Phytochemicals from the Leaves of Cyclocarya paliurus and their 11β-HSD1 Enzyme Inhibitory Effects

Article abstract of DOI:10.1002/cbdv.202000772

Two new dammarane-type triterpenoid saponins, 3β-(α-l-arabinopyranosyloxy)-24,25-dihydroxydammar-20-en-12α-yl 6-deoxy-β-d-glucopyranoside (1) and (24R)-3β-[(4-O-acetyl-α-l-arabinopyranosyl)oxy]-25-hydroxy-20,24-epoxydammaran-12β-yl 6-deoxy-β-d-glucopyrano

Full text of DOI:10.1002/cbdv.202000772

DOI: 10.1002/cbdv.202000772
FULL PAPER
Phytochemicals from the Leaves of Cyclocarya paliurus and their
11β-HSD1 Enzyme Inhibitory Effects
Huan Yan,a Xiang Li,a, b Wei Ni,a Qing Zhao,b Ying Leng,c and Hai-Yang Liu*a
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, and Yunnan Key Laboratory of
Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
P. R. China, e-mail: haiyangliu@mail.kib.ac.cn
Yunnan University of Chinese Medicine, Kunming 650500, P. R. China
b
c
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P. R. China
Two new dammarane-type triterpenoid saponins, 3β-(α-L-arabinopyranosyloxy)-24,25-dihydroxydammar-20-
en-12α-yl 6-deoxy-β-D-glucopyranoside (1) and (24R)-3β-[(4-O-acetyl-α-L-arabinopyranosyl)oxy]-25-hydroxy-
20,24-epoxydammaran-12β-yl 6-deoxy-β-D-glucopyranoside (2), and fourteen known triterpenoids were isolated
from the 70% MeOH extract of the leaves of Cyclocarya paliurus. Their structures were established based on
analyses of spectroscopic data. All compounds were tested for their inhibitory activities against the 11β-HSD1
enzyme. Hederagenin (13) exhibited moderate inhibitory effect for mouse 11β-HSD1 with an IC50 value of 0.16�
0.04 μM.
Keywords: Cyclocarya paliurus, dammarane-type triterpenoid saponins, 11β-HSD1 enzyme, inhibitory activity.
food resource for maritime people.[8] Furthermore, the
leaves of C. paliurus is an essential ingredient of Tujia
Introduction
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) ethnomedicine in P. R. China, in light of treating
is the enzyme primarily responsible for the regulation diabetes mellitus.[9,10] Previous research shows that C.
of intracellular cortisol levels. Inhibition of 11β-HSD1 is paliurus ethanol extract displayed antihyperglycemic
a new strategy for the treatment of metabolic disorder and improved insulin resistance bioactivities.[11] Be-
diseases, such as type 2 diabetes.[1,2] In modern sides, structurally diverse secondary metabolites, such
medicine, a number of synthetic 11β-HSD1 inhibitors as seco-dammarane triterpenoids, lignans, flavonoids,
have been reported,[3] however, natural products are and phenolic compounds, have been isolated from C.
rarely.[4,5] Herein, we report the isolation and structure paliurus so far.[12] These results inspired our great
determination of two new compounds from Cyclo- interest in exploring the bioactive phytochemicals of
carya paliurus and their inhibitory activities against this plant. As a result, two dammarane-type triterpe-
11β-HSD1.
noid saponins, named 3β-(α-L-arabinopyranosyloxy)-
Cyclocarya paliurus (Batalin) Iljinsk., a Chinese 24,25-dihydroxydammar-20-en-12α-yl 6-deoxy-β-D-glu-
endemic plant, belongs to the family Juglandaceae.[6,7] copyranoside (1) and (24R)-3β-[(4-O-acetyl-α-L-arabino-
Because of the sweet taste of its leaves, this species pyranosyl)oxy]-25-hydroxy-20,24-epoxydammaran-
have long been used as the herbal tea known as 12β-yl 6-deoxy-β-D-glucopyranoside (2), along with 14
‘sweet tea tree’, In addition, C. paliurus can serve as a known triterpenoids (Figure 1) were obtained. The
dietary supplement for trace elements and a good known compounds were identified by detailed com-
parison of their NMR and MS date with those reported
in previous literatures as (20S,24R)-epoxydammarane-
3β,12β,25-trihydroxy-12-O-β-D-quinvopyranosyl-3-O-α-
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202000772
L-arabinpyranoside (3),[13] cyclocarioside (4),[14]
I
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Chem. Biodiversity 2021, 18, e2000772
Figure 1. Chemical structures of 1–16 isolated from C. paliurus.
