Xanthine oxidase inhibitory activity of constituents of Cinnamomum cassia twigs

Article abstract of DOI:10.1016/j.bmcl.2012.05.051

A methanol extract of the twigs of Cinnamomum cassia was found to inhibit xanthine oxidase. Purification of the methanol extract afforded three new phenolic glycosides, cinnacasolide A-C (11-13), together with 10 known compounds (1-10). The structures of

Full text of DOI:10.1016/j.bmcl.2012.05.051

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4625–4628
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Xanthine oxidase inhibitory activity of constituents of Cinnamomum
cassia twigs
Tran Minh Ngoc a,b, Nguyen Minh Khoi a, Do Thi Ha a, Nguyen Xuan Nhiem b, Bui Huu Tai b, Dao Van Don c,
Hoang Van Luong c, Doan Cao Son d, KiHwan Bae b,
⇑
a National Institute of Medicinal Materials, Hanoi, Viet Nam
b College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
c Vietnam Military Medical University, Hanoi, Viet Nam
d National Institute of Drug Quality Control, Hanoi, Viet Nam
a r t i c l e i n f o
a b s t r a c t
Article history:
A methanol extract of the twigs of Cinnamomum cassia was found to inhibit xanthine oxidase. Purification
of the methanol extract afforded three new phenolic glycosides, cinnacasolide A–C (11–13), together with
10 known compounds (1–10). The structures of the three new compounds were determined by interpre-
tation of spectroscopic data. Cinnamaldehyde derivatives 1ꢀ5 and 7 were significant inhibitors of xan-
Received 19 March 2012
Revised 10 May 2012
Accepted 14 May 2012
Available online 18 May 2012
thine oxidase, with IC50 values ranging from 7.8 to 36.3 lg/mL. The results indicate that the acyl group
of these cinnamaldehyde derivatives plays an important role in the inhibition of xanthine oxidase.
Keywords:
Cinnamomum cassia
Cinnacasolide
Ó 2012 Elsevier Ltd. All rights reserved.
Cinnamaldehyde derivatives
Xanthine oxidase inhibitor
Ramulus cinnamomi, the dried young stem of Cinnamomum cas-
sia (Lauraceae), is distributed in Vietnam, Myanmar, Laos and the
southern part of mainland China. Ramulus cinnamomi has been
used as traditional Chinese medicines for treating dyspepsia, gas-
tritis, blood circulation disturbances, and inflammatory diseases.1
Previous investigations on the bark and the twig of C. cassia
showed the presence of a variety of monoterpenoids, sesquiterpe-
noids, diterpenoids, sterols, cinnamaldehyde and its analogues, and
flavan-3-ols and their oligomers as well as coumarins, and poly-
phenols.2,3 Pharmacological investigations showed that the crude
extract or compounds isolated from this species possessed a wide
variety of activities including anti-allergy, insecticidal, antimicro-
bial, antiulcer, anti-inflammatory, vasodilatory, immune-suppres-
sive, and neuronal death prevention, tyrosinase inhibition and
anticancer, antioxidant and free radical scavenging, as well as anti-
diabetic and aldose reductase inhibition activities.4–9 In the course
of our program to screening for xanthine oxidase inhibitors from
medicinal plants, it was found that a methanol extract of the twigs
of C. cassia exhibited a strong inhibitory activity (>85% inhibition at
XOD inhibitors, allopurinol, oxypurinol, and febuxostat have been
used widely for the treatment of hyperuricemia and gout. The inhi-
bition of XOD reduces both vascular oxidative stress and circulat-
ing levels of uric acid. In addition, the superoxide anion radicals
generated by XOD are involved in various pathological processes
such as hepatitis, inflammation, aging, carcinogenesis, and ische-
mia-reperfusion. Thus, XOD inhibitors may be useful for treatment
of many other diseases.10,11 The present study describes the isola-
tion and characterization of thirteen phenolic compounds, includ-
ing three new phenolic glucosides (11–13) and ten known
compounds (1–10), from the MeOH extract of the twigs of C. cassia
and their inhibitory activity against XOD.
