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New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from “Tugancao” (Derris eriocarpa How) Hongxia Zhang, Guo Sun, Jianlong Gu, and ZhiZhi Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00137 • Publication Date (Web): 25 Feb 2017 Downloaded from http://pubs.acs.org on February 27, 2017
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Journal of Agricultural and Food Chemistry
New Sweet-Tasting Oleanane-Type Triterpenoid Saponins from “Tugancao” (Derris eriocarpa How) Hong-Xia Zhang†, Guo Sun†,‡,, Jian-Long Gu§, Zhi-Zhi Du*,†
†
Key Laboratory of Economic Plants and Biotechnology, Yunnan Key Laboratory for
Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China ‡
University of Chinese Academy of Sciences, Beijing 100049, China
§
Yunnan Tobacco Quality Inspection & Supervision Station, Kunming 650106, China
Corresponding Author * Phone: +86-871-65223224; Fax: +86-871-65216335 E-mail address:
[email protected] 1
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ABSTRACT
2
Aiming to investigate the sweet-tasting compounds in Derris eriocarpa How (a
3
substitute for licorice in “Zhuang” and “Dai” ethnopharmacy in Guangxi and Yunnan
4
province of China) as well as to ascertain why the stem of D. eriocarpa can be used to
5
substitute for licorice in sweetness taste aspect, taste sensory-guided fractionation was
6
conducted to isolate sweet constituents from the extract of D. eriocarpa. Four
7
sweet-tasting triterpenoid saponins were obtained, including Millettiasaponin A (1)
8
and three new ones named as derrisaponins A-C (2-4). The sweetness potency was
9
evaluated by a human sensory panel test. The sweetness intensities of compounds 1-4
10
were determined to be approximately 150, 80, 2 and 0.5 times relative to sucrose at
11
the concentration 1%, respectively, of which 1 and 2, with a free carboxyl group at the
12
C-30 position, showed more potent sweetness intensity. In addition, 1 and 2 showed
13
no acute toxic activity in dose of 250 mg/kg bw and 400 mg/kg bw respectively
14
assessed through caudal veins injection to ICR mice. The contents of the sweetest
15
compounds in stems were analyzed quantitatively as 352.80 mg/kg for 1 and 1887.60
16
mg/kg for 2 respectively performed by UPLC–MS/MS.
17 18
KEYWORDS
19
Derris eriocarpa, sweet-tasting, triterpenoid saponins, sensory evaluation, acute
20
toxicity
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INTRODUCTION
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Human can detect at least five basic tastes including sweet, bitter, salty, sour and
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umami tastes. Among them, sweetness plays a central role for it is close relationship
24
to appetitive sensations in food recognition, and is usually and naturally originated
25
from sugars. However, with the excessive intake of sugar, more and more people are
26
suffered from diseases, such as dental caries, hypertension, hyperglycemia,
27
cardiovascular diseases and obesity.1-3 Accordingly, there has been an increasing
28
demand for new highly sweet and noncaloric sucrose substitutes. The compounds, in
29
being so-called “high-potency (HP) sweeteners” and “low-calorie sweeteners” with
30
these metrics: safety, taste quality, stability, solubility, cost, and patentability,
31
especially natural sweeteners, attract many researchers’ attentions.4, 5 In addition to
32
the development of synthetic HP sweeteners, a number of highly sweet natural
33
compounds have been discovered from green plants, but only a relatively few
34
sweet-tasting plant-derived natural products have been successfully applied as sucrose
35
substitutes to date.6 Efforts to find more highly sweet plant constituents have been
36
stimulated both by a public demand for natural flavors to tackle problems of toxicity
37
and taste quality of existing synthetic HP sweeteners. By following up ethnobotanical
38
leads to assist in the selection of candidate sweet-tasting plant, particularly those used
39
medicinally by indigenous cultures, it is possible to discover new potent sweet natural
40
products.5
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During our ethnobotanical field survey and sensory evaluation of characteristic
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edible and medical plants in traditional medicine market on “Dragon boat festival”, an 3
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ethnic edible and medical plant called “Tugancao” was of obvious sweet taste.
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Traditional medicine market on “Dragon boat festival” of Jingxi County in Guangxi
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province has been going on for more than 700 years with more than 500 species of
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traditional medicinal herbs for sale.7 The abundant resources of traditional edible and
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medicinal herbs provide a great material basis for our researches. By means of the
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ethnobotanical methods (Key Informantion Interview), we got that the local people
49
immerse the “Tugancao” with other medicinal herbs into the white spirit to make
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herbal liquor. Informants also mentioned that the herbal liquor is not only to relieve a
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cough but also become a little licorice-like sweetness and pleasant to take.
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The plant was taxonomically identified as Derris eriocarpa How, belonging to the
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Leguminosae family, commonly known as “Tugancao” in “Zhuang” and “Dai”
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ethnopharmacy in Guangxi and Yunnan provinces of China.8, 9 The “Tugancao”, the
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stem of D. eriocarpa is used to substitute for licorice due to its similar sweet taste and
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medicinal properties in folk medicine in Guangxi province. 10-11 In previous chemical
57
investigation, some isoflavonoids, stibenoids and coumarins were reported.12-14
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However, to date there are no investigations on sweet-tasting components from this
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plant.
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Aiming to identify the sweet-tasting compounds and ascertain why the D.
61
eriocarpa can be used to substitute for licorice from the sweetness point, we carried
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out a sensory-guided fractionation and purification of the crude extract of this plant.
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In the present study, four triterpenoid saponins were isolated from the extract of D.
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eriocarpa stems and they were proposed mine chemical constituents responsible for 4
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the sweet taste of the plant.
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MATERIALS AND METHODS Chemicals. The following materials were used: and
D-glucose
D-glucuronic
acid,
D-galactose,
69
L-rhamnose
(J&K Scientific Ltd. Guangzhou, China),
L-cysteine
70
methyl hydrochloride (Sigma-aldrich, Shanghai, China), N-trimethylsilylimidazole
71
(Sangon Biotech, Shanghai, China), n-hexane (Damao, Tianjin, China), HPLC
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acetonitrile (Merck, Shanghai, China), ethyl acetate (Jige, Tianjin, China), acetic
73
anhydride (Damao, Tianjin, China), sulphuric acid (Xilong Chemical Co. Ltd,
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Guangdong, China), hydrochloric acid (Xilong Chemical Co. Ltd), ferric chloride
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(Damao, Tianjin, China), chloroform (Rionlon, Tianjin, China), dioxane (Sinopharm
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chemical reagent Co. Ltd., Shanghai, China), sodium dicarbonate (Damao, Tianjin,
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China).