(20S,24R)-20,24-epoxy-25-hydroxy-12β-(β-D-quinovo-
pyranosyloxy)-3,4-seco-dammar-4(28)-en-3-oic
counmaroyloxy-2α,3β-dihydroxyplen-12-en-28-acid
acid (16),[26] respectively. In conclusion, This article reports
methyl ester (5),[15] cyclocarioside III (6),[16] cyclocario- the isolation, structural elucidation, and 11β-HSD1
side F (7),[17] cyclocarioside J (8),[18] cyclocariol D (9),[19] enzyme activities of these triterpenoids.
2α,3β,23-trihydroxylurs-12,20(30)-dien-28-oic acid-β-D-
glucopyranoside (10),[20] actinidia acid (11),[21] 2α-
hydroxyursolic acid (12),[22] hederagenin (13),[23] mas-
linic acid (14),[24] chebuloside II (15),[25] and 23-trans-p-
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Chem. Biodiversity 2021, 18, e2000772
correlations (Figure 2) revealed six structural fragments
by correlations of H2-1/H2-2/H-3, H-5/H2-6/H2-7, H-9/
Results and Discussion
Compound 1, white amorphous powder, had the H2-11/H-12/H-13/H-17, H2-16/H2-15/H-17, H2-22/H2-23/
molecular formula of C41H70O12 as established by HR- H-24, H-1’/H-2’/H-3’/H-4’/H-5’ and H-1’’/H-2’’/H-3’’/H-
EI-MS data (m/z 754.4867 [M]+), indicating seven 4’’/H-5’’/H3-6’’. Besides, ten key HMBCs signals, H3-19
1
unsaturated degrees. The H-NMR spectrum (Table 1) (δH 1.39) with C-1/C-5/C-9/C-10, H3-18 (δH 1.04) with C-
of 1 exhibited eight methyl singlets at δH 1.58 (3H, s, 7/C-8/C-9/C-14, H3-30 (δH 0.60) with C-8/C-14/C-15, H2-
CH3-6’’), 1.52 (3H, s, CH3-27), 1.48 (3H, s, CH3-26), 1.39 21 (δH 4.91 and 4.90) with C-17/C-20/C-22, H3-26 (δH
(3H, s, CH3-19), 1.25 (3H, s, CH3-28), 1.04 (3H, s, CH3- 1.48) with C-24/C-25, H3-27 (δH 1.52) with C-24/C-25,
18), 0.98 (3H, s, CH3-29) and 0.60 (3H, s, CH3-30); one H3-28 (δH 1.25) with C-4/C-5,and H3-29 (δH 0.98) with
pair of terminal olefinic protons at δH 4.91 (1H, br. s, H- C-3/C-4, a anomeric proton H-1’ (δH 4.72) with C-3, and
21) and 4.90 (1H, br. s, H-21); and partially overlapped another anomeric proton at H-1’’ (δH 4.87) with C-12,
multiples of methylene and methine protons were were observed. Overall, the above spectral data
observed between δH 0.94 and 2.70. Combined with indicated that compound 1 was a dammarane-type
two anomeric protons at δH 4.88 (d, J=11.6, H-1’’) and triterpenoid saponin similar to cyclocarioside Q,[27] and
4.72 (d, J=6.4, H-1’), with other oxygen-bearing their major differences are located at the side chains.
methine and one oxymethylene protons of the sugar The terminal double bond at C-20 and C-21 was
units, these spectroscopic data suggested that 1 was a elucidated by HMBCs of H2-21 (δH 4.91 and 4.90) with
glycoside with two glycosyl moieties. Acid hydrolysis C-20/C-17/C-22 (Figure 2). The position of two hydroxy
of 1, one set of carbon signals for an L-arabinopyr- groups were supported by the key HBMC correlations
anose and another set of carbon signals for a D- of H3-26 (δH 1.48) with C-24/C-25. Thus, the planar
quinovopyranose were identified by comparison on structure of 1 was elucidated as shown in Figure 2.