The twigs of C. cassia was purchased from Yenbai Province, Viet-
nam in November 2010, and authenticate by Professor Pham
Thanh Ky, Department of Pharmacognosy, Hanoi College of Phar-
macy, Vietnam where the voucher specimens were deposited.
The MeOH extract of the twigs of C. cassia was partitioned into n-
hexane-, EtOAc-, and BuOH-soluble fractions. Repeated column
chromatography of those fractions resulted in the purification of
compounds 1ꢀ13 (Fig. 1). The 10 known compounds were identi-
fied as cinnamaldehyde (1),12 2-methoxycinnamaldehyde (2),13
2-hydroxycinnamaldehyde (3),14 cinnamic acid (4),15 coniferalde-
hyde (5),16 cinnamic alcohol (6),17 O-coumaric acid (7),18 dihy-
dromelilotoside (8),19 methyl dihydromelilotoside (9),20 and
rosavin (10).21 Three new metabolites cinnacasolide A (11), cinnac-
asolide B (12), and cinnacasolide C (13) were determined by
100 lg/mL).
Xanthine oxidase (XOD), which catalyzes the oxidation of xan-
thine and hypoxanthine into uric acid, plays a vital role in disor-
ders such as hyperuricemia and gout. Among the many known
⇑
Corresponding author. Tel.: +82 42 821 5925; fax: +82 42 821 6566.
E-mail address: baekh@cnu.ac.kr (K.H. Bae).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.051

4626
T. M. Ngoc et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4625–4628
Figure 1. Structures of isolated compounds 1–13.
spectroscopic data interpretation such as UV, IR, 1H NMR, 13C NMR,
HMQC, HMBC, COSY, and ROESY, as well as high-resolution mass
spectrometry (HREIMS).
and between the b-
osyl group at C-60. Consequently, compound 11 was methyl 3-hy-
doxybenzoyl-3-O-b- -apiofuranosyl-(1?6)-b- -glucopyranoside
and was named cinnacasolide A.
Compound 12 was purified as an amorphous solid. The molec-
ular formula, C20H28O11, was determined from a molecular ion
peak at m/z 443.1645 [M]ꢀ in the HREIMS spectrum (cacld for
D-apiofuranosyl group and the b-D-glucopyran-
D
D
Compound 11 was obtained as a colorless powder, with ½a D25
ꢁ
+27.5. Its infrared (IR) absorbance spectrum showed a band at
1675 cmꢀ1 due to a carboxyl group and strong absorption bands
at 3445 and 1123 cmꢀ1, which suggest a glycosidic structure. The
molecular formula was established as C19H26O12 from a molecular
ion peak at m/z 469.1326 [M+Na]+ in the HREIMS spectrum (cacld
for C19H26O12Na, 469.1322). 1H and 13C NMR spectra (Table 1) of 11
C20H28O11, 443.16348). The IR spectrum showed strong bands at
3885 and 1112 cmꢀ1, suggestive of a glycosidic structure. Com-
bined analyses of the 1H, 13C, DEPT and HMQC NMR spectra of
12 revealed the presence of an o-hydroxycinnamic alcohol skele-
ton. The data further contained an oxygenated methylene signal,
two trans-olefinic resonances, and o-hydroxyphenyl signals. The
1H and 13C NMR data (Table 1) also suggested the presence of
indicated the presence of a b-D-glucopyranosyl moiety, a b-D-apio-
furanosyl moiety, and methyl 3-hydroxybenzoate resonances. The
downfield shift of C-60 (dC 68.9) indicated a linkage to C-100 of an
apiofuranosyl moiety. Analysis of the HBMC spectrum of 11
showed long-range correlations between H-10 and C-3 and be-
tween H-100 and C-60 (Fig. 2), indicative of connections between
two sugar moieties, a b-D-glucopyranosyl unit and a b-D-apiofur-
anosyl unit, similar to those in 11. The HMBC spectrum revealed
the b-
D-glucopyranosyl group and the 3-hydroxy benzoyl at C-3
key correlations between the aromeric proton H-10 and C-2 and
Table 1
NMR spectroscopic data for 11–13 (in MeOD)
No.
