New Aromatic Compounds from the Fruiting Body of Sparassis crispa (Wulf.) and Their Inhibitory Activities on Proprotein Convertase Subtilisin/Kexin Type 9 mRNA Expression

Article abstract of DOI:10.1021/acs.jafc.7b02657

Successive chromatography of EtOAc-soluble extracts of the fruiting body of Sparassis crispa (Wulf.) resulted in isolation of four new aromatic compounds, sparoside A (1) and sparalides A-C (3-5), two new naturally occurring compounds, 2 and 6, and eight known compounds, 7-14. The chemical structures were determined by interpretation of nuclear magnetic resonance and mass spectrometry spectroscopic data. Extract, solvent-soluble fractions of the extract, and all of the pure compounds isolated from the fractions were subjected to the mRNA expression assay for proprotein convertase subtilisin/kexin type 9 (PCSK9). Among them, sparoside A (1), hanabiratakelide A (8), adenosine (11), and 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3β,7β-diol (14) exhibited potent inhibitory activities on PCSK9 mRNA expression, with IC₅₀ values of 20.07, 7.18, 18.46, and 8.23 μM, respectively (berberine, positive control, IC₅₀ = 8.04 μM), suggesting that compounds 1, 8, 11, and 14 are suitable for use in supplements to the statins for hyperlipidemia treatments.

Full text of DOI:10.1021/acs.jafc.7b02657

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Article
New Aromatic Compounds from the Fruiting Body of Sparassis crispa
Wulf.) and Their Inhibitory Activities on PCSK9 mRNA Expression
(
Sunghee Bang, Hee-Sung Chae, Changyeol Lee, Hyun Gyu Choi, JiYoung Ryu,
Wei Li, Hanna Lee, Gil-Saeng Jeong, Young-Won Chin, and Sang Hee Shim
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02657 • Publication Date (Web): 09 Jul 2017
Downloaded from http://pubs.acs.org on July 9, 2017
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Page 1 of 26
Journal of Agricultural and Food Chemistry
New Aromatic Compounds from the Fruiting Body of Sparassis crispa (Wulf.)
and Their Inhibitory Activities on PCSK9 mRNA Expression
†
‡
†
†
†
§
Sunghee Bang, Hee-Sung Chae, Changyeol Lee, Hyun Gyu Choi, JiYoung Ryu, Wei Li,
#
┴
‡
†,*
Hanna Lee, Gil-Saeng Jeong, Young-Won Chin, and Sang Hee Shim
OH
O
NH2
N
H CO
OH
3
N
N
OCH3
CH3
O
O
N
O
H CO
O
3
OH
OH
O
OH
OH
OH
11
8
OH
1
PCSK9 mRNA expression
IC (μΜ)
50
1
8
20.07
7.18
18.46
8.23
8.04
H
1
1
HO
OH
O
14
berberine
1
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New Aromatic Compounds from the Fruiting Body of Sparassis crispa (Wulf.)
and Their Inhibitory Activities on PCSK9 mRNA Expression
3
†
‡
†
†
†
§
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5
Sunghee Bang, HeeꢀSung Chae, Changyeol Lee, Hyun Gyu Choi, Jiyoung Ryu, Wei Li,
#
┴
‡
†,*
Hanna Lee, GilꢀSaeng Jeong, YoungꢀWon Chin, and Sang Hee Shim
†
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8
College of Pharmacy, Duksung Women’s University, Samyangꢀro, Dobongꢀgu, Seoul 01369,
Republic of Korea; Eꢀmails: scbsh4331@hanmail.net (SH. Bang), jaber29@naver.com (CY.
Lee), chgꢀꢀꢀ@hanmail.net (H.G. Choi), ryuji@hanmail.net (J. Ryu).
‡
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0
1
College of Pharmacy and Integrated Research Institute for Drug Development, Dongguk
1
1
UniversityꢀSeoul, 32, Donggukꢀlo, Goyangꢀsi, Gyeonggiꢀdo 10326, Republic of Korea; Eꢀ
mails: chaeheesung83@gmail.com (H.ꢀS. Chae), f2744@dongguk.edu (Y.ꢀW. Chin).
§
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1
1
1
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KM Application Center, Korea Institute of Oriental Medicine, Daegu, Republic of Korea;
Eꢀmail: liwei1986@kiom.re.kr (W. Li).
#
National Development Institute of Korean Medicine, Gyeongsan 38540, Republic of Korea;
Eꢀmail: iiihanna@nikom.or.kr (HN. Lee).
┴
1
1
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College of Pharmacy, Keimyung University, Daegu 42601, Republic of Korea; Eꢀmail:
gsjeong@kmu.ac.kr (G.ꢀS. Jeong).
