A new picrotoxane sesquiterpene from the berries of Baccaurea ramiflora with antifungal activity against Colletotrichum gloeosporioides

Article abstract of DOI:10.1080/14786419.2014.999335

(Image Presented) Three picrotoxane sesquiterpenes including one new glycoside and two known constituents, sapidolide A (2) and picrotoximaesin (3), were isolated from the berries of Baccaurea ramiflora. The structure of the new sesquiterpene glycoside, ramifloside (1), was elucidated as 2-one-6α-hydroxy-13-nor-11-picrotoxen-3(15β)-olide 10-O-β-d-glucopyranoside on the basis of extensive spectroscopic analysis. Compounds 1-3 exhibited antifungal activity against Colletotrichum gloeosporioides with MICs of 12.5, 12.5 and 50 g/mL.

Full text of DOI:10.1080/14786419.2014.999335

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A new picrotoxane sesquiterpene from
the berries of Baccaurea ramiflora
with antifungal activity against
Colletotrichum gloeosporioides
Zheng-Hong Pana, De-Sheng Ninga, Si-Si Huanga, Yun-Fei Wub, Tao
Dinga & Lei Luob
a Guangxi Key Laboratory of Functional Phytochemicals Research
and Utilization, Guangxi Institute of Botany, Guangxi Zhuang
Autonomous Region and Chinese Academy of Sciences, Guilin
541006, P.R. China
Click for updates
b Faculty of Chemistry and Chemical Engineering, Yunnan Normal
University, Kunming 650500, P.R. China
Published online: 13 Jan 2015.
To cite this article: Zheng-Hong Pan, De-Sheng Ning, Si-Si Huang, Yun-Fei Wu, Tao Ding & Lei Luo
(2015) A new picrotoxane sesquiterpene from the berries of Baccaurea ramiflora with antifungal
activity against Colletotrichum gloeosporioides, Natural Product Research: Formerly Natural
Product Letters, 29:14, 1323-1327, DOI: 10.1080/14786419.2014.999335
To link to this article: http://dx.doi.org/10.1080/14786419.2014.999335
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Natural Product Research, 2015
Vol. 29, No. 14, 1323–1327, http://dx.doi.org/10.1080/14786419.2014.999335
A new picrotoxane sesquiterpene from the berries of Baccaurea ramiflora
with antifungal activity against Colletotrichum gloeosporioides
Zheng-Hong Pana*, De-Sheng Ninga, Si-Si Huanga, Yun-Fei Wub, Tao Dinga and Lei Luob
aGuangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute
of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006,
P.R. China; bFaculty of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming
650500, P.R. China
(Received 12 October 2014; final version received 11 December 2014)
OH
O
HO
HO
O
O
H
O
OH
OH
O
Three picrotoxane sesquiterpenes including one new glycoside and two known
constituents, sapidolide A (2) and picrotoximaesin (3), were isolated from the berries of
Baccaurea ramiflora. The structure of the new sesquiterpene glycoside, ramifloside (1),
was elucidated as 2-one-6a-hydroxy-13-nor-11-picrotoxen-3(15b)-olide 10-O-b-D-
glucopyranoside on the basis of extensive spectroscopic analysis. Compounds 1–3
exhibited antifungal activity against Colletotrichum gloeosporioides with MICs of 12.5,
12.5 and 50 mg/mL.
Keywords: Baccaurea ramiflora; picrotoxane; sesquiterpene glycoside; antifungal
activity; ramifloside
1. Introduction
Baccaurea ramiflora Lour., a tall evergreen tree in the family Euphorbiaceae, is widely
distributed in the Karst region of southwest China (Li 1994). The whole plant has been used as a
folk medicine in China to treat rheumatoid arthritis, cellulitis, apostema, injury from fall, etc.
(Lin et al. 2003). Previous chemical investigations of B. ramiflora have showed the presence of
phenolic, sesquiterpenoid and a series of volatile components (Yang et al. 2007, 2010; Xu, Guan,
et al. 2007; Xu, Lin, et al. 2007). As a part of our search for naturally occurring bioactive
metabolites from Karst plants, we investigated the chemical constituents of the berries of
B. ramiflora, which led to the isolation of a new nor-picrotoxane sesquiterpene glycoside, named
ramifloside (1), along with two picrotoxane sesquiterpenes: sapidolide A (2) and
picrotoximaesin (3) (Bordoloi et al. 1996; Tane et al. 1996) (Figure 1). To the best of our
*Corresponding author. Email: pan7260@126.com
q 2015 Taylor & Francis