11
12
No.
13
dH (J in Hz)
dC, mult
dH (J in Hz)
dC, mult
dH (J in Hz)
dC, mult
1
2
3
4
5
6
7
8
—
122.4
119.2
154.7
123.8
135.4
132.2
168.4
53.0
—
104.0
75.1
77.7
71.5
—
—
128.4
156.2
117.3
130.5
123.7
127.4
126.5
130.5
64.2
102.9
75.1
77.1
71.7
78.2
1
2
3
4
5
6
—
152.2
102.1
155.1
141.2
120.7
107.8
56.6
143.1
149.5
103.9
152.9
110.8
116.2
56.7
104.6
75.2
78.2
71.6
78.4
62.8
104.0
75.1
78.0
71.8
7.39 d (2.4)
—
7.57 dt (1.6, 8.0)
7.13 t (8.0)
7.76 dd (1.6, 8.0)
—
3.89 s
—
4.86 d (7.2)
3.48 m
3.56 m
3.31 m
3.45 m
4.01 d (10.0); 3.60 m
5.00 d (2.4)
3.91 d (2.4)
6.46 d (2.7)
—
—
7.00 d (8.7)
6.30 dd (2.7, 8.7)
3.81 s
7.18 t (8.0)
7.23 dt (1.6, 8.0)
6.99 t (7.2)
7.51 dd (1.2, 7.2)
7.07 d (16.0)
6.34 td (5.6, 16.0)
4.24 dd (1.6, 5.6)
4.86 d (7.2)
3.48 m
3.56 m
3.31 m
3.45 m
4.01 d (10.0); 3.60 m
4.89 d (2.4)
7-OCH3
10
—
—
9
20
10
20
30
40
50
60
100
200
300
400
500
30
6.81 d (2.7)
—
6.58 dd (2.7, 8.7)
6.69 d (8.7)
3.82 s
4.70 d (7.5)
3.44 m
3.56 m
3.31 m
3.45 m
3.90 d (10.5); 3.66 m
4.74 d (7.5)
3.45 m
40
50
60
78.1
68.9
111.1
77.3
80.6
700-OCH3
100
68.9
111.1
78.2
80.7
75.1
200
3.91 d (2.4)
—
3.96 d (9.6); 3.74 d (9.6)
3.57 s
300
—
400
3.96 d (9.6); 3.74 d (9.6)
3.57 s
75.1
65.5
500
65.7
600
1000
2000
3000
4000
5000
6000
3.56 m
3.31 m
3.45 m
3.68 d (10.5); 3.65 m
78.3
62.7

T. M. Ngoc et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4625–4628
4627
Figure 2. Selected HMBC correlations of 11–13.
between H-100 and C-60. These indicated linkages between b-
furanosyl unit and C-60 and between the b-
-glucopyranosyl group
and C-2 (Fig. 2). Accordingly, 12 was elucidated as 2-hydroxy cin-
namic alcohol 2-O-b- -apiofuranosyl-(1?6)-b- -glucopyranoside
D-apio-
Table 2
Inhibition effects of isolated compounds 1–13 against XOD
D
Compounds
Xanthine oxidasea
D
D
% Inhibition at (50
lg/mL)
IC50 (lg/mL)
and was named cinnacasolide B.