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* Corresponding author (Tel: +82 2 901 8774; Fax: +82 2 901 8386: Eꢀmail:
sangheeshim@duksung.ac.kr)
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ABSTRACT
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Successive chromatography of EtOAcꢀsoluble extracts of the fruiting body of Sparassis
crispa (Wulf.) resulted in isolation of four new aromatic compounds, sparoside A, 1, and
sparalides AꢀC, 3ꢀ5, two new naturallyꢀoccurring compounds, 2 and 6, in addition to eight
known compounds, 7ꢀ14. The chemical structures were determined by interpretation of NMR
and MS spectroscopic data. Extract, solventsꢀsoluble fractions of the extract, and all the pure
compounds isolated from the fractions were subjected to mRNA expression assay for
proprotein convertase subtilisin/kexin type 9 (PCSK9). Among them, sparoside A, 1,
hanabiratakelide A, 8, adenosine, 11, and 5α,6αꢀepoxyꢀ(22E,24R)ꢀergostaꢀ8(14),22ꢀdieneꢀ
3β,7βꢀdiol, 14, exhibited potent inhibitory activities on PCSK9 mRNA expression with IC50
values of 20.07, 7.18, 18.46 and 8.23 ꢁM, respectively (berberine, positive control, IC50
=
8.04 ꢁM), suggesting that compounds 1, 8, 11, and 14 are suitable for use in supplements to
the statins for hyperlipidemia treatments.
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KEYWORDS: Sparassis crispa, proprotein convertase subtilisin/kexin type 9 (PCSK9),
phthalide, sparoside A.
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INTRODUCTION
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Sparassis crispa (Wulf.) is an edible/medicinal mushroom belonging to the family of
Sparassidaceae and is called "cauliflower mushroom" due to its appearance. The mushroom,
which mostly grows on the stubs of coniferous trees, is distributed throughout northern
1
temperature zones of the world. The fruiting bodies of S. crispa produce various bioactive
2
3
4
substances, including βꢀglucan, benzoate derivatives, sesquiterpenoids, and maleic acid
5
derivatives. In particular, βꢀglucan is a major constituent, present in more than 40% of S.
crispa. They have been demonstrated to show a variety of pharmacological activities,
6
ꢀ8
9
10
including antitumor, hematopoietic responseꢀenhancing, wound healing, antimetastatic,
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antihypertensive, and antidiabetic effects.
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Proprotein convertase subtilisi/kexin type 9 (PCSK9) is noted to interfere with the
function of lowꢀdensity lipoprotein receptor (LDLR) on the liver cell surface that transport
LDLꢀcholesterol (LDLꢀC) into the liver for metabolism, leading to high levels of LDLꢀC.
Thus, PCSK9 inhibitor was proposed to be a new LDLꢀC lowering agent. When PCSK9
inhibitor was used in combination with a statin, known as a HMGꢀCoA reductase inhibitor, it
has been shown to dramatically lower LDLꢀC levels by up to 60%. Therefore, PCSK9
inhibitor has recently emerged as a new strategy to treat hyperlipidemia. To date two PCSK9
inhibitors (evolocumab and alirocumab) have been approved by FDA to treat familial
12
hypercholesterolemia and several PCSK9 inhibitors are currently under clinical trial. In
addition, several natural compounds such as berberine and curcumin have been reported to
13
inhibit PCSK9 mRNA expression. For these reasons, more investigation is required to
discover new PCSK9 inhibitors, which could be good supplements to statin treatment due to
their effects on PCSK9 mRNA.
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The extracts of the cauliflower mushroom were reported to exhibit antihypertensive and
antidiabetic activities related to hyperlipidemia. To our knowledge there are no reports on
effects of cauliflower mushroom and its constituents on PCSK9 mRNA expression. We
therefore examined the effects of the extracts and its pure compounds on PCSK9 mRNA
expression to see if their hypocholesterolemic effects could be partly explained by additional
effects on the PCSK9 mRNA expression. We found that some compounds from the
cauliflower mushroom extracts inhibit the PCSK9 mRNA expression. Therefore, we herein
report the isolation of compounds, their structural determination, and their PCSK9 inhibitory
activities from the extract of S. crispa.
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MATERIALS AND METHODS
General experimental procedures.
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The HRESIMS data were obtained on a UHR ESI QꢀTOF mass spectrometer (Bruker,
1
Billerica, MA). The NMR spectra were acquired with a 300 Ultra shield spectrometer ( H:
13
1
13
300 MHz/ C: 75 MHz) (Bruker), a NMR systems 500 MHz ( H: 500 MHz/ C: 125 MHz)
1
13
(Varian, Palo Alto, CA), and a DD2 700 spectrometer ( H: 700 MHz/ C: 175 MHz) (Agilent
Technologies, Santa Clara, CA) using the solvent signals (δ 2.50/δ 39.51 for DMSOꢀd ; δ
H
H
C
6
C 3
3.31/δ 49.15 for CD OD) (Cambridge Isotope Laboratories, Inc., Tewksbury, MA) as
internal standards; chemical shifts are indicated as δ values. The analytical high performance
liquid chromatography (HPLC) was carried out on a 1260 infinity HPLC system (Agilent
Technologies) supplied with a G1311C quaternary pump, a G1329B autosampler, a G1315D
PDA detector, and a G1316A oven for column. The column used was a 150 mm x 4.6 mm i.d.,
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5 µm, ZORBAX SBꢀC18 (Agilent Technologies). Semiꢀpreparative HPLC was operated on a
600 controller (Waters, Milford, MA) with a 996 PDA detector using the column ZORBAX
SBꢀC18 (250 mm x 21.2 mm i.d., 5 µm, Agilent Technologies). Column chromatography was
operated over silica gel 60 (70ꢀ230 mesh) (Merck, Darmstadt, Germany). Silica gel 60 F254
and RPꢀ18 F254S plates (Merck) were used for analysis by thin layer chromatography (TLC)
under detection of UV and 10% H SO reagent to visualize the compounds. Analytical grade
2
4
of solvents were used for the whole experiments.