78
General Experiment Procedures. Optical rotations were measured with a Horiba
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Sepa-300 polarimeter (Horiba, Tokyo, Japan). UV spectra were obtained using a
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Shimadzu UV-2401A spectrophotometer (Shimadzu, Tokyo, Japan). A tensor 27
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spectrophotometer (Bruker, Bremen, Germany) was used for scanning IR
82
spectroscopy using KBr pellets. 1D and 2D NMR spectra were measured on Bruker
83
AVANCE III 500MHz and AV Ⅲ 800 MHz spectrometers (Bruker, Bremen,
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Germany) at 296 K. Unless otherwise specified, chemical shifts (δ) were expressed in
85
ppm with reference to the solvent signals. ESIMS data were obtained on a Bruker
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HCT Esquire spectrometer (Bruker, Bremen, Germany). HRESIMS data were 5
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recorded on an Agilent G6230 TOF MS spectrometer (Agilent Technologies, Santa
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Clara, America).
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Column chromatography (CC) was done using silica gel (200-300 mesh, Qingdao
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Marine Chemical Co., Ltd., China), RP-18 gel (40-63µm, Merck, Germany), Diaion
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HP-20 (Mitsubishi Chemical Corporation, Japan), resin D101 column (Shanghai
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YuanYe Bio-Technology Co., Ltd., China) and Sephadex gel LH-20 (GE Healthcare
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Bio-Sciences AB, Sweden). TLC was performed on silica gel GF254 (Qingdao Marine
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Chemical Co., Ltd., China), and spots were visualized by heating silica gel plates
95
sprayed with 10% H2SO4 in ethanol. Quantitation by UPLC-MS/MS was performed
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on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA). The
97
lyophilizer (Virtis Benchtop K, America) was used to dry the samples and eliminate
98
the residual solvents.
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Plant Material. The fresh stems of D. eriocarpa were collected from Jingxi
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County of Guangxi Province, China, and identified by Prof. Li-Song Wang. A voucher
101
specimen (No.11-32219) has been deposited in the Herbarium of Kunming Institute of
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Botany (KUN), Chinese Academy of Sciences.
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Animals. Male and female ICR mice (3-4 weeks, Institute of Cancer Research),
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certified specific pathogen-free, were purchased from Kunming medical university
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under the license number SCXK (DIAN) K2011-0004. Mice were kept in an IVC
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animal room under the license No.: SYXK (DIAN) K2013-004, conditioned at a
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temperature of 25±3℃, a relative humidity of 40-70% and 12h light/dark cycle. All
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animals had a one-week acclimatization period before experiment started and received 6
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basic feed and fresh water freely during the experiment period.15 Extraction, Sensory-guided Fractionation and Purification of the
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Sweet-Tasting Compounds. The air-dried and powdered stems (6 kg) of D.
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eriocarpa were extracted 3 times with 80% aqueous MeOH at room temperature.
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After evaporation of MeOH under reduced pressure, the aqueous residue was
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partitioned with CHCl3, EtOAc and n-BuOH to yield CHCl3 portion (150 g), EtOAc
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portion (12 g), n-BuOH portion (80 g) and the aqueous layer. The n-BuOH portion
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(80 g) and the aqueous layer showed sweet taste in sensory evaluation tests.
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The n-BuOH portion (80 g) was subjected to a macroporous resin D101 column
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chromatography (2 kg), eluted with H2O, 30%, 50% , 70% EtOH-Water, and EtOH
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successively (6 L each). The elutant eluted with water was abandoned. The other
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elutant was evaporated to remove organic solvent in reduced pressure and lyophilized
121
to give fraction B1 (about 11 g, eluted with 30% aqueous ethanol), fraction B2 (49 g,
122
eluted with 50% and 70% aqueous ethanol), fraction B3 (2 g, eluted with ethanol).
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Fraction B2 was identified as the sweet-tasting fraction according to sensory
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evaluation results.
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Fraction B2 (49 g) was chromatographed on silica gel column, eluted with
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CHCl3-MeOH-Water (C/M/W: 7/3/0, 7/3/0.5, 6/4/1) to get 4 fractions on the basis of
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TLC analysis. According to the sensory evaluation, fraction B2-a (9.3 g) and B2-b
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(8.5 g) showed non-sweet taste, fraction B2-c and B2-d showed interesting sweet taste.
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Fraction B2-c (15 g) was subjected to CC over silica gel (C/M/W:7/3/0.5) to yield
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three sub-fractions based on TLC analysis. B2-c-1 and B2-c-3 were non-sweet tasting 7
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sub-fractions, and B2-c-2 was sweet tasting sub-fraction. B2-c-2 (3.5 g) afforded 1
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(800 mg) after purified by CC over silica gel (C/M/W: 7/3/0.5). Fraction B2-d (16 g)
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was subjected to CC over silica gel (C/M/W:7/3/0.5) to yield four sub-fractions based
134
on TLC analysis. B2-d-1 to B2-c-3 were non-sweet tasting sub-fractions, and B2-d-4
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was sweet tasting sub-fraction. B2-d-4 (2.2 g) was subjected to experiments of CC
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over silica gel (C/M/W: 7/3/0.5) to afford 2 (120 mg).
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The aqueous layer was concentrated under vacuum to get a fraction named WP
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(about 850 g). The fraction WP (850 g) was subjected to macroporous resin column
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chromatography (Diaion HP-20, 2 kg) eluted with water, 30%, 50%, 70%
140
MeOH-Water, and MeOH, successively (6 L each). The elutant eluted by water was
141
abandoned. The other elutant was evaporated to remove organic solvent in reduced
142
pressure and lyophilized to give fraction WP1 (about 11.3 g, eluted with 30% aqueous
143
methanol), fraction WP2 (18.2 g, eluted with 50% aqueous methanol), fraction WP3
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(55.5 g, eluted with 70% aqueous methanol), and fraction WP4 (25.5 g, eluted with
145
methanol). According to the sensory evaluation, only fraction WP3 showed interesting
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sweet taste. Fraction WP3 was subjected to macroporous resins column
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chromatography (Diaion HP-20, 2 kg) eluted with water, 50%, 70% MeOH-Water,
148
and MeOH, successively (6 L each). The solution eluted by water was abandoned.
