SUPPLEMENTARY MATERIAL
Chemical constituents from Piper wallichii
Yan-Ni Shi,a,b Lian Yang,a Jin-Hua Zhao,a Yi-Ming Shi,a,b Yan Qu,a
Hong-Tao Zhu,a Dong Wang,a Chong-Ren Yang,a Xing-Cong Li,c Min
Xu,a,* and Ying-Jun Zhang a,*
a
State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
People's Republic of China
b
University of Chinese Academy of Sciences, Beijing 100049, People's Republic of
China
c
National Center for Natural Products Research, School of Pharmacy, University of
Mississippi, Mississippi 38677, United States
Chemical constituents from Piper wallichii
Fifteen known compounds, including four triterpenoids (1–4), one sterol
(5), one diketopiperazine alkaloid (6), and nine phenolics (7–15) were
isolated from the stems of Piper wallichii. Their structures were
elucidated by means of spectroscopic analysis, and acidic acid
hydrolysis in case of the 2-oxo-3β,19α,23-trihydroxyurs-12-en-28-oic
acid β-D-glucopyranosyl ester (1). The structure of compound 1 was
fully assigned by 1D- and 2D-NMR experiments for the first time. All
isolates were tested for their antibacterial, antifungal, anti-inflammatory,
and antiplatelet aggregation bioactivities.
Keywords: Piper wallichii; triterpenoid glycoside; miscellaneous
constituents
Pa
ge
General experimental procedures
1
Plant material
1
Extraction and isolation
2
3
Acid hydrolysis of compound 1
Antibacterial and antifungal bioassays
3
Anti-inflammatory bioassay
4
Antiplatelet aggregation bioassays
4
5
Physical-chemical properties of compound 1
1
5
Figure S1. H NMR spectrum of compound 1
13
6
Figure S2. C NMR spectrum of compound 1
6
Figure S3. HSQC spectrum of compound 1
7
Figure S4. HMBC spectrum of compound 1
1
1
7
Figure S5. H- H COSY spectrum of compound 1
8
Figure S6. ROESY spectrum of compound 1
8
Figure S7. ESI spectrum of compound 1
9
Figure S8. HRESI spectrum of compound 1
9
Figure S9. UV spectrum of compound 1
10
Figure S10. IR spectrum of compound 1
1
13
Table S1. H and C NMR spectroscopic data for compound 1 in 11
pyridine-d5a
Reference
12
Content
General experimental procedures
Optical rotations were performed on a P-1020 polarimeter (JASCO, Tokyo, Japan). IR
spectra were detected on a Bruker Tensor 27 spectrometer with KBr pellets. 1D- and
2D-NMR spectra were recorded on Bruker DRX-500 and AV-600 instruments
operating at 500 and 600 MHz for 1H NMR, and 125 and 150 MHz for
13
C NMR.
Coupling constants are expressed in Hz, and chemical shifts are given on a ppm scale
with tetramethylsilane as internal standard. UV data were obtained on a Shimadzu
UV2401PC spectrophotometer. ESIMS data were recorded on Waters Xevo TQ-S.
HRESIMS were recorded on an API Qstar Pulsa i spectrometer.
Column chromatography (CC) was performed with silica gel (200–300 mesh,
Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of China),
Sephadex LH-20 (25–100 μm, Pharmacia Fine Chemical Co. Ltd. Japan), Lichroprep
RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), MCI gel (75–150 μm,
Mitsubishi Chemical Corporation, Tokyo, Japan), and Diaion HP20SS (Mitsubishi
Chemical Co., Tokyo, Japan). Thin-layer chromatography (TLC) was carried out on
silica gel H-precoated plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao,
People’s Republic of China). Spots were detected by spraying with 10% H2SO4 in
EtOH followed by heating. Preparative HPLC (p-HPLC) was performed on a Gilson
liquid chromatography with a 7 μm Zorbax SB-C18 (21.2 × 250 mm) column. Semi
p-HPLC separation was performed on an Agilent 1260 liquid chromatography with a
5 μm Thermo BDS HYPERSIL-C18 column (10 × 250 mm) column.