1324
Z.-H. Pan et al.
OH
6'
4'
O
HO
5'
1'
O
O
10
9
2'
HO
H
H
H
3'
OH
8
14
7
O
O
6
1
O
2
O
O
HO
OH
OH
OH
HO
O
O
O
15
5
3
4
11
12
2
3
1
Figure 1. Structure of compounds 1–3.
knowledge, the new compound was the first nor-picrotoxane glycoside from plant origin, and the
two known constituents were reported for the first time from this plant. This article describes the
isolation, structural elucidation and antifungal activity of these picrotoxane sesquiterpenes.
2. Results and discussion
Compound 1 was obtained as a white amorphous solid. The molecular formula of 1 was
established to be C20H28O10 by HR-ESI-MS at m/z 451.1576 [M þ Na]þ (calcd for
C20H28O10Na, 451.1575), indicating seven degrees of unsaturation. Its IR spectrum implied
the presence of hydroxyl (3424 cm21) and carbonyl (1794 and 1711 cm21) groups. The 13C
NMR and DEPT spectra exhibited 20 carbon signals due to 1 methyl, 5 methylene (including 2
oxygenated and 1 olefinic), 10 methine (including 6 oxygenated and 1 olefinic), 2 quaternary
carbon (including 1 oxygenated) and 2 carbonyl resonances. The 1H NMR spectrum revealed the
presence of a terminal double bond at dH 5.16 (2H, dd, J ¼ 17.5, 10.5 Hz) and dH 6.07 (ddd,
J ¼ 17.5, 10.5, 10.5 Hz), and a tertiary methyl singlet at dH 1.29. The six characteristic 13C NMR
signals at dC 104.3, 77.5, 77.5, 74.9, 71.1 and 62.3 suggested the presence of a glucose moiety.
Gas chromatography (GC) analysis of hydrolyzate of 1 established the D-configuration of the
sugar moiety. Careful analysis of NMR data of 1 revealed that the remaining signals were very
similar to those of sapidolide A (2), except for the presence of the above sugar signals and an
extra ketonic signal (dC 209.1) in 1 and the absence of C-2 signal (dC 103.0) in 2. The HMBC
correlations of a ketonic carbon at dC 209.1 with H-3 (dH 4.53), H-4 (dH 3.65), H-9 (dH 2.22) and
H-14 (dH 1.29) indicated that the ketonic group was attached to C-2 (see Supplementary
material, Figure S1). The glucose moiety located at C-10 was confirmed by the HMBC
correlation between H-10 (dH 4.13) and C-10 (dC 70.8) and has a b-anomeric orientation
determined by the large coupling constant (J ¼ 9.0 Hz) of the anomeric proton H-10. In order to
better determine the stereochemistry of 1, the NMR spectra of 1 recorded in DMSO-d6 solvent
were determined (see Supplementary material, Figures S8–S11). The relative configuration of
the aglycone of 1 was characterised on the basis of the NOESY spectrum, in which NOE
correlations were found between H-11 and H-3, H-5, Me-14, OH-6; H-9 and Me-14, OH-6; and
H-3 and Me-14. Furthermore, the NMR data of 1 were in very close accord with those of
sapidolide A (2), which was also isolated from the same plant, and its stereochemistry was
confirmed by X-ray diffraction experiment (Bordoloi et al. 1996). Therefore, the structure of
compound 1 was elucidated as 2-one-6a-hydroxy-13-nor-11-picrotoxen-3(15b)-olide 10-O-b-
D-glucopyranoside, named as ramifloside.

