Bioactivity-Guided Isolation and Identification of Antiadipogenic Compounds in Shiya Tea (Leaves of Adinandra nitida)

Article abstract of DOI:10.1021/acs.jafc.9b01326

Obesity is a worldwide epidemic contributing to a higher risk of developing maladies such as type 2 diabetes, heart disease, and cancer. Shiya tea (leaves of Adinandra nitida), a traditional Chinese tea, is widely consumed due to its palatable flavor and various curative effects, such as reducing blood pressure and blood lipids, as well as anti-inflammation, etc. However, no relevant research on the antiobesity effects of Shiya tea has been reported. In particular, no health-benefiting compounds, other than flavonoids, in Shiya tea have been reported. Thus, 3T3-L1 preadipocytes have been used as a bioactivity-guided identification model to verify the inhibitory effects of Shiya tea on adipogenesis, as well as to identify antiadipogenic compounds. Four triterpenoid saponins (1-4), including one new compound (2α,3α-dihydroxyursolic acid 28-O-β-d-glucopyranosyl ester, compound 1), and a flavonoid (5) have been identified using NMR (1D and 2D NMR) and liquid chromatography (LC)-MS techniques. Compound 1, the major antiadipogenic constituent with an IC₅₀ value of 27.6 μg/mL, has been identified for the first time in Shiya tea. To understand the structure-activity relationship, three hydrolytic compounds (1s, 2s, and 5s) were obtained to provide an inhibitory effect on lipid accumulation during 3T3-L1 adipocyte differentiation. The inhibitory effect of the triterpenoid (1s) possessing no sugar group decreased significantly, while the flavonoid (5s) also without a sugar group showed increased activity. In addition, the hydroxyl group position may also play a role in inhibitory efficacy.

Full text of DOI:10.1021/acs.jafc.9b01326

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Bioactive Constituents, Metabolites, and Functions
Bioactivity-guided isolation and identification of anti-
adipogenic compounds in Shiya tea (leaves of Adinandra nitida)
Chunmao Yuan, Linhua Huang, Joon Hyuk Suh, and Yu Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01326 • Publication Date (Web): 29 May 2019
Downloaded from http://pubs.acs.org on May 30, 2019
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Journal of Agricultural and Food Chemistry
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Bioactivity-guided isolation and identification of anti-adipogenic
compounds in Shiya tea (leaves of Adinandra nitida)
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Chunmao Yuan,†, ‡,⊥ Linhua Huang,†, ||,⊥ Joon Hyuk Suh, Yu Wang,†,*
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Citrus Research and Education Center, Food Science and Human Nutrition, University of
Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850, United States
‡
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State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical
University, 3491 Baijin Road, Guiyang 550014, People's Republic of China
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Citrus Research Institute, Southwest University, Xiema, Beibei, Chongqing 400712, People's
Republic of China
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*Corresponding Author:
Yu Wang, Ph.D.
Department of Food Science and Human Nutrition
Citrus Research and Education Center
University of Florida
700 Experiment Station Rd, Lake Alfred, FL 33850 USA
Tel: +1-863-956-8673
E-mail: yu.wang@ufl.edu
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Abstract
Obesity is a worldwide epidemic contributing to a higher risk of developing maladies such
as type 2 diabetes, heart disease, and cancer. Shiya tea (leaves of Adinandra nitida), a
traditional Chinese tea, is widely consumed due to its palatable flavor and various curative
effects, such as reducing blood pressure and blood lipids, as well as anti-inflammation, etc.
However, no relevant research on the anti-obesity effects of Shiya tea has been reported. In
particular, no health benefiting compounds, other than flavonoids, in Shiya tea have been
reported. Thus, 3T3-L1 preadipocytes have been used as a bioactivity-guided identification
model to verify the inhibitory effects of Shiya tea on adipogenesis, as well as identify anti-
adipogenic compounds. Four triterpenoid saponins (1–4), including one new compound (2α,
3α-dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester, compound 1), and a flavonoid
(5) have been identified using NMR (1D and 2D NMR) and LC−MS techniques. Compound
1, the major anti-adipogenic constituent with an IC value of 27.6 μg/mL, has been identified
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for the first time in Shiya tea. In order to understand the structure-activity relationship, three
hydrolytic compounds (1s, 2s, and 5s) were obtained to provide an inhibitory effect on lipid
accumulation during 3T3-L1 adipocyte differentiation. The inhibitory effect of the triterpenoid
(1s) possessing no sugar group decreased significantly, while the flavonoid (5s) also without a
sugar group showed increased activity. In addition, the hydroxyl group position may also play
a role in inhibitory efficacy.
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KEYWORDS: Shiya tea, anti-adipogenesis, bioactivity-guided isolation, triterpenoid saponins,
structure-activity relationship
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INTRODUCTION
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Obesity is a worldwide epidemic contributing to a higher risk of developing maladies
such as type 2 diabetes, heart disease, and cancer.1-3 According to the World Health
Organization (WHO), nearly two billion adults worldwide are overweight and over
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500,000,000 are obese. Previously, only five drugs have been approved by the FDA to treat
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obesity, but reports indicate various side effects from their usage. Therefore, searching for
alternative therapies or using natural resources to prevent obesity with little or no side effects
has become a necessity.
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Adinandra nitida (Theaceae), a shrub or tree, is mainly distributed in the southern part of
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China, such as Guangdong, Guangxi, Guizhou and Yunnan Provinces. The leaves of A. nitida,
have long been consumed as a traditional tea (Shiya tea) beverage in China, 6, 7 due to its
palatable flavor and many curative effects, such as reducing blood pressure and blood lipids,
as well as anti-inflammation, etc.8,9 Previous studies indicated that Shiya tea is rich in
flavonoids, such as camellianin A, camellianin B, and apigenin, with great radical scavenging
ability for 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antitumor activities.7,8 Flavonoids in
Shiya tea are considered the major active and functional chemical constituents.10-13 However,
there has been no studies indicating additional compounds, other than flavonoids, in Shiya tea
demonstrate any bioactivities, in particular anti-obesity effects. In order to explore other
potential bioactive compounds in Shiya tea, as well as potential to prevent obesity, its inhibitory
effects of adipogenesis using 3T3-L1 preadipocytes has been conducted, and the bioactivity-
guided identification of anti-adipogenic compounds has also been performed. Consequently,
bioactivity-guided isolation of the methanol extract led to the isolation and identification of
four triterpenoid saponins (1–4), including one new compound (1), and a flavonoid (5) using
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NMR (1D and 2D NMR) and LC-MS techniques. The new triterpenoid saponin (1), the major
active constituent in Shiya tea, reduced intracellular lipid accumulation in 3T3-L1 adipocytes.