Plant material
The stems of P. wallichii were collected from Henan Province, People’s Republic of
China, in June 2012, and were identified by Mr. Xiao-Ming Fang from South China
Botanical
Garden,
Chinese
Academy of
Sciences.
A
voucher
specimen
(HITBC_015077) was deposited at the State Key Laboratory of Phytochemistry and
1
Plant Resources in West China, Kunming Institute of Botany (KIB), Chinese
Academy of Sciences (CAS).
Extraction and isolation
The air-dried and powdered stems of Piper wallichii (19.0 kg) were extracted by
MeOH at 60 oC. After removal of the solvent under reduced pressure, the crude
extract (2.1 kg) was suspended in H2O and partitioned with EtOAc (3 × 20 L). The
EtOAc extract (600 g) was subjected to silica gel CC, eluting with a CHCl3–MeOH
(99:1–5:1) to give fractions A–H. Fr. B (5.5 g) was subjected to a RP-18 column using
a step gradient of MeOH–H2O (70–100%) to afford Fr.B1-Fr.B3. Fr.B1 (78 mg) was
purified by semi p-HPLC and preparative TLC (p-TLC) (petroleum ether: acetone,
2:1, v/v) to yield 6 (9.6 mg). Fr.B3 (1.3 g) was recrystallized in MeOH to yield 5
(36.0 mg). Fr.E (65 g) was separated by RP-18 column using a step gradient of
MeOH–H2O (40–90%) to afford Fr. E1-E8. Fr. E2 (499 mg) was chromatographed
over a silica gel column, eluting with petroleum ether–acetone (5:1), and purified by
semi p-HPLC to afford 3 (7.8 mg) and 4 (44.7 mg). Fr. E3 (3.8 g) was
chromatographed on Sephadex LH-20 column eluting with MeOH, followed by
RP-18 and purified by semi p-HPLC to afford 7 (194.6 mg), 8 (6.6 mg), 9 (3.8 mg),
and 10 (4.7 mg). Fr.F (35 g) was subjected to Diaion HP20SS CC using a step
gradient of MeOH–H2O (40–100%) to afford thirteen subfractions, Fr. F1-F13. Fr. F8
(1.5 g) was separated by Sephadex LH-20 (MeOH) and followed by p-HPLC to yield
compound 12 (5.9 mg). Fr. F9 (2.8 g) was separated by Sephadex LH-20 and followed
by RP-18 column with 40% MeOH–H2O to afford compound 11 (6.4 mg). Fr. G (3.3
g) was isolated by a silica gel column, eluting with a CHCl3–MeOH (15:1) to yield
compound 2 (113.8 mg). Fr.H (340 g) was subjected to Diaion HP20SS CC using a
step gradient of MeOH–H2O (20–80%) to afford Fr. H1–H6. Fr. H3 (13 g) was
separated by RP-18 using a step gradient of MeOH–H2O (20–100%) to afford four
subfractions. Fr. H3C (280 mg) was isolated by Sephadex LH-20 (MeOH) and
followed by silica gel (CHCl3–MeOH, 15:1) CC to obtain compound 15 (1.0 mg). Fr.
2
H3B (548 mg) was subjected to Sephadex LH-20 (MeOH) and followed by semi
p-HPLC to yield compound 13 (14.7 mg). Fr. H3D (3.5 g) was separated by Sephadex
LH-20 (MeOH) and followed by silica CC, eluting with CHCl3–MeOH (15:1) to
obtain compounds 1 (43.9 mg) and 14 (1.1 mg).
Acid hydrolysis of compound 1
Compound 1 (20 mg) was hydrolyzed with 1.5 N HCl (2 mL) at 70 °C for 5 h. The
reaction mixture was extracted with CHCl3 (3 × 3 mL). After neutralized with NaOH
(1 N), the aqueous layer was applied to a Sephadex LH-20 CC, eluting with MeOH, to
afford D-glucose (2.88 mg), [α]D25 +45.0 (c 0.28, H2O), which was comparable to that
of the standard D-(+)-glucose ([α]D23 +52.3 (c 1.19, H2O), Sigma).
