New phenolic constituents from the fruit juice of Phyllanthus emblica

Article abstract of DOI:10.1248/cpb.49.537

Six new phenolic constituents, L-malic acid 2-O- (1), mucic acid 2-O- (5), mucic acid 1,4-1actone 2-0- (6), 50- (8), 3-0- (10), and 3,5-di-0- (11) gallates, were isolated from the fruit juice of Phyllanthus emblica together with their methyl esters (2-4,

Full text of DOI:10.1248/cpb.49.537

May 2001
Chem. Pharm. Bull. 49(5) 537—540 (2001)
537
New Phenolic Constituents from the Fruit Juice of Phyllanthus emblica
a
a
b
,a
Ying-Jun ZHANG, Takashi TANAKA, Chong-Ren YANG, and Isao KOUNO*
a
Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo Machi 1–14, Nagasaki 852–8521, Japan and
b
Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China.
Received October 25, 2000; accepted January 18, 2001
Six new phenolic constituents, L-malic acid 2-O- (1), mucic acid 2-O- (5), mucic acid 1,4-lactone 2-O- (6), 5-
O- (8), 3-O- (10), and 3,5-di-O- (11) gallates, were isolated from the fruit juice of Phyllanthus emblica together
with their methyl esters (2—4, 7, 9), and their structures were determined by spectral and chemical methods.
Compounds 5, 6, and 8, the major phenolic constituents of the juice, were present as an equilibrium mixture in
aqueous solution.
Key words Phyllanthus emblica; Euphorbiaceae; organic acid gallate; mucic acid
9
)
10)
Phyllanthus emblica L. (Euphorbiaceae) is a shrub or tree chebulic acid, and 1-O-galloyl-b-D-glucose, respectively.
growing in subtropical and tropical areas of the People’s Re-
Compound 1 was obtained as a white amorphous powder
public of China, India, Indonesia, and the Malay Peninsula. and showed a dark blue coloration with ferric chloride
The whole plant, especially the fruit has been used as an anti- reagent. Its molecular formula was assigned as C H O on
1
1
10
9
1
3
inflammatory and antipyretic drug in many local traditional the basis of the C-NMR spectral data, the negative-ion
medicines: Chinese herbal medicine, Tibetan medicine, and FAB-MS [m/z 285, (MϪH) ], and elemental analysis. The
Ϫ
1
)
13
Ayurvedic medicine. Several reports about this plant have
shown that the fruits are rich in vitamin C, mucic acid, and carbons at d 170.9 and 170.6, an oxygen bearing methine at
C-NMR spectrum of 1 showed signals due to two carboxyl
2
—6)
7,8)
tannins.
In our previous work,
a novel highly oxy- d 69.4 and a methylene at d 36.5, along with a set of carbons
genated norbisabolane together with its methyl ester and arising from a galloyl group. Hydrolysis of the galloyl ester
three ester glycosides were isolated from the roots of the with tannase yielded gallic acid and L-malic acid ([a]D
plant along with 15 tannins and related compounds. As part Ϫ2.79°). In addition, the appearance of the methine proton
of our continuing studies on the plant, we report herein the signal of the malic acid moiety at low field (d 5.60) indicated
isolation and structural elucidation of new phenolic con- the galloyl group was attached to this position. Hence, the
stituents, L-malic acid 2-O- (1), mucic acid 2-O- (5), mucic structure of 1 was determined to be L-malic acid 2-O-gallate.
acid 1,4-lactone 2-O- (6), 5-O- (8), 3-O- (10), 3,5-di-O- (11)
gallates, and their methyl esters (2—4, 7, 9) from the fruit C H O based on the C-NMR spectral data, the positive-
The molecular formula of compound 2 was shown to be
1
3
1
5
18 12
ϩ
juice of P. emblica.
ion FAB-MS [m/z 391, (MϩH) ], and elemental analysis.
