Neolignan Constituents with Potential Beneficial Effects in Prevention of Type 2 Diabetes from Viburnum fordiae Hance Fruits

Article abstract of DOI:10.1021/acs.jafc.8b03772

Nine new neolignan glycosides (1-9), viburfordosides A-I, two new neolignans, fordianes A and B (10, 11), and seven known analogues (12-18) have been isolated and identified from the fruits of Viburnum fordiae Hance. The structures and absolute configurations of undescribed neolignan constituents were identified by chemical methods and spectroscopic analyses. The α-glucosidase inhibitory, ABTS^?+ and DPPH^? scavenging, and anti-inflammatory activities of these secondary metabolites were evaluated. Some of them exhibited significant potency in inhibiting α-glucosidase and scavenging free radicals. Among the 14 metabolites that were found to have the capacity to inhibit NO production in LPS-stimulated RAW264.7 macrophage cells, compounds 2, 4, 6, 10, 11, 14, 17, and 18 were potent with IC₅₀ values of 10.88-41.10 μM. These results support that V. fordiae fruits possessing the neolignan compounds may serve as both a functional food and a medicinal resource to prevent and treat type 2 diabetes (T2D).

Full text of DOI:10.1021/acs.jafc.8b03772

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Bioactive Constituents, Metabolites, and Functions
Neolignan Constituents with Potential Beneficial Effects in
Prevention of Type 2 Diabetes from Viburnum fordiae Hance Fruits
Chunchao Zhao, Jia Chen, Jianhua Shao, Jie Shen, Kehuan Li, Wenyan Gu, Sihui Li, and Judi Fan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03772 • Publication Date (Web): 19 Sep 2018
Downloaded from http://pubs.acs.org on September 25, 2018
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Page 1 of 33
Journal of Agricultural and Food Chemistry
Neolignan Constituents with Potential Beneficial Effects in Prevention of Type 2 Diabetes from
Viburnum fordiae Hance Fruits
Chunchao Zhao,*,†,‡ Jia Chen,† Jianhua Shao,*,† Jie Shen,† Kehuan Li,‡ Wenyan Gu,‡ Sihui Li,‡ and
Judi Fan§
†Jiangsu Key Laboratory of Crop Genetics and Physiology/CoꢀInnovation Center for Modern
Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China
‡Joint International Research Laboratory of Agriculture & AgriꢀProduct Safety of Ministry of
Education of China, Yangzhou University, Yangzhou 225009, China
§School of Pharmacy, Guizhou Medical University, Guiyang 550004, China
* Corresponding authors. Tel: +86ꢀ514ꢀ87991556. Eꢀmail: cczhao@yzu.edu.cn, jhshao@yzu.edu.cn.
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ABSTRACT: Nine new neolignan glycosides (1−9), viburfordosides A−I, two new neolignans,
fordianes A and B (10, 11), and seven known analogues (12−18) have been isolated and identified
from the fruits of Viburnum fordiae Hance. The structures and absolute configurations of undescribed
neolignan constituents were identified by chemical methods and spectroscopic analyses. The
αꢀglucosidase inhibitory, ABTS•+ and DPPH• scavenging and antiꢀinflammatory activities of these
secondary metabolites were evaluated. Some of them exhibited significant potency in inhibiting
αꢀglucosidase and scavenging free radicals. Among fourteen metabolites that were found to have the
capacity to inhibit NO production in LPSꢀstimulated RAW264.7 macrophage cells, compounds 2, 4,
6, 10, 11, 14, 17 and 18 were potent with IC50 values of 10.88 to 41.10 ꢀM. These results support that
V. fordiae fruits possessing the neolignan compounds may serve as both functional food and
medicinal resource to prevent and treat type 2 diabetes (T2D).
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KEYWORDS: Adoxaceae, Viburnum fordiae Hance, berry fruits, neolignans, αꢀglucosidase
inhibition, radical scavenging, antiꢀinflammatory
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INTRODUCTION
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Type 2 diabetes (T2D), representing over 90% of diabetes, has reached pandemic proportions
throughout the world, and is now recognized as a leading risk factor for cardiovascular events that
are responsible for most of the deaths in diabetic patients, the proportion of which is up to 65%.1−3
T2D is characterized by hyperglycemia. Prevention of hyperglycemia is important, especially
following a meal when plasma glucose levels are highest.1 Sustained postprandial hyperglycemia
(PPHG) can result in postprandial oxidative stress and inflammation, and in turn, continued oxidative
stress and inflammation in T2D mediate the action of acute hyperglycemia that is involved in the
development of diabetes complications such as cardiovascular disease (CVD).4−7
Scientific evidence has revealed that PPHG can be effectively regulated by inhibiting
carbohydrateꢀhydrolyzing enzymes to retard absorption of glucose.8 Thus, αꢀglucosidase inhibitors,
for example acarbose and miglitol, play a vital role in controlling T2D. However, their clinical
application is limited due to their gastroꢀintestinal side effects and high cost.9 Recently, more
attention has been paid to plantꢀbased foods, particularly some fruits and vegetables. In
epidemiological research, the plantꢀbased foods have been considered as the critical ingredients of
dietary patterns that can effectively prevent chronic diseases such as T2D and CVD.10,11 Polyphenols,
abundant in fruits, vegetables and tea, have been reported to possess therapeutic potential in the
management of T2D in humans.12,13 These natural compounds have the inhibitory activity against
αꢀglucosidase in concert with acarbose for effective glycemic control, and can provide protective
effects on organs and tissues against oxidative stress and inflammation.14−16 Therefore, some fruits
and vegetables, especially berries, rich in polyphenols have been proposed for the prevention and
treatment of T2D and CVD.
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Viburnum fordiae Hance, also called “man shan hong” in Chinese due to its crimson berries, is
distributed throughout South China region, particularly in mountain areas.17 In recent years, the
cultivation of V. fordiae is increasing in China due to its high economic values. Its stems, roots and
leaves, known as traditional Chinese medicine, have been commonly used owing to their therapeutic
efficacy in various inflammatory diseases.17 And its fruits, containing large amounts of nutritional
ingredients, such as vitamins, microelements and amino acids, are a type of edible berry, and usually
used to make beverages in Jiangxi and Guizhou provinces, China.18 Previous studies, including those
from our group, have resulted in the identification of essential oils, terpenoids, neolignans and other
phenolics from the stems, leaves and roots of V. fordiae, some of which exhibited weak or moderate
antioxidant, antiꢀinflammatory and αꢀglucosidase inhibitory properties.19−22 As an edible berry with
abundant nutrient ingredients, the fruits of V. fordiae may be more suitable to be utilized as a food
material than its stems, leaves and roots. However, to our knowledge, no investigations, involving
biological activities and chemical components of V. fordiae fruits, have been reported. In our search
for potential food materials for prevention of T2D, the nꢀbutanol fraction from V. fordiae fruits
showed significant αꢀglucosidase inhibitory, free radical scavenging and NO inhibitory activities.