Antibacterial and antifungal bioassays
All the organisms were obtained from the American Type Culture Collection
(Manassas, VA) and included Candida albicans ATCC 90028, C. glabrata ATCC
90030, C. krusei ATCC 6258, Cryptococcus neoformans ATCC 90113, and
Aspergillus fumigatus ATCC 204305, and the bacteria Staphylococcus aureus ATCC
29213, methicillin-resistant S. aureus ATCC 33591 (MRS), Escherichia coli ATCC
35218, Pseudomonas aeruginosa ATCC 27853, and Mycobacterium intracellulare
ATCC 23068. Susceptibility testing was performed using a modified version of the
CLSI (formerly NCCLS) methods (NCCLS 2002b) (NCCLS 2002a). M.
intracellulare was tested using a modified method of Franzblau et al (Franzblau et al.
1998). All the samples were serially diluted in 20% DMSO/saline and transferred in
duplicate to 96-well flat bottom microplates. Microbial inocula were prepared by
correcting the OD630 of the microbe suspensions in incubation broth to afford final
target inocula after addition to the samples. All organisms were read spectrometrically
prior to and after incubation. The detailed protocol has been described in a previous
article (Samoylenko et al. 2009).
3
Anti-inflammatory bioassay
Murine monocytic RAW 264.7 macrophages were dispensed into 96-well plates (2 ×
105 cells/well) containing RPMI 1640 medium (Hyclone) with 10% fetal bovine
serum under a humidified atmosphere of 5% CO2 at 37 ° C. After 24 h pre-incubation,
cells were treated with serial dilutions of the compounds with the maximum
concentration of 25 μM in the presence of 1 μg/mL LPS for 18 h. Each compound
was dissolved in DMSO and further diluted in medium to produce different
concentrations. NO production in each well was assessed by adding 100 μL of Griess
reagent (reagent A and reagent B, respectively, Sigma) to 100 μL of each supernatant
from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5
min incubation, the absorbance was measured at 570 nm with a 2104 Envision
multil-abel plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA).
MG-132 was used as a positive control (Fan et al. 2010).
Antiplatelet aggregation bioassays
Turbid metric measurements of platelet aggregation of all the isolates and GB were
performed in a Chronolog Model 700 Aggregometer (Chronolog Corporation,
Havertown, PA, USA) according to Born’s method (Born 1962, Born & Cross 1963).
The blood from the rabbits by cardiac puncture, were anticoagulated with 3.8%
sodium citrate (9:1, v/v). Platelet-rich plasma (PRP) was prepared shortly after blood
collection by spinning the sample at 180 g for 10 min at 22 °C. The PRP was
carefully removed and the remaining blood were centrifuged at 2400 g for 10 min to
obtain platelet-poor plasma (PPP). The centrifugation temperature was maintained at
22 °C. Platelet counts were adjusted by the addition of PPP to the PRP to achieve a
count of 500 × 109 L-1. Platelet aggregation studies were completed within 3 h of the
preparation of PRP. Immediately after the preparation of PRP, 250 L was transferred
into each of prepared test tubes, with 250 L PPP set as a control. Before addition of
inducers, compounds were incubated with PRP at 37 °C for 5 min. The aggregation of
PRP was stimulated by PAF 40 ng/mL. The change of optical density as a result of
4
platelet aggregation was recorded, and inhibition percentage of compounds was
calculated according to the formula:
Inhibition of aggregation (%) = (A–B)/A × 100% [A: maximum change of turbidity in
DMSO added. B: maximum change of turbidity in sample added.]
2-Oxo-3β,19α,23-trihydroxyurs-12-en-28-oic acid β-D-glucopyranosyl ester (1)
White amorphous powder; [α]D16.2 +27.8 (c 3.48, MeOH); UV (MeOH) max (log ε)
279.8 (2.32), 202.4 (3.13) nm; IR (KBr) νmax 3432, 1710, 1634 cm-1; HRESIMS m/z
663.3745 [M – H]– (calcd 663.3744); positive ESIMS m/z 687 [M + Na]+; 1H and 13C
NMR data, see Table 1.
