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LH-20 (CHCl3–MeOH, 1:1) to yield 2 (500 mg) and 3 (60 mg). Fraction 3 was further subjected
to silica gel column chromatography using petroleum ether–Me2CO (4:1 to 0:1) as eluent to
provide subfractions 3.1–3.3. Subfraction 3.2 was applied to an RP-18 gel column with a
gradient elution (H2O–MeOH, 40–80%), and further purified by silica gel column
chromatography (CHCl3–Me2OH, 9:1) to obtain compound 1 (260 mg).
3.4.1. Ramifloside (1)
White amorphous solid; ½aꢀD 2 8.8 (c ¼ 0.11, MeOH); IR (KBr) nmax cm21: 3424, 2922,
12:4
1794, 1711, 1635, 1382, 1315, 1075, 1019; HR-ESI-MS (positive mode) m/z: 451.1576
[M þ Na]þ (calcd for C20H28O10Na, 451.1575). 1H NMR data (MeOH; 500 MHz), d: 4.53 (1H,
d, J ¼ 5.5 Hz, H-3), 3.65 (1H, m, H-4), 2.87 (1H, d, J ¼ 4.5 Hz, H-5), 2.33 (1H, dd, J ¼ 6.5, 12.5
Hz, H-7a), 1.84 (1H, m, H-7b), 1.91 (1H, m, H-8a), 1.35 (1H, m, H-8b), 2.22 (1H, m, H-9), 3.88
(1H, dd, J ¼ 4.5, 9.5 Hz, H-10a), 3.16 (1H, br d, J ¼ 9.5 Hz, H-10b), 6.07 (1H, ddd, J ¼ 10.5,
10.5, 17.5 Hz, H-11), 5.16 (2H, dd, J ¼ 10.5, 17.5 Hz, H2-12), 1.29 (3H, s, H3-14), 4.13 (1H, d,
J ¼ 9.0 Hz, H-10), 3.13 (1H, dd, J ¼ 7.5, 9.0 Hz, H-20), 3.32 (1H, m, H-30), 3.28 (1H, m, H-40),
3.24 (1H, m, H-50), 3.82 (1H, dd, J ¼ 12.0, 2.0 Hz, H-60a), 3.68 (1H, m, H-60b). 13C NMR data
(MeOH; 125 MHz), d: 59.6 (C-1), 209.1 (C-2), 85.0 (C-3), 49.6 (C-4), 54.8 (C-5), 82.7 (C-6),
41.2 (C-7), 28.0 (C-8), 55.1 (C-9), 70.8 (C-10), 132.4 (C-11), 120.0 (C-12), 24.8 (C-14), 176.5
(C-15), 104.3 (C-10), 74.9 (C-20), 77.5 (C-30), 71.1 (C-40), 77.5 (C-50), 62.3 (C-60).
3.5. Acid hydrolysis of compound 1 and determination of sugar component
Compound 1 (1.0 mg) was hydrolysed with 1 M HCl–dioxane (1:1, 1 mL) at 808C for 4 h. The
reaction mixture was partitioned between EtOAc and H2O three times. The aqueous layer was
neutralised with 2 M NaHCO3 and evaporated in vacuo. The residue was dissolved in pyridine
(0.5 mL), to which L-cysteine methyl ester hydrochloride in pyridine (0.1 M, 0.5 mL) was added.
After reacting at 608C for 1 h, trimethylsilylimidazole (0.5 mL) was added to the reaction
mixture and kept at 608C for another 30 min. The mixture was partitioned between n-hexane and
H2O, and the n-hexane extract was analysed by GC–MS. By comparison of the retention time of
the authentic sample, the monosaccharide of compound 1 was determined to be D-glucose
(tR ¼ 19.25).
3.6. Antifungal activity against C. gloeosporioides
Antifungal assay was performed by the microdilution method in 96-well flat-microtitre plates
using PD medium (Li et al. 2012). The compounds were made up to 2 mg/mL in DMSO.
Carbendazim was used as positive control and the equal concentration of DMSO was used as a
negative control. The fungus was incubated in the PD medium for 18 h at 28 ^ 0.58C at 150 rpm,
and spores of different microorganism concentrations were diluted to approximately 1 £ 106
CFU with PD medium. In flat-microtitre plates, tested compounds, fungal suspension and sterile
water were added to make up final concentrations of the compounds in the range of 1.54–
200 mg/mL. After incubation at 28 ^ 0.58C for 48 h, MIC was determined as the lowest
concentrations that produce complete growth inhibition of the tested microorganisms. All
experiments were repeated three times.
4. Conclusion
In conclusion, three picrotoxane sesquiterpenes were isolated from the berries of B. ramiflora,
including one new glycoside named as ramifloside (1). Compounds 1–3 exhibited antifungal
activity against C. gloeosporioides with MICs of 12.5, 12.5 and 50 mg/mL.