Therefore, it was subjected to phytochemical investigation and further bioactivity screening. In this
paper, eleven undescribed neolignan constituents (1−11) and seven known analogues (12−18) from
the fruits of V. fordiae were identified and their bioactivities were also evaluated in vitro.
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MATERIALS AND METHODS
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General Experimental Procedures. A JASCO (Tokyo, Japan) Jꢀ810 circular dichroism (CD)
spectrometer, connected with Peltier temperature controller, was used to acquire CD data. A SGWꢀ2
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digital polarimeter (Yidian Wuli Guangxue Company, Shanghai, China) was used for experimental
optical rotation measurements. UV–vis–NIR measurements were obtained by a Cary 5000
spectrophotometer (Varian, Palo Alto, USA). The Fourier transform infrared (FTIR) spectroscopy
measurements were conducted using a 610/670 infrared microspectrometer (Varian, Palo Alto,
USA), with the prepared powders diluted in KBr pellets. HRꢀESIMS spectra were acquired by a
UHR–TOF maxis instrument (Bruker, Bremen, Germany). A Bruker (Rheinstetten, Germany)
Avance 600 MHz spectrometer was used to acquire 1D/2D NMR spectra.
Macroporous resin (HPDꢀ100), ODSꢀAꢀHG, Sephadex LHꢀ20, silica gel, and MCIꢀgel CHP20P
applied to column chromatography (CC) were provided by Bonherb Technology Company (Hebei,
China), YMC (Kyoto, Japan), GE Healthcare Biosciences (Uppsala, Sweden), Haiyang Chemical
Company (Qingdao, China), and Mitsubishi Chemical Industries (Tokyo, Japan), respectively.
β
ꢀGlucosidase, trolox, indomethacin, and acarbose were available from SigmaꢀAldrich (St. Louis,
USA). Semiꢀpreparative HPLC was accomplished on a YMCꢀpacked column (5 ꢀm, 250 mm × 10
mm i.d.) with an LC3000 system (Chuangxin Tongheng Company, Beijing, China).
Plant Material. The fresh fruits of V. fordiae, used in this study, were picked in Guiyang city,
Guizhou province, China, in October 2011. A voucher specimen (201102) was stored in Medicinal
Plant Biotechnology Laboratory, Yangzhou University and identified by Professor Huyin Huai from
Yangzhou University.
Extraction and Isolation. Air dried V. fordiae fruits (16.5 kg) were well mashed and then
extracted with 95% ethanol three times. The extract was evaporated to obtain a residue. This residue
was dissolved in distilled water, and then extracted in sequence with petroleum ether and nꢀbutanol.
The nꢀbutanol part (830.0 g) was chromatographed over HPDꢀ100 macroporous resin and orderly
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eluted with H2O and 95% ethanol to afford two portions. The 95% ethanol eluate (492.0 g) was
loaded onto silica gel column with gradient solvents of MeOH/CHCl3 (0:100 → 100:0) as the eluent,
to acquire seven fractions (F1–F7).
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Fraction F2 was applied to the open column packed with MCI gel using the sequential elution with
MeOH/H2O (90:10) and acetone. The portion eluted by MeOH/H2O (90:10) was applied to ODS CC
using MeOH/H2O (20:80 → 100:0) as the eluent, to yield six subfractions (F2ꢀ1–F2ꢀ6). F2ꢀ2 was loaded
onto Sephadex LHꢀ20 column and eluted with MeOH/CHCl3 (1:2) to give a mixture of compounds
15 and 16. This mixture was fractionated by semiꢀprep. HPLC (MeOH/H2O, 44:56, 1.5 mL/min) to
produce compounds 15 (24 mg, tR = 17.1 min) and 16 (10 mg, tR = 21.1 min). F2ꢀ3 was
chromatographed over Sephadex LHꢀ20 with MeOH/CHCl3 (1:2) as the eluent, to yield compound
14 (58 mg) and a mixture, which was isolated by semiꢀprep. HPLC (MeOH/H2O, 31:69, 1.5 mL/min)
to give compounds 10 (178 mg, tR = 67.4 min) and 11 (132 mg, tR = 77.3 min). Another subfraction
F2ꢀ4 was applied to Sephadex LHꢀ20 CC eluting with MeOH/CHCl3 (1:2), and further separated by
semiꢀprep. HPLC (MeOH/H2O, 43:57, 1.5 mL/min), to obtain compounds 17 (11 mg, tR = 71.1 min)
and 18 (12 mg, tR = 76.5 min).
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Fraction F5 was separated into six subfractions (F5ꢀ1–F5ꢀ6) through ODS CC eluting with
MeOH/H2O (5:95 → 100:0) and two consecutive Sephadex LHꢀ20 CC with MeOH/CHCl3 (1:2) and
MeOH as the eluent, respectively. F5ꢀ4 was applied to Sephadex LHꢀ20 CC with MeOH/H2O (45:55)
as the eluent, to yield fractions F5ꢀ4ꢀ1–F5ꢀ4ꢀ4. F5ꢀ4ꢀ2 was subjected to MCI gel CC using MeOH/H2O
(10:90 → 100:0) as the eluent, to yield fourteen fractions (F5ꢀ4ꢀ2ꢀ1–F5ꢀ4ꢀ2ꢀ14). F5ꢀ4ꢀ2ꢀ6 was loaded onto
Sephadex LHꢀ20 column and eluted with MeOH, to give 1 (128 mg). F5ꢀ4ꢀ2ꢀ7 was fractionated by
Sephadex LHꢀ20 CC eluting with MeOH, to give 5 (16 mg). F5ꢀ4ꢀ2ꢀ9 was loaded onto Sephadex
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LHꢀ20 column using MeOH/CHCl3 (1:2) as the eluent, to afford a mixture of 7, 8, 9, and 12. The
mixture was separated by semiꢀprep. HPLC (MeOH/H2O, 32:68, 1.5 mL/min) to give 7 (42 mg, tR =
64.1 min), 8 (30 mg, tR = 68.6 min), 9 (25 mg, tR = 72.7 min), and 12 (15 mg, tR = 66.0 min). F5ꢀ4ꢀ2ꢀ11
was applied to Sephadex LHꢀ20 CC with MeOH/CHCl3 (1:2) as the eluent, to produce a mixture,
which was fractionated by semiꢀprep. HPLC (MeOH/H2O, 33:67, 1.5 mL/min) to yield 3 (12 mg, tR
= 84.4 min) and 2 (19 mg, tR = 96.3 min). F5ꢀ4ꢀ2ꢀ13 was fractionated by Sephadex LHꢀ20 CC eluting
with MeOH and then subjected to semiꢀprep. HPLC (MeOH/H2O, 42:58, 1.5 mL/min) to give 4 (14
mg, tR = 31.8 min) and 6 (21 mg, tR = 35.7 min). Compound 13 (10 mg, tR = 82.5 min) was acquired
from F5ꢀ4ꢀ3 on Sephadex LHꢀ20 column eluting with MeOH, in combination with semiꢀprep. HPLC
(MeOH/H2O, 33:67, 1.5 mL/min).