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According to TLC analysis, the 50% and 70% MeOH-Water portions showed the
150
same color as the sweet compounds 1 and 2 isolated from the n-BuOH portion. And
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these two portions were combined and the combination (recorded as WP3-a, 20 g)
152
was chromatographed on silica gel column, eluted with CHCl3-MeOH-Water (8/2/0, 8
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7/3/0, 7/3/0.5, 6/4/1) to get 5 fractions on the basis of TLC analysis. According to
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sensory evaluation, fractions WP3-a-1 to WP3-a-4 were non-sweet fractions, while
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fractions WP3-a-5 was sweet-tasting fraction and. Fractions WP3-a-5 (10.5 g) was
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chromatographed on RP-18 silica gel reduced pressure column to get 4 fractions (30%,
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35%, 40%, 45%, 50% MeOH-Water, 500 ml each, φ 2.50 × 6.5 cm; 20 g RP-18 silica
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gel, Merck, Germany). Fraction WP3-a-5-b and WP3-a-5-d showed sweet taste on the
159
basis of sensory evaluation. WP3-a-5-b (2.4 g) was chromatographed on RP-18 silica
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gel reduced pressure column (33%, 38%, 40% MeOH-Water, 300 ml each) to yield 4
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(230 mg, eluted by 38% MeOH-Water). WP3-a-5-d (2.3 g) was chromatographed on
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RP-18 silica gel reduced pressure column (35%, 38%, 40%, 45% MeOH-Water, 300
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ml each) to yield 3 (150 mg, eluted by 40% MeOH-Water). All of those extractions,
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fractions and compounds were lyophilized for two times to eliminate the residual
165
solvent prior to the sensory experiments.
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Millettiasaponin A (1): white amorphous powder; the 1H NMR (Pyridine-d5, 500 13
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MHz) and
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Tables 1 and 2; the positive ESI-MS m/z 1037, [M+Na]+, 1059, [M+2Na–H]+; and the
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negative ESI-MS m/z 1013, [M–H]–, 587, [M–2H]2–.
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C NMR (Pyridine-d5, 125 MHz) spectroscopic data are presented in
Derrisaponin A (2): white amorphous powder; [α]22.6 –10.4 (c 0.18, H2O); UV (H2O) D
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λmax(nm) (log ε): 190 (3.74); IR (KBr) νmax cm–1: 3423, 2972, 2934, 1713, 1615, 1265,
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1074, 1048; the 1H NMR (Pyridine-d5, 500 MHz) and
173
MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS m/z 1175 [M–H]–;
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HR-ESI-MS m/z 1175.5480, [M–H]– (calcd for C56H87O26–, 1175.5486).
13
C NMR (Pyridine-d5, 125
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Derrisaponin B (3): white amorphous powder; [α]22.6 –11.7 (c 0.12, H2O); UV (H2O) D
176
λmax(nm) (log ε): 190 (3.77); IR (KBr) νmax cm–1:3424 (OH), 2969, 2932, 1718, 1615,
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1383, 1261, 1072, 1048; the 1H NMR (Pyridine-d5, 800 MHz) and
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(Pyridine-d5, 200 MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS
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m/z 1175 [M–H]–; HR-ESI-MS m/z 1175.5482, [M–H]– (calcd for C56H87O26–,
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1175.5486.
13
C NMR
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Derrisaponin C (4): white amorphous powder; [α]22.8 –4.3 (c 0.15, H2O); UV (H2O) D
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λmax(nm) (log ε): 190 (3.71); IR (KBr) νmax cm–1:3424 (OH), 2968, 2932, 1720, 1619,
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1383, 1263, 1073, 1049; the 1H NMR (Pyridine-d5, 800 MHz) and
184
(Pyridine-d5, 200 MHz) spectroscopic data are presented in Tables 1 and 2; ESI-MS
185
m/z 1337 [M–H]–, 1175 [M–Glc–H]–; HR-ESI-MS m/z 1337.6017, [M–H]– (calcd for
186
C62H97O31–, 1337.6014).
13
C NMR
187
Acute Toxicity Test. Three groups of mice (5 males and 5 females) fasted
188
overnight but allowed access to water. The parenteral acute toxicity tests of the
189
compounds were carried out by the method of previous investigation.18 Compounds 1
190
and 2 were dissolved in normal saline and injections were made into the caudal veins
191
one time in 24 h, at the dose of 250 mg/kg.bw and 400 mg/kg. bw for 1 and 2,
192
respectively. The animals were observed for toxic signs (convulsion, salivation,
193
diarrhoea, lethargy, sleep, coma, nervousness and so on) and deaths regularly
194
throughout the first day, then daily for at least 14 day. In these tests, animals were
195
dosed once at a time. At the end of the observation, surviving animals were sacrificed
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cervical by taking off the cervical spine and organs (heart, lung, spleen, liver, kidney, 10
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uterus and right ovary) were removed, weighed and examined macroscopically.
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Sensory Evaluation. The taste threshold and sweetness intensity relative to sucrose
199
were evaluated by a human sensory panel. Ten evaluation panelists, which were
200
sensitive to sweetness, were screened from more than 30 employees and graduate
201
students invited from Kunming Institute of Botany (KIB), CAS, according to
202
Givandan’s panelist selection procedure (taste intensity ranking test). All of them were
203
trained following ISO norms.16
204
The extracts and sub-fractions were dissolved in distilled water to prepare a water
205
solution at the concentration 0.2% (w/v). The sensory panelist consisting of seven
206
sweet sensitive taster (four women and three men, age from 24-45, Chinese only)
207
were asked to taste the sample solutions to evaluate the taste character as previously
208
described,17 and modified as needed, so that to find the sweet portions or
209
sub-fractions.
210
The compounds were dissolved in distilled water to prepare a stock solution at the
211
concentration of 0.1% (w/v). Fold dilution each stock solution to prepare a series of
212
lower concentration solutions from 0.1 to 0.0002% (w/v). The geometric mean of the
213
last and the second last concentration were calculated and taken as the individual
214
recognition threshold. Seven different panelists evaluated the threshold value in two
215
independent sessions. The sucrose solutions were prepared at concentrations 1%, 2%
216
and 4%. The panelists were asked to taste a sample solution and to estimate its
217
sweetness intensity relative to that of the sucrose solution of appropriate concentration.
