Q. Han et al.
Phytochemistry 185 (2021) 112674
afford 5 (12.1 mg, tR 41.9 min).
4.6. Enzymatic hydrolysis of 7
20
Julibroside K (1): white, amorphous powder; [
α
]
ꢀ 43 (c 0.1,
D
MeOH); UV (MeOH) λmax (log
ε
) 213 (4.6) nm; IR (KBr) νmax 3445, 2929,
Compound 7 (0.7 mg) and snailase (2.0 mg) were dissolved in ace-
tate buffer (0.2 M, pH 5.0, 1.0 mL) and incubated at 55 ◦C for 72 h. The
reaction mixture was extracted with CH2Cl2 to give compound 8
1707, 1648, 1599, 1567, 1515, 1454, 1384, 1133, 1025, 842, 676,
579 cmꢀ 1
;
1H and 13C NMR data, see Tables 1–3; MALDITOFMS m/z
2193.7686 [M
+
Na]+(calcd for
C
102H162O49Na, 2194.0083),
(0.3 mg)
2209.7573 [M + K]+ (calcd for C102H162O49K, 2209.9822).
Compound 8: [
α
]
+ 17 (c 0.11, CHCl3); 1H NMR (400 MHz,
20
CDCl3): δH 6.89 (1H, t, JD= 7.2 Hz, H-3), 5.91 (1H, dd, J = 10.6, 17.4 Hz,
H-7), 5.24 (1H, d, J = 17.2 Hz, H-8a), 5.10 (1H, d, J = 10.8 Hz, H-8b),
2.24 (2H, m, H-4), 1.83 (3H, s, H-9), 1.66 (2H, m, H-5), 1.32 (3H, s, H-
10).
20
Julibroside L (2): white, amorphous powder; [
α
]
ꢀ 20 (c 0.13,
D
MeOH); UV (MeOH) λmax (log ε) 213 (4.6) nm; IR (KBr) νmax 3445, 2929,
1707, 1648, 1599, 1567, 1515, 1454, 1384, 1133, 1025, 842, 676,
579 cmꢀ 1
;
1H and 13C NMR data, see Tables 1–3; MALDITOFMS m/z
2310.0022 [M
+ Na]+(calcd for C107H170O52Na, 2310.0556),
2325.9697 [M + K]+ (calcd for C107H170O52K, 2326.1107).
4.7. Strong alkaline hydrolysis of 6
20
Julibroside M (3): white, amorphous powder; [
α
]
ꢀ 30 (c 0.12,
D
MeOH); UV (MeOH) λmax (log ε) 212 (4.6) nm; IR (KBr) νmax 3372, 2930,
Compound 6 was hydrolyzed with 3% NaOH (1.0 mL) and MeOH
(1.0 mL) for 10 h at room temperature. After adjusting the pH to 5.0 with
2M HCl, the reaction mixture was extracted successively with EtOAc and
n-BuOH. The EtOAc extract of 6 was purified by preparative HPLC using
31% CH3CN (2.5 mL/min) to yield compound 10 (0.2 mg), the n-BuOH
extract was separated by preparative HPLC using 28% CH3CN (2.5 mL/
min) to afford compound 9 (0.3 mg).
1736, 1693, 1386, 1229, 1073, 591 cmꢀ 1
;
1H and 13C NMR data, see
Tables 1–3; MALDITOFMS m/z 2029.8301 [M + Na]+ (calcd for
C
C
91H146O48Na, 2029.8882), 2045.8099 [M
91H146O48K, 2045.9386).
+
K]+ (calcd for
20
Julibroside N (4): white, amorphous powder; [
α
]
ꢀ 30 (c 0.1,
MeOH); UV (MeOH) λmax (log
ε
) 213 (4.6) nm; IR (KBDr), νmax 3416,
2922, 1722, 1676, 1654, 1439, 1384, 1152, 1028, 876, 664, 580 cmꢀ 1
;
1H and 13C NMR data, see Table 1–3; MALDITOFMS m/z 1867.7755 [M
+ Na]+ (calcd for C85H136O43Na, 1867.8353), 1883.7560 [M + K]+
4.8. Cytotoxicity assays
(calcd for C85H136O43K, 1883.9438).
20
Julibroside O (5): white, amorphous powder; [
α
]
ꢀ 30 (c 0.09,
D
Human lung cancer (A549), colorectal cancer (HCT116), gastric
cancer (BGC-823), and liver cancer (HepG2) cell lines were obtained
from the State Key Laboratory of Natural and Biomimetic Drugs, Peking
University Health Science Center (Beijing, People’s Republic of China).
Cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM,
M&C Gene Technology., Ltd, Beijing, China) and supplemented with
10% fetal bovine serum (PAN-Biotech, Aidenbach, Germany), 100 U/mL
MeOH); UV (MeOH) λ
(log ε) 213 (4.6) nm; IR (KBr), νmax 3394,
max
2936, 1751, 1686, 1638, 1459, 1388, 1229, 1073, 648 cmꢀ 1; 1H and 13
C
NMR data, see Tables 1–3; MALDITOFMS m/z 2027.8531 [M + Na]+
(calcd for C92H148O47Na, 2027.9089), 2043.8329 [M + K]+ (calcd for
C
85H136O43K, 2044.0174).
penicillin, and 100 μ
g/mL streptomycin at 37 ◦C with 5% CO2.
4.4. Determination of the absolute configuration of the monosaccharides
Cytotoxic activity was investigated using the MTT colorimetric
method (Alley et al., 1988). Briefly, cells were seeded in a 96-well plate
An HPLC-UV-based method was performed for monosaccharide
determination (Tanaka et al., 2007). Compound 1 (2.0 mg) was dis-
solved in 2M HCl–H2O (2.0 mL) and heated at 95 ◦C for 10 h. The re-
action mixture was extracted with CH2Cl2 3 times, the aqueous layer
contained sugars was evaporated under vacuum to furnish a neutral
residue. L-Cysteine methyl ester hydrochloride (2.0 mg) and anhydrous
pyridine (1.0 ml) were added to the mixture and stirred at 60 ◦C for 1 h.
at a density of 5 × 103 cells per well in 180
μL of medium for 12 h, fol-
lowed by exposure to the test compounds or positive control. After 48 h
exposure, 20
incubated at 37 ◦C for 4 h. Supernatant fractions were aspirated, and
100 L of DMSO was added to each well. The absorbance was measured
μL of MTT (5 mg/mL) was added and the cells were further
μ
at 570 nm using a microplate reader (EnVision, PerkinElmer Life-
sciences, USA). Cytotoxic activity was expressed as IC50 values (con-
centration inhibiting the proliferation rate of tumor cells by 50%,
compared to untreated control cells). All experiments were carried out
in quadruplicate. Taxol was used as the positive control.
The product was mixed with 10 μL o-tolylisothiocyanate and kept at
60 ◦C for another 1 h. The final reaction mixture was analyzed by HPLC
under the following conditions: an Agilent 1260 chromatograph
equipped with a phenomenex column (5 μm, 4.6 × 250 mm); column
temperature: 30 ◦C; mobile phase: isocratic elution of 23% CH3CN–H2O
containing 0.2% formic acid for 50 min and subsequent washing of the
column with 90% CH3CN–H2O for 10 min, flow rate: 0.8 mL/min; in-
Associated content
jection volume: 10 μL; UV detection wavelength: 250 nm. From the acid
Supporting information available.
hydrolysate of 1, L-arabinose, L-rhamnose, D-fucose, D-quinovose,
D-xylose, and D-glucose were confirmed by comparison of the retention
times of their derivatives with those standard sugars derivatized in a
similar way, which showed retention times of 27.16, 40.68, 33.08,
38.08, 28.07, and 24.24 min, respectively. The constituent sugars of
compounds 2–5 were also identified by the same method.
NMR, MS and IR spectra of compounds 1–5 (PDF)
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.5. Mild alkaline hydrolysis of 1
Solution of compound 1 (5.0 mg) with saturated NaHCO3 in MeOH
(2.0 mL) were refluxed for 1 h, respectively. The reaction mixtures were
concentrated in vacuo to dryness. Then, the residues were dissolved in
water and partitioned successively with EtOAc and n-BuOH respectively.
The EtOAc extract of 1 was purified by preparative HPLC using 31%
CH3CN (2.5 mL/min) respectively to yield compound 7 (0.7 mg, tR
22.2 min), the n-BuOH extract was separated by preparative HPLC using
30% CH3CN (2.5 mL/min) to afford compound 6 (1.0 mg, tR 20 min).
Acknowledgements
This study was financially supported by The Drug Innovation Major
Project (2018ZX09711001-008-003). We are grateful to Prof. Mingying
Shang (Peking University Health Science Center) for identifying the
plant material. We thank the State Key Laboratory of Natural and Bio-
mimetic Drugs, Peking University Health Science Center, for offering
cancer cell lines and certain spectroscopic measurements.
7