The structure-activity relationship of triterpenoid saponins is also discussed.
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MATERIALS AND METHODS
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Materials. Analytical (250 μm thickness, 2−25 μm particle size) TLC plates were purchased
from Macherey-Nagel company (Macherey-Nagel, Germany). Silica gels for column
chromatography (100−200 or 200−300 mesh, (Thermo Scientific, CA, USA) and Sephadex
LH-20 (Thermo Scientific, CA, USA) were obtained from Thermo Scentific. Methoxyamine,
N-methyl-N-(trimethylsily)trifluoracetamide, and pyridine were used for preparing derivative
of the glucose (Sigma-Aldrich, USA). LC-MS grade acetonitrile, water, and formic acid and
ACS-grade n-hexane, ethyl acetate, acetone, chloroform and methanol were purchased from
Sigma-Aldrich. Shiya tea samples were produced in Zhaoping of Guangxi Province, China in
May 2018.
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LC−MS Analysis. LC−MS analysis was conducted using a Vanquish UHPLC system
(UHPLC+ focused pump, autosampler, and column compartment) coupled with a Q Exactive
mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) via an electrospray
ionization (ESI) interface. Chromatographic separation for crude extract and isolates were
performed using an ACQUITY UPLC BEH C18 column (150 mm × 2.1 mm id, 5 μm, Waters,
Milford, MA, USA). The mobile phase consisted of 0.1% formic acid in water (solvent A) and
0.1% formic acid in acetonitrile (solvent B). The sample was eluted with the following linear
gradient: 25–65% B from 0–12 min, 65–98% B from 12–15 min, and maintain at 98% from
15–22 min. The flow rate was 0.2 mL/min, and the injection volume was set at 2 μL. The
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column temperature was maintained at 40 °C. The MS parameters were as follows: negative
ionization mode, capillary temperature of 250 °C, sheath gas (N ) flow rate of 45 psi, aux gas
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(N ) flow rate of 10 psi, ion spray voltage of 2.5 kV, and S-lens RF level of 50. Full MS scans
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in the FT cell were acquired in the range of m/z 65−975 with a mass resolution of 70,000, AGC
target of 1,000,000 and maximum IT of 246 ms. The MS/MS experiments were set as data
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dependent scan (dd-MS ) with a mass resolution of 17,500, AGC target of 200,000 and
maximum IT of 54 ms. The isolation window was set at m/z 1.0, and the stepped collision
energy was used at 20, 40 and 60 eV. Xcalibur (Ver. 4.0) was employed for data collection and
analysis.
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NMR Analysis. H (600 or 400 MHz), C (150 or 100 MHz), H− H COSY (correlation
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spectroscopy), H− C HMQC (heteronuclear multiple quantum correlation), H− H ROESY
(rotating frame overhauser effect spectroscopy), and HMBC (heteronuclear multiple bond
correlation) NMR spectra were acquired on a Bruker 600 MHz instrument. Compounds 1–4,
1s, and 2s were analyzed in CD OD, while compounds 5 and 5s were measured in pyridine-d .
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Purification of Compounds 1−5. Shiya tea (4.0 kg) was powdered and soaked in MeOH (20
L) three times at 25 °C. The combined extract was filtered and concentrated with a rotary
evaporator to yield crude extract (0.7 kg), which was suspended in water and then extracted
with EtOAc (3 × 5 L) resulting in the corresponding extracts: EtOAc (200 g) and water layer
(500 g). The anti-adipogenic effects were evaluated by measuring inhibition of adipogenic
differentiation in 3T3-L1 preadipocytes. The active EtOAc fraction was applied to a silica gel
CC (7.62 cm × 65 cm id) and eluted with petroleum ether/acetone (50:1, 20:1, 10:1, 5:1, 2:1,
1:1, and 0:1 v/v, each 25 L) to create four fractions (Fr. 1–Fr.4), in which only Fr. 3 showed
better anti-adipogenic activity. Then, Fr. 3 (40 g) was subjected to an ODS CC (6.35 cm × 48
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cm id, MeOH/H O, 40, 50, 60, 70, 80, and 100% v/v, each 300 mL) resulting in five fractions
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(Fr. 3A–Fr.3E), in which only Fr.3D was active. Next, Fr.3D (20 g) was fractionated using a
gradient of CH Cl /MeOH (50:1, 30:1, 10:1 v/v, each 3 L) to obtain subfractions. Fr. 3D.1–
2
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Fr.3D.5. Fr.3D.1 (7 g) was further purified on a silica gel CC using CH Cl /acetone (3:2, 1:1
2
2
v/v, each 10 L) to furnish 1 (2.0 g) and 2 (1.0 g). Fr.3D.2 (4 g) was subjected to a Sephadex
LH-20 CC (145 cm × 4.5 cm id) flushed with MeOH giving rise to five subfractions (Fr.
3D.2.1–Fr. 3D.2.5), and Fr.3D.2.3 was then purified by a semi-preparative HPLC (52%
MeOH/H O for 50 min, v/v, 4 mL/min) with an X-bridge column (250 mm × 20 mm, 5 μm) to
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afford 3 (200 mg) and 4 (210 mg) with retention times of 37.0 and 44.5 min, respectively.
Fr.3D.5 (5 g) was repeatedly recrystallized in EtOAc to afford 5 (3.0 g).