218
The ratio of the concentration of sucrose solution and that of compounds solution is 11
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the sweetness intensity relative to sucrose.
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Samples were coded and randomly presented to panelists of 10 mL in each cup and
221
the total cups less than 20 at ambient temperature. The panelists were asked to rinse
222
their mouths with water in between samples and rest for some time after tasting
223
several cups of samples. The assays were performed at least in triplicate on separate
224
occasions.
225
Acid Hydrolysis of Compounds. Compounds 1-4 (each 2 mg) were dissolved in 2
226
M HCl (dioxane-H2O, 1:1, 2 mL) and heated at 95 °C for 2 h, respectively. After
227
cooling and evaporated to dryness under reduced pressure, the reaction mixture was
228
extracted with EtOAc for three times. The aqueous layer was neutralized with
229
NaHCO3 solution and evaporated under vacuum to furnish a neutral residue.19 The
230
residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine
231
methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 2 h, and
232
after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added;
233
the mixture was kept at 60°C for another 2 h.20 The reaction mixture was partitioned
234
between n-hexane and H2O (2 mL each), and the n-hexane extract was analyzed by
235
Agilent 7890Agas chromatography (Agilent Technologies, Santa Clara, America)
236
with a flame ionization detector (FID) under the following conditions: HP-5 capillary
237
column (50 m × 0.32 mm i.d., with a 0.52 µm film thickness, Agilent, Santa Clara,
238
America); FID temp., 250 °C; injector temp., 250 °C; over program, initial temp.
239
160 °C, then raised to 280 °C at 5 °C /min; carrier gas, helium; flow rate: 1.28
240
mL/min. Under these conditions, the following sugar units were confirmed by 12
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comparison of the retention times of their derivatives with those of authentic sugars
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derivatized in a similar way: tR (min) 21.6 (D-glucuronic acid), 20.5 (D-galactose),
243
14.6 (L-rhamnose), and 21.7 (D-glucose), respectively.
244
Quantitation by UPLC–MS/MS. Standard solutions were prepared in methanol at
245
concentrations of 23.300, 116.500, 233.000 and 466.000 µg/mL for 1, and 8.200,
246
49.000, 80.000, and 160.000 µg/mL for 2 to make working curves respectively. The
247
stems of D. eriocarpa were weighed 5.000 g precisely to the conical flasks (100 mL),
248
to which 60 mL methanol was added. The samples were extracted for 30 min on the
249
ultrasonic conditions and the supernatants were filtrated through a 0.45 µm filter
250
membrane to make tested solutions used.
251
Chromatographic analysis was performed on a Waters Acquity UPLC system
252
(Waters Corp., Milford, MA, USA), consisting of a binary pump solvent
253
management system, an online degasser, and an autosampler. MassLynxTM
254
software (version 4.1, Waters, Milford, MA, USA) was used to control the
255
instruments, and for data acquisition and processing. The separation was
256
performed on a reversed phase column (ZORBAX SB–C18, 1.8 µm, 2.1×50 mm,
257
Agilent, America), which was maintained at 30 ℃ . The mobile phase was
258
composed of acetonitrile/0.1% formic acid (9:1 at 0–5 min, 5:5 at 5–6 min, 9:1 at
259
6–8 min, v/v) with a flow rate set at 0.2 mL/min. The auto-sampler was
260
conditioned at 5℃ and each injection volume was 1 µL.
261
Mass spectrometry detection was performed using a Xevo Triple Quadrupole MS
262
(Waters Corp., Milford, MA, USA) equipped with an electrospray ionization source 13
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(ESI). The ESI source was set in negative ionization mode. The parameters in the
264
source were set as follows: capillary voltage, 2.0 kV; cone Voltages, 70 V; collision
265
gas, argon; collision energy, 55 eV; desolvation gas, nitrogen; desolvation gas flow,
266
600 L/h; desolvation temperature, 250℃; cone gas flow, 150 L/h. The analytes were
267
quantitated by the multiple reactions monitoring (MRM). The mass spectra of the two
268
compounds (1 and 2) showed a relative abundant [M–H]– at m/z 1013.5 both for 1 and
269
2, while MS/MS of the ion indicated that the ions at m/z 487.41 for 1 and 645.28 for 2
270
were the most abundant product ions.
271 272 273
RESULTS AND DISCUSSION Isolation and Elucidation of Sweet Compounds. According to the sensory
274
bioassay-guided investigation, the n-BuOH portion and aqueous portion of 80%
275
aqueous MeOH extract, both of which showed licorice-like sweetness, were subjected
276
to isolation and purification on repeated column chromatography to obtain 4
277
sweet-tasting triterpenoid saponins (1–4), of which compounds 2-4 are new ones,
278
while compound 1 is identified for the first time from the genus Derris.
279
Millettiasaponin A (1) was obtained as a white amorphous powder. The molecular
280
formula was C50H78O21, with 12 degrees of unsaturation, in agreement with the
281
positive ESI-MS spectrum (m/z 1037, [M+Na]+; 1059, [M+2Na–H]+) and the negative
282
ESI-MS (m/z 1013, [M–H]–, 2027, [2M–H]–) , as well as 13C NMR spectroscopic data.
283
Obvious signals observed in the 1H NMR spectrum (Tables 1 and 2) of 1 were seven
284
tertiary methyls [δH 0.64, 0.87, 0.96, 1.30, 1.31, 1.39, 2.07 (each 3H, s)], a secondary 14
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methyl [δH 1.79 (d, J=6.5)], a hydroxymethyl [δH 4.22, 3.23 (each d, J=11.5), a
286
olefinic bond [δH 5.52 (br s)], and three anomeric signals [δH 4.97 (d, J=8.0), 5.70 (d,
287
J=7.5), 6.17 (s)]. The 13C NMR spectrum (Tables 1 and 2) exhibited characteristic
288
carbon signals of an oleanane-type triterpenoid aglycone with a hydroxymethyl
289
group.21, 22 Detailed analysis of 13C NMR data with those of millettiasaponins A,21
290
which was isolated from Millettia speciosa, revealed that that it was almost the same
291
as those of millettiasaponins A. Further analysis of HSQC, HMBC, 1H-1H COSY, and
292
ROESY spectra of 1, it was determined as millettiasaponin A unambiguously. Here,
293
the 1H NMR data were assigned completely.