Cinnamaldehyde (1)
2-Methoxycinnamaldehyde (2)
2-Hydroxycinnamaldehye (3)
Cinnamic acid (4)
Coniferaldehyde (5)
Cinnamic alcohol (6)
o-Coumaric acid (7)
Dihydromelilotoside (8)
Methyldihydromelilotoside (9)
Rosavin (10)
96.7 2.3
95.8 1.6
94.2 2.1
93.4 2.3
72.5 2.5
47.6 3.4
73.2 1.6
45.4 2.3
22.4 2.0
18.7 2.1
22.2 2.0
35.4 1.7
28.2 2.1
N.D
7.8 1.1
13.8 1.5
14.6 2.0
26.4 1.2
36.3 1.9
>50
32.2 2.1
>50
>50
>50
>50
>50
>50
Compound 13 was obtained as a colorless powder with ½a D25
ꢁ
36.5. The molecular formula C26H34O15 was established from an
HREIMS ion peak at m/z 609.1869 [M+Na]+ (cacld for C19H26O12Na,
609.1862). The IR spectrum contained absorption peaks at 3354
and 1087 cmꢀ1, indicative of a glycosidic structure. The 1H NMR
spectrum of 13 contained peaks corresponding to two individual
b-glucopyranosyl groups with aromeric protons at dH 4.70 (H-10)
and 4.74 (H-100) and signals corresponding to a 1,3,4-trisubstituted
benzene ring at dH 6.46 (d, J = 2.7 Hz, H-2), 7.00 (d, J = 8.7 Hz, H-5),
and 6.30 (dd, J = 2.7, 8.7 Hz, H-6). Also evident were a 1,2,4-trisub-
tituted benzene ring at dH 6.81 (d, J = 2.7 Hz, H-30), 6.58 (dd, J = 2.7,
8.7 Hz, H-50), 6.69 (d, J = 8.7 Hz, H-60) and two resonances corre-
sponding to methoxy groups at dH 3,81 and 3.82 for 7-OCH3 and
70-OCH3, respectively. The 13C NMR spectrum of 13 indicated
twelve aromatic carbon atoms (6 CH and 6 C), two methoxy carbon
atoms, and two b-glucopyranose units. Two de-shielded 13C signals
at dC 152.2 (C-1) and 143.1 (C-10) in benzyl rings suggested the
presence of a C–O–C moiety. NMR data (Table 1) indicated that
13 is an arbutin dimer derivative with a structure very similar to
that of pyrocallianthaside A.22,23 HMQC, COSY, and HMBC spectra
revealed the linkage positions of the methoxy and glucose groups
to aromatic rings. Correlations were observed between 7-OCH3
and C-3, and between 70-OCH3 and C-20, indicating bonds between
Cinnacasolide A (11)
Cinnacasolide B (12)
Cinnacasolide C (13)
Allopurinolb
0.53 0.07
a
Values present mean S.D. of triplicate experiments.
Positive control. N.D.: Not determined.
b
and chemical structure. Cinnamaldehyde derivatives 1–3 and 5
were strong inhibitors of XOD with IC50 values ranging from 7.8
to 36.3
l
g/mL. The cinnamic acids 4 and 7 showed remarkable
g/mL,
degrees of inhibition, with IC50 values of 26.4 and 32.2
l
respectively. Other compounds, including the six phenolic gluco-
sides 8–13 and cinnamic alcohol (6) showed only weak inhibitory
effects, with percent inhibition values from 18.7% to 47.6% at a
dose of 50 lg/mL. These data suggest that an acyl group is an
3
two OCH3 groups and C-3 and C-20, respectively. The JC,H correla-
essential structural component that helps determine the XOD
inhibitory activity of a given compound. An aldehyde group played
an important role in the inhibition of XOD, as indicated by a com-
parison of cinnamic aldehyde (1) (IC50 7.8
(7) (IC50 26.4 g/mL), and cinnamic alcohol (10) (IC50 >50
These data indicate that cinnamaldehyde derivatives, isolated from
twigs of C. cassia, can significantly inhibit the activity of xanthine
oxidase.