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25
26
27
28
2α, 3α-Dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester (1). White, amorphous
powder; [α]D25 +27 (c 0.4, MeOH); IR νmax 3397, 2939, 2864, 1753, 1458, 1379, 1067, 1027,
−
1
1
13
605, 583 cm ; H and C NMR data, see Table 1; HR-ESI-MS: m/z calcd. for C H O [M
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59 11
–
+ HCOO] 679.4057 found 679.4064.
Kajiichigoside F1 (2). White amorphous powder; HR-ESI-MS: m/z calcd. for C H O [M +
1
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–
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HCOO] 695.4007 found 695.4014. H NMR (400 HMz, CD OD): δ 0.79 (s, 3H), 0.89 (s,
3
H
3H), 0.95 (d, 3H, J = 5.2 Hz, H-30), 1.01 (s, 3H), 1.01 (s, 3H), 1.23 (s, 3H), 1.37 (s, 3H), 3.70,
(dd, 1H, J = 11.8, 4.2 Hz, H-6a), 3.82, (dd, 1H, J = 11.8, 4.2 Hz, H-6b), 3.95, (dt, 1H, J = 11.8,
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3.5 Hz, H-2), 5.33 (t, 1H, J = 2.9 Hz, H-12), 5.34 (d, 1H, J = 8.6 Hz, H-1'); C NMR (100
HMz, CD OD): δ 15.2 (CH , C-30), 15.6 (CH , C-25), 16.3 (CH , C-26), 17.9 (CH , C-6),
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C
3
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3
2
21.1 (CH , C-24), 23.4 (CH , C-11), 25.1 (CH , C-27), 25.7 (CH , C-16), 25.8 (CH , C-21),
3
2
3
2
2
27.9 (CH , C-29), 28.3 (CH , C-15), 29.3 (CH , C-23), 32.6 (CH , C-7), 36.9 (CH , C-22), 38.0
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2
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2
2
(C, C-10), 38.1 (C, C-4), 40.0 (C, C-8), 41.2 (C, C-14), 41.4 (CH, C-20), 41.6 (CH , C-1), 46.8
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(CH, C-9), 47.9 (C, C-17), 48.1 (CH, C-5), 53.6 (CH, C-18), 61.0 (CH, C-6'), 65.8 (CH, C-2),
69.7 (CH, C-4'), 72.5 (CH, C-2'), 72.3 (CH, C-19), 76.9 (CH, C-5'), 77.2 (CH, C-3'), 78.7 (CH,
C-3), 94.4 (CH, C-1'), 128.2 (CH, C-12), 138.3 (C, C-13), 177.1(C, C-28)
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Glucosyl tormentate (3). White amorphous powder; HR-ESI-MS: m/z calcd. for C H O
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–
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[M + HCOO] 695.4007 found 695.4014; H NMR (400 HMz, CD OD): δ 0.80 (3H, s), 0.83
3
H
(3H, s), 0.95 (3H, d, J = 6.0 Hz), 1.03 (3H, s), 1.22 (3H, s), 1.35 (3H, s), 5.33 (1H, s, H-12),
1
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5.34 (1H, d, J = 8.5, H-1 of glucose); C NMR (100 HMz, CD OD): δ 15.2 (CH , C-30), 15.7
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C
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(CH , C-26), 16.0 (CH , C-25), 16.2 (CH , C-24), 18.3 (CH2, C-6), 23.3 (CH2, C-11), 23.4
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(CH , C-27), 25.1 (CH , C-16), 25.7 (CH , C-21), 25.8 (CH , C-29), 27.9 (CH , C-15), 28.2
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2
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(CH , C-23), 32.7 (CH , C-7), 36.9 (CH , C-22), 37.8 (C, C-10), 39.1 (C, C-8), 39.9 (C, C-4),
3
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2
41.3 (CH, C-20), 41.6 (C, C-14), 46.8 (CH, C-9), 48.0 (CH , C-1), 48.2 (C, C-17), 53.6 (CH,
2
C-18), 55.3 (CH, C-5), 61.1 (CH, C-6'), 68.1 (CH , C-2), 69.7 (CH, C-4'), 72.2 (C, C-19), 72.5
2
(CH, C-2'), 76.9 (CH, C-5'), 77.2 (CH, C-3'), 83.2 (CH, C-3), 94.4 (CH, C-1'), 138.4 (CH, C-
12), 128.1 (C, C-13), 177.1 (C, C-28).
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2α,3α,19α-Trihydroxy-olean-12-en-28-oic acid-28-O-β-D-glucopyranoside (4). White
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amorphous powder; HR-ESI-MS: m/z calcd. for C H O [M + HCOO] 695.4007 found
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1
695.4019. H NMR (400 HMz, CD OD): δ 0.77 (s, 3H, H-24), 0.83 (s, 3H, H-30), 0.96 (d,
3
H
3H, H-25), 0.97 (d, 3H, H-29), 1.02 (s, 3H, H-26), 1.03 (s, 3H, H-23), 1.31 (s, 3H, H-27), 3.70,
(dd, 1H, J = 11.8, 4.2 Hz, H-6a), 3.84, (dd, 1H, J = 11.8, 4.2 Hz, H-6b), 3.95, (dt, 1H, J = 11.8,
1
3
3.5 Hz, H-2), 5.35 (t, 1H, J = 2.9 Hz, H-12), 5.40 (d, 1H, J = 8.6 Hz, H-1'); C NMR (400
HMz, CD OD): δ 15.6 (CH , C-25), 16.0 (CH , C-26), 16.4 (CH , C-6), 18.3 (CH , C-24),
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C
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23.5 (CH , C-11), 23.6 (CH , C-30), 23.8 (CH , C-27), 27.2 (CH , C-16), 27.9 (CH , C-29),
2
3
3
2
3
28.0 (CH , C-21), 28.0 (CH , C-15), 31.9 (CH , C-23), 32.4 (CH , C-7), 34.6 (CH , C-22), 38.0
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(C, C-20), 38.2 (C, C-9), 39.1 (C, C-10), 39.1 (C, C-4), 39.5 (C, C-8), 41.3 (C, C-14), 43.7
(CH , C-1), 45.7 (CH, C-18), 46.7 (C, C-17), 48.4 (CH, C-5), 61.0 (CH, C-6'), 68.1 (CH, C-2),
2
69.7 (CH, C-4'), 72.5 (CH, C-2'), 77.0 (CH, C-5'), 77.3 (CH, C-3'), 81.0 (CH, C-3), 83.2 (CH,
C-19), 94.4 (CH, C-1'), 123.4 (CH, C-12), 142.6 (C, C-13), 177.2 (C, C-28).