HPLC with authentic sample. The 13C-NMR and DEPT
The relative orientation of H-3 and H-12 were
spectra of 1 displayed 41 carbon signals, 11 of the 41 established by the ROESY experiment (Figure 3), in
carbons were assigned to the glycosyl groups, and which correlations of H-3/H-5α, H-12/H3-18/H3-19/H-
other 30 carbons to aglycone which have seven 13β revealed that H-3 and H-12 shared α- and β-
methyl groups, nine methylene groups (one olefinic), orientation, respectively. Based on the aforementioned
seven methine groups (three oxygenated), and six data, the structure of 1 was deduced as 3β-(α-L-
quaternary carbons (one olefinic). The 1H,1H-COSY
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of 1 and 2 in (D5)pyridine (δ in ppm, J in Hz).
No.
1
2
No.
1
2
δH
δC
35.6 (t)
δH
δC
δH
δC
32.1 (t)
δH
δC
34.2 (t)
1
2
3.09–3.12 (m)
2.01–2.08 (m)
1.88–1.92 (m)
1.48–1.54 (m)
3.58–3.62(m)
3.13–3.17 (m)
2.09–2.15 (m)
1.73–1.77 (m)
1.54–1.58 (m)
3.57–3.60 (m)
35.6 (t)
22
23
2.64–2.70 (m)
2.24–2.31 (m)
2.04 (overlap)
1.79–1.85 (m)
3.75–3.78 (m)
1.73 – 1.78 (m)
1.55 – 1.60 (m)
2.05 (overlap)
1.94 (overlap)
3.94–3.97 (m)
30.0 (t)
26.8 (t)
31.3 (t)
26.4 (t)
3
4
5
6
81.4 (d)
38.1 (s)
51.1 (d)
18.3 (d)
81.6 (d)
38.0 (s)
51.0 (d)
18.4 (t)
24
25
26
27
28
29
30
78.5 (d)
72.7 (s)
26.0 (q)
26.1 (q)
30.0 (q)
23.2 (q)
16.1 (q)
84.3 (d)
71.3 (s)
26.0 (q)
27.7 (q)
23.2 (q)
30.0 (q)
16.8 (q)
1.57 (overlap)
1.51–1.53 (m)
1.42–1.45 (m)
1.44–1.51 (m)
1.12 (d, J=11.6)
1.61 (overlap)
1.50–1.53 (m)
1.46–1.47 (m)
1.50 (overlap)
1.19–1.20 (m)
1.48 (s)
1.52 (s)
1.25 (s)
0.98 (s)
0.60 (s)
1.45 (s)
1.41 (s)
1.00 (s)
1.27 (s)
0.60 (s)
7
36.6 (t)
36.5 (d)
8
9
10
11
41.6 (s)
54.0 (d)
40.0 (s)
32.1 (t)
41.6 (s)
53.9 (d)
40.0 (s)
34.6 (t)
Ara(p)
1’
1.79–1.86 (m)
1.87 (overlap)
4.72 (d, J=6.4)
4.35–4.40 (m)
4.20 (dd, J=3.2, 12)
4.33–4.35 (m)
4.29 (dd, J=3.2, 8.0)
3.76 (overlap)
101.9 (d)
72.5 (d)
74.7 (d)
69.2 (d)
66.4 (t)
4.72 (d, J=7.6)
4.24–4.26 (m)
4.01 (overlap)
5.36–5.42 (m)
4.30–4.33 (m)
3.52 (t, J=10.6)
102.4 (d)
75.1 (d)
75.1 (d)
73.4 (d)
63.4 (t)
2’
2.50–2.53 (m)
1.40 (overlap)
4.38–4.43 (m)
1.93 (overlap)
2.87–2.90 (m)
1.48–1.50 (m)
4.36–4.43 (m)
1.80–1.84 (m)
3’
4’
12
13
14
15
76.8 (d)
44.1 (d)
49.6 (s)
31.3 (t)
77.5 (d)
41.3 (d)
50.1 (s)
31.6 (t)
5’
COOMe
COOMe
Qui
1’’
170.8 (s)
21.0 (q)
1.43–1.48 (m)
0.98 (overlap)
1.82–1.87 (m)
1.80–1.87 (m)
2.23–2.28 (m)
1.04 (s)
1.34–1.37 (m)
0.95–0.98 (m)
2.08–2.12 (m)
1.90–1.93 (m)
1.86–1.91 (m)
