd (11.4, 5.4)

3.58, dd (11.4, 5.4)

3′

76.8

3.12

76.8

3.12

76.8

3.12

76.8

3.12

4′

70.0

3.05

70.0

3.05

70.0

3.05

70.0

3.05

5′

76.9

3.05

76.9

3.05

76.9

3.05

76.9

3.05

6′

61.0

3.64, br. d (10.2)

61.0

3.65, br. d (9.6)

61.0

3.65, dd (11.4, 5.4)

61.0

3.65, dd (11.4, 5.4)

3.43 a

1.92

Page 22 of 33

3.42

3.43

means signals could be interchangeable with the corresponding position in one compound; All multiple and overlapping peaks were not distinguished.

22


3.43

Page 23 of 33


Table 2. NMR Spectroscopic Data (600 MHz for 1H and 150 MHz for 13C, in DMSO-d6) of Compounds 5 and 6 5

6

5

No. δC 8, 8′

9, 9′

δH, J (HZ)

11, 11′

12, 12′

13, 13′

14, 14′

15, 15′

19, 19′

20, 20′

1′′

δC

δH, J (HZ)

δC

δH, J (HZ)

8-Gen

δC

δH, J (HZ)

166.2

166.2

169.1

166.2

1

94.5

5.42, d (7.8)

94.5

5.42, d (7.8)

125.2

125.3

2

72.5

3.23

72.5

3.23

127.0 10, 10′

6

No.

125.3

8-Gen

3

76.2

3.27

76.2

3.26

139.9

7.33, d (10.8)

139.9

7.35, d (10.8)

4

69.2

3.23

69.2

3.24

138.0

7.21, d (11.4)

139.9

7.34, d (10.8)

5

76.2

3.43

76.2

3.43

6

68.0

3.95, br. d (10.8)

68.0

123.8

6.64

123.9

6.67, t (13.2)

124.2

6.62

123.9

6.65, t (13.2)

144.6

6.76, d (15.0)

144.6

6.82, d (15.0)

1′

103.0

4.25, d (7.8)

103.0

4.25, d (7.8)

143.3

6.73, d (15.0)

144.6

6.77, d (15.0)

3.62, dd (10.8, 4.8)

3.98, br. d (10.2) 3.60, dd (12.0, 6.0)

2′

73.4

3.01

73.4

3.01

136.9

136.9

3′

76.5

3.17

76.5

3.17

136.6

136.9

4′

69.8

3.17

69.8

3.17

a

136.0

6.50, br. d (12.0)

136.0

6.53, br. d (10.2)

5′

73.8

3.36

73.8

3.36

135.3

6.50, br. d (12.0)

135.9a

6.50, br. d (10.2)

6′

63.5

4.36,br. d (10.8)

63.5

4.36, br. d (10.8)

132.0

6.84, dd (10.2, 2.4)

132.0

6.86, dd (7.8, 2.4)

131.6

6.84, dd (10.2, 2.4)

132.0

6.86, dd (7.8, 2.4)

12.7

1.95, s

12.7

1.96, s

1

94.5

5.42, d (7.8)

12.8

1.92, s

12.7

1.97, s

2

72.5

3.23

4.18, dd (12.0, 6.0)

4.18, br. d (12.0) 8′-Gen

12.6

1.97, s

12.5

1.98, s

3

76.3

3.26

12.5

1.98, s

12.6

2.00, s

4

69.2

3.24

5

76.3

3.43

6

67.9

166.7

166.7

2′′

114.8

6.57, d (15.6)

114.8

6.57, d (15.6)

3′′

145.5

7.55, d (15.6)

145.5

7.55, d (15.6)

4′′

124.4

5′′

106.3

124.4 7.03, s

106.3

3.98, br. d (10.2) 3.60, dd (12.0, 6.0)

7.03, s

1′

103.1

4.17, d (7.8)

2′

73.5

2.96

23



Page 24 of 33

6′′

148.0

148.0

3′

76.8

3.12

7′′

138.3

138.3

4′

70.0

3.05

8′′

148.0

148.0

5′

76.9

3.05

9′′

106.3

7.03, s

106.3

7.03, s

6′

61.0

3.65, (11.4, 6.0)