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Plant Material.
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Dried fruiting bodies of S.crispa were provided by Gyeongshin Bio Co. (Euiwang,
South Korea) in August 2016. This sample was botanically identified by the corresponding
author (S.H. Shim). A voucher was deposited at the pharmacognosy laboratory of College of
Pharmacy, Duksung Women’s University (specimen No.: NPCꢀ16ꢀ08).
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Extraction and Isolation.
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Dried fruiting bodies of S. crispa (1 kg) were extracted with 100% MeOH (3.0 L) under
reflux three times to afford 153.0 g of the extracts. The extracts were suspended in distilled
1
1
1
1
00
01
02
03
water (1.0 L) and partitioned using nꢀhexane (3 x 1.0 L), CH
L), and nꢀBuOH (3 x 1.0 L) consecutively, yielding nꢀhexane (17.6 g), CH
(9.8 g), nꢀBuOH (15.0 g), and H O (107.9 g) layers, respectively. The EtOAcꢀsoluble layer
2
Cl
2
(3 x 1.0 L), EtOAc (3 x 1.0
2
Cl (2.7 g), EtOAc
2
2
(9.8 g) was set apart by vacuum liquid chromatography (VLC) (40 x 9 cm) over silica gel
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04
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10
11
12
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14
15
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using gradient solvents of nꢀhexane/EtOAc/MeOH (10:1:0, 2.5:1:0, 1.5:1:0, 1:1:0.2; each 5
L), CHCl /MeOH/H O (10:1:0, 5:1:0.1; each 5 L), and 100% MeOH (3 L) to obtain seven
3
2
fractions (Fr. E1 ~ Fr. E7). Silica gel column chromatography was employed to fraction E2
with the elution of nꢀhexane/acetone gradient solvents (20:1, 15:1; each 0.2 L) to afford
compound 2 (8.6 mg). Fraction E4 (1.4 g) was fractionated on silica gel column
chromatography (15 x 8 cm) with gradient solvents of CHCl
each 1 L) and 100% MeOH (1 L) to afford six fractions (Fr. E4ꢀ1 ~ Fr. E4ꢀ6). Fraction E4ꢀ1
was further purified with reversedꢀphase HPLC using a H O/acetonirile (60:40 → 45:55, v/v)
3
/acetone (65:1, 20:1, 10:1, 7:1;
2
gradient to yield compounds 12 (15 mg), 13 (18.5 mg), and 14 (1.5 mg). Compounds 1 (2.4
mg), 5 (2.9 mg), 6 (1.7 mg), 7 (1.8 mg), 8 (5.6 mg), 9 (7.3 mg), and 10 (4.1 mg) were
obtained from fraction E5 using reversedꢀphase HPLC with a gradient of H
→ 0:100, v/v). Fraction E6 was subjected to reversedꢀphase HPLC with gradient solvents of
O/MeOH (95:5 → 50:50, v/v) to furnish compounds 3 (13.0 mg), 4 (6.5 mg), and 11 (2.9
2
O/MeOH (80:20
H
2
mg).
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Sparoside A (2ꢀhydroxyꢀ3ꢀmethoxyꢀ6ꢀmethyl benzoic acid methyl ester 4ꢀOꢀαꢀDꢀriboside, 1).
+
1
1
1
1
1
20
21
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Yellowish amorphous solid; (+) HRESIMS m/z 367.1003 [M + Na] (calcd for
1
13
C
15
H
20NaO
9
, 367.1000); H and C NMR (Table 1); HMBC correlations (CD
3
OD, H → C)
Hꢀ5 → Cꢀ1, Cꢀ3, Cꢀ4, and CH ; Hꢀ1' → Cꢀ4, Cꢀ2', Cꢀ3', and Cꢀ4'; Hꢀ2' → Cꢀ1'; Hꢀ3' → Cꢀ1',
3
Cꢀ2', Cꢀ4', and Cꢀ5'; H
COOCH
2
3 3 3
ꢀ5' → Cꢀ3' and Cꢀ4'; CH → Cꢀ5 and Cꢀ6; OCH → Cꢀ3; COOCH →
3
.
1
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Sparalide A (5ꢀmethoxyꢀphthalide 7ꢀOꢀαꢀDꢀriboside, 3).
+
1
1
1
1
1
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White amorphous powder; (+) HRESIMS m/z 335.0738 [M + Na] (calcd for
1
13
C
14
H
16NaO
8
, 335.0737); H and C NMR (Table 1); HMBC correlations (DMSOꢀd
Hꢀ3 → Cꢀ1, Cꢀ3a, Cꢀ4, Cꢀ5, and Cꢀ7a; Hꢀ4 → Cꢀ3, Cꢀ5, Cꢀ6, Cꢀ7, and Cꢀ7a; Hꢀ6 → Cꢀ1, Cꢀ4,
Cꢀ5, Cꢀ7, and Cꢀ7a; Hꢀ1' → Cꢀ3', Cꢀ4', and Cꢀ7'; Hꢀ3' → Cꢀ1'; Hꢀ4' → Cꢀ3'; H ꢀ5' → Cꢀ3' and
Cꢀ4'; OCH → Cꢀ5.