1.09 (s)
1.97 (s)
16
21.9 (t)
22.1 (t)
4.88 (d, J=11.6)
3.92 (t, J=8.4)
4.06 (t, J=8.4)
3.64 (overlap)
3.64 (overlap)
1.57 (d, J=4.7)
101.8 (d)
75.6 (d)
78.3 (d)
76.9 (d)
72.7 (d)
18.6 (q)
5.02 (d, J=7.6)
3.94–3.98 (m)
4.17 (t, J=9.0)
3.68 (t, J=9.0)
3.80–3.86 (m)
1.61 (d, J=6.0)
101.9 (d)
75.6 (d)
78.5 (d)
76.9 (d)
72.9 (d)
18.7 (q)
2’’
17
18
19
20
21
48.3 (d)
17.2 (q)
16.8 (q)
153.2 (s)
108.3 (d)
49.2 (d)
17.1 (q)
16.8 (q)
86.2 (s)
24.5 (q)
3’’
4’’
1.39 (s)
1.41 (s)
5’’
6’’
4.91 (br. s)
4.90 (br. s)
1.17 (s)
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Figure 2. The 1H,1H-COSY and key HMBC data of 1 and 2.
ever, the results showed that only 13 had moderate
bioactivity for mouse 11β-HSD1 with an IC50 value of
0.16�0.04 μM, compared with positive control glycyr-
rhizinic acid (6.60�1.49 nM).
Conclusions
The phytochemical study of C. paliurus let to the
isolation of two new dammarane-type triterpenoid
saponins 1 and 2, along with fourteen known
triterpenoids including seven dammarane-type triter-
penoids 3-9, three ursane-type triterpenoids 10-12,
and four oleanane-type triterpenoid lactones 13-16.
Figure 3. Key ROESY correlations for the aglycone moiety of 1.
arabinopyranosyloxy)-24,25-dihydroxydammar-20-en-
12α-yl 6-deoxy-β-D-glucopyranoside.
Compound 2 was obtained as colorless flake The isolates were tested for their inhibitory activities
crystals. Its molecular formula, C43H72O13, was con- against the 11β-HSD1 enzyme. Compound 13 exhib-
firmed by the HR-EI-MS peak at m/z 796.4978 [M]+ ited moderate inhibitory effect for mouse 11β-HSD1
(calc. for C43H72O13, 796.4973). Detailed inspection of with an IC50 value of 0.16�0.04 μM.
1
the H-NMR and 13C-NMR data (Table 1) of 2 disclosed
that it was also a dammarane-type triterpenoid
derivative with two sugar moieties. Comparison the Experimental Section
NMR data of 2 with the data of (24R)-3β-(α-L-
arabinopyranosyloxy)-25-hydroxy-20,24-epoxydam-
General
maran-12β-yl 6-deoxy-β-D-glucopyranoside[12] indi- Optical rotations were measured with a JASCO P-1020
cated that they shared the same aglycone, except for digital polarimeter. IR Spectra were recorded on a
the occurrence of additional signals for an acetyl Bruker-Tensor-27 infrared spectrophotometer with KBr
group. The acetyl group was assigned to be placed at pellets. NMR Spectra were obtained on DRX-500 and
C-4’ of the arabinopyranosyl unit by the observed AM-400 instruments with TMS as internal standard.