6′′, 8′′-OCH3

56.1

3.80, s

56.1

3.80, s

a

3.44


24


Page 25 of 33


Table 3. NMR Spectroscopic Data (600 MHz for 1H and 150 MHz for 13C, in DMSO-d6) of Compounds 7-9, 13 and 14 7

8

9

13

14

No. δC 8, 8′

9, 9′

10, 10′

11, 11′

12, 12′

13, 13′

15, 15′

δC

166.2

166.2

167.4

194.5

δC

δC

9.44, s

δH, J (HZ)

δC

δH, J (HZ)

δC

166.2

166.1

169.3

169.3

166.1

166.2

125.2

125.4

125.1

127.2

128.1

126.2

136.5

127.3

125.8

125.0

139.9

7.35, d (11.4)

139.9

138.4

7.24, d (11.4)

123.8

6.66

124.0

6.62

δH, J (HZ)

7.36, d (10.8)

139.9

7.35, d (11.4)

140.1

7.45

140.0

7.36

149.2

7.17, d (10.8)

137.9

7.20, d (10.8)

137.9

7.21

137.9

7.38, d, (10.8)

124.1

6.68, dd (15.0, 11.4)

123.7

6.67

124.9

6.64

125.5

6.64

123.9

6.82

124.3

6.63

124.0

6.62

123.5

6.60

144.6

6.82, d (14.4)

144.5

6.83

144.7

6.81, d (15.0)

136.8

7.47

135.3

7.36

143.9

6.77, d (15.0)

145.7

6.89

143.2

6.72, d (15.0)

143.2

6.71, d, (15.3)

144.7

6.81, d, (14.4)

136.9a 136.8

14, 14′

δH, J (HZ)

136.8

a

137.0

137.3

135.1

136.6

135.2

136.4

136.2

136.0

6.53, d (10.2)

135.9

6.54, d (10.2)

136.0

6.52, d (9.6)

134.5

6.38, d (11.7)

133.7

6.34, d (11.7)

135.6

6.51, d (10.2)

137.0

6.61, d (10.8)

135.2

6.49, d (9.6)

134.8

6.44, d (11.7)

136.2

6.47, d, (11.7)

132.0

6.85

131.9

6.91

132.1

6.83

130.8

7.15

131.1

7.14

131.7

6.85

132.6

6.89

131.5

6.84

130.6

6.76

130.3

6.74

12.7

1.97, s

12.7

1.97, s

12.7

1.97, s

12.7

1.96, s

12.7

1.96, s

12.8

1.95, s

9.4

1.82, s

12.9

1.92, s

12.8

1.91, s

12.8

1.92, s

12.6

1.99, s

12.6

2.00, s

12.5

1.98, s

20.0

1.97, s

20.1

1.97, s

12.5

1.98, s

12.5

2.02, s

12.6

1.98, s

12.5

1.99, s

12.5

1.98, s

1′′

60.1

4.15, q (7.2)

2′′

14.2

1.24, t (7.2)

19, 19′

20, 20′

8-Gen

8-Gen

8-Gen

8-Gen

8’-Gen

1

94.5

5.42, d (7.8)

94.5

5.42, d (8.4)

94.5

5.41, d (7.8)

94.5

5.45, d (8.1)

94.5

5.42, d (7.2)

2

72.5

3.22

72.4

3.22

72.4

3.21

72.5

3.22

72.4

3.21

25



3

76.3

3.26

76.2

3.25

76.2

3.25

76.2

3.25

76.2

3.25

4

69.2

3.24

69.2

3.23

69.3

3.18

69.2

3.21

69.2

3.20

5

76.3

3.43

76.3

3.42

76.4

3.39

76.3

3.44

76.3

3.43

6

67.9

3.99, br. d (10.2)

67.9

3.99, br. d (11.4)

68.0

3.92, br. d (10.2)

67.9

4.00, d (8.1)

67.9

3.59, dd (11.4, 5.4)

3.59, dd (11.4, 5.4)

3.57, dd (11.4, 5.4)

3.59, dd (11.7, 5.4)

3.99, d (9.9) 3.59, dd (11.7, 5.4)

1′

103.1

4.17, d (7.8)

103.1

4.17, d (7.8)

103.7

4.14, d (7.8)

103.1

4.17, d (8.1)

103.1

4.17, d (8.1)

2′

73.5

2.96, t (7.8)