Viburfordoside A (1). White powder; HRꢀESIMS m/z 735.2486 [M + Na]+ (calcd forC33H44O17Na,
735.2471); CD (MeOH) λ (ꢁε) 216 (+2.02), 221 (–4.15), 234 (–7.38), 271 (+5.60) nm; UV (MeOH)
λmax (log ε) 208 (4.89), 274 (4.46) nm; [α]2D3 –24.0 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR
(KBr) vmax 3401, 1599, 1498, 1463, 1127, 1074, 1022, 890, 636 cmꢀ1.
Viburfordoside B (2). White powder; HRꢀESIMS m/z 885.2795 [M + Na]+ (calcd forC41H50O20Na,
885.2788); CD (MeOH) λ (ꢁε) 217 (–6.77), 235 (+4.35), 272 (–6.79) nm; UV (MeOH) λmax (log ε)
213 (4.28), 268 (3.97) nm; [α]2D3 –50.4 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR (KBr) vmax
3400, 1703, 1598, 1500, 1463, 1123, 1072, 824, 765, 629 cmꢀ1.
Viburfordoside C (3). White powder; HRꢀESIMS m/z 915.2897 [M + Na]+ (calcd forC42H52O21Na,
915.2893); CD (MeOH) λ (ꢁε) 235 (+8.96), 275 (–13.39) nm; UV (MeOH) λmax (log ε) 276 (4.78)
nm; [α]2D3 –16.2 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR (KBr) vmax 3406, 1704, 1600, 1499,
1462, 1120, 1073, 822, 765, 669 cmꢀ1.
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Viburfordoside D (4). White powder; HRꢀESIMS m/z 885.2790 [M + Na]+ (calcd forC41H50O20Na,
885.2788); CD (MeOH) λ (ꢁε) 224 (+3.99), 231 (+4.59), 277 (–12.28), 285 (–12.64) nm; UV
(MeOH) λmax (log ε) 277 (4.64) nm; [α] 2D3 –13.3 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR (KBr)
vmax 3391, 1706, 1605, 1513, 1463, 1114, 1074, 1030, 863, 765, 669 cmꢀ1.
Viburfordoside E (5). White powder; HRꢀESIMS m/z 707.2524 [M + Na]+ (calcd forC32H44O16Na,
707.2522); CD (MeOH) λ (ꢁε) 241 (+2.58), 290 (+1.17) nm; UV (MeOH) λmax (log ε) 282 (3.92) nm;
[α]2D3 +14.8 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR (KBr) vmax 3392, 1608, 1498, 1076, 1033,
897, 633 cmꢀ1.
Viburfordoside F (6). White powder; HRꢀESIMS m/z 857.2829 [M + Na]+ (calcd forC40H50O19Na,
857.2839); CD (MeOH) λ (ꢁε) 218 (+2.49), 233 (–4.48), 277 (–2.17) nm; UV (MeOH) λmax (log ε)
266 (4.45) nm; [α] 2D3 –35.9 (c 0.10, MeOH); 1D NMR data, Tables 1, 3; IR (KBr) vmax 3436, 1703,
1599, 1514, 1280, 1076, 1032, 856, 763 cmꢀ1.
Viburfordoside G (7). White powder; HRꢀESIMS m/z 575.2087 [M + Na]+ (calcd forC27H36O12Na,
575.2099); CD (MeOH) λ (ꢁε) 208 (+1.79), 229 (–3.66), 253 (+0.55), 281 (–0.42) nm; UV (MeOH)
λmax (log ε) 267 (4.11) nm; [α]2D3 –18.8 (c 0.10, MeOH); 1D NMR data, Tables 2, 4; IR (KBr) vmax
3391, 1600, 1511, 1464, 1266, 1074, 1032, 899, 669 cmꢀ1.
Viburfordoside H (8). White powder; HRꢀESIMS m/z 575.2090 [M + Na]+ (calcd forC27H36O12Na,
575.2099); CD (MeOH) λ (ꢁε) 236 (–1.66), 283 (+0.58) nm; UV (MeOH) λmax (log ε) 266 (4.38) nm;
[α]2D3 –21.2 (c 0.10, MeOH); 1D NMR data, Tables 2, 4; IR (KBr) vmax 3384, 1600, 1511, 1463, 1264,
1073, 1031, 897, 667 cmꢀ1.
Viburfordoside I (9). White powder; HRꢀESIMS m/z 575.2085 [M + Na]+ (calcd for C27H36O12Na,
575.2099); CD (MeOH) λ (ꢁε) 215 (–4.60), 224 (+1.90), 279 (–1.75) nm; UV (MeOH) λmax (log ε)
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266 (4.11) nm; [α]2D3 –17.8 (c 0.10, MeOH); 1D NMR data, Tables 2, 4; IR (KBr) vmax 3390, 1600,
1511, 1465, 1265, 1074,1030, 898, 668 cmꢀ1.
Fordiane A (10). Yellow oil; HRꢀESIMS m/z 397.1280 [M + Na]+ (calcd for C20H22O7Na,
397.1258); CD (MeOH) λ (ꢁε) 212 (+2.13), 216 (–1.54), 224 (+0.82), 231 (+0.80) nm; UV (MeOH)
λmax (log ε) 226 (4.20), 337 (4.12) nm; [α]2D3 +38.5 (c 0.10, MeOH); 1D NMR data, Tables 2, 4; IR
(KBr) vmax 3391, 2939, 2839, 1662, 1596, 1510, 1465, 1272, 1135, 1031, 972, 815, 599 cmꢀ1.
Fordiane B (11). Yellow oil; HRꢀESIMS m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na,
397.1258); CD (MeOH) λ (ꢁε) 212 (–1.00), 217 (+0.39), 225 (–0.47), 235 (+0.32) nm; UV (MeOH)
λmax (log ε) 223 (3.91), 335 (3.95) nm; [α]2D3 –16.9 (c 0.10, MeOH); 1D NMR data, Tables 2, 4; IR
(KBr) vmax 3389, 2925, 2852, 1656, 1597, 1512, 1460, 1272, 1137, 1029, 814, 592 cmꢀ1.
Enzymatic Hydrolysis of 1, 5, 7−9, and 12. The enzymatic hydrolysis reaction was conducted in
accordance with a previously reported method.23 Briefly, at 37 °C for 12 or 24 h, compounds 1, 5,
7−9, and 12 were separately hydrolyzed by βꢀglucosidase (10 mg) in H2O (3 mL), and then extracted
by EtOAc for three times. The EtOAc phases from 1, 5, 7−9, and 12 were isolated and purified to
give aglycones, 1a, 5a, 7a−9a and 12a, respectively. The aqueous phases were isolated and purified
to obtain glucose possessing [α]D values in range from +42.5 to +53.9 (c = 0.15 → 0.30, H2O). The
nꢀBuOH/CH3COOH/H2O(4:1:5) was used as developing solvent, and the glucose was identified by
the paper chromatography with authentic Dꢀglucose.