correlations of H-4’ (δH 5.39) with C=O/C-3’/C-5’ in the ESI-MS and HR-ESI-MS spectra were measured by
HMBC spectrum (Figure 2). Thus, the structure of 2 was Bruker HTC/Esquire and API-Qstar-Pulsar spectrome-
elucidated to be (24R)-3β-[(4-O-acetyl-α-L-arabinopyr- ters, respectively. Semi-preparative HPLC was carried
anosyl)oxy]-25-hydroxy-20,24-epoxydammaran-12β-yl
6-deoxy-β-D-glucopyranoside.
out by an Agilent 1100 apparatus with a Zorbax SB-C18
column (9.4 mm×25 cm, 5 μm, Agilent) under the
Compounds 1-16 were evaluated for their inhib- detect guidance of 203 nm. Column chromatography
itory activity on murine and human 11β-HSD1. How- (CC) was performed on silica gel (SiO2, 200–300 mesh,
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Qingdao Marine Chemical Inc., China) or SiO2 H (10– silica gel column (200–300 mesh, AcOEt/MeOH, 7:1 to
40 μm, Qingdao Marine Chemical Inc., China), MCI-gel 1:1, v/v) and purified by semi-preparative HPLC
CHP20P (75–150 μm, Mitsubishi Chemical Co., Japan), (MeCN/H2O, 40:60 to 80:20, v/v, flow rate: 3.0 mL/
RP-18 (50 μm, Merck, Germany), and Sephadex LH-20 min) to give 1 (4 mg, tR =6.26 min), 10 (6 mg, tR =
(GE Healthcare, USA). Fractions were monitored by TLC 12.1 min), and 15 (30 mg, tR =10.2 min).
and spots were detected with a UV254 lamp followed
by heating SiO2 plates sprayed with 10% H2SO4 in
EtOH.
3β-(α-L-Arabinopyranosyloxy)-24,25-dihydroxy-
dammar-20-en-12α-yl
6-Deoxy-β-D-glucopyrano-
side (1). White amorphous powder. [α]D22 =À 30.2 (c=
0.1, CH3OH). IR (KBr): νmax 3440, 2935, 1633, 1066 cmÀ 1.
1H-NMR (500 MHz, (D5)pyridine) and 13C-NMR
Plant Material
The leaves of C. paliurus collected in Xinning County, (125 MHz, (D5)pyridine): see Table 1. HR-EI-MS: m/z
Hunan Province, P. R. China, in August 2013. The 754.4882 ([M]+, calc. for C41H70O12, m/z 754.4867).
materials were identified by Dr. Yun-Heng Ji from the
Kunming Institute of Botany. A voucher specimen (No.
(24R)-3β-[(4-O-Acetyl-α-L-arabinopyranosyl)oxy]-
HY201307) has been deposited at the Sate Key 25-hydroxy-20,24-epoxydammaran-12β-yl
6-De-
Laboratory of Phytochemistry and Plant Resources in oxy-β-D-glucopyranoside (2). White and flake crystals.
West China, Kunming Institute of Botany, Chinese [α]D22 =À 20.0 (c=0.12, CH3OH). IR (KBr): νmax 3429,
Academy of Sciences, Kunming, China.
2937, 1638, 1066 cmÀ 1. 1H-NMR (500 MHz, (D5)
pyridine) and 13C-NMR (125 MHz, (D5)pyridine): see
Table 1. HR-EI-MS: m/z 796.4978 ([M]+, calc. for
C43H72O13, m/z 796.4973).