73.4

2.96, td (7.8, 4.8)

73.3

2.95

73.4

2.96, t (7.8)

73.5

2.96, t (7.8)

3′

76.8

3.12

76.7

3.11

76.6

3.07, t (8.4)

76.8

3.12

76.8

3.12, t, (8.4)

4′

70.0

3.05

69.9

3.05

69.5

3.24

70.0

3.05

70.0

3.05

5′

76.9

3.05

76.9

3.05

65.7

3.67, dd (11.4, 5.4)

76.9

3.05

76.9

3.05

6′

61.0

3.65, br. d (10.2)

61.0

3.65, dd (10.8, 5.4)

2.97

61.0

3.65, d, (11.7)

61.0

3.65, d (9.9)

3.4 a

Page 26 of 33

3.42

3.44


26


3.43

Page 27 of 33


Table 4. Neuroprotective Activities of the Crocins on SH-SY5Y Cells

Compound

1 2 3/4 5 6 7 8 9 10 11 12 13/14 15 16 17/18 19 20

Cell Viability against Hydrogen Peroxide (H2O2) Induced Injury Concentrations (mol/L)

Cell Viability against L-Glutamic Acid Induced Injury Concentrations (mol/L)

10-5

10-6

10-7

10-5

10-6

10-7

10.39±4.85 8.82±6.57 5.49±5.57 18.60±3.43* 36.38±5.27*** 36.60±3.81* 9.43±1.47 33.92±2.30* 2.20±3.81 0.00±0.00 40.19±8.41 5.08±6.24 0.00±0.00 0.00±0.00 5.66±4.90 44.30±4.23* 10.98±14.08

0.17±0.29 0.00±0.35 0.00±0.00 6.83±3.63 26.02±3.62** 5.09±4.50 0.67±1.13 16.98±3.74 4.29±4.42 0.00±0.00 24.24±7.44 3.75±6.50 0.00±0.00 0.00±0.00 3.90±6.76 19.66±3.23* 4.84±1.61

0.00±0.00 0.00±0.00 0.25±0.08 2.42±4.19 14.56±11.22* 2.28±3.95 0.00±0.00 6.28±5.68 2.27±2.82 0.00±0.00 3.72±3.30 0.09±0.15 0.00±0.00 0.00±0.00 0.00±0.00 5.80±1.71 5.31±1.77

40.03±3.91** 16.29±14.12 12.95±4.01** 74.93±15.36* 55.02±0.60** 41.28±7.52* 46.12±8.45* 35.20±13.61* 4.80±1.28 0.00±0.00 39.43±7.49 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 7.59±13.14 0.00±0.00

27.63±5.36* 7.69±9.01 2.78±3.24 57.05±10.87** 48.75±9.49** 7.39±7.08 38.02±5.15* 33.15±12.55* 7.02±4.12 0.00±0.00 21.48±6.91 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

9.89±2.93 6.68±11.57 1.51±2.62 42.60±5.40** 24.51±13.70* 0.00±0.00 18.26±5.02 18.26±5.02 9.09±4.62 0.00±0.00 8.82±1.94 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

27



Page 28 of 33

Table. 5 Crocins Identified by UPLC-Q/TOF-MS in the Chromatogram of GJ-4 Mass error tR