6
, H → C)
2
3
1
1
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Sparalide B (6ꢀmethoxyꢀ4,5,7ꢀtrihydroxy phthalide, 4).
+
1
1
1
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35
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Yellow amorphous solid; (+) HRESIMS m/z 235.0212 [M + Na] (calcd for C
9
H
8
NaO
6
,
1
13
235.0213); H and C NMR (Table 1); HMBC correlations (DMSOꢀd
Cꢀ3a, Cꢀ4, Cꢀ5, Cꢀ7, and Cꢀ7a; OCH → Cꢀ6.
6
, H → C) H
2
ꢀ3 → Cꢀ1,
3
1
1
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Sparalide C (5,6ꢀdihydroxyꢀ7ꢀmethoxy phthalide, 5).
+
1
1
1
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Colorless amorphous solid; (+) HRESIMS m/z 219.0268 [M + Na] (calcd for
1
13
C
9
H
8
NaO
5
, 219.0264); H and C NMR (Table 1); HMBC correlations (CD
3
OD, H → C)
→ Cꢀ7.
H
2
ꢀ3 → Cꢀ1, Cꢀ3a, Cꢀ4, Cꢀ5, and Cꢀ7a; Hꢀ4 → Cꢀ3, Cꢀ5, Cꢀ6, and Cꢀ7a; OCH
3
1
1
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Acid hydrolysis of compounds 1 and 3.
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Each 1 mg of compounds 1 and 3 was hydrolyzed with 1N HCl (1 mL) at 80 °C for 2 h
to afford aglycone and sugar moieties. The reaction mixtures were extracted with EtOAc to
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separate a sugar moietyꢀcontaining aqueous fraction from the aglyconeꢀcontaining fraction.
The aqueous fraction was evaporated and then analyzed on silica gel TLC plates with a
2
gradient of acetone/H O for the comparison with authentic Dꢀribose and Lꢀribose (Sigmaꢀ
Aldrich, St. Louis, MO).
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1
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Determination of absolute configuration of ribose.
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To determine absolute configuration of the ribose in compounds 1 and 3, derivatives of
1
4
the sugar moieties were analyzed. Acid hydrolysis of each compound afforded aglycone and
sugar moieties. The sugar moieties (each 0.5 mg) were dissolved in pyridine (100 ꢁL),
supplemented with Lꢀcysteine methyl ester hydrochloride (0.5 mg), and placed at 60 °C for 1
h for reaction. Ten microliters of oꢀtolyl isothiocyanate was then added to the mixture to
allow for a reaction at 60 °C for 1 h. Each of the mixture was evaporated and subjected to
reverseꢀphase HPLC for analysis; ZORBAX SBꢀC18 column (Agilent Technologies, 250 x
4.6 mm i.d., 5 µm); column temperature 35 °C; mobile phase H O/acetonitrile (75:25, v/v)
2
for 30 min; flow rate 0.8 mL/min; detection wavelength at 250 nm. Both authentic
and ꢀribose were reacted in the same manner as described above. The absolute configuration
of ribose in compounds 1 and 3 was assigned by comparing their retention times with those
of the authentic derivatives (t ꢀribose derivative 12.68 min, ꢀribose derivative 8.20 min).
Dꢀribose
L
R
:
D
L
1
1
1
64
65
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PCSK9 mRNA Expression Assay.
RNA extraction procedure provided by the TRIzol (Life Technologies, Carlsbad, CA)
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67
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was employed for total RNA extraction. In brief, cDNA was prepared by adding reverse
transcriptase (200 U) and oligoꢀdT primer (500 ng) to total RNA (1 ꢁg) in 50 mM TrisꢀHCl
2
(pH 8.3), 75 mM KCl, 3 mM MgCl , 10 mM DTT, and 1 mM dNTPs at 42 °C for 1 h.
Incubation of the solution at 70 °C for 15 min terminated the reaction and an aliquot of the
cDNA mixture (1 ꢁL) was adopted for enzyme amplification. Polymerase chain reactions
were conducted by cDNA (1 ꢁL), master mix (9 ꢁL) containing iQ SYBR Green Supermix
(BioꢀRad, Hercules, CA), 5 pmol of forward primer, and 5 pmol of reverse primer using a
CFX384 RealꢀTime PCR System (BioꢀRad) with the following conditions: 3 min at 95 °C,
subsequently 40 cycles for 10 s at 95 °C, then 30 s at 55 °C, and finally plate reading. The
fluorescence which was generated using SYBR Green I DNA dye was quantified in the
course of the annealing. Specificity of the amplification was ascertained through a melting
curve analysis. CFX Manager Software (BioꢀRad) was used for acquisition of data, which
were presented as the cycle threshold (C
T
). Then, relative abundance of an interesting gene
ꢀ(ꢂꢂCT)
was standardized to that of glyceraldehyde 3ꢀphosphate dehydrogenase (ꢂꢂC
T
). The 2
1
5
method was employed for calculation of mRNA abundance of the sample. Specific primer
sets used in this study were as follows (5′–3′): GAPDH: GAAGGTGAAGGTCGGAGTCA
(forward), AATGAAGGGGTCATTGATGG (reverse), PCSK9:
GGGCATTTCACCATTCAAAC (forward), TCCAGAAAGCTAAGCCTCCA (reverse).