1
13
The H-, C-NMR, and heteronuclear single quantum coher-
Results and Discussion
ence (HSQC) spectra of 2 indicated the presence of two car-
A 60% aqueous acetone extract of the powdered form of boxyl carbons (d 170.6, 174.8), four oxygen bearing me-
fruit juice of P. emblica was subjected to MCI gel CHP 20P thines [d 73.4, 71.9, 71.0, 70.6, corresponding to d 5.55
C
H
column chromatography to afford five fractions. Fraction 1 (d, Jϭ2.0 Hz), 4.10 (dd, Jϭ1.5, 10.0 Hz), 4.62 (d, Jϭ1.5 Hz),
obtained by elution with H O was further chromatographed 4.40 (dd, Jϭ2.0, 10.0 Hz), respectively], two methoxyl
2
successively over Sephadex LH-20, MCI gel CHP20P, Toy- groups [d 52.7, 52.5, corresponding to dH 3.79, 3.75 (each
opearl HW-40F, and Cosmosil 75C18 OPN to afford com- 3H, s)], and a galloyl group [d 7.24 (2H, s)]. In the het-
H
pounds 1—11 and three known compounds. By comparison eronuclear multiple bond connectivity (HMBC) spectrum,
of the physical and spectral data with those of authentic sam- the two methoxyl groups (d 3.79, 3.75) were correlated with
ples, the known compounds were identified as gallic acid, the two carboxyl carbons at d 170.6 (C-1) and d 174.8 (C-6),
Chart 1
∗
To whom correspondence should be addressed. e-mail: ikouno@net.nagasaki-u.ac.jp
© 2001 Pharmaceutical Society of Japan

5
38
Vol. 49, No. 5
respectively. In addition, correlations of a doublet methine position, this observation suggested that the mucic acid moi-
proton at d 5.55 (H-2) with the C-1 and the carboxyl carbon ety in 6 existed as a 1,4-lactone form. Although locations of
(
d 166.7) of galloyl group indicated that 2 was a 2-O-gallate the two esters could not be determined by the HMBC experi-
1
of 2,3,4,5-tetrahydroxyladipic acid (mucic acid) dimethyl ment, this was achieved by comparison of the H-NMR spec-
ester. The dimethyl ester was obtained by hydrolysis of 2 trum with that of D-saccharic acid 1,4-lactone (12), which is
with tannase and identified as mucic acid dimethyl ester by a C-4 epimer of mucic acid with the same relative configura-
direct comparison with an authentic sample. Accordingly, the tions at C-2 and C-3 positions. The H-2 signal of 12 ap-
structure of 2 was established as mucic acid dimethyl ester 2- peared as a doublet signal with large coupling constant
O-gallate.
(J ϭ8.7 Hz, J ϭ7.8 Hz, J ϭ2.8 Hz), and the J value coin-
2,3 3,4 4,5
ϩ
Compounds 3 and 4 showed the same (MϩH) ion peak at cided with that observed for the methine signal at d 5.97 of 6
m/z 377 in the positive-ion FAB-MS, which was 14 mass where the galloyl group was located. This indicated that the
units less than that of 2 and suggested the lack of a methoxyl galloyl group of 6 was attached to the hydroxyl group adja-
group. The H- and C-NMR spectral data of 3 and 4 closely cent to the lactone carbonyl carbon. Based on the above evi-
resembled those of 2, except for the absence of one of the dence, the structure of 6 was concluded to be mucic acid 1,4-
1
13
two methoxyl signals. The presence of a mucic acid core in lactone 2-O-gallate.
1
their molecules was confirmed by tannase hydrolysis fol-
The H-NMR spectrum of compound 7 was almost super-
lowed by methylation affording mucic acid dimethyl ester. In imposable on that of 6, except for the appearance of an addi-
the HMBC spectra of 3 and 4, H-2 (d 5.58 for 3, d 5.55 for tional methyl signal at d 3.81(s). The positive-ion FAB-MS
ϩ
4
) was correlated with C-1 (d 170.6 for 3, d 170.2 for 4) and of 7 showed the (MϩH) ion peak at m/z 359, which was 14
the carboxyl carbon of galloyl group (d 166.2 for 3, d 166.3 mass units more than that of 6, indicating the occurrence of a
for 4). However, the methoxyl proton (d 3.71) of 3 was cor- methyl group in 7. Accordingly, 7 was determined as mucic
related with the C-6 (d 174.6), while the methoxyl proton (d acid 1,4-lactone 6-methyl ester 2-O-gallate.
3
.73) of 4 was coupled with the C-1 (d 170.2). This observa-
Compound 8 was obtained as a white amorphous powder.