–
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1
1
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Camellianin A (5). White amorphous powder; HR-ESI-MS: m/z calcd. for C H O [M – H]
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31 15
1
619.1663 found 619.6154; H NMR (400 HMz, pyridine-d ): δ 1.66 (3H, d, J = 6.0 Hz, H-
5
H
6'''), 1.90 (3H, s, H-8''), 6.41 (1H, s, H-6), 6.73 (1H, s, H-3), 6.97 (1H, s, H-8), 7.18 (2H, d, J =
1
3
8.6 Hz, H-3' and H-5'), 7.80 (2H, d, J = 8.6 Hz, H-2' and H-6'); C NMR (100 HMz, CD OD):
3
δ 20.1 (CH , C-6'''), 21.8 (CH , C-8''), 65.1 (CH , C-6''), 71.4 (CH, C-5'''), 72.8 (CH, C-2'''),
C
3
3
2
73.8 (CH, C-4'''), 73.9 (CH, C-3'''), 75.9 (CH, C-2''), 76.3 (CH, C-5''), 78.7 (CH, C-3''), 80.3
(CH, C-4''), 98.8 (C, C-8), 100.6 (CH, C-1'''), 102.2 (CH, C-6), 102.6 (CH, C-1''), 108.2 (CH,
C-3), 109.8 (C, C-10), 118.0 (CH, C-3' and C-5'), 123.7 (C, C-1'), 129.8 (CH, C-2' and C-6'),
159.9 (C, C-5), 161.3 (C, C-9), 162.8 (C, C-7) , 164.0 (C, C-4'), 165.5 (C, C-2), 172.0 (C, C-
7''), 178.5 (C, C-4).
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1
1
1
1
1
1
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Verification of D-glucose isomer for compound 1. Compound 1 (2 mg) was heated with 2 M
HCl (water, 2 mL) at 80 ℃ for 2 h. After cooling, the reaction mixture was extracted with
CHCl (3 × 2 mL). The aqueous layer was then evaporated to dryness using MeOH until
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neutral. The dried residue was mixed with 90 μL of methoxyamine hydrochloride (20 mg/mL
in pyridine) and vortexed for 2 h at room temperature. Afterward, 240 μL of N-methyl-N-
(trimethylsily)trifluoracetamide were added to the sample, and the mixture was vortexed for an
additional 30 min.15
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GC-MS analysis of the derivatized sample was conducted using a Clarus 680 GC (Perkin-
Elmer, Inc., Waltham, MA) equipped with a Clarus SQ 8T MS in EI mode with 70 eV. The
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sample solution (1 μL) was injected into an Rxi-5 MS capillary column (30 m × 0.25 mm, 0.25
μm film thickness). The oven temperature was programmed at 70 °C for 5 min, rised to 270 °C
at a rate of 4 °C/min, and then increased to 320 °C (5 min hold) at a rate of 20 °C/min. The
flow rate of the carrier gas, helium, was 1.1 mL/min. The configuration of D-glucose for
compound 1 was established by comparing the retention time and molecular weight of the
corresponding derivative to that of the standard, D-glucose.15
1
1
1
1
1
1
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91
92
93
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Hydrolysis of compounds 1, 2 and 5 for structure-activity study. A solution of 1 (30 mg)
or 2 (30 mg) in 5 mL MeOH was added to 1 M HCl (5 mL) and stirred at 70 °C for 2 h. The
mixture solution was next added to 10 mL water and then extracted with ethyl acetate (3 × 30
mL). The organic layer was concentrated using a rotary evaporator and then purified using
flash chromatography with a gradient of CH Cl /acetone (9:1, 8:2, 6:4 v/v, each 50 mL) to
2
2
obtain the pure compounds 1s (20 mg) or 2s (21 mg).14
1
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2 M HCl (water, 3 mL) and compound 5 (30 mg) in MeOH (3 mL) were heated at 80 ℃
1
1
1
2
2
2
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02
for 2 h. After cooling, the reaction mixture was extracted with ethyl acetate (3 × 10 mL). The
organic layer was concentrated using a rotary evaporator, which then was purified using flash
chromatography with a gradient of CH Cl /acetone (8:2, 6:4:1 v/v, each 50 mL) to yield 5s (18
2
2
mg). The spectroscopic data of compounds 1s, 2s, and 5s are listed below. The compounds 1,
2, and 5 and their hydrolyzed compounds were applied to 3T3-L1 cell cultures to understand
the structure-activity relationship.
2
2
2
2
03
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05
06
2α, 3α-Dihydroxy-ursolic acid (1s). White amorphous powder; HR-ESI-MS: m/z calcd. for
–
1
C H O [M – H] 471.3474 found 471.3484; H NMR (400 HMz, CD OD): δ 0.86 (s, 3H,
3
0
47
4
3
H
H-26), 0.89 (s, 3H, H-24), 0.91 (br s, 3H, H-29), 1.00 (d, 3H, J = 6.8 Hz, H-30), 1.01 (s, 3H,
H-23), 1.03 (s, 3H, H-25), 1.16 (s, 3H, H-27), 2.23 (d, 1H, J = 11.0 Hz, H-18), 3.34 (m, 1H,
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H-3), 3.95 (dt, 1H, J = 11.8, 3.5 Hz, H-2), 5.26 (t, 1H, J = 3.6 Hz, H-12); C NMR (100 HMz,
CD OD): δ 15.6 (CH , C-25), 16.3 (CH , C-29), 16.4 (CH , C-26), 17.7 (CH , C-6), 20.2
3
C
3
3
3
2
(CH , C-30), 21.1 (CH , C-24), 22.8 (CH , C-27), 23.0 (CH , C-11), 23.9 (CH , C-16), 27.8
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2
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(CH , C-23), 27.9 (CH , C-15), 30.4 (CH , C-21), 32.8 (CH , C-7), 36.7 (CH , C-22), 38.0 (C,
3
2
2
2
2
C-10), 38.1 (C, C-4), 39.0 (CH, C-20), 39.0 (CH, C-19), 39.6 (C, C-8), 41.2 (CH , C-1), 42.0
2
(C, C-14), 47.2 (CH, C-9), 47.9 (CH, C-5), 48.2 (C, C-17), 52.9 (CH, C-18), 65.7 (CH, C-2),
78.7 (CH, C-3), 125.4 (CH, C-12), 138.3 (C, C-13), 180.2 (C, C-28).