In summary, three new phenolic glycosides, cinnacasolide A
(11), cinnacasolide B (12), and cinnacasolide C (13), and 10 known
compounds (1 – 10), were isolated from the twigs of C. cassia and
evaluated for their inhibition of xanthine oxidase. Cinnamaldehyde
derivatives 1ꢀ5 and 7 were the most potent inhibitors.
tions between H-100 and C-4 and between H-1000 and C-40 support
the respective linkage of the two b-D-glucopyranosyl units to C-4
and C-40 (Fig. 2). Accordingly, compound 13 was identified as
1-O,10-didehydrobis[3,20-dimethoxy phenyl b-glucopyranoside]
and was named cinnacasolide C.
l
g/mL), cinnamic acid
g/mL).
l
l
All of the isolated compounds 1–13,24 together with allopurinol
as a positive control,11 were examined for their inhibitory effects
on XOD activity in an in vitro assay.25 Table 2 shows the percent
inhibitory activity of each compound at a dose of 50 lg/mL. Com-
pounds 1–4, obtained from the hexane-soluble fraction of the ex-
tract, exhibited strong potential activity with percentages of
inhibition greater than 90%. Compounds 5–7 were purified from
the EtOAc-soluble fraction and also displayed significant inhibition
of XOD with percent inhibition values of 72.5 for 5, 47.6 for 6, and
73.2 for 7. Conversely, the phenolic glycosides 8–13, which were
isolated from the BuOH-soluble fraction, yielded weak or negligible
inhibitory activity against XOD. The isolated compounds 1–13
showed significant differences with regard to biological activity
Acknowledgments
This work was supported by a grand from the National Founda-
tion for Science and Technology Development, Vietnam (NAFO-
STED 2011–2012).

4628
T. M. Ngoc et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4625–4628
hexaneꢀEtOAc (10:1), and then purification by medium-pressure liquid
m), eluted with
MeOHꢀH2O (60:40), to give 3 (1.2 g). Compound 4 (2.0 g) was isolated from
fraction H7 using YMC-ODS column chromatography, with MeOHꢀH2O
(60:40) as eluent and and then purified with MPLC (YMC-ODS 15 ꢂ 300 mm,
Supplementary data
chromatography (MPLC) (YMC-ODS 15 ꢂ 300 mm,
5 l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
051.
5
lm) and MeOHꢀH2O (50:50) as mobile phase. The EtOAc-soluble fraction
(175 g) was chromatographed on a silica gel column with a stepwise gradient
of CHCl3 and MeOH (60:1 to 0:1), to separate eight fractions E1ꢀE8.
Subfraction E2 was chromatographed on a silica gel column with mixtures of
hexaneꢀEtOAc (10:1 to 2:1) as the eluting solvent systems, and preparative
References and notes
HPLC (YMC-ODS, 10 ꢂ 200 mm, 5 lm), MeOHꢀH2O (40:60) as eluting mobile-
1. Loi, D. T. Vietnamese Medicinal Plants and Ingredients; Medical Publishing
House: Hanoi, 2004. p 862.
2. Nohaza, T.; Kashiwada, Y.; Murakami, K.; Tomimasu, T.; Kido, M.; Yagi, A.;
Hishioka, I. Chem. Pharm. Bull. 1981, 29, 2451–2459.
3. Yazaki, K.; Okudu, T. Phytochemistry 1990, 29, 1559.
4. Singh, H. B.; Srivastava, M.; Singh, A. B.; Srivastava, A. K. Allergy 1995, 50, 995.
5. Kwon, B. M.; Lee, S. H.; Choi, S. U.; Park, S. H.; Lee, C. O.; Cho, Y. K.; Sung, N. D.;
Bok, S. H. Arch. Pharm. Res. 1998, 21, 147.
6. Kurokawa, M.; Kumeda, C. A.; Yamamura, T.; Shiraki, K. Eur. J. Pharm. 1998, 348,
45.