tion indicated that these two compounds were positional iso- Its molecular composition C H O was identical to that of
1
3
12 11
mers differing in the location of their methoxyl group. 6. Coupling patterns of four methine signals observed in the
1
Hence, the structures of 3 and 4 were established as mucic
acid 6-methyl ester 2-O-gallate (3) and mucic acid 1-methyl H-5 of 8 resonated at lower field [d 5.57 (d, Jϭ2.1 Hz)] com-
ester 2-O-gallate (4), respectively. pared with that of 6 [d 4.49 (d, Jϭ1.8 Hz)], and instead, the
The molecular formula of compound 5 was assigned as H-2 was shifted to higher field [d 4.71 (d, Jϭ8.7 Hz) for 8, d
H-NMR spectrum were similar to those of 6; however, the
H
1
3
C H O based on the C-NMR spectral data, the negative- 5.97 (d, Jϭ8.7 Hz) for 6]. This observation clearly indicated
ion FAB-MS [m/z 361, (MϪH) ], and elemental analysis. that the galloyl group was attached to C-5 in 8, which was
Chemical shifts and coupling patterns of the H-NMR spec- also supported by the lower field shift of C-5 (d 74.4) and the
trum of 5 as well as its C-NMR spectral data were very upper field shift of C-2 (d 74.0) compared to those of 6 [d
similar to those of 2—4, except for the absence of methyl 68.2 (C-5), d 75.5 (C-2)]. According to the above evidence,
signals, indicating that this compound is the free acid form of the structure of 8 was established as mucic acid 1,4-lactone
1
3
14 12
Ϫ
1
1
3
2
2
—4. Thus, the structure of 5 was determined as mucic acid 5-O-gallate.
-O-gallate. Compound 9 exhibited the (MϩH) ion peak at m/z 359,
Compound 6 was obtained as a white amorphous powder. and H-NMR spectrum was very similar to that of 8 except
ϩ
1
1
13
Its H-, C-NMR, and HSQC spectra revealed the presence for occurrence of an additional methyl signal at d 3.80, sug-
of two carboxyl groups (d 171.0, 173.0), four oxygen bear- gesting that 9 was the methyl ester of 8. Therefore, 9 was de-
ing methines [d 82.0, 75.5, 72.4, 68.2, corresponding to d
termined to be mucic acid 1,4-lactone 6-methyl ester 5-O-
C
H
4
.76 (dd, Jϭ1.8, 8.4 Hz), 5.97 (d, Jϭ8.7 Hz), 4.87 (dd, gallate.
Jϭ8.7, 8.4 Hz), 4.49 (d, Jϭ1.8 Hz), respectively], and a gal- Compound 10 was found to be an isomer of 6 and 8 by
loyl group [d 7.18 (2H, s)]. The composition of 6 was simi- analysis of the C-NMR spectral data and the result of posi-
1
3
H
Ϫ
1
lar to that of 5; however, their chemical shifts and coupling tive-ion FAB-MS [m/z 343, (MϪH) ]. The H-NMR spec-
constants were significantly different. The core alcohol of 6 trum showed a large down field shift of H-3 [d 5.73 (dd,
was shown to be mucic acid because it became an equilib- Jϭ7.2, 6.6 Hz)] instead of H-2 of 6, indicating that the gal-
rium mixture with 5 in aqueous solution and even in the loyl group was attached to the C-3 position in 10. Thus, the
NMR sample tube (acetone-d ϩD O). The positive-ion FAB- structure of 10 was established as mucic acid 1,4-lactone 3-
MS of 6 showed the (MϩH) ion peak at m/z 345, which O-gallate.
was 18 mass units less than that of 5, indicating that 6 is a Compound 11 was obtained as a white amorphous powder,
lactone form of 5. Among the above-mentioned four methine and its H- and C-NMR spectra revealed the presence of
protons, two doublet signals at d 5.97 and 4.49 were assigna- two galloyl groups (d 7.23, 7.17), and a mucic acid core.
ble to H-2 or H-5 of the mucic acid core. In the HMBC spec- This was supported by the positive-ion FAB-MS showing the
6
2
ϩ
1
13
Ϫ
1
trum, one of the doublet signals at d 5.97 was correlated with (MϪH) peak at m/z 495. In the H-NMR spectrum, the sig-
two carboxyl signals at d 171.0 and 166.5, the latter being nals due to H-3 and H-5 were appeared at the lower field [d
assignable to the carboxyl carbon of the galloyl group, indi- 5.62 (dd, Jϭ7.5, 6.9 Hz) and d 5.65 (d, Jϭ1.8 Hz), respec-
cating that the galloyl group was located at this position. Fur- tively], while H-2 and H-4 signals remained at the upper field
thermore, the chemical shift of the other doublet signal indi- [d 5.10 (d, Jϭ7.5 Hz), d 5.14 (dd, Jϭ1.8, 6.9 Hz)]. Hence,
cated that this position was not acylated. Since the lactone the structure of 11 was determined to be mucic acid 1,4-lac-
formation usually occurs at g (1,4-lactone) or d (1,5-lactone) tone 3,5-di-O-gallate.