Acid Hydrolysis of 2−4 and 6. The acid hydrolysis reaction was performed in accordance with a
previously reported method.24 The EtOAc phases were isolated and purified by silica gel CC, using
MeOH/CHCl3 (1:8) as the eluent, for the hydrolyzates from 2 and 6 to yield vanillic acid, and from 3
and 4 to give syringic acid. These phenolic acids were confirmed by comparing with authentic
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samples through CoꢀTLC, melting point and HPLC data. The aqueous phases of 2−4 and 6 were
identified according to the enzymatic hydrolysis method.
αꢀGlucosidase Inhibitory Assay. The assay for evaluating inhibitory ability of all isolates toward
αꢀglucosidase was set up as formerly reported.25 The positive control used was acarbose. The
αꢀglucosidase inhibitory effect of tested compounds was qualified in terms of IC50 value, which
denoted the sample concentration demanded for exhibiting 50% of antiꢀαꢀglucosidase activity.
Radical Scavenging Assay. The ABTS•+ and DPPH• assays of isolated compounds were
conducted in accordance with previously described.26 The positive control used was trolox. The
calculated formula of radical scavenging activity (RSA) was determined as RSA%=[(ODcontrol
–
ODsample)/(ODcontrol – ODblank)] ×100 %. The IC50 values represented the concentrations of tested
compounds in which 50% of ABTS+ or DPPH radicals could be scavenged.
LPSꢀInduced Nitric Oxide Production Inhibition Assay. The measurement of NO in RAW264.7
cells was performed in accordance with a previously reported method using the Griess reaction.27
Nitrite levels in the supernatants were determined by comparing with the calibration curve prepared
with sodium nitrite standards. The positive control used in this assay was indomethacin. The nitric
oxide production inhibitory activity of the tested samples was determined as IC50 value.
Statistical Analysis. Each concentration of tested compounds was performed in triplicate. Data
from the experiments were described as the mean ± SD and evaluated with SPSS version 20.0. The
difference (p < 0.05) was statistically significant.
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RESULTS AND DISCUSSION
Identification of Compounds. The nꢀbutanol part from the 95% EtOH extract of V. fordiae fruits
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was submitted to HPLC and other column chromatography through HPDꢀ100 macroporous resin,
ODS, Sephadex LHꢀ20, and MCI gel to afford eighteen different secondary metabolites. Compounds
1−11 were identified as new compounds.
Viburfordoside A (1), white powder, had a molecular formula C33H44O17 by positive HRꢀESIMS.
1
Hydrolysis of 1 with
β
ꢀglucosidase produced
D
ꢀglucose with [α]2D3 = +48.6 (c 0.20, H2O). The H
NMR spectrum of 1 suggested the presence of two 1,3,4,5ꢀtetrasubstituted benzene rings [δH 6.97
(br.s, Hꢀ2', 6') and 6.68 (s, Hꢀ2, 6)]. Two C3ꢀunits at Cꢀ7, ꢀ8, ꢀ9 [δH 5.50 (d, J = 6.6 Hz, Hꢀ7), 3.50 (m,
Hꢀ8), 3.66 (m, Hꢀ9b), 3.75 (m, Hꢀ9a)] and Cꢀ7', ꢀ8', ꢀ9' [δH 6.57 (d, J = 15.9 Hz, Hꢀ7'), 6.22 (dt, J =
15.9, 6.0 Hz, Hꢀ8'), 4.18 (dd, J = 13.6, 6.0 Hz, Hꢀ9'b), 4.40 (dd, J = 13.6, 6.0 Hz, Hꢀ9'a)] were
determined according to 1Hꢀ1H COSY and HSQCꢀTOCSY correlations (Figure 2). The 1H NMR data
of 1 were closely identical to those of compound 13 except for signals of a glucose group and a
methoxyl residue. In the HMBC spectrum (Figure 2), the cross peaks from Hꢀ1''' (δH 4.91) to Cꢀ4 (δC
134.2) and Hꢀ1'' (δH 4.21) to Cꢀ9' (δC 68.7) showed that the linkage points of two glucose moieties
were at Cꢀ4 and Cꢀ9', respectively. Additionally, the HMBC cross peaks between three methoxy
protons and Cꢀ5/Cꢀ3/Cꢀ3' were observed. Consequently,
1
was confirmed to be
5ꢀmethoxydehydrodiconiferyl alcohol 4,9'ꢀdiꢀOꢀ ꢀglucopyranoside. Because of the coupling
β
ꢀD
constant (J7,8 = 6.6 Hz) of Hꢀ7 and Hꢀ8, the relative configuration of Hꢀ7 and Hꢀ8 of 1 was identified
as transꢀform.28 This was also confirmed by the cross peaks between Hꢀ7 and Hꢀ9, as well as
between Hꢀ2/Hꢀ6 and Hꢀ8 in the NOESY spectrum. The absolute configuration of 1 was confirmed
to be 7S, 8R by a positive Cotton effect near 271 nm in the CD spectrum.29 Thus, viburfordoside A
was elucidated to have the structural formula 1 as shown in Figure 1.
The molecular formula C41H50O20 of viburfordoside B (2) was exhibited in positive HRꢀESIMS.
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Acid hydrolysis of 2 produced two
Dꢀglucose groups with positive optical rotations. The βꢀanomer of
D
ꢀglucose was confirmed by the coupling constant (J = 7.8 Hz or 6.8 Hz).30 The 1D NMR spectra of
2 exhibited good similarity to those of 1 except for signals of a vanilloyl moiety. Comparison of the
13C NMR data of 1 and 2 showed that the glucosyl Cꢀ6''' of 2 was characterized by a 2.7 ppm
downfield shift, which suggested that the vanilloyl group was linked to Cꢀ6''' of 2. This was also
confirmed by the HMBC cross peaks between Hꢀ6''' (δH 4.45, 4.15) and Cꢀ7'''' (δC 165.4). The
relative configuration of Hꢀ7 and Hꢀ8 of 2 was implied as trans form by the coupling constant (J7,8 =
6.7 Hz) and the NOESY cross peaks from Hꢀ8 (δH 3.45) to Hꢀ2/Hꢀ6 (δH 6.64/6.64), and from Hꢀ9 (δH
3.73, 3.65) to Hꢀ7 (δH 5.49). Meanwhile, a negative Cotton effect near 272 nm indicated that the
absolute configuration of 2 was determined as 7R, 8S.26 Therefore, viburfordoside B was
unambiguously confirmed to have the structural formula 2 as shown in Figure 1.
Viburfordoside C (3), white powder, had a molecular formula C42H52O21 by positive HRꢀESIMS.
The 1D NMR signals of 3 were close to those of 2 except that the NMR signals of a vanilloyl moiety
in 2 were replaced by those attributed to a syringoyl unit in 3. This was also confirmed by HMBC
cross peaks from Hꢀ1''' (δH 4.89) to Cꢀ4 (δC 134.1), from Hꢀ1'' (δH 4.21) to Cꢀ9' (δC 68.7), from Hꢀ6'''
(δH 4.47, 4.19) to Cꢀ7'''' (δC 165.4), and from an additional OCH3 (δH 3.76) to Cꢀ5'''' (δC 147.5). A
trans configuration of Hꢀ7 and Hꢀ8 of 3 was determined by the J7,8 (6.5 Hz) and the NOESY cross
peaks from Hꢀ8 (δH 3.41) to Hꢀ2/Hꢀ6 (δH 6.63/6.63), and from Hꢀ9 (δH 3.71, 3.65) to Hꢀ7 (δH 5.47).