Ion Type

Mea. mass

Cal. mass

Formula

Fragmentation

Identification

(ppm) 5.79

[M+Na]+

976.3770, 999.3676; 652.2716, 675.2599; 999.3676

999.3685

-0.9

C44H64O24

19 329.1764, 311.1641, 293.1528

7.35

[M+Na]+

814.3245, 837.3118; 675.2667; 837.3118

837.3157

-4.7

C38H54O19

16 329.1737, 311.1633, 293.1537

9.15

[M+Na]+

652.2706, 675.2614; 513.2049; 675.2614

675.2629

-2.2

C32H44O14

15 329.1725, 311.1632, 293.1535

9.25

[M+Na]+

1182.4335,1183.4384, 1205.4243; 881.3331; 1205.4243

1205.4264

-1.7

C55H74O28

6 675.2614; 531.1690, 369.1180, 207.0654

11.71

[M+Na]+

976.3772, 999.3680, 1953.7651; 652.2731; 999.3680

999.3685

-0.5

C44H64O24

20 329.1740, 311.1644, 293.1530

13.41

[M+Na]+

814.3242, 837.3129; 837.3129

837.3157

-3.3

C38H54O19

17 329.1747, 311.1649, 293.1537

13.52

[M+Na]+

814.3256, 837.3130; 837.3130

837.3157

-3.2

C38H54O19

18 329.1751, 311.1654, 293.1543

13.56

[M+H]+

988.3539, 989.3597, 1976.7084; 989.3597

989.3654

-5.8

C48H60O22

1 665.2532; 827.3113

13.61

[M+H]+

988.3622, 989.3635; 665.2552; 827.2856; 989.3635

989.3654

-1.9

C48H60O22

2 311.1626, 293.1410

15.09

[M+Na]+

652.2720, 675.2620; 675.2620

675.2629

-1.3

C32H44O14

12 329.1744, 311.1635, 293.1530

16.12

[M+Na]+

622.2648, 645.2529; 645.2529

645.2523

0.9

C31H42O13

9 329.1751, 311.1656, 293.1543

17.06

[M+H]+

988.3561, 989.3600, 1977.7115; 989.3600

989.3654

-5.5

C48H60O22

3 665.2578; 827.3083; 311.1651, 293.1537

17.08

[M+Na]+

652.2731, 675.2632; 675.2632

675.2629

0.4

C32H44O14

13 329.1759, 311.1651, 293.1541

17.16

[M+H]+

988.3575, 989.3602, 1977.7135, 989.3602

989.3654

-5.3

C48H60O22

4 665.2574; 827.3069; 311.1650, 293.1553

17.51

[M+Na]+

652.2675, 675.2625; 675.2625

675.2629

-0.6

C32H44O14

14 329.1741, 311.1641, 293.1539

18.03

[M+Na]+

490.2192, 513.2098, 1003.4275; 329.1747, 513.2098

513.2101

-0.6

C26H34O9

11 311.1643, 293.1534

18.39

[M+Na]+

858.3277, 881.3243; 531.1615, 369.1183, 881.3203

881.3208

-0.6

C43H54O18

5 207.0655

20.47

[M+Na]+

703.2919

703.2942

-3.3

C34H48O14

680.3058, 703.2919; 357.2047; 1383.5914

28


7

Page 29 of 33


Fig. 1 Crocins isolated from gardenia (Gardenia jasminoides Ellis) fruits

O

OH O

HO O

O OH HO HO

HO HO

O

O

O

O

OH

O

OH

HO

OH O

HO

O HO O HO HO

O OH HO HO

O

O

OH

O HO

O

OH

O

OH

O HO

O

HO

Fig. 2 Key HMBC (→) and COSY (▬) correlations of compounds 1 and 2

29



Fig. 3 Chromatogram of the crocin-rich fraction by UPLC-Q/TOF-MS. MS parameters: capillary voltage 2.0 kV, source temperature 100°C, desolvation gas N2 at 600 L/h, drying gas temperature 300°C, m/z 50 - 2000. Chromatographic parameters: RP-18 column (1.7 µm, ϕ 3.0 × 150 mm; BEH), mobile phases (A) 0.1% formic acid in water, (B) 0.1% formic acid in acetonitrile; flow rate 0.6 mL/min; gradient program: 0 - 0.5 min isocratic at 20% B; 0.5 - 19 min – linear gradient to 50% B; 19 to 20 min – linear gradient to 100% B; 20 to 23 min – isocratic at 100% B.

30


Page 30 of 33

Page 31 of 33


Fig. 4 Major peaks in the crocin-rich fraction identified in HPLC-ELSD (above) and UPLC-DAD (440 nm) (below). HPLC parameters: RP-18 column (5 µm, ϕ 4.6 × 250 mm; COSMOSIL) column oven 30°C, mobile phases (A) 0.1% acetic acid in water and (B) methanol at 1.0 mL/min, gradient program: 0 - 40 min – linear gradient from 50% B to 90% B; 40 - 45 min – linear gradient to 100% B, chromatograph at 440 nm was shown in the figure. UPLC experimental parameters were as depicted in Fig. 3.

31



Fig. 5 Proposed fragmentation pathways of compounds 1 and 6

32


Page 32 of 33

Page 33 of 33


33


Gardeniae Fructus and Their Neuroprotective - ACS Publications

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