May 2001
539
Extraction and Isolation The fruit juice powder (5.0 kg) was extracted
with 60% aq. acetone four times at room temperature to give an extract
(
(
4.8 kg). A portion (920 g) of the extract was subjected to MCI gel CHP 20P
H O–MeOH, 1 : 0—0 : 1, and 50% acetone) to afford five fractions (frac-
2
tions 1—5). One quarter of fraction 1 was further separated successively by
Sephadex LH-20 (H O–MeOH, 1 : 0—0 : 1 and then 50% acetone), MCI gel
2
CHP 20P (H O–MeOH, 1 : 0—0 : 1) and Toyopearl HW-40F (H O–MeOH,
2
2
1
: 0—0 : 1) to give compounds 2 (371 mg), 3 (26 mg), 11 (15 mg), gallic
acid (600 mg), and 1-O-galloyl-b-D-glucose (963 mg). Since most of the re-
maining compounds in this fraction were deduced to be salts and were not
adsorbed by the chromatography, these compounds were combined and the
aqueous solution was acidified to ca. pH 2. Repeated chromatography of the
mixture over Sephadex LH-20 and Cosmosil 75C18 OPN column gave com-
pounds 1 (230 mg), 5 (400 mg), 4 (114 mg), 6 (122 mg), 7 (10 mg), 8 (141
mg), 9 (34 mg), 10 (15 mg), and chebulic acid (35 mg).
2
2
L-Malic Acid 2-O-Gallate (1) White amorphous powder, [a] Ϫ0.4°
D
1
(
cϭ0.24, MeOH). H-NMR (acetone-d6, 300 MHz) d: 3.03 (1H, d,
Fig 1. Equilibrium Relationships between Compounds 2—9
Jϭ8.1 Hz, H-3a), 3.06 (1H, d, Jϭ4.5 Hz, H-3b), 5.60 (1H, dd, Jϭ8.1,
1
3
4
.5 Hz, H-2), 7.17 (2H, s, galloyl H-2Ј, 6Ј). C-NMR (acetone-d , 75 MHz)
6
d 36.5 (C-3), 69.4 (C-2), 110.5 (galloyl C-2Ј, 6Ј), 120.9 (C-1Ј), 139.1 (C-4Ј),
46.0 (C-3Ј, 5Ј), 165.8 (C-7Ј), 170.6, 170.9 (COOH). FAB-MS m/z: 285
These mucic acid gallates were unstable in the aqueous so-
lution and became an equilibrium mixture. Actually, treat-
1
Ϫ
(
MϪH) , 133. Anal. Calcd for C H O : C, 46.17; H, 3.52. Found: C,
11 10 9
ment of 2 with H O/MeOH (4 : 1) at room temperature for 45.91; H, 3.63.
2
3
6 h afforded compounds 2—9, and 5 also gave a mixture of
Enzymatic Hydrolysis of 1 with Tannase A solution of 1 (20 mg) in
water (1 ml) was incubated with tannase (2 mg) at room temperature
overnight. The reaction mixture was subjected to Chromatorex ODS column
compounds 2—9 under similar conditions, confirming the
equilibrium between these compounds (Fig. 1). Taking the
equilibrium into account, the fact that these gallates are opti-
chromatography with H O–MeOH (1 : 0—4 : 1) to give gallic acid (3 mg)
2
25
1
and L-malic acid (2.4 mg): [a]D Ϫ2.79° (cϭ0.22, H O). H-NMR (CD OD,
2
3
cally active implied the occurrence of an enantiospecific gal- 300 MHz) d: 2.65 (1H, dd, Jϭ7.8, 16.2 Hz, H-3a), 2.81 (1H, dd, Jϭ4.5,
1
3
1
7
6.2 Hz, H-3b), 4.49 (1H, dd, Jϭ7.8, 4.5 Hz, H-2). C-NMR (CD OD,
loylation at C-2 position of the optically inactive mucic acid
in P. emblica. Interestingly, the mucic acid core of 8 corre-
sponds to the enantiomer of 6.