–
2
2
2
2
2
2
2
2
2
2
14
15
16
17
18
19
20
21
22
23
Euscaphic acid (2s). HR-ESI-MS: m/z calcd. for C H O [M – H] 487.3423 found 487.3419.
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5
1
H NMR (400 HMz, CD OD): δ 0.81 (s, 3H, H-24), 0.89 (s, 3H, H-25), 0.95 (d, 3H, J = 5.2
3
H
Hz, H-30), 1.01 (s, 3H, H-26), 1.01 (s, 3H, H-23), 1.22 (s, 3H, H-29), 1.37 (s, 3H, H-27), 3.35,
1
3
(m, 1H, H-3), 3.95, (dt, 1H, J = 11.8, 3.5 Hz, H-2), 5.32 (t, 1H, J = 4.0 Hz, H-12); C NMR
(100 HMz, CD OD): δ 15.2 (CH , C-30),15.5 (CH , C-25), 16.1 (CH , C-26), 17.9 (CH , C-
3
C
3
3
3
2
6), 21.1 (CH , C-24), 23.3 (CH , C-11), 23.5 (CH , C-27), 25.2 (CH , C-16), 25.7 (CH , C-21),
3
2
3
2
2
25.9 (CH , C-29), 27.9 (CH , C-15), 28.2 (CH , C-23), 38.0 (C, C-10), 38.1 (C, C-4), 39.9 (C,
3
2
3
C-8), 32.7 (CH , C-7), 37.6 (CH , C-22), 41.1 (CH, C-20), 41.3 (CH , C-1), 41.7 (C, C-14),
2
2
2
46.8 (CH, C-9), 47.3 (C, C-17), 47.9 (CH, C-5), 53.7 (CH, C-18), 65.8 (CH, C-2), 72.2 (CH,
C-19), 78.7 (CH, C-3), 128.0 (CH, C-12), 138.7 (C, C-13), 180.9 (C, C-28).
–
2
2
2
2
2
2
24
25
26
27
28
29
Apigenin (5s). White amorphous powder; HR-ESI-MS: m/z calcd. for C H O [M – H]
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1
269.0450 found 269.0449; H NMR (400 HMz, pyridine-d ): δ 6.75 (1H, s, H-6), 6.84(1H, s,
5
H
H-3), 6.90 (1H, s, H-8), 7.23 (2H, d, J = 8.6 Hz, H-3' and H-5'), 7.90 (2H, d, J = 8.6 Hz, H-2'
1
3
and H-6'); C NMR (100 HMz, CD OD): δ 94.7 (C, C-8), 99.8 (CH, C-6), 103.7 (CH, C-3),
3
C
104.8 (C, C-10), 116.6 (CH, C-3' and C-5'), 122.0 (C, C-1'), 128.7 (CH, C-2' and C-6'), 158.3
(C, C-5), 162.5 (C, C-9), 162.9 (C, C-7) , 164.3 (C, C-4'), 165.7 (C, C-2), 182.5 (C, C-4).
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Cell culture and adipocyte differentiation. 3T3-L1 cells (American Type Culture Collection;
ATCC CL-193, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% bovine calf serum (BS), 100 units/mL penicillin, and 50 μg/mL
streptomycin in an incubator with 5% CO at 37 °C. For differentiation experiments, 3T3-L1
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preadipocytes were cultured in 24-well plates until confluent, and then maintained for two
additional days. Afterwards, differentiation was induced with a mixture of 1 μM
dexamethasone (Dex), 5 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), and the
sample in DMEM containing 10% fetal bovine serum (FBS). Two days later, medium was
replaced with fresh DMEM supplemented with 10% FBS and 5 μg/mL insulin for 2 days. Then,
the medium for the maturing adipocytes was replaced with 10% FBS in DMEM for another 2
days. Finally, cells were stained with Oil Red O. All samples were dissolved in dimethyl
sulfoxide (DMSO) before apply to preadipocytes. The final concentration of DMSO was less
than 0.2%.
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Oil Red O staining. Differentiated 3T3-L1 adipocytes were fixed with 10% formaldehyde in
phosphate-buffered saline (PBS) for 30 min at room temperature, and washed twice with PBS.
Fixed cells were stained with Oil Red O solution (0.3% Oil Red O in 60% isopropyl alcohol)
for 20 min, followed by two PBS rinses. Stained lipid droplets of the 3T3-L1 adipocytes were
photographed using an Olympus IX73 microscope (Tokyo, Japan) at 200 × magnification.
Finally, the dye retained in the cells was dissolved in isopropyl alcohol and quantified by
measuring the absorbance at 490 nm using a microplate reader (FlexStation 3™, Molecular
Devices, Sunnyvale, CA, USA).
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Cytotoxicity assay. The cytotoxic effects of samples on 3T3-L1 cells were estimated using an
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells
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were cultured in 96-well plates until the full confluence, and incubated with the presence or
absence of sample in differentiation medium for 4 days, and then exposed to 10% FBS in
DMEM for an additional day without the sample. The effective fractions and samples didn’t
show cytotoxicity. Then, the MTT was added into each well at a final concentration of 0.5
mg/mL and placed in an incubator for 4 hours. After removal of medium and MTT solution,
the formazan was dissolved using 200 μL DMSO and quantified by measuring the absorbance
at 570 nm by using a microplate reader.