294
Derrisaponin A (2) was obtained as a white amorphous powder. The molecular
295
formula of 2 was deduced to be C56H88O26, with 12 degrees of unsaturation, through
296
the HRESI-MS spectrum (m/z 1175.5480, [M–H]–), which was in agreement with the
297
results of the ESI-MS (m/z 1175, [M–H]–; 587, [M–2H]2–) and 13C NMR data analysis.
298
The IR spectrum suggested the presence of hydroxyl group (3423 cm–1), carbonyl
299
group (1713 cm–1), and olefinic group (1615 cm–1). Acid hydrolysis of 2 afforded
300
D-glucuronic
301
NMR spectra data (Tables 1 and 2) of 2 with 1, it was inferred that 3 and 1 shared the
302
same aglycone, except for the presence of the additional signals corresponding to a
303
glucosyl of C6H10O5 based on the molecular weight with162 more than that of 1. The
304
C3Gal-O-Glc was deduced based on the glycosidation shift of C-3Gal from δ 76.6 to δ
305
84.9, combined with the HMBC correlations. The assignment of all the sugar and
306
aglycone residues was determined by the HSQC, HMBC, 1H-1HCOSY and ROESY
acid, D-galactose, L-rhamnose, D-glucose. Comparing the 1H and 13C
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307
experiments. The linkage sites of sugar units and the aglycone were determined by the
308
HMBC correlations from H-1GluA (δH 4.96) to C-3 (δC 91.8), from H-1Ga l δH (5.70) to
309
C-2GluA (δC 77.0), from H-1Rha (δH 6.10) to C-2GalδC (77.0), and from H-1Glc δH (5.04)
310
to C-3Gal (δC 84.9). From all the analysis above, the structure of 2 was established as
311
22β-acetyloxy-3β, 24-dihydroxy-olean-12-en-30-oic acid
312
3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranosyl
313
-(1→2)-β-D-glucuronopyranoside, and named derrisaponin A.
314
Derrisaponin B (3) was obtained as a white amorphous powder. The molecular
315
formula of 3 was deduced to be C56H88O26, with 13 degrees of unsaturation, on the
316
basis of HRESI-MS spectrum (m/z 1175.5482, [M–H]–), which was compatible with
317
the results of ESI-MS (m/z 1175, [M–H]–) and NMR data analysis. The IR spectrum
318
suggested the presence of hydroxyl group (3424 cm–1), carbonyl groups (1718 cm–1),
319
and olefinic group (1615 cm–1). The molecular formula (C56H88O26) suggested the
320
same molecular formula of 3 with 2. And acid hydrolysis of 3 yielded the same sugar
321
with 2. Detailed analysis of the NMR spectra data (Tables 1 and 2) for the aglycone of
322
3 and 1, indicated that 3 and 1 shared the same aglycone and C-3 sugar linkage, and
323
the significant difference between them was that the carboxyl group at C-30 was
324
glycosylated by a glucosyl on the basis of the glycosidation shift of C-30 from δ 179.9
325
to δ 177.0. The assignment of all the sugar residues and the aglycone moiety was
326
determined by the HSQC, HMBC, 1H-1H COSY and ROESY experiments. The
327
linkage of the sugar units and the aglycone were determined by the HMBC
328
correlations from H-1GluA (δH 4.96) to C-3 (δC 91.8), from the proton at δH 5.69 16
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(H-1Gal) to C-2GluA (δC 76.9), from H-1Rha (δH 6.18) to the C-2Gal (δC 77.7), and from
330
H-1Glc (δH 6.10) to C-30 (δC 177.0). Therefore, the structure of 3 was determined as
331
22β-acetyloxy-3β, 24-dihydroxy-olean-12-en-30-oic acid
332
3-O-α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyrano
333
syl-30-O-β-D-glucopyranoside, and named derrisaponin B.
334
Derrisaponin C (4) was obtained as a white amorphous powder. The molecular
335
formula of 4 was deduced to be C62H98O31, with 14 degrees of unsaturation, by
336
HRESI-MS spectrum (m/z 1337.6017, [M–H]–), which was compatible with the
337
results of ESI-MS (m/z 1337, [M–H]– and NMR data analysis. The IR spectrum
338
suggested the presence of hydroxyl group (3424 cm–1), carbonyl group (1720 cm–1),
339
and olefinic group (1619 cm–1). Acid hydrolysis of 4 yielded D-glucuronic acid,
340
D-galactose, L-rhamnose, D-glucose.
341
for 4 with compounds 1-3 was indicated by the analysis of 1H and 13C NMR data
342
(Tables 1 and 2). Detailed analysis of 13C NMR data of 4 with those of 2 indicated
343
that the significant difference between them was that the carboxyl group at C-30 was
344
glycosylated by a glucosyl on the basis of the glycosidation shift of C-30 from δ 180.0
345
to δ 177.1. And the C3Gal-O-glucosyl and C30-O-glucosyl can be confirmed by the
346
glycosidation shifts of C-3Gal from δ 76.6 to δ 84.8, and C-30 from δ 179.9 to δ 177.1.
347
The assignment of all the sugar residues and linkage sites of sugar units and the
348
aglycone were determined by the HSQC, HMBC, 1H-1H COSY and ROESY
349
experiments. Therefore, the structure of 4 was determined as 22β-acetyloxy-3β,
350
24-dihydroxy-olean-12-en-30-oic acid
The same aglycone of oleanane-type triterpenoid
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351
3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-galactopyranosyl
352
-(1→2)-β-D-glucuronopyranosyl-30-O-β-D-glucopyranoside, and named derrisaponin
353
C.
354
Acute Toxicity Test. The acute toxic activities of 1 and 2 were assessed through
355
caudal veins injection to ICR mice. After injection, one of the animals administered
356
by compound 2 (at dose of 400 mg/kg bw) showed diaphragm spasm, but return to
357
normal 10 min later, and the other animals in this group and the other two groups
358
animals (administered by compound 1 at the dose of 250 mg.kg bw and solvent
359
respectively) did not show any unmoral signs. No treatment-related clinical signs of
360
toxicity or mortality were observed for the 14 days observation. In addition, all
361
internal organs examined at necropsy were free from any gross pathological changes
362
at the end of observation. This indicates that the maximum tolerated doses (MTD) of
363
compounds 1 and 2 are 250 mg/kg and 400 mg/kg respectively.