3
5 MHz) d: 40.0 (C-3), 68.7 (C-2), 172.9, 174.0 (COOH).
Mucic Acid Dimethyl Ester 2-O-Gallate (2) White amorphous pow-
22
1
der, [a]D Ϫ51.0° (cϭ0.41, MeOH). H-NMR (acetone-d , 500 MHz) d:
6
Most of the methyl esters were probably artifacts gener-
3
.75 (3H, s, 6-OCH ), 3.79 (3H, s, 1-OCH ), 4.10 (1H, dd, Jϭ1.5, 10.0 Hz,
3
3
ated during the separation; however, direct analysis of the H-4), 4.40 (1H, dd, Jϭ2.0, 10.0 Hz, H-3), 4.62 (1H, d, Jϭ1.5 Hz, H-5), 5.55
1
3
(
1
(
(
1H, d, Jϭ2.0 Hz, H-2), 7.24 (2H, s, galloyl H-2Ј, 6Ј). C-NMR (acetone-d6,
6
0% aq. acetone extract of the juice by TLC and HPLC indi-
25 MHz) d: 52.5 (6-OCH ), 52.7 (1-OCH ), 70.6 (C-3), 71.0 (C-5), 71.9
C-4), 73.4 (C-2), 110.0 (galloyl C-2Ј, 6Ј), 120.2 (C-1Ј), 139.2 (C-4Ј), 145.7
C-3Ј, 5Ј), 166.7 (C-7Ј), 170.6 (C-1), 174.8 (C-6). FAB-MS m/z: 391
3
3
cated the occurrence of small amounts of the methyl esters.
Because of the equilibrium, purification of the mucic acid
ϩ
gallates was extremely difficult, and the absolute configura- (MϩH) , 153. Anal. Calcd for C H O ·5/4H O: C, 43.64; H, 5.01.
1
5
18 12
2
tion has not been determined. Although isolation yields were Found: C, 43.69; H 4.79.
Enzymatic Hydrolysis of 2 with Tannase A solution of 2 (25 mg) in
water (1 ml) was incubated with tannase (5 mg) at room temperature
overnight. The reaction mixture was subjected to Chromatorex ODS column
not high, HPLC analysis showed that compounds 5, 6, and 8
were the major phenolic constituents of the juice together
with 1-O-galloyl-b-D-glucose, and these galloyl esters may
chramatography with H O–MeOH (1 : 0—4 : 1) to give gallic acid (6.1 mg)
2
1
play an important role as antioxidants in the juice together and mucic acid dimethyl ester (12.6 mg): H-NMR (C D N, 300 MHz) d:
5
5
13
with vitamin C.
3.58 (6H, s, OCH
3
), 5.03 (2H, s, H-2, 5), 5.39 (2H, s, H-3, 4). C-NMR
(
C D N, 75 MHz) d: 50.2 (OCH ), 70.8 (C-2, 5), 71.5 (C-3, 4), 174.2
5
5
3
Experimental
(COO). An authentic sample of mucic acid dimethyl ester was prepared by
Optical rotations were measured with a JASCO DIP-370 digital polarime- methylation of mucic acid (Nacalai Tesque) with CH N /ether.
2
2
1
13
ter. H- and C-NMR spectra were recorded with Varian Unity plus 500 and
Mucic Acid 6-Methyl Ester 2-O-Gallate (3) White amorphous pow-
Varian Gemini 300 spectrometers operating at 500 and 300 MHz for H-, der, [a]D Ϫ43.9° (cϭ0.28, MeOH). H-NMR (acetone-d , 500 MHz) d:
and 125 and 100 MHz for C-, respectively. Coupling constants were ex- 3.71 (3H, s, 6-OCH ), 4.08 (1H, dd, Jϭ1.5, 10.0 Hz, H-4), 4.60 (1H, d,
1
22
1
6
1
3
3
pressed in Hz, and chemical shifts were given on a d (ppm) scale with Jϭ1.5 Hz, H-5), 4.44 (1H, dd, Jϭ2.0, 10.0 Hz, H-3), 5.58 (1H, d, Jϭ2.0 Hz,
1
3
tetramethylsilane as an internal standard. MS were recorded on a JEOL JMS H-2), 7.24 (2H, s, galloyl H-2Ј, 6Ј). C-NMR (acetone-d , 125 MHz) d:
6
DX-303 spectrometer. Column chromatographies were performed with 52.3 (6-OCH ), 71.3 (C-5), 71.4 (C-3), 72.5 (C-4), 73.1 (C-2), 110.3 (galloyl
3
MCI-gel CHP 20P (75—150 mm, Mitsubishi Chemical Co.), Sephadex LH- C-2Ј, 6Ј), 121.4 (C-1Ј), 138.9 (C-4Ј), 145.9 (C-3Ј, 5Ј), 166.2 (C-7Ј), 170.6
ϩ
2
(
0 (25—100 mm, Pharmacia Fine Chemical Co. Ltd.), Toyopearl HW-40F (C-1), 174.6 (C-6). FAB-MS m/z: 377 (MϩH) . Anal. Calcd for C H O ·
14
16 12
37—70 mm, Tosoh Co.), Cosmosil 75C -OPN (Nacalai Tesque, Inc.), and 3/2H O: C, 41.70; H, 4.75. Found: C, 41.48; H, 4.92.