7. Lin, C. C.; Wu, S. J.; Chang, C. H.; Ng, L. T. Phytother. Res. 2003, 17, 726.
8. Kim, H. O.; Park, S. W.; Park, H. D. Food Microbiol. 2004, 21, 105.
9. Cheng, S. S.; Liu, J. Y.; Tsai, K. H.; Chen, W. J.; Chang, S. T. J. Agric. Food Chem.
2004, 52, 4395.
10. Nguyen, M. T. T.; Awale, S.; Tezuka, Y.; Tran, Q. L.; Kadota, S. Chem. Pharm. Bull.
2005, 53, 984.
11. Higgins, P.; Ferguson, L. D.; Walters, M. R. Expert Rev. Cardiovasc. Ther. 2011, 9,
399.
12. Choi, J.; Lee, K. T.; Ka, H.; Jung, W. T.; Jung, H. J.; Park, H. J. Arch. Pharm. Res.
2001, 24, 418.
phase, to afford 5 (10 mg). Subfraction E4 was supplied to silica gel column
chromatography, using hexane–EtOAc (4:1 ꢃ2:1) as the eluting gradient
systems, and then purification by MPLC (YMC-ODS, 15 ꢂ 300 mm,
5 lm),
eluted with MeOHꢀH2O (60:40) to give
6 (250 mg). Subfraction E5 was
subjected to a silica gel column chromatography, eluted with hexane–acetone
(2:1) to give 7 (36 mg) after purifying by YMC ODS column chromatography,
eluting with MeOH–H2O (50:50). The n-BuOH-soluble fraction (85 g) was
suspended in H2O and then applied to a Diaion HP-20 column with a H2O and
MeOH gradient eluent to give fractions B1ꢀB8. Fraction B5 was subjected to
passage over silica gel, using CHCl3–MeOHꢀH2O (10:1:0.1 to 1:1:0.1) for
elution, to yield nine subfractions B5.1–B5.9. Sub-fraction B5.5 was applied to a
Sephadex LH-20 column, eluted with MeOHꢀH2O (1:5), and then YMC-ODS
column chromatography with the mobile phase system, MeOHꢀH2O (1:4), to
give 12 (5 mg). Fraction B5.6 was chromatographed on a Sephadex LH-20
column with MeOHꢀH2O (1:5) as solvent, and then purified by reversed-phase
HPLC (YMC-ODS, 10 ꢂ 250 mm, 5
and yielded (15 mg),
l
m), with MeOHꢀH2O (1:5) as mobile phase,
8
9
(10 mg), 10 (42 mg). Fraction B7 was
chromatographed on a silica gel column, using CHCl3–MeOHꢀH2O (10:1:0.1
to 3:1:0.1) to give five subfractions B7.1–B7.5. Subfraction B7.3 was subjected
to passage over LH-20 column with MeOHꢀH2O (1:5) as solvent, and then
13. Geirsson, J. K. F.; Arnadottir, L.; Jonsson, S. Tetrahedron 2004, 60, 9149.
14. Byoung, M. K.; Young, K. C.; Seung, H. L.; Ji, Y. N.; Song, H. B.; Soo, K. C.; Jeong, A.
K.; Ihn, R. L. Planta Med. 1996, 62, 183.
15. Huong, D. T. L.; Jo, Y. S.; Lee, M. K.; Bae, K. H.; Kim, Y. H. Nat. Prod. Sci. 2000, 6,
16.
purified by reversed-phase HPLC (YMC-ODS, 10 ꢂ 250 mm,
5 lm), with
MeOHꢀH2O (1:4) as mobile phase to give 11 (7.5 mg), and 13 (2.9 mg).