364
Sweetness Intensities of Compounds 1-4. The sweetness intensities of 1-4 were
365
evaluated at near sweetness detection threshold concentrations by seven
366
sweet-sensitive panelists and the results are reported. Compounds 1 and 2, with a
367
carboxyl group at C-30, showed potent sweetness (150 and 80 times, sweeter than that
368
of sucrose, and the threshold value is 0.05 mg/ml and 0.0625 mg/ml, respectively),
369
while the sweetness of 3 and 4, with a carboxyl group glycosylated by a glucose
370
group, showed only 2 and 0.5 times sweeter than that of sucrose, respectively (The
371
threshole value is 1 mg/ml and 5 mg/ml, respectively). Moreover, 1 and 2 showed the
372
most licorice-like sweetness. Accordingly, a free carboxyl group at the C-30 position 18
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seems to be essential to the potent sweetness for this type of compounds.
374
Quantitation by UPLC–MS/MS. The quantitation of compounds 1 and 2 in the
375
stems of D. eriocarpa were analyzed by means of UPLC–MS/MS. The interesting
376
contents of 352.80 mg/kg for 1 and 1887.60 mg/kg for 2 respectively were calculated
377
from the tested data (Table 3). The regression equations of 1 and 2 were y = 316.72x –
378
2467.9 (r2 = 0.9991) and y = 10.882x – 40.715 (r2 = 0.9993), respectibely.
379
Based on the experimental data, it can be inferred that these four oleanane-type
380
triterpenoid saponins are major sweet-tasting components in the stem of this plant and
381
they are responsible for the licorice-like sweetness of D. eriocarpa. These studies
382
provide solid evidence that the stems of D. eriocarpa can be used for the alternatives
383
of licorice in the taste aspect. In addition, compounds 1 and 2 showed no acute
384
toxicity and the maximum tolerated doses (MTD) of them are 250 mg and 400 mg
385
respectively, suggesting that they may be potential natural sweeteners.
386 387
ASSOCIATED CONTENT
388
Supporting Information
389 390 391
The 1H NMR,
13
C NMR, and 2D-NMR (HSQC, HMBC, 1H-1H COSY, and
ROESY) and MS spectra of compounds 1-4 The Supporting information is available free of charge on the ACS Publications
392
website at DOI:
393
AUTHOR INFORMATION
394
Corresponding Author 19
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Journal of Agricultural and Food Chemistry
395
* Phone: +86-871-65223224; E-mail address:
[email protected] 396
Funding
397
This work was sponsored by grants from the Natural Science Foundation of Yunnan
398
Province (2013FB065), the 45th Scientific Research Foundation for the Returned
399
Overseas Chinese Scholars from State Education Ministry and National S&T Basic
400
Work Program of China (2012FY110300).
401
Note
402
The authors declare no competing financial interests.
403 404
ACKNOWLEDGMENTS
405
We thank Professor Lisong Wang for identification of the plant. The authors
406
appreciate the efforts of all panelists participating in the sensory tests. The authors
407
appreciate the sacrifice of all ICR mice.
20
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REFERENCES
409
(1) Temussi, P. The history of sweet taste: not exactly a piece of cake. J. Mol.
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Recognit. 2006, 19, 188-199.
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(2) Vos, M. B.; Kaar, J. L.; Welsh, J. A. Added sugars and cardiovascular disease risk
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in children: a scientific statement from the American Heart Association. Circulation
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2016, 134, 1-18.
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(3) Yang, Q. H.; Zhang, Z. F.; Gregg, E. W. et al. Added sugar intake and
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cardiovascular diseases mortality among US adults. JAMA Intern. Med. 2014, 174,
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516-524.
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(4) Kim, N. C.; Kinghorn, A.D. Highly sweet compounds of plant origin. Arch. Pharm.
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Res. 2002, 25, 725-746.
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(5) Kinghorn, A. D.; Kennelly, E. J. Discovery of highly sweet compounds from
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natural sources. J. Chem. Educ. 1995, 72, 676-680.
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(6) Kinghorn A. D.; Chin Y. W.; Pan L.; Jia Z. H. Natural products as sweeteners and
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sweetness modifiers. In Comprehensive natural products II; Chemistry and Biology;
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Development & Modification of Bioactivity; Elsevier: Oxford, U.K, 2010; vol. 3, pp.
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269-314.
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(7) Huang Y. L.; Guo Z.Y.; Liu Y. J.; Wang Y. L.; Luo B. S.; Long C. L. Indigenous
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Botanical Nomenclature Used by the Zhuang People in Jingxi County, Guangxi. Plant
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Diversity and Resour. 2013, .35, 443-452.
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(8) Yunnan Food and Drug Administration. The Standards of Chinese Medicinal
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Materials in Yunnan Province, Dai Ethnophamacy ( Ⅱ ). Yunnan Science & 21
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Technology Press: Kunming, 2005, vol. 5, pp. 7, 115.
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(9) Guangxi Institute of Chinese Medicine & Pharmaceutical Science. Medicinal
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Plants Directory of Guangxi. Guangxi People's Publishing House: Nanning, 1986, pp.
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232.
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(10) Editoria Committee of the Administration Bureau of Traditional Chinese
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Medicine. Chinese Matrria Medica (Zhonghua Bencao). Shanghai Press of Science
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and Technology: Shanghai, 1998, vol. 4, pp. 442.
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(11) Yang, D. A.; Guo, L. C. Review of five Tugancao medicines with same name in
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Guangxi. Cent. South Pharm. 2013, 11, 282-284.
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(12) Wang, L. X.; Wu, H. G.; Zhang, H.; Lou, H. Y.; Liang, G. Y.; Jiang, W. W.; Yang,
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Z. C.; Pan, W. D. Studies on flavonoids from Derris eriocarpa. Chin. J. Chin. Mater.
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Med. 2015, 40, 3009-3012
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(13) Yang, L. F.; Wang, K.; Jiang M. G.; Liu, H. C.; Wang, X.; Qin, P. Y.; Ouyang, Q.
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L. Isolation and characterization of a new bioactive isoflavone from Derris eriocarpa.
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J. Asian. Nat. Prod. Res. 2015, 17, 1002-1009.
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(14) Zhang, H. X.; Lunga, P. K.; Li, Z. J.; Dai, Q.; Du, Z. Z. Flavonoids and
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stilbenoids from Derris eriocarpa. Fitoterapia 2014, 95, 147-153.