18
2
Chromatorex ODS (100—200 mesh, Fuji Silysia Chemical Ltd.). TLC was
Mucic Acid 1-Methyl Ester 2-O-Gallate (4) White amorphous pow-
2
2
1
performed on precoated Kieselgel 60 F254 plates (0.2 mm thick, Merck), der, [a]D Ϫ38.1° (cϭ0.22, MeOH). H-NMR (acetone-d , 500 MHz) d:
6
using solvent systems of benzene–ethyl formate–formic acid (1 : 7 : 1), and
3.73 (3H, s, 1-OCH ), 4.12 (1H, br d, Jϭ10.0 Hz, H-4), 4.36 (1H, dd, Jϭ2.0,
3
spots were detected by ultraviolet (UV) illumination and by spraying 2% 10.0 Hz, H-3), 4.57 (1H, d, Jϭ1.0 Hz, H-5), 5.55 (1H, d, Jϭ2.0 Hz, H-2),
1
3
ethanolic ferric chloride and 10% sulfuric acid reagent. HPLC analysis was 7.23 (2H, s, galloyl H-2Ј, 6Ј). C-NMR (acetone-d , 125 MHz) d: 52.3 (1-
6
performed on a Tosoh CCPM solvent delivery system, a JASCO UV-970 OCH ), 70.8 (C-5), 71.7 (C-3), 72.4 (C-4), 73.6 (C-2), 110.3 (galloyl C-
3
spectrometer and
a Cosmosil 5C -AR (Nacalai Tesque) column 2Ј,6Ј), 121.3 (C-1Ј), 139.1 (C-4Ј), 146.1 (C-3Ј, 5Ј), 166.3 (C-7Ј), 170.2 (C-
18
ϩ
(
2.5ϫ25 mm) [mobile phase, acetonitrile–50 mM H PO aqueous solution 1), 175.2 (C-6). FAB-MS m/z: 377 (MϩH) . Anal. Calcd for C H O ·
3 4
14 16 12
(
gradient elution of 0%→30% acetonitrile for 30 min); flow rate, H O: C, 42.65; H, 4.60. Found: C, 42.51; H, 4.40.
2
0
.8 ml/min; detection, 280 nm]. Mucic acid and D-saccharic acid 1,4-lactone
Hydrolysis of 4 A solution of 4 (13.5 mg) in water (1 ml) was incubated
with tannase (2 mg) at room temperature overnight. After evaporation in
were purchased from Nacalai Tesque.
Plant Material The powdered fruit juice of Phyllanthus emblica L. was vacuo, the residue was dissolved in MeOH (2 ml) and treated with
bought in Yunnan Province, China. A voucher specimen of this plant is de-
CH N /ether. The mixture was subjected to silica gel column with CHCl –
posited in the Herbarium of Kunming Institute of Botany, Chinese Academy MeOH–H O ( 1 : 0 : 0—8 : 2 : 0.2) to give mucic acid dimethyl ester (3.2 mg).
2
2
3
2
22
of Sciences.
Mucic Acid 2-O-Gallate (5) Off-white amorphous powder, [a]D

5
40
Vol. 49, No. 5
1
Ϫ25.3° (cϭ0.28, H O). H-NMR (D O, 300 MHz) d: 4.08 (1H, br d, Jϭ9.3
Mucic Acid 1,4-Lactone 3,5-Di-O-gallate (11) White amorphous pow-
2
2
2
2
1
Hz, H-4), 4.29 (1H, br d, Jϭ9.3 Hz, H-3), 4.52 (1H, br s, H-5), 5.51(1H, s, der, [a]D Ϫ96.5° (cϭ0.20, MeOH); H-NMR (acetone-d , 300 MHz) d:
6
1
3
H-2), 7.31 (2H, s, galloyl H-2Ј, 6Ј) C-NMR (D O, 75 MHz) d: 70.4, 71.6
5.10 (1H, d, Jϭ7.5 Hz, H-2), 5.14 (1H, dd, Jϭ1.8, 6.9 Hz, H-4), 5.62 (1H,
C-3, 4, 5), 74.3 (C-2), 110.4 (galloyl C-2Ј, 6Ј), 120.2 (C-1Ј), 139.9 (C-4Ј), dd, Jϭ7.5, 6.9 Hz, H-3), 5.65 (1H, d, Jϭ1.8 Hz, H-5), 7.17, 7.23 (each 2H, s,
2
(
1
3
1
(
45.0 (C-3Ј, 5Ј), 167.6 (C-7Ј), 172.2, 174.2 (C-1, 6). FAB-MS m/z: 361
MϪH) . Anal. Calcd for C H O ·3/2H O: C, 40.11; H, 4.40. Found: C,
galloyl H-2Ј, 2Љ, 6Ј, 6Љ). C-NMR (acetone-d , 75 MHz) d: 71.1 (C-5), 72.4
6
Ϫ
1
3
14 12
2
(C-2), 76.3 (C-3), 79.1 (C-4), 110.1 (galloyl C-2Ј, 2Љ, 6Ј, 6Љ), 120.2, 119.8
3
9.55; H, 4.05.