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Statistical analysis. All results of bioactivity testing were expressed as the mean ± standard
deviation (SD) (n = 3). Statistical comparisons were determined by student T-test. A P-value
of < 0.05 was considered significant.
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RESULTS AND DISCUSSION
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Bioassay-guided Isolation of Potential Anti-adipogenic Compounds from Shiya tea. Each
fraction isolated from MeOH extracts of Shiya tea was screened for potential anti-adipogenic
effects (Figure 1). Only the active fractions were used for the further isolation. The ethyl acetate
layer was subjected to a silica gel column to obtain different fractions, and only Fr.3 was active.
Fractions 3B and 3B1, which were fractionated/sub-fractionated from Fr. 3, significantly
inhibited intracellular lipid accumulation at 100 μg/mL during 3T3-L1 adipocyte
differentiation without cytotoxicity. From Fr. 3B1, five compounds were isolated.
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Structure Elucidation of Compounds (1−5) from Shiya tea. Bioactivity-guided isolation of
MeOH extracts of Shiya tea led to the isolation of four triterpenoid saponins (1–4), including
one new compound (1), and a flavonoid (5) (Figure 2), which were elucidated using
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spectroscopic methods (1D, 2D NMR, and LC−Orbitrap−MS). Four known compounds,
1
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kajiichigoside F1 (2), glucosyl tormentate (3), 2α, 3α, 19α-trihydroxy-olean-12-en-28-oic
1
9
20
acid-28-O-beta-D-glucopyranoside (4), and camellianin A (5) were identified by comparing
their spectroscopic data with those reported in the literature.
2
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2α, 3α-Dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester (1), of which structure has
been not reported, was white amorphous powder with a molecular formula of C H O based
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9
–
on m/z 679.4064 [M + HCOO] (calcd. for C H O , 679.4057) with 8 degrees of unsaturation.
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1
The H NMR spectra (Table 1) revealed the presence of seven methyls [δ 0.85 (s), 0.88 (s),
H
0.92 (d, J = 6.2 Hz), 0.99 (d, J = 5.2 Hz), 1.00 (s), 1.03 (s), 1.15 (s)], and an olefinic proton
at δ 5.28 (d, J = 2.9 Hz). The 1D NMR data along with the HMQC spectra showed 36 carbon
H
signals, including a characteristic glucose, two hydroxyls, a double bond and a carbonyl. The
above-mentioned information indicated that 1 was an ursane-type triterpenoid saponin.
2
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2
2
2
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Further analysis of the 1D and 2D NMR data of 1 suggested a high similarity between 1
and glucosyl tormentate (3) except for the relative configuration of OH-3 and absence of a
1
7
1
1
hydroxyl at C-19. In the H- H COSY spectrum (Figure 3), two isolated spin systems were
observed as H -1/H-2/H-3 and Me-29/H-19/H-20/Me-30, which revealed the presence of two
2
fragments as indicated by the highlighted bold bonds in Figure 3. The fragments were further
confirmed from the HMBC correlations of Me-23 to C-3, C-4, and C-5, Me-29 to C-18, C-19,
and C-20, and Me-30 to C-19, C-20, and C-21 (Figure 3). The glucose was connected to the
carbonyl of C-28 by the key HMBC correlation from the anomeric proton of H-1' (δ 5.36, d,
H
J = 8.6 Hz) to C-28 (δ 176.6), and the coupling constant indicated that the glucopyranose was
C
β-oriented. D-glucose was obtained from acid hydrolysis of 1, which was determined by GC
1
4
analysis of corresponding derivatives. Thus, the planar structure of 1 was assigned.
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The relative configuration of 1 was assigned from the ROESY correlations and coupling
constant, in which cross-peaks of H-2/H-3, H-2/Me-25, H-3/Me-24, Me -25/ M-26, and Me-
29/H-20 implied that those groups were cofacial and randomly assigned as β-oriented (Figure
3). The coupling constant between H-2 and H-3 (J = 3.5 Hz) along with the chemical shift of
1
7,19
C-2 (δ 65.8) and C-3 (δ 78.8) further confirmed the β-orientation of H-2 and H-3.
In
C
C
addition, H-5, H-9, H-19, and Me-30 were α-oriented due to the ROESY correlations of H-
5/H-9, H-9/Me-27, and Me-30/H-19. Therefore, the relative configuration of 1 was constructed
as 2α, 3α-dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester.
3
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MS Fragmentation Patterns of Authentic Isolates (1–5). Four triterpenoid saponins had the
−
−
predominant [M − H] and [M + HCOO] precursor ions due to the presence of formic acid in
the mobile phase (Table 2). The cleavage of sugar moieties from the compound structures were
the characteristic fragmentation in all isolates (Table 2). Compounds (1–4) generated fragment
ions with the loss of glucose (m/z 162), and compound (5) formed a fragment ion with the loss
of glucose (m/z 162) and rhamnose (m/z 146). Compound 1, the new triterpenoid saponin,
–
2
without the hydroxyl at C-19 had only one fragment ion [M – Glu – H] in the MS spectra,
while others were found to generate more fragments, such as the loss of a carboxylic acid at C-
28 and H O. The LC−MS chromatogram of the isolates (1–5) in crude extract was shown in
2
Figure 4.
3
3
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3
3
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Potential Anti-adipogenic Effects of Purified Compounds and their hydrolyzed
compounds. All the isolates (1−5) were evaluated by measuring the inhibitory effect on lipid
accumulation in 3T3-L1 adipocytes (Figure 5). The intracellular lipid accumulation and lipid
droplets were significantly suppressed by 1 in a dose-dependent manner (Figure 6) with an IC50
value of 27.6 μg/mL without cytotoxicity. As for the ursane- and oleanane-type triterpenoids
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(1–4), the absence of the hydroxyl at C-19 was important for inhibitory effect on lipid
accumulation in 3T3-L1 adipocytes. Therefore compound 1, without a hydroxyl at C-19
revealed the most effective inhibition of 3T3-L1 adipocyte differentiation. In order to discuss
the effect of a sugar group on the bioactivity, three aglycones of 2α, 3α-dihydroxy-ursolic acid
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1
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(1s), euscaphic acid (2s) , and apigenin (5s) were obtained by hydrolysis of compounds 1,
2, and 5, respectively. When β-D-glucopyranose was removed by acid hydrolysis from
compound 1, the percentage of lipid accumulation increased from 47.6 % to 85.4% at 40 μg/mL.