Extraction and Isolation
The air-dried and powdered leaves of C. paliurus
(5.0 kg) were extracted three times with 70% ethanol
under reflux. The filtrate was concentrated under
vacuum to give a residue, which was suspended in
Acid Hydrolysis of Compounds 1–2 and Determination
of the Absolute Configuration of the Sugars by HPLC
H2O and extracted with ethyl acetate (AcOEt) for three Compounds 1 (1.0 mg) and 2 (2.0 mg) in 4 M
times. The AcOEt fraction was submitted to an MCI-gel CF3COOH (1,4-dioxane/H2O 1:1, 2.0 mL) were heated
°
CC eluted with MeOH/H2O (1:1 to 1:0, v/v) to yield six at 99 C for 3 h, respectively. The reaction mixture was
fractions A-H. Fr. C (190 g) was subjected to a silica gel diluted with H2O (1 mL) and then, extracted with
(200–300 mesh) column eluted with CH3Cl/MeOH CHCl3 (3×2 mL). Next, each aqueous layer was
(30:1 to 1:7, v/v) to afford six fractions (Frs. C1–C6). evaporated to dryness using rotary evaporation. Each
Frs. C2 (3.0 g) and C3 (5.0 g) were separately applied dried residue was dissolved in pyridine (1.0 mL) mixed
to Rp-C18 gel (MeOH/H2O, 40:60 to 90:10, v/v), with L-cysteine methyl ester hydrochloride (1.0 mg)
°
Sephadex LH-20 (MeOH), silica gel column (200– (Aldrich, Japan) and heated at 60 C for 1 h. Then, O-
300 mesh, petroleum ether/acetone, 1:1 to 1:2, v/v; tolyl isothiocyanate (5.0 μL) (Tokyo Chemical Industry
CHCl3/MeOH, 60:1 to 0:1, v/v), and semi-preparative Co., Ltd., Japan) was added to each mixture, this being
°
HPLC (MeCN/H2O, 30:70 to 65:35, v/v, flow rate: heated at 60 C for 1 h. Each reaction mixture was
3.0 mL/min), to give 11 (10 mg, tR =12.0 min), 12 directly analyzed by reversed-phase HPLC following
(6 mg, tR =17.1 min), 13 (10 mg, tR =19.0 min) and 14 the above procedure. The reaction mixture was
(20 mg, tR =17.9 min) from Fr. C2 as well as 9 (50 mg, directly analyzed by analytical HPLC on a Poroshell
tR =18.9 min) and 16 (4 mg, tR =20.1 min) from Fr. C3. 120 SB-C18 column (100×4.6 mm, 2.7 μm, Agilent)
Fr. C4 (15 g) was purified successively by an Rp-C18 gel using an elution of CH3CN/H2O (20:75!40:60, v/v) at
CC (MeOH/H2O, 40:60 to 90:10, v/v) and a Sephadex a flow rate of 0.6 mL/min. Under UV detection at
LH-20 (MeOH) column to give 2 (1.5 g), 5 (200 mg), 254 nm, retention time for the standard monosacchar-
and 6 (1.5 g). Likewise, Fr. C5 (26 g) was submitted to ides:
L-arabinose
(13.079 min),
D-quinovose
diverse CC (Rp-C18 gel, MeOH/H2O, 45:55 to 90:10, v/ (15.748 min) was used for comparison with retention
v; silica gel, 200–300 mesh, CHCl3/MeOH, 25:1 to 0:1, times from reaction mixtures for the saponin. The
v/v; Sephadex LH-20, MeOH) to give 3 (1 g), 4 absolute configurations of the sugars for 1 and 2 were
(100 mg), 7 (100 mg), and 8 (15 mg). Fr. C6 was identified as L-arabinose (13.077 min) and D-quinovose
submitted to Sephadex LH-20 (MeOH) to provide three (15.752 min).
fractions: Frs. C6.1–C6.3. Fr. C6.1 was separated by a
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The inhibitory activities of the isolated compounds on
human or mouse 11β-HSD1 was evaluated using the
scintillation proximity assay (SPA). The full-length
cDNAs of human or murine11β-HSD1 was isolated
from the cDNA libraries provided by NIH Mammalian
Gene Collection. Then, the cDNAs were cloned into
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11β-HSD1 assay. The product, [3H] cortisol, was
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a monoclonal antibody
coupled to protein A-coated SPA beads. All tests were
performed for three independent replicates with
glycyrrhizinic acid as a positive control. The %
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Acknowledgments
This work was financially supported by the National
Natural Science Foundation of China (U1802287) and
(32000280), the Ten Thousand Talents Plan of Yunnan
Province for Industrial Technology Leading Talents, the
Major Biomedical Project of Yunnan Province
(2019ZF010), and the State Key Laboratory of Phyto-
chemistry and Plant Resources in West China (P2019-
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manuscript. Y. Leng carried out the bioassays. H.-Y. Liu
designed and supervised this research.
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Received September 17, 2020
Accepted November 18, 2020
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