25. Valentao, P; Frenandes, E; Carvalho, F; Andrade, B. P; Seabra, R. M; Bastos, M. L.
Bio. Pharm. Bull. 2002, 25, 1324. The inhibitory activity against xanthine
oxidase was evaluated by measuring the formation of uric acid from xanthine
16. Sy, L. K.; Brown, G. D. Phytochemistry 1999, 50, 781.
17. Mohammad, M.; Mojtahedi, E. A.; Sharifi, R.; Abaee, M. S. Org. Lett. 2007, 9,
2791.
by
contained 50 mM sodium carbonate buffer (pH 7.8), 50
0.1 mM EDTA in the presence or absence of tested compound. The
a
spectrophotometer at ambient temperature. The reaction mixture
l
M xanthine, and
18. Aldrich Library of 13C and 1H FT NMR Spectra, Sigma–Aldrich: milwaukee, 1992;
Vol. 2, 1046A, 1046B.
a
absorbance was read at 295 nm after 5 min. The inhibitory activity was
determined as the mean of triplicate measurements and is expressed as IC50
value from the formative uric acid of the control.
19. Taskova, R. M.; Gotfredsen, C. H.; Jensen, S. R. Phytochemistry 2005, 66, 1440.
20. Malakov, P. Y.; Papanov, G. Y.; De La Torre, M. C.; Rodriguez, B. Fitoterapia 1998,
69, 552.
21. Kishida, M.; Akita, H. Tetrahedron: Asymmetry 2005, 16, 2625.
22. Chen, Y. L.; Tan, C. H.; Tan, J. J.; Qu, S. J.; Wang, H. B.; Zhang, Q.; Jiang, S. H.; Zhu,
D. Y. Helv. Chim. Acta 2002, 90, 2421.
23. Meng, D.; Wu, J.; Zhao, W. Phytochemistry 2010, 71, 325.
24. The sliced twigs of C. cassia (20 kg) were extracted with hot MeOH (3 ꢂ 40 L),
and then the MeOH extract was concentrated. The residue (1260 g) was
suspended in H2O (4 L) and then fractionated successively with n-hexane, ethyl
acetat (EtOAc), and n-butanol (BuOH), producing a hexane-soluble (310 g),
EtOAc-soluble (175 g), and n-BuOH-soluble (85 g) fractions, respectively. The
hexane-soluble fraction (310 g) was subjected to silica gel column
chromatography, eluted with hexane and EtOAc gradient mixtures
(100:1 ? 0:100) to give seven fractions H1ꢀH7. Fraction H2 was
chromatographed on silica gel, eluting with hexane–EtOAc (25:1), as well as
ODS silica gel with MeOHꢀH2O (55:45), to afford 1 (3.0 g), and 2 (1.0 g).
Fraction H6 was subjected to silica gel column chromatography, eluting with
26. Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501. Acid Hydrolysis
of Compounds 11–13. Solutions of 11 (1.2 mg), 12 (1.1 mg) and 13 (1.0 mg) in
0.5 N H2SO4 (dioxaneꢀH2O, 1:1, 2 mL) were each heated at 100 oC for 2 h. Each
reaction mixture was diluted with H2O (3 mL) and extracted with CHCl3
(5 mL ꢂ 3 times). The H2O layer was dried in vacuo after neutralization with
1 N NaOH and passed through a Sep-Pak C18 cartridge (Waters, Milford, MA).
Sugars were identified as glucose (Rf 0.16), and apiose (Rf 0.38) by TLC in
CHCl3–MeOH–H2O (12:6:1), using the authentic samples,
apiose. The remaining eluate was concentrated to dryness, and the residue was
stirred with -cysteine methyl ester hydrochloride, hexamethyldisilazane and
trimethylsilylchloride in pyridine using the same procedures as in a previous
D-glucose, and D-
D
26
report.
After the reaction, the supernatant was analyzed by GC [column:
capillary column BD-5, 0.25 mm ꢂ 30 m, detector: FID, detector temperature:
300 °C, injector temperature: 270 °C, carrier gas, N2; column temperature,
210 °C]. The peaks corresponding to the
D-glucosyl and D-apiosyl derivatives
appeared with tR 8.55 and 4.03 min, respectively.