Customꢀsynthesized geneꢀspecific primers were provided by Bioneer (Daejeon, Korea).
1
1
1
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87
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Statistical Analyses.
Data were expressed as the mean ± SEM. Analysis of variance (ANOVA) determined
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the level of statistical significance and Dunnett's tꢀtest was used for multiple comparison
procedures. P values (calculated probability) less than 0.05 were regarded to be significant.
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RESULTS AND DISCUSSION
Structural Elucidation.
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A series of chromatographic methods carried out on the extract of S. crispa led to the
isolation of fourteen compounds, which include four new aromatic compounds, 1 and 3ꢀ5,
two new naturallyꢀoccurring compounds, 2 and 6, and eight known compounds, 7ꢀ14 (Figure
1).
1
1
2
2
2
2
2
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02
03
04
The known compounds were identified to be methyl 2,4ꢀdihydroxyꢀ3ꢀmethoxyꢀ6ꢀ
1
6
17
methylbenzoate, 2, 5ꢀhydroxyꢀ7ꢀmethoxyphthalide, 6, 5ꢀmethoxyꢀ7ꢀhydroxyphthalide,
17
18
19
20
7, hanabiratakelide A, 8, nicotinamide, 9, 5′ꢀdeoxyꢀ5′ꢀmethylthioadenosine, 10,
21
22
23
adenosine, 11, ergosterol, 12, ergosterol peroxide, 13, and 5α,6αꢀepoxyꢀ(22E,24R)ꢀ
24
ergostaꢀ8(14),22ꢀdieneꢀ3β,7βꢀdiol, 14 by comparing their NMR and MS data with those in
the reference. Of these, although compounds 2 and 6 have been reported as synthetic
intermediates, they have been reported for the first time in nature in this study.
2
2
2
2
2
05
06
07
08
09
Compound 1 was obtained as a yellowish amorphous solid. Positive HRESIMS
1
suggested its molecular formula to be C15
aromatic proton at δ
at δ 3.93 (3H, s, COOCH
CH
H
20
O
9
. The H NMR spectrum of 1 displayed an
H
6.66 (1H, s, Hꢀ5), one sugar unit at δ
) and 3.85 (3H, s, 3ꢀOCH ), and methyl group at δ
). The C NMR spectrum of 1 suggested existence of a carbonyl group (δ
H
5.73ꢀ3.65, two methoxyl groups
2.43 (3H, s, 6ꢀ
172.81), six
H
3
3
H
1
3
3
C
1
0
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2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
aromatic carbons (δ
C
156.45, 154.57, 137.40, 137.22, 112.34, and 111.09), one sugar unit (δ
C
102.64, 88.46, 73.82, 71.23, and 63.30), two methoxyl groups (δ 61.63 and 52.68), and a
C
1
13
C
methyl group (δ 23.28). One sugar unit was confirmed to consist of a ribose by H and C
NMR data, which was further supported by chemical reaction. Acid hydrolysis of 1 followed
by TLC with authentic ribose supported that the sugar was ribose. Moreover, the 4.4 Hz of
coupling constant for the anomeric proton at δ
H
5.73 suggested an αꢀconfiguration. HMBC
correlation of the methoxyl protons at δ 3.93 with the carbonyl carbon at δ 172.81 indicated
H
C
that the methyl carboxylate was attached to the aromatic ring. The positions of the methoxyl,
methyl group and ribose at the aromatic ring were assigned by analysis of the HMBC
spectrum. HMBC correlations of the methoxyl protons at δ
137.22 and the anomeric proton at δ 5.73 with the aromatic carbon at δ
for the assignment of the carbons bearing the methoxyl group and the ribose. HMBC
correlation of the methyl protons at δ 2.43 with the aromatic methine carbon at δ 112.34
and the nonꢀprotonated aromatic carbon at δ 137.40 and the aromatic methine proton at δ
6.66 with the carbons at δ 111.09, 137.22, and 154.57 indicated the methyl, the methyl
H
3.85 with the aromatic carbon at
δ
C
H
C
154.57 allowed
H
C
C
H
C
carboxylate, the methoxyl, and the ribose were attached to the Cꢀ6, Cꢀ1, Cꢀ3, and Cꢀ4
positions, respectively. Determination of the absolute configuration of the ribose was
conducted by comparing retention time of
derivative of acidꢀhydrolysate with those for authentic Dꢀ/Lꢀ ribose derivatives in HPLCꢀUV.