A negative Cotton effect near 275 nm showed that 3 could be identified as 7R, 8S configuration.
Consequently, viburfordoside C was identified to have the structural formula 3 as shown in Figure 1.
Viburfordoside D (4), white powder, was assigned a molecular formula C41H50O20 by positive
HRꢀESIMS. The NMR spectroscopic features of 4 were close to those of 3, except that the NMR
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signals of a 1,3,4,5ꢀtetrasubstitued aromatic ring in 3 were replaced by those attributed to a
1,3,4ꢀtrisubstituted aromatic ring in 4. This was further confirmed by HMBC cross peaks between
Hꢀ1''' (δH 4.96) and Cꢀ4 (δC 146.1), Hꢀ1'' (δH 4.21) and Cꢀ9' (δC 68.7), Hꢀ6''' (δH 4.57, 4.19) and Cꢀ7''''
(δC 165.5), together with Hꢀ5 (δH 7.06) and Cꢀ1 (δC 135.3). The J7,8 (6.1 Hz), the NOESY cross peaks
from Hꢀ8 (δH 3.37) to Hꢀ2/Hꢀ6 (δH 6.95/6.67), and the negative Cotton effects at 277 and 285 nm
showed that compound 4 possessed 7R, 8S configurations at Cꢀ7 and Cꢀ8. Thus, viburfordoside D
was established to have the structural formula 4 as shown in Figure 1.
Viburfordoside E (5), white powder, was given a molecular formula of C32H44O16 based on
positive HRꢀESIMS. The 1D NMR spectra of 5 were similar to those of glochidioboside31 except for
a set of glucosyl signals. The HMBC cross peak between Hꢀ1''' (δH 4.24) and Cꢀ9 (δC 70.1) showed
that a glucose group was linked to Cꢀ9. Additionally, the HMBC cross peak between Hꢀ1'' (δH 4.12)
and Cꢀ9' (δC 68.0) established that the other glucose group was attached to Cꢀ9'. The J7,8 (7.1 Hz), the
NOESY cross peak from Hꢀ8 (δH 3.58) to Hꢀ2 (δH 6.97), and a positive Cotton effect at 290 nm
indicated that viburfordoside E was concluded to have the structural formula 5 as shown in Figure 1.
The molecular formula of viburfordoside F (6) was deduced to be C40H50O19 by positive
HRꢀESIMS. The 1D NMR signals of 6 exhibited similarity with those of 5 except for signals of a
vanillic acid moiety. By comparing the 13C NMR data of 5 and 6, the upfield shifts of the Cꢀ4 (∆δ 0.3)
and Cꢀ9 (∆δ 7.1), and the downfield shifts of Cꢀ1 (∆δ 3.6) and Cꢀ6''' (∆δ 2.8) were observed in 6.
This indicated that two glucopyranosyl moieties were located at Cꢀ4 and Cꢀ9', and a vanilloyl moiety
was linked to Cꢀ6''', which was also confirmed by the HMBC cross peaks from Hꢀ1''' (δH 4.97) to Cꢀ4
(δC 146.0), from Hꢀ1'' (δH 4.11) to Cꢀ9' (δC 67.9), and from Hꢀ6''' (δH 4.55, 4.16) to Cꢀ7'''' (δC 165.4).
The J7,8 (6.4 Hz), the NOESY cross peaks of Hꢀ2/Hꢀ6 (δH 6.96/6.75) and Hꢀ8 (δH 3.37), and a
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negative Cotton effect near 277 nm suggested that 6 could be confirmed as 7R,8Sꢀconfiguration.
Consequently, viburfordoside F was defined to have the structural formula 6 as shown in Figure 1.
Viburfordoside G (7), white powder, was given a molecular formula C27H36O12 by positive
HRꢀESIMS. Hydrolysis of 7 with
βꢀglucosidase yielded 7a and Dꢀglucose. 7 exhibited NMR
spectroscopic data almost identical to those of glehlinoside H (12). A small coupling constant (J7,8
=
1
4.7 Hz) was observed in the H NMR spectrum of 7a (Table S98), thus the relative configuration of
Hꢀ7 and Hꢀ8 of 7a was determined as the erythroꢀform.32 In the 13C NMR spectrum, ∆δC8–C7 value of
7 (12.0) was smaller than those of 8 (13.0) and 9 (13.0), which suggested that 7 should possess a
relative stereochemistry of 7,8ꢀerythro, while 8 and 9 should be determined to be in the threoꢀform.33
The R configuration at Cꢀ8 of 7 was confirmed by a negative Cotton effect near 229 nm.34 Thus,
viburfordoside G was determined to have the structural formula 7 in Figure 1.
The spectroscopic data of viburfordoside H (8) (Tables 2 and 4) indicated that it was a
threoꢀisomer of 7. Hydrolysis of 8 with
βꢀglucosidase gave Dꢀglucose and 8a. A large coupling
1
constant J7,8 = 8.0 Hz, in the H NMR spectrum of 8a (Table S98), showed that the relative
configuration of 8a was assigned as the threoꢀform of Hꢀ7 and Hꢀ8.32 A negative Cotton effect near
236 nm demonstrated that the absolute configuration of 8 was 7R, 8R as shown in Figure 1.
Viburfordoside I (9) showed spectroscopic data (Tables 2 and 4) completely identical to those of 8.
However, 8 and 9 were fractionated by semiꢀprep. HPLC with retention times of 68.6 min and 72.7
min, respectively. Hydrolysis of 9 with βꢀglucosidase yielded 9a and Dꢀglucose. The J7,8 (J = 8.0 Hz)
(Table S98) of the aglycone (9a) and a positive Cotton effect near 224 nm of 9 showed that the
absolute configuration of viburfordoside I was identified to be 7S, 8S as shown in Figure 1.
Fordiane A (10), yellow oil, was assigned a molecular formula C20H22O7 by positive HRꢀESIMS.
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In the H NMR spectrum of 10 (Tables 2 and 4), besides two sets of ABX proton signals, an
aldehyde signal at δH 9.60 (d, J = 7.8 Hz, Hꢀ9') and transꢀolefinic protons at δH 6.76 (dd, J = 15.8,
7.8 Hz, Hꢀ8'), 7.60 (d, J = 15.8 Hz, Hꢀ7') established the presence of a transꢀpropenal group. And a
1,2,3ꢀpropanetriol moiety was confirmed by two oxymethine protons at δH 4.47 (m, Hꢀ8) and 4.70
(tꢀlike, J = 5.0 Hz, Hꢀ7), as well as two oxymethylene protons at δH 3.62 (m, Hꢀ9b) and 3.65 (m,
Hꢀ9a). The HMBC cross peak between Hꢀ8 (δH 4.47) and Cꢀ4' (δC 151.2) indicated that the aryl
glycerolꢀ8ꢀyloxy was linked to Cꢀ4'. The above evidence indicated that 10 had the same planar
structure as guaiacylglycerolꢀβ βꢀconiferyl
ꢀconiferyl aldehyde ether.35 However, guaiacylglycerolꢀ
aldehyde ether, with relative configuration determined, was reported to have ambiguous absolute
configuration.35,36 The erythroꢀform of Hꢀ7 and Hꢀ8 in 10 was identified by a small coupling constant
(J7,8 = 5.0 Hz, in CDCl3, Table S98). Furthermore, a positive Cotton effect near 231 nm showed that
10 had a S configuration at Cꢀ8. Thus, fordiane A was delineated to have the structural formula 10 as
shown in Figure 1.