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(15) Ren W. K.; Chen S.; Yin J.; Duan J. L.; Li T. J.; Liu G.; Feng Z. M.; Tan B.; Yin
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Y. L.;, Wu G. Y. Dietary arginine supplementation of mice alters the microbial
449
population and activates intestinal innate immunity. J. Nutr. 2014, 144, 988-995.
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(16) ISO 8586: 2012. Sensory Analysis—General guidelines for the selection, training
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and monitoring of selected assessors and expert sensory assessors, 2012. 22
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(17) Jia, Z. H.; Yang, X. G. A minor, sweet cucurbitane glycoside from Siraitia
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grosvenorii. Nat. Prod. Commun. 2009, 4, 769-772.
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(18) Fairhurst, S.; Marrs, T. C.; Parker, H. C.; Scawin J. W.; Swanston D. W. Acute
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toxicity of T2 toxin in rats, mice, guinea pigs, and pigeons. Toxicology 1987, 43,
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31-49.
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(19) Wang J. S.; Yang X. W.; Di Y. T.; Wang Y. H.; Shen Y. M,; Hao, X. J. Isoflavone
458
Diglycosides from Glycosmis pentaphylla. J. Nat. Prod. 2006, 69, 778-782.
459
(20) Liang, D.; Hao, Z. Y.; Zhang, G. J.; Zhang, Q. J.; Chen, R. Y.; Yu, D. Q.
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Cytotoxic triterpenoid saponins from Lysimachia clethroides. J. Nat. Prod. 2011, 74,
461
2128-2136.
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(21) Uchiyama, T.; Furukawa, M.; Isobe, S.; Makino, M.; Akiyama, T.; Koyama, T.;
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Fujimoto, Y. New oleanane-type triterpene saponins from Millettia speciosa.
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Heterocycles 2003, 60, 655-661.
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(22) Mahato S. B.; Kundu A. P. 13C NMR spectrumof pentacylclic triterpenoids — a
466
compilation and some salient features. Phytochemistry 1994, 37, 1517-1575.
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Figure captions
468
Figure 1. The structure of compounds 1-4
469
Figure 2. The selected HMBC (H
470
Figure 3. ROESY correlations of the aglycone moiety
C) and 1H-1H COSY (
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Table 1. 1H and 13C NMR spectroscopic data of the aglycone moieties of compounds 1-4 in pyridine-d5 (δ in ppm, J in Hz) 1
2
3
4
NO. 1
δC
δH
δC
δH
δC
δH
δC
δH
38.9, t
1.29 (m), 0.76 (m)
39.0, t
1.33 (m), 0.79 (m)
38.8, t
1.34 (m), 0.84 (m)
38.9, t
1.37 (m), 0.86 (m)
2
27.0, t
2.09 (m), 1.74 (m)
27.1, t
2.10 (m), 1.95 (m)
26.9, t
2.13 (m), 1.84 (m)
27.0, t
2.14 (m), 1.84 (m)
3
91.5, d
3.35 (dd, 11.5, 3.5)
91.8, d
3.35 (dd, 11.5, 4.0)
91.4, d
3.35 (dd, 12.0, 4.0)
91.7, d
3.36 (dd, 11.2, 4.0)
4
44.2, s
/
44.3, s
/
44.2, s
/
44.2, s
/
5
56.3, d
0.80 (m)
56.5, d
0.84 (m)
56.2, d
0.81 (m)
56.3, d
0.84 (m)
6
18.8, t
1.51 (m), 1.20 (m)
19.0, t
1.57 (m), 1.29 (m)
18.8, t
1.52 (m), 1.22 (m)
19.0, t
1.56 (m), 1.27 (m)
7
33.1, t
1.45 (m), 1.24 (m)
33.3, t
1.50 (m), 1.33 (m)
33.1, t
1.43 (m), 1.21 (m)
33.1, t
1.45 (m), 1.26 (m)
8
40.5, s
/
40.6, s
/
40.3, s
/
40.4, s
/
9
48.0, d
1.57 (m)
48.1, d
1.59 (m)
47.9, d
1.54 (m)
48.0, d
1.55 (m)
10
36.8, s
/
36.9, s
/
36.7, s
/
36.9, s
/
11
24.4, t
1.76 (2H)
24.5, t
1.79 (2H)
24.2
1.70 (2H)
24.4, t
1.72 (2H)
12
123.6, d
5.52 (brs)
123.7, d
5.52 (brs)
123.7, d
5.40 (brs)
123.8, d
5.42 (brs)
13
144.5, s
/
144.6, s
/
144.0, s
/
144.2, s
/
14
42.3, s
/
42.4, s
/
42.1, s
/
42.1, s
/
15
26.6, t
1.74 (m), 0.98 (m)
26.8, t
1.77 (m), 1.01 (m)
26.5, t
1.68 (m), 0.94 (m)
26.5, t
1.70 (m), 0.96 (m)
16
26.6, t
1.93 (m), 0.98 (m)
26.7, t
1.95 (m), 1.01 (m)
26.4, t
1.83 (m), 0.94 (m)
26.4, t
1.83 (m), 0.96 (m)
17
36.6, s
/
36.7, s
/
36.6, s
/
36.7, s
/
18
44.4, d
2.93 (dd, 12.5, 3.5)
44.6, d
2.93 (dd, 12.5, 4.0)
44.1, d
2.84 (dd, 12.0, 4.0)
44.1, d
2.84 (dd, 12.0, 4.0)
19
42.0, t
20
41.2, s
2.30 (d, 11.5) 1.82 (m) /
42.2, t 41.3, s
2.30 (d, 10.5) 1.82 (m) /
41.3, t 41.4, s
25
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2.17 (d, 12.0) 1.75 (m) /
41.4, t 41.5, s
2.19 (dd, 11.2, 5.6) 1.75 (m) /
Journal of Agricultural and Food Chemistry
2.81 (d, 14.0),
2.82 (d, 13.0)
22
78.6, d
4.83 (brs)
78.5, d
4.84 (brs)
77.4, d
4.88 (brs)
77.6, d
4.90 (brs)
23
23.3, q
1.39 (s)
23.4, q
1.38 (s)
23.2, q
1.40 (s)
23.2, q
1.39 (s)
24
63.8, t
25
16.1, q
0.64 (s)
16.1, q
0.67 (s)
16.0, q
0.67 (s)
26
17.1, q
0.87 (s)
17.3, q
0.90 (s)
17.0, q
0.85 (s)
17.1, q
0.88 (s)
27
27.1, q
1.30 (s)
27.0, q
1.33 (s)
26.8, q
1.23 (s)
26.9, q
1.25 (s)
28
21.8, q
0.96 (s)
21.9, q
0.98 (s)
21.6, q
0.86 (s)
21.7, q
0.88 (s)
29
30.2, q
1.31 (s)
30.3, q
1.33 (s)
30.0, q
1.27 (s)
30.0, q
1.29 (s)
30
179.9, s
/
180.0, s
/
177.0, s
/
177.1, s
/
171.1, s
/
171.0, s
/
171.4, s
/
171.6, s
/
21.6, q
2.07 (s)
21.5, q
2.07 (s)
22.0, q
2.38 (s)
22.1, q
2.39 (s)
4.22 (d, 11.5); 3.23 (d, 11.5)
64.0, t
4.23 (m) 3.29 (d, 11.5)
63.6, t
26
ACS Paragon Plus Environment
1.71 (m)
4.22 (m) 3.23 (d, 11.2)
35.7, t
2.66 (d, 13.6)
35.6, t
1.78 (m)
35.7, t
2.64 (d, 13.6)
21
1.77 (m)
35.7, t
Page 26 of 33
63.7, t 16.1, q
1.73 (m)
4.24 (m) 3.30 (d, 11.2) 0.70 (s)
Page 27 of 33
Journal of Agricultural and Food Chemistry
Table 2. 1H and 13C NMR spectroscopic data of the sugar moieties of compounds 1-4 in pyridine-d5 (δ in ppm, J in Hz) NO.