Figure S1. 1H NMR spectrum of compound 1
5
Figure S2. 13C NMR spectrum of compound 1
Figure S3. HSQC spectrum of compound 1
6
Figure S4. HMBC spectrum of compound 1
Figure S5. 1H-1H COSY spectrum of compound 1
7
Figure S6. ROESY spectrum of compound 1
Figure S7. ESI spectrum of compound 1
8
Figure S8. HRESI spectrum of compound 1
Figure S9. UV spectrum of compound 1
9
Figure S10. IR spectrum of compound
10
Table S1. 1H and 13C NMR spectroscopic data for compound 1 in pyridine-d5a
position
δC, mult.
δH (mult., J in Hz)
position
δC, mult.
δH (mult., J in Hz)
1
53.8, CH2
2.21 (d, 12.0, Hα)
2.52 (d, 12.0, Hβ)
212.9, C
77.7, CH
50.0, C
46.4, CH
18.8, CH2
54.4, CH
72.6, C
42.1, CH
26.6, CH2
2.91, (s)
2
3
4
5
6
18
19
20
21
22
37.7, CH2
23
64.8, CH2
24
25
26
27
28
29
30
Glc-1'
2'
3'
4'
5'
6'
14.0, CH3
17.0, CH3
17.1, CH3
24.4, CH3
177.0, C
26.9, CH3
16.7, CH3
95.9, CH
74.1, CH
79.4, CH
71.2, CH
79.0, CH
62.2, CH2
7
8
9
10
11
32.8, CH2
40.9, C
47.3, CH
43.6, C
24.0, CH2
12
13
14
15
127.8, CH
139.4, C
42.2, C
29.3, CH2
16
26.0, CH2
17
48.6, C
aData
5.02 (s)
2.44 (m)
1.43 (m)
1.79 (m)
1.41 (m)
1.80 (overlap)
2.21 (brd, 12.0)
1.94 (m)
2.01 (m)
5.51 (brs)
1.19 (m)
2.46 (m)
1.96 (m)
3.07 (td, 3.4, 12.8)
1.34 (overlap)
1.19 (overlap)
1.94 (overlap)
1.81 (m)
2.01 (m)
3.74 (d, 11.0)
4.04 (d, 11.0)
0.82 (s)
0.99 (s)
1.17 (s)
1.62 (s)
1.36 (s)
1.05 (d, 6.5)
6.31 (d, 8.2)
4.24 (t, 8.6)
4.05 (m)
4.40 (m)
4.32 (t, 8.9)
4.42 (dd, 4.1, 12.0)
4.49 (d, 12.0)
were measured at 600 MHz for 1H NMR spectrum and 150 MHz for 13C NMR spectrum
11
Reference
Born GVR. 1962. Aggregation of Blood Platelets by Adenosine Diphosphate and Its
Reversal. Nature. 194:927.
Born GVR, Cross MJ. 1963. Aggregation of Blood Platelets. Journal of
Physiology-London. 168:178-195.
Fan J, Su J, Peng Y, Li Y, Li J, Zhou Y, Zeng G, Yan H, Tan N. 2010. Rubiyunnanins
C-H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric
oxide production and NF-κB activation. Bioorg Med Chem. 18:8226-8234.
Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan
MT, Cook MB, Quenzer VK, Ferguson RM, et al. 1998. Rapid,
low-technology MIC determination with clinical Mycobacterium tuberculosis
isolates by using the microplate Alamar Blue assay. J Clin Microbiol.
36:362-366.
NCCLS. 2002a. Reference method for broth dilution antifungal susceptibility testing
of filamentous fungi, approved standard. 22:M38-A.
NCCLS. 2002b. Reference method for broth dilution antifungal susceptibility testing
of yeasts; approved standard. 22:M27-A22.
Samoylenko V, Ashfaq MK, Jacob MR, Tekwani BL, Khan SI, Manly SP, Joshi VC,
Walker LA, Muhammad I. 2009. Indolizidine, Antiinfective and Antiparasitic
Compounds from Prosopis glandulosa var. glandulosa. J Nat Prod. 72:92-98.
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