(C-1Ј, 1Љ), 138.3 (C-4Ј, 4Љ), 146.0 (C-3Ј, 3Љ, 5Ј, 5Љ), 166.6, 165.6 (C-7Ј, 7Љ),
Ϫ
Mucic Acid 1,4-Lactone 2-O-Gallate (6) White amorphous powder, 168.2 (C-6), 172.9 (C-1). FAB-MS m/z: 495 (MϪH) . Anal. Calcd for
2
2
1
[a] Ϫ9.5° (cϭ0.26, MeOH). H-NMR (acetone-d , 500 MHz) d: 4.49 (1H,
C H O ·5/2H O: C, 44.37; H, 3.91. Found: C, 44.23; H, 4.22.
D
6
20 16 15 2
d, Jϭ1.8 Hz, H-5), 4.76 (1H, dd, Jϭ1.8, 8.4 Hz, H-4), 4.87 (1H, dd, Jϭ8.7,
Equilibrium of Compounds 2—9 A solution of 2 (150 mg) in
8
.4 Hz, H-3), 5.97 (1H, d, Jϭ8.7 Hz, H-2), 7.18 (2H, s, galloyl H-2Ј, 6Ј). H O/MeOH (4 : 1, 10 ml) was left to stand at room temperature for 36 h. Re-
2
13
C-NMR (acetone-d , 125 MHz) d: 68.2 (C-5), 72.4 (C-3), 75.5 (C-2), 82.0 peated chromatography of the mixture over Sephadex LH-20 and Cosmosil
6
(
C-4), 110.2 (galloyl C-2Ј, 6Ј), 120.1 (C-1Ј), 139.5 (C-4Ј), 146.1 (C-3Ј,5Ј), 75C18 OPN column afforded compounds 3 (20 mg), 4 (17 mg), 5 (30 mg), 6
ϩ
1
66.5 (C-7Ј), 171.0 (C-1), 173.0 (C-6). FAB-MS m/z: 345 (MϩH) . Anal. (10 mg), 7 (3 mg), 8 (30 mg), 9 (7 mg), and a recovery of 2 (14 mg). These
1
13
Calcd for C H O ·H O: C, 43.10; H, 3.90. Found: C, 43.05; H, 4.19.
compounds were identified by comparison of the H-, C-NMR spectral
13
12 11
2
Mucic Acid 1,4-Lactone Methyl Ester 2-O-Gallate (7) White amor- data and the [a]D values with those of the authentic samples. A similar ex-
2
2
1
phous powder, [a] Ϫ13.0° (cϭ0.19, MeOH). H-NMR (acetone-d6, periment was also carried out for compound 5: a solution of 5 (1 mg) in
D
3
00 MHz) d: 3.81 (3H, s, OCH ), 4.54 (1H, d, Jϭ1.8 Hz, H-5), 4.73 (1H, dd, H O/MeOH (4 : 1, 10 ml) was left to stand at room temperature for 48 h and
Jϭ1.8, 8.4 Hz, H-4), 4.89 (1H, dd, Jϭ8.7, 8.4 Hz, H-3), 5.96 (1H, d, Jϭ8.7 it became a mixture of compounds 2—9, which were identified directly by
Hz, H-2), 7.18 (2H, s, galloyl H-2Ј, 6Ј). FAB-MS m/z: 359 (MϩH) . Anal. TLC comparison with the authentic samples (benzene–ethyl formate–formic
3
2
ϩ
Calcd for C H O ·11/4H O: C, 41.24; H, 4.82. Found: C, 40.89; H, 4.36.
acid, 1 : 7 : 1).
14
14 11
2
Mucic Acid 1,4-Lactone 5-O-Gallate (8) White amorphous powder,
2
2
1
[
(
a] Ϫ29.3° (cϭ0.33, MeOH). H-NMR (acetone-d , 300 MHz) d: 4.46
Acknowledgements The authors are grateful to Mr. K. Inada and Mr. N.