The spectroscopic data of fordiane B (11) (Tables 2 and 4) indicated that it was a threoꢀisomer of
10. A large coupling constant (J7,8 = 7.7 Hz, in CDCl3, Table S98) showed that 11 had the threoꢀform
of Hꢀ7 and Hꢀ8. Meanwhile, a positive Cotton effect near 235 nm indicated that 11 possessed the 7S,
8S configuration. Consequently, fordiane B was defined to have the structural formula 11 as shown
in Figure 1.
The other compounds were confirmed as glehlinoside H (12),37 dehydrodiconiferyl alcohol
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9'ꢀOꢀ
(7S,8R)ꢀ4,9'ꢀdihydroxylꢀ3,3'ꢀdimethoxylꢀ7,8ꢀdihydrobenzofuranꢀ1'ꢀpropylneolignan
(7R,8S)ꢀguaiacylglycerolꢀ8ꢀOꢀ4'ꢀ(synapyl alcohol) ether
β
ꢀ
D
ꢀglucopyranoside
(13),38
(14),39
(15),40,41
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(7S,8S)ꢀguaiacylglycerolꢀ8ꢀOꢀ4'ꢀ(synapyl alcohol) ether (16),41 lappaol A (17),42 and isolappaol A
(18).42
αꢀGlucosidase Inhibitory Activity. All isolates were assayed for intestinal αꢀglucosidase
inhibitory activity. In comparison with positive control, these compounds exhibited potential
inhibitory activity toward αꢀglucosidase (Table 5). Among them, 10 and 11 demonstrated the most
potent inhibitory activity toward αꢀglucosidase (IC50 = 14.17 and 15.56 ꢀM, respectively). The IC50
values obtained showed that it was indefinite to discover the obvious differences in the structure–
activity relationships of 4',7ꢀepoxyꢀ8,5'ꢀneolignan constituents, however, it was easy to find out that
the changes in configurations at Cꢀ7/Cꢀ8 of compounds 7−9, 12 were of negligible importance in
inhibiting intestinal αꢀglucosidase activity.
Free Radical Scavenging Activity. Eighteen different secondary metabolites were assayed for
free radical scavenging activities. The tested compounds exhibited varying ABTS•+ and DPPH•
scavenging activities except compounds 1, 7−9 and 12 as shown in Table 5. These obtained results
showed the fact that the free phenolic hydroxyl played a key role in free radical scavenging
activities.43 Additionally, comparing with the DPPH• assay, the ABTS•+ assay was significantly
powerful for fruits, which was consistent with the previous report.44 Compounds 10 and 11
demonstrated potent ABTS•+ scavenging activities (IC50 = 10.80 and 18.65 ꢀM, respectively), which
were stronger than trolox (IC50 = 26.30 ꢀM). In the DPPH• assay, the IC50 values of compounds 10
and 11 were 17.98 and 30.56 ꢀM, respectively, which were comparable to that of trolox (IC50 = 38.68
ꢀM). Interestingly, among 8,4'ꢀoxyneolignan stereoisomers, compound 11 (7S) was weaker than 10
(7R), and 16 (7S) was weaker than 15 (7R) in ABTS and DPPH radical scavenging tests, which
implied that R configuration at Cꢀ7 of these compounds may play a key role in exerting free radical
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scavenging activity. Meanwhile, the weak activity of compounds 1−9, 12, and 13 indicated that the
reduction of the radical scavenging effect may be correlated to glycosylation.
Inhibitory Effects on LPSꢀinduced NO Production. All isolates were examined for their
cytotoxic activity on RAW264.7 macrophage cells by the MTT assay and showed no toxicity at the
dose evaluated (100 ꢀM), and then assayed for the inhibition on LPSꢀinduced NO production.
Compounds 2−12, 14, 17 and 18 exhibited differential inhibitory effects against NO production (IC50
= 10.88 → 90.43 ꢀM) in comparison with indomethacin (IC50 = 49.46 ꢀM) (Table 5), and among
which, 10, 11 and 14 exhibited the strongest inhibitory effect with IC50 values of 10.88, 15.50 and
12.54 ꢀM, respectively. Comparing with compounds 1 and 5, an additional vanilloyl or syringoyl
moiety in compounds 2−4 and 6, located at Cꢀ6''' of glucosyl group appeared to reduce LPSꢀinduced
NO production. Compound 5 exhibited significantly weaker inhibitory effect on NO production than
14, which attributed to two additional bulky glucose moieties in 5. Similarly, comparing 3 (IC50 =
54.43 ꢀM) with 2 (IC50 = 41.10 ꢀM) and 4 (IC50 = 39.38 ꢀM), an additional methoxyl group located
at Cꢀ5 or Cꢀ5'''' could clearly reduce the inhibitory effect on NO production.
In conclusion, our research resulted in eleven undescribed neolignan constituents (1−11) and seven
known analogues (12−18) isolated from V. fordiae fruits. Among these secondary metabolites, new
compounds 2−4 and 6 are unusual 4',7ꢀepoxyꢀ8,5'ꢀneolignan glycosides with a phenolic glucosyl
group. The neolignan constituents are firstly reported from the edible fruits of Viburnum species. In
bioactivity screening, these neolignans exhibited various αꢀglucosidase inhibitory, free radical
scavenging and NO inhibitory activities. Interestingly, relative to the other tested neolignans,
compounds 10 and 11 exhibited the most potent bioactivities in all three assays, which was probably
associated with α, βꢀunsaturated aldehyde group in their structures. Therefore, these research results
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not only revealed that the neolignans were major bioactive constituents of V. fordiae fruits, but also
supported the edible berry of V. fordiae as functional food source for prevention of PPHG. Moreover,
two new neolignans 10 and 11, because of their potent bioactivities, may be developed as natural
healthꢀpromoting food ingredients or further act as potential lead molecules for development of
therapeutic agents for T2D.
355
ASSOCIATED CONTENT
356
357
358
Supporting Information
HRꢀESIMS, IR, UV, CD, 1D/2D NMR spectra for 1−11, 1H NMR spectra for 7a−9a, ORD values of
7a–9a, 12a.
359
AUTHOR INFORMATION
360
361
362
363
364
365
366
367
368
369
Corresponding Authors
* Eꢀmail: cczhao@yzu.edu.cn.
* Eꢀmail: jhshao@yzu.edu.cn.