The derivatives of the authentic ꢀribose and ꢀribose eluted at t 12.68 ( ꢀribose) and 8.20
ꢀribose) min, respectively on the isocratic HPLC. Since the derivative of compound 1
eluted at t 11.93 min, the ribose in 1 was confirmed to have configuration. Thus, the
structure of 1 was determined to be 2ꢀhydroxyꢀ3ꢀmethoxyꢀ6ꢀmethyl benzoic acid methyl ester
Lꢀcysteine methyl ester and oꢀtolyl isothiocyanate
D
L
R
D
(
L
D
R
1
1
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33
4ꢀOꢀαꢀDꢀriboside and was named as sparoside A.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
Compound 3 was obtained as a white amorphous powder. Its positive HRESIMS data
1
suggested the molecular formula to be C14
aromatic methine protons at δ
5.28 (2H, s, Hꢀ3), one sugar unit at δ
H
16
O
8
. The H NMR spectrum displayed two
H
6.82 (1H, s, Hꢀ4) and 6.81 (1H, s, Hꢀ6), an oxymethylene at
5.95ꢀ3.45 and a methoxyl group at δ 3.85 (3H, s, 5ꢀ
). The C NMR spectrum of 3 suggested the presence of a carbonyl group (δ 168.89),
six aromatic carbons (δ 166.11, 156.23, 151.68, 107.05, 102.59, and 100.49), one pentose
δ
H
H
H
13
OCH
3
C
C
moiety (δ
C
101.47, 87.99, 71.74, 69.77, and 61.48), an oxymethylene (δ
C
68.98), and a
methoxyl carbon (δ
C
56.19). It was presumed to be bicyclic compound to meet seven
unsaturations obtained from its molecular formula. The presence of an aromatic ring, an
oxymethylene group, together with carboxyl carbon indicated that compound 3 has a
phthalide skeleton, which has previously been reported in S. crispa. The pentose was
presumed to be ribose, based on the carbon chemical shifts and proton resonances. The
coupling constant (3.9 Hz) of the anomer proton suggested that the ribose was attached to the
aglycone with an αꢀconfiguration. The presence of ribose was verified by the direct
comparison of acid hydrolysate of 3 with commercially available authentic ribose. The
HMBC correlations of the oxymethylene at δ 5.28 with the carboxyl carbon (δ 168.89) and
H
C
C
the aromatic carbon (δ 151.68 and 107.05) supported the idea that 3 had a γꢀlactonylated
aromatic compound, known as a phthalide. The singlet aromatic methine protons suggested
the methoxyl group and the ribose moiety were not attached to the adjacent carbon atoms of
the aromatic ring. Their positions were confirmed by HMBC data. HMBC correlations of the
anomeric proton (δ 5.95) with the carbon (δ 156.23) and methoxyl protons (δ 3.85) with
H
C
H
C
the carbon (δ 166.11) indicated that the ribose and the methoxyl groups were attached to
1
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2
2
2
2
2
2
2
56
57
58
59
60
61
62
63
carbons at δ
C
156.23 and 166.11, respectively. HMBC correlations of the Hꢀ4 methine proton
at δ 6.82 of the phthalide with the methoxylated carbon (δ 166.11), another aromatic
H
C
methine carbon (δ
C
C
102.59), and the nonꢀprotonated aromatic carbon (δ 107.05) indicated
that the methoxyl group and the ribose were attached to Cꢀ5 and Cꢀ7, respectively. Thus a
planar structure could be established. Assignment of the absolute configuration of ribose was
conducted as described above. Since the retention time of the ribose derivative of acid
hydrolysate for 3 was same as that for the
Dꢀribose derivative, structure of compound 3 was
determined as 5ꢀmethoxyꢀphthalide 7ꢀOꢀαꢀ
D
ꢀriboside, named sparalide A.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
64
65
66
67
68
69
70
71
72
73
74
75
76
77
Compound 4 was obtained as a yellow amorphous solid, in which its molecular formula
1
was established as C
9
H
8
O
6
on the basis of positive HRESIMS. In its H NMR spectrum, one
oxymethylene and one methoxyl signal appeared at δ
H
5.09 (2H, s, Hꢀ3) and 3.79 (3H, s, 6ꢀ
OCH ), respectively. Six aromatic carbons (δ 141.94, 139.30, 139.19, 135.44, 124.99, and
3
C
106.25), an oxymethylene carbon (δ
C
C
66.79), and a carbonyl group (δ 168.90) shown in the
1
3
C NMR spectrum suggested that 4 had a phthalide skeleton, similar to compound 3. The
position of the methoxyl group was assigned to be attached to Cꢀ6 on the ground that the
methoxyl protons (δ 3.79) showed HMBC correlations with Cꢀ6 (δ 139.31) while the
H
C
oxymethylene protons (δ 5.09) showed HMBC correlations with Cꢀ7 (δ 141.94), Cꢀ5 (δ
H
C
C
4
139.19), Cꢀ4 (δ
C
135.44), Cꢀ3a (δ
C
124.99), and Cꢀ7a (δ
C
106.25) (Figure 2). JCH HMBC
3
correlations between H
2
ꢀ3 and Cꢀ5 and Cꢀ7 in addition to JCH HMBC correlations between
the H
2
ꢀ3 and Cꢀ4 ascertained that the methoxyl group was attached to Cꢀ6 rather than Cꢀ4, Cꢀ
5 or Cꢀ7. From these evidences, the structure of 4 was determined to be 6ꢀmethoxyꢀ4,5,7ꢀ
trihydroxy phthalide, named sparalide B.