Natural Product Research
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Because detailed 1H and 13C NMR spectral data of sapidolide A (2) were not reported in the
literature (Bordoloi et al. 1996), we performed the complete assignments of 1D NMR spectra of
2 (see Supplementary material, Table S1). The other known compound was identified as
picrotoximaesin (3) by analysis of the spectroscopic data and comparison with the literature
values (Tane et al. 1996). Compounds 2 and 3 were isolated from B. ramiflora for the first time.
Since sapidolide A (2) was reported to exhibit antifungal activity (Bordoloi et al. 1996), the
three isolates were tested for the antifungal activity against the phytopathogen Colletotrichum
gloeosporioides, using carbendazim as a positive controls (MIC ¼ 6.25 mg/mL). Compounds 1
and 2 exhibited significant antifungal activities against C. gloeosporioides with minimum
inhibitory concentrations (MICs) of 12.5 and 12.5 mg/mL, while compound 3 exhibited weak
antifungal activity with MIC of 50 mg/mL. The above results suggest that the terminal double
bond in 1 and 2 plays an important role in the antifungal activity.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured with a Horiba SEPA-300 polarimeter (Horiba, Tokyo, Japan).
IR spectroscopy was measured in a Bio-Rad FTS-135 spectrometer (Bio-Rad, Richmond, CA,
USA) with KBr pellets. MS were performed on an Agilent 6500 Q-TOF spectrometer (Agilent
Technologies, Santa Clara, CA, USA). 1D NMR and 2D NMR spectra were recorded on DRX-
500 spectrometer (Bruker Co., Ettlingen, Germany) with TMS as internal standard. Unless
specified, chemical shifts (d) are expressed in ppm with reference to the solvent signals. Column
chromatography was performed either on silica gel (100–200 and 200–300 mesh; Qingdao
Marine Chemical Inc., Qingdao, China), Lichroprep RP-18 gel (40–63 mm; Merck, Darmstadt,
Germany) or on Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden). Fractions
were monitored by TLC, and spots were visualised by heating silica gel plates sprayed with 10%
H2SO4 in EtOH. Solvents were distilled before use.
3.2. Plant material
The berries of B. ramiflora were collected from Longzhou County of Guangxi province, China,
in August 2013. Voucher specimens (GXIB20130801) were deposited at the Guangxi Key
Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany
and were identified by one of the authors (Associate Professor Tao Ding).
3.3. Fungal material
The fungal strain C. gloeosporioides was purchased from the Agricultural Culture Collection of
China, Beijing, China. It was deposited at the Guangxi Key Laboratory of Functional
Phytochemicals Research and Utilization, Guangxi Institute of Botany. This fungus was
incubated in the potato dextrose (PD) medium.
3.4. Extraction and isolation
The air-dried and powdered berries of B. ramiflora (2.7 kg) were extracted with EtOH (3 £ 10 L,
each 24 h) at room temperature and filtered. The filtrate was evaporated to give a residue, which
was suspended in H2O (3 L) and then extracted with EtOAc (4 £ 2 L). The EtOAc extract (580 g)
was subjected to silica gel column chromatography with a step gradient system (petroleum
ether–Me2CO, 1:0 to 0:1) to obtain fractions 1–4. Fraction 2 was subjected to silica gel column
chromatography (petroleum ether–Me2CO, 4:1 to 0:1) and then further purified by Sephadex

1326
Z.-H. Pan et al.
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.

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Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and
Figures S1–S15.
Funding
This work was supported financially by the National Natural Science Foundation of China [number
21102023] and the funds of Guangxi Key Laboratory of Functional Phytochemicals Research and
Utilization [number ZRJJ2012-8], [number ZRJJ2013-8].
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