Both aglycones of 1s and 2s showed no obvious anti-adipogenic activity indicating the effect
of the position of a hydroxyl was not conclusive in the absence of a sugar group. Ursolic acid,
which exhibited a better inhibitory effect of preadipocyte differentiation, possesses a similar
2
3
structure as compound 1s with the beta-hydroxyl at C-3 instead of alfa-hydroxyl. Thus, in
addition to glycoside, the numbers and the orientation of the hydroxyl groups play an important
role in the inhibitory effect of ursolic acid type tritepenoid on lipid accumulation. In addition,
ursane-type triterpenoids exhibited multiple biological functions, such as antioxidant,
antimicrobial, antiplatelet aggregation, and reducing serum total cholesterol activities.24-27
Apigenin (5s), an aglycone of compound 5, showed an enhanced inhibitory effect with an IC50
value of 34.6 μg/mL when compared to compound 5. This complements the literature in that
2
8
flavonoids without a sugar group provide stronger anti-adipogenic effects. However, this
result opposes that of triterpenoid saponin. Therefore, the triterpenoid saponin (1) and
flavonoid (5s) both showed anti-adipogenic potential for obesity prevention.
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In conclusion, bioactivity-guided isolation of Shiya tea led to the isolation and
identification of four triterpenoid saponins (1–4), including one new compound, and a
flavonoid (5) using NMR (1D and 2D NMR) and tandem liquid chromatography mass
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spectrometry (HPLC-ESI/MS ) techniques. A comprehensive profile of isolates in Shiya tea
was outlined by LC-MS. Compound 1 could reduce intracellular lipid accumulation in 3T3-L1
adipocytes in a dose-dependent manner with an IC value of 27.6 μg/mL. Previous studies
5
0
indicated that flavonoids in Shiya tea are considered the major active and functional chemical
constituents. However, this study found a new triterpenoid, saponin, to be the major anti-
adipogenic constituent in Shiya tea. Three hydrolytic compounds (1s, 2s, and 5s) were obtained
and screened for their effect on lipid accumulation inhibition. The inhibitory activity of the
triterpenoid (1s) without a sugar group decreased significantly, while the flavonoid (5s) without
a sugar showed great increased activity.
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ASSOCIATED CONTENT
Supporting Information
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The 1D, 2D NMR, HRESIMS of compound 1; Table of anti-adipogenic effects of the
fractions.
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AUTHORS INFORMATION
Corresponding Authors
*Telephone: +1-863-956-8673. E-mail: yu.wang@ufl.edu
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Author Contributions
⊥These authors contributed equally to this work.
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ORCID
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Yu Wang: 0000-0002-2003-270X
Notes
The authors declare no competing financial interest.
Acknowledgements
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We thank Laura Reuss (Citrus Research and Education Center, University of Florida) for
editing the manuscript.
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from defatted pepper (Capsicum annuum L.) seeds. J. Func. Foods 2015, 14, 670-675.
(17) Guang, Y. L.; Gray, A. I.; Waterman, P. G. Pentacyclic triterpenes from the fruits of Rosa
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(18) Lontsi, D.; Sondengam, B. L.; Ayafor, J. F.; Tsoupras, M. G.; Tabacchi, R. Further
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Yao Xue Xue Bao 2008, 43, 504-8.
(20) Liu, B.; Ma, Y.; Liu, Y.; Yang, Z.; Zhang, L. Ultrasonic-assisted extraction and
antioxidant activity of flavonoids from Adinandra nitida leaves. Trop. J. Pharm. Res. 2013,
12, 1045-1051.
(21) Bhandari, S. P. S.; Garg, H. S.; Agrawal, P. K.; Bhakuni, D. S. Ursane triterpenoids from
Nepeta eriostachia. Phytochemistry 1990, 29, 3956-8.
(22) Gong, S. S.; Liu, Y. L.; Li, Y.; Li, X.R.; Feng, Y. L.; Xu, Q. M.; Yang, S. L. Chemical
constituents from whole plant of Gynura procumbens (I). Zhongcaoyao 2016, 47, 1856-1860.
(23) He, Y.; Li, Y.; Zhao, T.; Wang, Y.; Sun, C. Ursolic acid inhibits adipogenesis in 3T3-L1
adipocytes through LKB1/AMPK Pathway. Plos One 2013, 8, e70135.
(24) Yang, H. M.; Yin, Z. Q.; Zhao, M. G.; Jiang, C. H.; Zhang, J.; Pan, K. Pentacyclic
triterpenoids from Cyclocarya paliurus and their antioxidant activities in FFA-induced HepG2
steatosis cells. Phytochemistry 2018, 151, 119-127.
(25) Bisoli, E.; Garcez, W. S.; Hamerski, L.; Tieppo, C.; Garcez, F. R. Bioactive pentacyclic
triterpenes from the stems of Combretum laxum. Molecules 2008, 13, 2717-2728.
(26) Yang, B.; Zhu, J. P.; Rong, L.; Jin, J.; Cao, D.; Li, H.; Zhou, X. H.; Zhao, Z. X.
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145, 179-186.
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Sun, Y. Pedunculoside, a novel triterpene saponin extracted from Ilex rotunda, ameliorates
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(28) Yang, Z. G.; Wen, X. F.; Li, Y. H.; Matsuzaki, K.; Kitanaka, S. Inhibitory Effects of the
Constituents of Hippophae rhamnoides on 3T3-L1 cell differentiation and nitric oxide
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production in RAW264.7 Cells. Chem. Pharm. Bull. 2013, 61, 279-285.