2
78
Compound 5 was a colorless amorphous solid and the molecular formula was confirmed
1
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Journal of Agricultural and Food Chemistry
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2
2
2
2
2
2
2
2
2
2
2
2
2
79
to be C
9
H
8
O
5
by positive HRESIMS. The H NMR spectrum of 5 showed an aromatic proton
80
81
82
83
84
85
86
87
88
89
90
91
at δ 6.68 (1H, s, Hꢀ4), an oxymethylene at δ 5.15 (2H, s, Hꢀ3), and a methoxyl group at δ
H
H
H
1
3
4.01 (3H, s, 7ꢀOCH
3
). Its C NMR spectrum exhibited six aromatic carbons (δ
70.46), a carboxyl
62.65). The H and C NMR data presented
similarities to those for 4, except for an additional aromatic proton at δ 6.68. Thus, it was
C
155.49,
147.44, 142.32, 139.62, 109.50, and 104.67), an oxymethylene carbon (δ
C
1
13
C C
carbon (δ 172.14), and a methoxyl carbon (δ
H
assumed that two hydroxyl groups and a methoxyl group were attached to the aromatic ring
of the phthalide skeleton, which was also supported by the observation of HRꢀESIꢀMS. The
positions of the aromatic methine carbon and the methoxylated carbon were assigned to be Cꢀ
4 and Cꢀ7, respectively, supported by HMBC correlations of Hꢀ4 (δ
70.46), Cꢀ5 (δ 155.49), Cꢀ6 (δ 139.62), Cꢀ3a (δ 142.32), and Cꢀ7a (δ
methoxyl proton (δ 4.01) with Cꢀ7 (δ 147.44) (Figure 2). Using these data, the structure of
H
6.68) with Cꢀ3 (δ
C
C
C
C
C
109.50) and the
H
C
5 was established to be 5,6ꢀdihydroxyꢀ7ꢀmethoxy phthalide, named sparalide C.
2
2
92
93
Evaluation of PCSK9 mRNA expression.
2
2
2
2
2
2
3
94
95
96
97
98
99
00
The MeOH extract and polarityꢀbased solventꢀsoluble layers of S. crispa were tested on
inhibitory activity of PCSK9 mRNA expression using a HepG2 cells. The EtOAcꢀsoluble
layer showed the most potent inhibitory activities at 10 µg/mL, which prompted us to
elucidate the active compounds from the EtOAcꢀsoluble layer (Figure 3). Bioactivityꢀguided
fractionation led to isolation of fourteen compounds, 1ꢀ14, from this layer. All the isolated
compounds were assessed for their PCSK9 mRNA expression. The results demonstrated that
compounds 1, 8, 11, and 14 were found to potentially inhibit PCSK9 mRNA expression with
1
4
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3
3
3
3
3
01
02
03
04
05
IC50 values of 20.07, 7.18, 18.46, and 8.23 ꢁM, respectively, at the concentration of 20 ꢁM,
whereas the IC of berberine, positive control, was 8.04 ꢁM at the same concentration as the
50
isolated compounds. When compared with the positive control, compound 8
(hanabiratakelide A) was found to be a stronger PCKS9 inhibitor than berberine, which is
known to be one of the most potent PCSK9 inhibitors in nature far.
3
3
3
3
3
3
3
3
3
06
07
08
09
10
11
12
13
14
Concerning the structureꢀactivity relationship, the results showed that 4,5,6ꢀtrioxygenated
pattern in phthalide moiety seemed to be important for the PCSK9 inhibitory activity rather
than diꢀoxygenation or tetraꢀoxygenation pattern as shown in compound 8. In the case of
simple benzoic acid derivatives (compounds 1 and 2), ribose moiety seemed to be important
in the activity as shown for compound 1. The ribose moiety seemed also to play an important
role in adenosine derivatives (compounds 10 and 11), where the OH group at Cꢀ5 of ribose
moiety seemed to be effective rather than SCH . In the case of ergosterol derivatives, the OH
3
group and epoxide substituents on the B ring seemed to be more effective than when they had
endoperoxide or diene system.
3
3
3
3
15
16
17
18
The results suggest that hanabiratakelide A, 8, and 5α,6αꢀepoxyꢀ(22E,24R)ꢀergostaꢀ
8(14),22ꢀdieneꢀ3β,7βꢀdiol, 14, as well as the extract of S. crispa could be good supplements
to statins for the treatment of hyperlipidemia. Moreover, further studies regarding the
mechanistic and in vivo efficacies for compounds 8 and 14 might be required.
3
19
Supporting Information
3
3
20
21
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
.
3
22
NMR and HRMS spectra of compounds 1 and 3ꢀ5 (PDF)
1
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23
ACKNOWLEDGMENT
3
3
3
3
3
24
25
26
27
28
This study was supported by Korea Institute of Planning and Evaluation for Technology
in Food, Agriculture, Forestry and Fisheries(IPET) through High Valueꢀadded Food
Technology Development Program, funded by Ministry of Agriculture, Food and Rural
Affairs (MAFRA, 116001ꢀ3) and also supported by the National Research Foundation (NRF)
of Korea (Grant No. NRFꢀ2015R1D1A1A01057914 and NRFꢀ2016R1A6A1A03007648).
3
29
1
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REFERENCES
3
3
31
32
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(6) Ohno, N.; Miura, N. N.; Nakajima, M.; Yadomae, T. Antitumor 1,3ꢀβꢀglucan from
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extracted from a culinaryꢀmedicinal mushroom, Sparassis crispa Wulf.:Fr.
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(8) Yamamoto, K.; Kimura, T.; Sugitachi, A.; Matsuura, N. Antiꢀangiogenic and antiꢀ
metastatic effects of βꢀ1,3ꢀDꢀglucan purified from hanabiratake, Sparassis crispa. Biol.
Pharm. Bull. 2009, 32, 259ꢀ263.