1
2
3
4
δC
δH
δC
δH
δC
δH
δC
δH
1
105.8
4.97 (d, 8.0)
105.8
4.96 (m)
105.7
4.96 (d, 7.2)
105.8
4.95 (d, 8.0)
2
77.0
4.52 (m)
77.0
4.52 (m)
76.9
4.52 (t, 8.8)
76.8
4.54 (t, 8.8)
3
78.3
4.67 (m)
78.6
4.57 (m)
78.2
4.66 (m)
78.3
4.65 (m)
4
74.2
4.44 (m)
74.2
4.42 (m)
74.1
4.44 (m)
74.1
4.45 (m)
5
78.0
4.67 (m)
78.0
4.63 (m)
77.9
4.66 (m)
78.0
4.65 (m)
6
172.8
/
172.9
/
172.6
/
172.8
/
1
102.1
5.70 (d, 7.5)
102.1
5.70 (d, 7.5)
102.0
5.69 (d, 7.2)
102.0
5.69 (d, 7.2)
2
77.8
4.49 (m)
77.0
4.48 (m)
77.7
4.49 (dd, 9.6, 7.2)
76.7
4.48 (m)
3
76.6
4.10 (dd, 9.5, 3.0)
84.9
4.08 (dd, 9.5, 2.0)
76.5
4.08 (dd, 9.6, 3.2)
84.8
4.05 (m)
4
71.3
4.39 (m)
71.3
4.72 (m)
71.2
4.40 (d, 3.2)
70.9
4.79 (m)
5
76.6
3.97 (m)
76.6
3.96 (m)
76.4
3.95 (t, 6.4)
76.5
4.03 (t, 6.4)
6
61.7
62.2
4.25 (2H, m)
61.7
3-O-GluA
Gal
4.37(dd, 9.5, 3.0) 4.30 (dd, 11.0, 6.0)
4.42 (2H, m), 4.34 (m)
61.7
4.30 (2H, m) 4.27 (m)
Rha 1
102.6
6.17 (s)
102.7
6.10 (s)
102.4
6.18 (s)
102.6
6.13 (s)
2
72.7
4.81 (m)
72.7
4.87 (brs)
72.6
4.83 (dd, 3.2, 1.6)
72.5
4.92 (d, 3.2)
3
72.8
4.80 (dd, 9.5, 3.0)
73.0
4.69 (m)
72.7
4.79 (dd, 9.6, 3.2)
72.7
4.79 (m)
4
74.7
4.36 (m)
74.7
4.30 (m)
74.5
4.36 (m)
74.6
4.34 (m)
5
69.8
5.01 (m)
69.8
4.93 (m)
69.6
5.0 (dq, 12.0, 6.0)
69.6
4.97 (m)
6
19.3
1.79 (d, 6.5)
19.3
1.72 (d, 6.0)
19.2
1.76 (d, 6.4)
19.3
1.73 (d, 5.6)
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Glc I 1
106.3
5.04 (d, 8.0)
106.3
5.06 (d, 7.2)
2
75.4
3.95 (m)
75.2
3.98 (t, 8.8)
3
78.7
4.21 (m)
78.5
4.32 (m)
4
71.8
4.12 (t, 9.5)
71.7
4.12 (t, 9.6)
5
78.7
3.86 (m)
78.7
3.92 (m)
6
62.7
4.40 (m), 4.29 (m)
62.3
4.34 (m), 4.27 (m)
30-O-Glc II 1
96.6
6.10 (d, 8.0)
96.7
6.11 (d, 8.0)
2
74.0
4.20 (m)
74.2
4.22 (m)
3
78.7
4.31 (m)
78.2
4.32 (m)
4
71.4
4.33 (m)
71.4
4.33 (m)
5
79.5
4.05 (m)
79.6
4.06 (m)
6
62.3
4.37 (m), 4.29 (m)
62.5
4.42 (2H, m)
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Table 3. Content of 1 and 2 in the stems of D. eriocarpa Trace (m/z) parent
daughter
RT (min)
1
1013.5
645.28
3.29
7288.672
29.4
352.8
2
1175.9
645.3
2.70
1659.176
157.3
1887.6
Compounds
Area
Content in sample solution (µg/ml)
Content in stem (mg/kg)
29
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Figure 1.
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Page 31 of 33
Journal of Agricultural and Food Chemistry
Figure 2.
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Figure 3.
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Journal of Agricultural and Food Chemistry
TOC graphic O 30
29
?
Licorice-like sweetness Substitute for licorice(The stems of Derris eriocarpa)
OR2 19 13
11 25
26
1
23
17
22
OAc 28
14 10
9 15
3
R1O
21 18
27 5
7
CH2OH
24
Sweet-Tasting Compounds 1-4
33
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