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Table and figure captions
1
13
Table 1. H and C NMR spectroscopic data of compound 1 in CD OD.
3
Table 2. MS precursor and fragment ions of the isolates (1−5) in Shiya tea.
4
4
4
4
4
4
48
49
50
51
52
53
Figure 1. Effects of the fractions of A. nitida on lipid accumulation during 3T3-L1 adipocyte
differentiation (quercetin was used as positive control. **p < 0.01 vs. control, *p < 0.05 vs.
control). Data are expressed as a mean ± standard deviation (n = 3). The cells were treated with
the extracts of ethyl acetate and water, the fractions of Fr.1–4 at 200 μg/mL, the fractions of
Fr.1–4, Fr.3A–3E, and Fr.3B.1–3B.4 at 100 μg/mL, and the fractions of Fr.2 and Fr.3A, and
quercetin at 50 μg/mL.
4
4
4
54
55
56
Figure 2. Chemical structures of 1−5.
Figure 3. Key 2D NMR correlations of 1
Figure 4. Total ion chromatogram (TIC) of the isolates (1–5) in Shiya tea extract.
4
4
4
57
58
59
Figure 5. Effect of compounds 1–5 on lipid accumulation during 3T3-L1 adipocytes
differentiation. (quercetin was used as positive control. **p < 0.01 vs. control) Data are
expressed as a mean ± standard deviation (n = 3).
4
4
4
4
60
61
62
63
Figure 6. Inhibitory effects of compound 1 and 5s on intracellular lipid accumulation and
lipid droplets during adipocytes differentiation. Quantification of intracellular lipid in Oil red
O stained adipocytes (A). Representative images of Oil red O staining after various amounts
of compounds were added (B). Data are expressed as a mean ± standard deviation (n = 3).
4
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For Table of Contents Only.
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Table 1. H and C NMR spectroscopic data of compound 1 in CD OD.
3
No.
1
δH (J in Hz)
α: 1.28, m
β:1.63, m
δC, type
41.3, CH2
2
3
4
5
3.94, dt (11.8, 3.5)
3.34, d (3.5)
65.8, CH
78.7, CH
38.1, C
47.9, CH
17.7, CH2
1.25, m
α: 1.46, m
β:1.39, m
α: 1.58, m
β:1.35, m
6
7
32.8, CH2
8
9
1
39.8, C
47.2, CH
37.9, C
1.72, t (8.4)
0
1
α: 1.98, m
β:1.99, m
5.28, t (2.9)
23.0, CH2
1
1
1
1
2
3
4
125.7, CH
137.8, C
42.0, C
α: 1.11, m
β:1.96, m
α: 1.78, m
β:2.10, td (12.5, 4.0)
27.9, CH2
1
1
5
6
23.8, CH2
1
1
1
2
7
8
9
0
48.1, C
2.26, d (11.0)
1.42, m
1.02, m
α: 1.53, m
β:1.37, m
α: 1.65, m
β:1.78, m
1.00, s
0.88, s
1.03, s
0.85, s
1.15, s
52.8, CH
39.1, CH
38.9, CH
30.3, CH2
2
2
1
2
36.1, CH2
2
2
2
2
2
2
2
3
1
2
3
4
5
3
4
5
6
7
8
9
0
'
'
'
'
'
27.9, CH3
21.1, CH3
15.7, CH3
16.5, CH3
22.7, CH3
176.6, C
16.2, CH3
20.1, CH3
93.4, CH
72.5, CH
77.2, CH
69.8, CH
76.9, CH
0.92, d (6.2)
0.99, d (5.2)
5.36, d (8.6)
3.34, m
3.35, m
3.38, m
3.42, m
2
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a:3.70, dd (11.8, 4.2)
b:3.82, dd (11.8, 2.0)
61.1, CH2
6
'
4
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Table 2. MS precursor and fragment ions of the isolate (1−5) in Shiya tea.
Com. [M – H] – [M + HCOOH – MS2
–
H]
–
1
2
633.3985 679.4064
649.3881 695.4014
633.3985/471.3471 [M – Glu – H]
–
649.3881/487.3424 [M – Glu – H] , 425.3475 [M – Glu
–
–
H]
CO – H O – H] , 407.3346 [M – Glu – CO – 2H O –
2 2 2 2
–
–
3
4
5
649.3881 695.4014
649.4072 695.4019
619.1654 Not detected
649.3881/487.3426 [M – Glu – H] , 469.3304 [M – Glu
–
–
H O – H]
2
–
649.4072/487.3427 [M – Glu – H] , 469.3325 [M – Glu
–
–
H O – H]
2
–
619.1654/601.1686 [M – H O – H] , 577.1569 [M – C
H CO – H] , 269.0456 [M – Glu – CH CO – Rha – H]
2
–
–
3
3
2
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Figure 1.
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R4
R5
OH
R3
2'
1'
4'
2
1
R2
HO
9 O
19
2
2
6'
13
17
7
2
11
25
26
O
4
3
1
H
R6
10
2
8
5
HO
R1
O
OR
O
1
5
0
15
3
H
27
7
5
R = -Rham-Glc-OCCH3
H
3
5
s R =
H
24
2
R1 R2 R3 R4 R5
R6
CH3 -D-Glucopyranosyl
CH3
1
-OH CH3
H
H
H
H

-OH CH3
H
1s

-OH CH3 OH H CH3 -D-Glucopyranosyl
2
2
3
4
s
-OH CH3 OH H CH3
-OH CH3 OH H CH3 -D-Glucopyranosyl
-OH H OH CH CH -D-Glucopyranosyl
H
3
3
4
4
71
72
Figure 2.
2
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30
20
2
30
1
27
18
20
29
3
25
22
OH
5
1
8
11
26
2
5
24
2'
1
3
23
O
28
OH
3'
1
23
14
1
O
'
HO
HO
16
8
O
5'
10
' OH
3
4
2
7
7
6
'
OH
23
24
4
4
73
74
Figure 3.
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1
5
2
4
4
4
76
77
Figure 4.
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Figure 5.
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A
B
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83
Figure 6.
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