D
6
1H, dd, Jϭ8.7, 8.4 Hz, H-3), 4.71 (1H, d, Jϭ8.7 Hz, H-2), 4.88 (1H, dd, Yamaguchi (Nagasaki University) for NMR and MS measurements. One of
Jϭ2.1, 8.4 Hz, H-4), 5.57 (1H, d, Jϭ2.1 Hz, H-5), 7.25 (2H, s, galloyl H-2Ј,
the authors (Y. J. Zhang) is grateful to the Japanese Government for the
13
6
7
5
Ј). C-NMR (acetone-d , 75 MHz) d: 69.7 (C-3), 74.0 (C-2), 74.4 (C-5), award of a scholarship.
6
9.8 (C-4), 110.0 (galloyl C-2Ј,6Ј), 119.9 (C-1Ј), 139.5 (C-4Ј), 146.0 (C-3Ј,
ϩ
Ј), 166.0 (C-7Ј), 168.9 (C-6), 174.1 (C-1). FAB-MS m/z: 345 (MϩH) .
References
Anal. Calcd for C H O ·3/2H O: C, 42.06; H, 4.07. Found: C, 42.30; H,
1) Xia Q., Xiao P.-G., Wang L.-W., Kong J., Zhongguo Zhongyao Zazhi,
22, 515—518 (1997).
13
12 11
2
4
.33.
Mucic Acid 1,4-Lactone Methyl Ester 5-O-Gallate (9) White amor-
2) Pillay P. P., Mahadeva Iyer K., Current Sci., 27, 266—267 (1958).
3) Soman R., Pillay P. P., Current Sci., 31, 13—14 (1962).
4) Srivastava R. P., Srivastava R. K., Sci. Cult., 30, 446—447 (1964).
5) Margaret Theresa Y., Sastry K. N. S., Nayudamma Y., Leather Sci., 12,
327—328 (1965).
22
1
phous powder, [a] Ϫ30.9° (cϭ0.58, MeOH). H-NMR (acetone-d , 300
D
6
MHz) d: 3.80 (3H, s, OCH ), 4.38 (1H, dd, Jϭ9.0, 8.4 Hz, H-3), 4.61 (1H, d,
3
Jϭ9.0 Hz, H-2), 4.73 (1H, dd, Jϭ2.4, 8.4 Hz, H-4), 5.52 (1H, d, Jϭ2.4 Hz,
ϩ
H-5), 7.15 (2H, s, galloyl). FAB-MS m/z: 359 (MϩH) . Anal. Calcd for
C H O ·5/2H O: C, 41.69; H, 4.75. Found: C, 41.87; H, 4.84.
6) El-Mekkawy S., Meselhy M. R., Kusumoto I. T., Kadota S., Hattori
M., Namba T., Chem. Pharm. Bull., 43, 641—648 (1995).
7) Zhang Y.-J., Tanaka T., Iwamoto Y., Yang C. R., Kouno I., Tetrahedron
Lett., 41, 1781—1784 (2000).
14
14 11
2
Mucic Acid 1,4-Lactone 3-O-Gallate (10) White amorphous powder,
2
2
1
[
(
a] Ϫ30.3° (cϭ0.16, MeOH). H-NMR (acetone-d , 300 MHz) d: 4.46
D
6
1H, d, Jϭ1.8 Hz, H-5), 4.88 (1H, br d, Jϭ6.6 Hz, H-4), 4.90 (1H, d,
Jϭ7.2 Hz, H-2), 5.73 (1H, dd, Jϭ7.2, 6.6 Hz, H-3), 7.20 (2H, s, galloyl H-
8) Zhang Y.-J., Tanaka T., Iwamoto Y., Yang C. R., Kouno I., J. Nat.
Prod., 63, 1507—1510 (2000).
9) Lin J. H., Ishimatsu M., Takana T., Nonaka G., Nishioka I., Chem.
13
2
3
3
Ј, 6Ј). C-NMR (acetone-d , 75 MHz) d: 69.7 (C-5), 72.7 (C-2), 76.3 (C-
6
), 80.8 (C-4), 110.0 (galloyl C-2Ј, 6Ј), 120.0 (C-1Ј), 139.5 (C-4Ј), 146.0 (C-
Ϫ
Ј, 5Ј), 166.7 (C-7Ј), 173.2 (C-1), 173.7(C-6). FAB-MS m/z: 343 (MϪH) .
Pharm. Bull., 38, 1218—1223 (1990).
Anal. Calcd for C H O ·2H O: C, 41.06; H, 4.24. Found: C, 41.05; H, 10) Schmidt O. T., Schmadel H., Justus Liebigs Ann. Chem., 649, 149—
13
12 11
2
4
.64.
152 (1961).