ORCID
Chunchao Zhao: 0000ꢀ0001ꢀ7941ꢀ8175
Jianhua Shao: 0000ꢀ0003ꢀ2519ꢀ6735
Funding
This work was funded by National Natural Science Foundation of China (31201563).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We thank Dr. Fengmin Zhang, Dr. Qiuxiang Yan and Dr. Chunliang Lu from Yangzhou University
for spectroscopic tests.
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510
511
512
513
514
515
Figure Captions
Figure 1. Structures of compounds 1–18 and 7a–9a.
Figure 2. Key 1Hꢀ1H COSY, HSQCꢀTOCSY, HMBC and NOESY correlations for compounds 1, 3, 7,
10.
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Page 26 of 33
Table 1. 1H NMR Data of Compounds 1–6a
Position
1
2
3
4
5
6
2
5
6
7
8
9
6.68 (s)
6.64 (s)
6.63 (s)
6.95 (d, 2.0)
7.06 (d, 8.5)
6.97 (d, 1.9)
6.74 (d, 8.2)
6.96 (d, 1.7)
7.08 (d, 8.5)
6.68 (s)
6.64 (s)
6.63 (s)
6.67 (dd, 8.5, 2.0) 6.79 (dd, 8.2, 1.9) 6.75 (dd, 8.5, 1.4)
5.50 (d, 6.6)
3.50 (m)
5.49 (d, 6.7)
3.45b
5.47 (d, 6.5)
3.41b
5.47 (d, 6.1)
3.37 (m)
3.69b
5.47 (d, 7.1)
3.58 (m)
5.45 (d, 6.4)
3.37 (m)
33.75 (m)
3.66 (m)
3.73 (m)
3.65 (m)
6.96 (s)
3.71 (m)
3.65 (m)
6.95 (s)
3.95 (dd, 9.8, 7,7) 3.71 (m)
3.62 (m)
6.96 (s)
3.73 (m)
3.60 (m)
6.71 (br.s)
6.72 (br.s)
2.58 (t, 7.3)
1.79 (m)
3.79 (m)
3.41 (m)
4.11 (d, 7.8)
2.97 (m)
3.07 (m)
3.05 (m)
3.14 (m)
3.66 (m)
3.44 (m)
4.97 (d, 7.3)
3.31b
2'
6'
7'
8'
9'
6.97 (br.s)
6.97 (br.s)
6.57 (d, 15.9)
6.84 (br.s)
6.73 (br.s)
2.58 (t, 7.6)
6.96 (s)
6.96 (s)
6.96 (s)
6.57 (d, 16.0)
6.57 (d, 15.9)
6.57 (d, 15.8)
6.22 (dt, 15.9, 6.0) 6.21 (dt, 16.0, 6.0) 6.22 (dt, 15.9, 6.0) 6.21 (dt, 15.8, 5.9) 1.80 (m)
4.40 (dd, 13.6, 6.0) 4.41 (dd, 14.0, 6.0) 4.40 (dd, 13.5, 6.0) 4.41 (dd, 13.7, 5.9) 3.79 (m)
4.18 (dd, 13.6, 6.0) 4.18 (dd, 14.0, 6.0) 4.17 (dd, 13.5, 6.0) 4.19 (d, 13.7, 5.9) 3.43 (m)
1''
2''
3''
4''
5''
6''
4.21 (d, 7.9)
3.00 (m)
3.09 (m)
3.06 (m)
3.15 (m)
3.69 (m)
3.45 (m)
4.91 (d, 7.3)
3.20b
4.21 (d, 7.8)
4.21 (d, 7.9)
4.21 (d, 7.8)
4.12 (d, 7.8)
3.00 (tꢀlike, 8.4) 3.00 (tꢀlike, 8.7) 3.00 (tꢀlike, 8.3) 2.99b
3.08 (m)
3.07 (m)
3.15 (m)
3.69 (m)
3.45b
3.09 (m)
3.06 (m)
3.15 (m)
3.69 (m)
3.45 (m)
4.89 (d, 7.1)
3.41b
3.26b
3.26b
3.26b
3.09 (m)
3.07 (m)
3.15 (m)
3.69b
3.09b
3.06b
3.14b
3.67b
3.46 (dd, 11.5, 5.8) 3.44b
1'''
2'''
3'''
4'''
5'''
6'''
4.88 (d, 6.8)
3.38 (m)
3.26b
3.26b
3.26b
4.96 (d, 7.1)
3.25b
3.25b
4.24 (d, 7.8)
2.99b
3.09b
3.06b
3.14b
3.67b
3.04 (m)
3.14 (m)
3.20b
3.31b
3.24 (m)
3.74 (m)
3.26 (m)
3.71 (m)
4.55 (br.d, 11.8)
3.59 (m)
3.41 (m)
4.45 (br.d, 11.8)
4.47 (dd, 11.9, 2.1) 4.57 (br.d, 11.6)
4.15 (dd, 11.8, 6.3) 4.19 (dd, 11.9, 5.9) 4.19 (dd, 11.6, 6.8) 3.44b
4.16 (dd, 11.8, 7.0)
7.42 (d, 1.8)
6.89 (d, 8.3)
7.46 (dd, 8.3, 1.8)
3.73 (s)
2''''
7.36 (d, 2.0)
6.86 (d, 8.5)
7.13 (s)
7.19 (s)
5''''
6''''
7.33 (dd, 8.5, 2.0) 7.13 (s)
7.19 (s)
3.72 (s)
3.81 (s)
3.76 (s)
3/5ꢀOMe
3'ꢀOMe
3''''/5''''ꢀOMe
3.74 (s)
3.82 (s)
3.66 (s)
3.81 (s)
3.78 (s)
3.66 (s)
3.81 (s)
3.76 (s)
3.75 (s)
3.77 (s)
3.78 (s)
3.79 (s)
a 1H NMR data (δ) were measured at 600 MHz in DMSOꢀd6. Proton coupling constants (J) in Hz were given in parentheses. The
assignments were based on 1Hꢀ1H COSY, NOESY, HSQCꢀTOCSY, HSQC, and HMBC experiments. b Overlapped with other signals.