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(9) Harada, T.; Miura, N.; Adachi, Y.; Nakajima, M.; Yadomae, T.; Ohno, N. Effect of SCG, 1,
3ꢀβꢀDꢀglucan from Sparassis crispa on the hematopoietic response in cyclophosphamide
induced leukopenia mice. Biol. Pharm. Bull. 2002, 25, 931ꢀ939.
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(10) Kwon, A. H.; Qiu, Z.; Hashimoto, M.; Yamamoto, K.; Kimura, T. Effects of medicinal
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(11) Kimura, T. Natural products and biological activity of the pharmacologically active
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(12) Zimmerman, M. P. How do PCSK9 inhibitors stack up to statins for lowꢀdensity
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(13) Pagliaro, B.; Santolamazza, C.; Simonelli, F.; Rubattu, S. Phytochemical compounds and
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method. Methods 2001, 25, 402ꢀ408.
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(17) ElꢀFeraly, F. S.; Cheatham, S. F.; Mcchesney, J. D. Total synthesis of notholaenic acid. J.
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(19) Shchepin, R. V.; Barskiy, D. A.; Mikhaylov, D. M.; Chekmenev, E. Y. Efficient synthesis
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5
of nicotinamideꢀ1ꢀ N for ultrafast NMR hyperpolarization using parahydrogen.
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3
3
3
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Konig, G. M. Novel amino acid derived natural products from the ascidian Atriolum
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(21) Chenon, M. T.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.; Townsend, L. B. A basic set
of parameters for the investigation of tautomerism in purines established from carbonꢀ13
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(22) Nowak, R.; Drozd, M.; Mendyk, E.; Lemieszek, M.; Krakowiak, O.; Kisiel, W.; Rzeski,
W.; Szewczyk, K. A new method for the isolation of ergosterol and peroxyergosterol as
active compounds of Hygrophoropsis aurantiaca and in vitro antiproliferative activity of
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93
isolated ergosterol peroxide. Molecules 2016, 21, 946ꢀ955.
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3
3
94
95
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(23) Krzyczkowski, W.; Malinowska, E.; Suchocki, P.; Kleps, J.; Olejnik, M.; Herold, F.
Isolation and quantitative determination of ergosterol peroxide in various edible
mushroom species. Food Chem. 2009, 113, 351ꢀ355.
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(24) Ishizuka, T.; Yaoita, Y.; Kikuchi, M. Sterol constituents from the fruit bodies of Grifola
frondosa (Fr.) S. F. Gray. Chem. Pharm. Bull. 1997, 45, 1756ꢀ1760.
2
0
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4
4
99
00
01
Legends:
Figure 1. Structures of compounds 1–14 from S. crispa.
Figure 2. Key HMBC correlations of compounds 1, 3, 4, and 5.
4
4
4
4
02
03
04
05
Figure 3. Effect of (A) MeOH extracts and solventꢀsoluble fractions of S. crispa and (B)
isolated compounds from EtOAcꢀsoluble fractions. Expression of PCSK9 mRNA was
assayed by qRTꢀPCR in cells treated with 10 µg/mL of solventꢀsoluble fractions and 20
µM of isolated compounds.
4
4
4
4
4
4
4
4
4
06
07
08
09
10
11
12
13
14
2
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15
Figure 1.
4
4
16
17
4
4
18
19
4
4
4
20
21
22
2
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23
Figure 2.
4
4
24
25
4
4
4
4
26
27
28
29
2
3
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30
Figure 3.
4
31
4
4
4
4
4
4
4
4
4
32
33
34
35
36
37
38
39
40
4
4
41
42
2
4
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1
13
4
43
Table 1. The H and C NMR Data of Compounds 1 and 3ꢀ5.
a
b
b
a
No.
1
3
4
5
δ
C
δ
H
multi
δ
H
multi
δ
H
multi
δ
H
multi
δ
C
δ
C
δ
C
(
J in Hz)
(J in Hz)
(J in Hz)
(J in Hz)
1
2
3
111.09
156.45
137.22
168.89
168.90
172.14
68.98 5.28 s
151.68
66.79 5.09 s
124.99
70.46
5.15 s
3
a
142.32
4
154.57
100.49 6.82 s
166.11
135.44
104.67 6.68 s
155.49
5
6
7
112.34 6.66 s
137.40
139.19
102.59 6.81 s
156.23
139.30
139.62
141.94
147.44
7
a
107.05
106.25
109.50
1'
2'
3'
4'
102.64 5.73 d (4.4)
101.17 5.95 d (3.8)
73.82 4.32 dd (4.5, 6.4) 71.74 4.10 dd (4.0, 5.8)
71.23 4.10 dd (2.8, 6.5) 69.77 3.89 d (5.6)
88.46 4.16 m
87.99 4.00 dd (3.6, 5.8)
3
3
.70 dd (3.4, 12)
.65 dd (3.9, 12)
5'
63.30
61.48 3.45 m
OCH
CH
3
61.63 3.85 s
23.28 2.43 s
172.81
56.19 3.85 s
61.60 3.79 s
62.65
4.01 s
3
COOCH
3
COOCH
3
52.68 3.93 s
a
4
4
44
45
Measured in CD OD
3
b
Measured in DMSOꢀd
6
2
5
ACS Paragon Plus Environment