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Journal of Agricultural and Food Chemistry
Table 2. 1H NMR Data of Compounds 7–11a
Position
7
8
9
10
11
2
5
6
7
8
9
7.10 (d, 1.9)
7.09 (d, 8.3)
6.95 (dd, 8.3, 1.9)
4.86 (d, 5.9)
4.38 (m)
7.15 (d, 1.9)
7.14 (d, 8.2)
6.99 (dd, 8.2, 1.9)
4.96 (d, 5.1)
4.36 (m)
7.13 (d, 1.8)
7.12 (d, 8.2)
6.98 (dd, 8.2, 1.8)
4.94 (d, 5.2)
4.34 (m)
7.00 (d, 1.9)
6.66 (d, 8.0)
6.78 (dd, 8.0, 1.9)
4.70 (tꢀlike, 5.0)
4.47 (m)
6.97 (d, 1.9)
6.68 (d, 8.0)
6.76 (dd, 8.0, 1.9)
4.71 (tꢀlike, 4.5)
4.44 (m)
3.85b
3.79 (dd, 11.9, 4.3)
3.52 (dd, 11.9, 5.5)
7.07 (d, 2.3)
6.98 (d, 8.3)
6.93 (dd, 8.3, 2.3)
6.57 (dt, 16.0, 1.5)
6.22 (dt, 16.0, 6.3)
4.09 (dd, 6.3, 1.5)
4.87 (d, 7.3)
3.50 (m)
3.77 (dd, 11.9, 4.4)
3.50 (dd, 11.9, 5.5)
7.05 (d, 1.6)
6.97 (d, 8.3)
6.91 (dd, 8.3, 1.6)
6.55 (br.d, 15.7)
6.20 (dt, 15.7, 6.3)
4.06 (br.d, 6.3)
4.86 (d, 7.1)
3.48 (m)
3.65 (m)
3.60 (m)
3.78 (dd, 11.9, 3.7)
7.00 (br.s)
6.87 (br.s)
6.87 (br.s)
6.54 (dt, 15.7, 1.4)
6.18 (dt, 15.7, 6.1)
4.06 (dd, 6.1, 1.4)
4.81b
3.62 (m)
3.29 (m)
2'
7.30 (d, 1.9)
7.07 (d, 8.5)
7.20 (dd, 8.5, 1.9)
7.60 (d, 15.8)
6.76 (dd, 15.8, 7.8)
9.60 (d, 7.8)
7.35 (d, 2.0)
7.11 (d, 8.3)
7.23 (dd, 8.3, 2.0)
7.62 (d, 15.8)
6.78 (dd, 15.8, 7.9)
9.61 (d, 7.9)
5'
6'
7'
8'
9'
1''
2''
3''
4''
5''
6''
3.47 (m)
3.38b
3.38b
3.4b
3.4b
3.39b
3.39b
3.45 (m)
3.85b
3.48 (m)
3.46 (m)
3.88 (m)
3.86 (br.d, 12.1)
3.69 (dd, 12.1, 3.8)
3.83 (s)
3.69 (dd, 11.9, 4.9)
3.81 (s)
3.71 (dd, 12.0, 5.1)
3.85 (s)
3ꢀOMe
3'ꢀOMe
9'ꢀOMe
3.73 (s)
3.77 (s)
3.73 (s)
3.83 (s)
3.80 (s)
3.88 (s)
3.87 (s)
3.36 (s)
3.38 (s)
3.36 (s)
a 1H NMR data (δ) were measured at 600 MHz in CD3OD for 7–9 and DMSOꢀd6 for 10, 11. Proton coupling constants (J) in Hz were given
in parentheses. The assignments were based on NOESY, HSQC, and HMBC experiments. b Overlapped with other signals.
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Table 3. 13C NMR Data of Compounds 1–6
Position
1
2
3
4
5
6
1
136.9 137.4 137.4 135.3 132.0 135.6
104.3 104.0 104.1 110.2 110.5 110.2
152.7 152.9 152.8 148.8 147.5 148.9
134.2 133.9 134.1 146.1 146.3 146.0
152.7 152.9 152.8 114.8 115.2 115.0
104.3 104.0 104.1 117.7 118.5 117.7
87.1 87.1 87.0 86.6 86.7 86.3
52.9 53.0 53.0 53.2 50.6 53.5
62.9 62.8 62.8 62.9 70.1 63.0
130.3 130.3 130.3 130.2 134.9 134.8
115.2 115.3 115.2 115.3 116.8 116.6
143.7 143.8 143.7 143.7 143.3 143.4
147.3 147.4 147.3 147.3 145.5 145.5
129.3 129.3 129.3 129.3 128.6 128.7
110.6 110.6 110.6 110.5 112.7 112.6
131.7 131.8 131.8 131.8 31.4 31.4
123.7 123.6 123.6 123.6 31.4 31.4
68.7 68.7 68.7 68.7 68.0 67.9
102.0 102.0 102.0 102.0 102.9 103.0
73.5 73.5 73.5 73.5 73.5 73.5
76.9 76.9 76.9 76.9 76.8 76.8
70.1 70.1 70.1 70.1 70.1 70.1
76.8 76.8 76.8 76.8 76.7 76.7
61.1 61.1 61.1 61.1 61.1 61.1
102.6 102.7 102.8 99.8 102.8 99.8
74.1 74.0 74.1 73.1 73.5 73.1
77.1 76.2 76.1 76.6 76.9 76.6
69.9 70.0 70.0 70.1 70.1 70.0
76.5 74.1 74.1 73.9 76.8 73.8
60.9 63.6 63.8 64.0 61.1 63.9
2
3
4
5
6
7
8
9
1'
2'
3'
4'
5'
6'
7'
8'
9'
1''
2''
3''
4''
5''
6''
1'''
2'''
3'''
4'''
5'''
6'''
1''''
120.4 119.3 120.0
112.5 107.0 107.2
147.4 147.5 147.7
151.7 140.7 140.3
115.2 147.5 147.7
123.4 107.0 107.2
165.4 165.4 165.5
120.5
112.6
147.4
151.6
115.2
123.5
165.4
2''''
3''''
4''''
5''''
6''''
7''''
3/5ꢀOMe
3'/5'ꢀOMe
3''''/5''''ꢀOMe
56.5 56.3 56.4 55.7 55.6 55.7
55.8 55.8 55.8 55.7 55.7 55.7
55.6 56.1 56.1
55.7
Measured in DMSOꢀd6 at 150 MHz.
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Journal of Agricultural and Food Chemistry
Table 4. 13C NMR Data of Compounds 7–11
Position
7
8
9
10
11
1
137.8
112.8
150.5
147.4
118.7
121.1
73.9
137.4
112.6
150.6
147.4
118.5
120.9
73.6
137.4
112.6
150.6
147.4
118.5
120.9
73.6
132.9
111.5
146.9
145.5
114.5
119.5
71.6
132.8
111.0
147.0
145.5
114.7
119.0
71.1
2
3
4
5
6
7
8
85.9
86.6
86.6
83.4
83.8
9
62.2
61.9
61.9
60.3
60.2
1'
132.7
111.4
151.9
149.1
117.7
120.8
133.7
125.3
74.2
132.7
111.4
151.7
149.4
117.7
120.6
133.7
125.3
74.2
132.8
111.4
151.7
149.4
117.7
120.6
133.7
125.3
74.2
126.6
111.4
149.6
151.2
114.5
123.3
153.5
126.3
194.0
126.7
111.3
149.6
151.5
114.4
123.4
153.6
126.4
194.0
2'
3'
4'
5'
6'
7'
8'
9'
1''
103.1
74.9
103.0
74.9
102.9
74.9
2''
3''
78.2
78.2
78.2
4''
71.4
71.4
71.4
5''
77.8
77.8
77.8
6''
62.5
62.5
62.5
3ꢀOMe
3'ꢀOMe
9'ꢀOMe
56.7
56.7
56.7
55.4
55.7
55.4
55.8
56.5
56.6
56.6
58.1
58.1
58.1
Measured in CD3OD for 7–9 and in DMSOꢀd6 for 10, 11 at 150
MHz.
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