Microbial transformation of 20(S)-protopanaxatriol-type saponins by Absidia coerulea

Article abstract of DOI:10.1021/np070053v

Three 20(S)-protopanaxatriol-type saponins, ginsenoside-Rg₁ (1), notoginsenoside-R₁ (2), and ginsenoside-Re (3), were transformed by the fungus Absidia coerulea (AS 3.3389). Compound 1 was converted into five metabolites, ginsenoside-Rh₄ (4), 3β,2β,25- trihydroxydammar-(E)-20(22)-ene-6-O-β-D-glucopyranoside (5), 20(S)-ginsenoside-Rh₁ (6), 20(R)-ginsenoside-Rh₁ (7), and a mixture of 25-hydroxy-20(S)-ginsenoside-Rh₁ and its C-20(R) epimer (8). Compound 2 was converted into 10 metabolites, 20(S)-notoginsenoside-R ₂ (9), 20(R)-notoginsenoside-R₂ (10), 3β,12β,25- trihydroxydammar-(E)-20(22)-ene-6-O-β-D-xylopyranosyl-(1→2) -β-D-glucopyranoside (11), 3β,12β-dihydroxydammar-(E)-20(22),24- diene-6-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside (12), 3β,12β,20,25-tetrahydroxydammaran-6-O-β-D-xylopyranosyl- (1→2)-β-D-glucopyranoside (13), and compounds 4-8. Compound 3 was metabolized to 20(S)-ginsenoside-Rg₂ (14), 20(R)-ginsenoside-Rg ₂ (15), 3β,12β,25-trihydroxydammar-(E)-20(22)-ene-6-O- α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (16), 3β,12β-dihydroxydammar-(E)-20(22),24-diene-6-O-α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranoside (17), 3β,12β,20, 25-tetrahydroxydammaran-6-O-α-L-rhamnopyranosyl-(1→2) -β-D-glucopyranoside (18), and compounds 4-8. The structures of five new metabolites, 10-13 and 16, were established by spectroscopic methods.

Full text of DOI:10.1021/np070053v

J. Nat. Prod. 2007, 70, 1203-1206
1203
Microbial Transformation of 20(S)-Protopanaxatriol-Type Saponins by Absidia coerulea
Guangtong Chen,†,‡ Min Yang,† Zhiqiang Lu,† Jinqiang Zhang,† Huilian Huang,† Yan Liang,† Shuhong Guan,† Yan Song,‡
Lijun Wu,‡ and De-an Guo*,†
Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institute for
Biological Sciences, Chinese Academy of Sciences, 199 Guo Shoujing Road, Zhangjiang, Shanghai 201203, People’s Republic of China, and
College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical UniVersity, 103 Wen Hua Road, Shenyang 110016, Liaoning,
People’s Republic of China
ReceiVed February 6, 2007
Three 20(S)-protopanaxatriol-type saponins, ginsenoside-Rg1 (1), notoginsenoside-R1 (2), and ginsenoside-Re (3), were
transformed by the fungus Absidia coerulea (AS 3.3389). Compound 1 was converted into five metabolites, ginsenoside-
Rh4 (4), 3â,2â,25-trihydroxydammar-(E)-20(22)-ene-6-O-â-D-glucopyranoside (5), 20(S)-ginsenoside-Rh1 (6), 20(R)-
ginsenoside-Rh1 (7), and a mixture of 25-hydroxy-20(S)-ginsenoside-Rh1 and its C-20(R) epimer (8). Compound 2 was
converted into 10 metabolites, 20(S)-notoginsenoside-R2 (9), 20(R)-notoginsenoside-R2 (10), 3â,12â,25-trihydroxydammar-
(E)-20(22)-ene-6-O-â-D-xylopyranosyl-(1f2)-â-D-glucopyranoside (11), 3â,12â-dihydroxydammar-(E)-20(22),24-diene-
6-O-â-D-xylopyranosyl-(1f2)-â-D-glucopyranoside (12), 3â,12â,20,25-tetrahydroxydammaran-6-O-â-D-xylopyranosyl-
(1f2)-â-D-glucopyranoside (13), and compounds 4-8. Compound 3 was metabolized to 20(S)-ginsenoside-Rg2 (14),
20(R)-ginsenoside-Rg2 (15), 3â,12â,25-trihydroxydammar-(E)-20(22)-ene-6-O-R-L-rhamnopyranosyl-(1f2)-â-D-glu-
copyranoside (16), 3â,12â-dihydroxydammar-(E)-20(22),24-diene-6-O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranoside
(17), 3â,12â,20,25-tetrahydroxydammaran-6-O-R-L-rhamnopyranosyl-(1f2)-â-D-glucopyranoside (18), and compounds
4-8. The structures of five new metabolites, 10-13 and 16, were established by spectroscopic methods.
Panax notoginseng (Burk.) F. H. Chen is one of the most
prominent herbal medicines used in traditional Chinese medicine
(TCM) for the treatment of cardiovascular disease, inflammation,
pain, trauma, and internal and external bleeding due to injury.1-3
Phytochemical and pharmaceutical studies have shown that dam-
marane-type saponins are the major bioactive principles, which are
the derivatives of protopanaxadiol and protopanaxatriol.4 Microbial
transformation is an important tool in the structural modification
and metabolism study of organic compounds, especially for natural
products, due to its significant regio- and stereoselectivities.5,6 In
the course of our research intended to expand the chemical diversity
of ginsenoside and notoginsenoside derivatives for further phar-
maceutical research, and to attain a better understanding of microbial
models of ginsenoside and notoginsenoside metabolism, we have
studied the microbial transformation of three dammarane-type
saponins. Ginsenosides-Rg1 (1) and -Re (2) and notoginsenoside-
R1 (3) are the three major 20(S)-protopanaxatriol-type saponins in
P. notoginseng and are the subject of the present work.
Preliminary screening of 42 fungal strains for the microbial
biotransformation of 1-3 was carried out in 250 mL Erlenmeyer
flasks containing 100 mL of liquid medium (Table S1, Supporting
Information). After 48 h of preculture, the substrates were added
into each flask. The incubation was continued for an additional 4
days, after which metabolites in the n-BuOH extract of the broth
were detected by HPLC. Absidia coerulea (AS 3.3389) showed an
ability to convert these substrates with high yield. Spectroscopic
data interpretation was used to elucidate the structures of five new
metabolites, 10-13 and 16. Ten known metabolites (4-9, 14, 15,
17, and 18) were identified by comparison of their spectroscopic
data with those published in the literature.7-9
signal at δH 5.33 (t, J ) 7.2 Hz), together with the two olefinic
carbon signals at δC 130.8 and 126.1 in its 13C NMR spectrum,
indicating the presence of a double bond at C-24(25). The H and
1
13C NMR spectra of 10 were similar to those of 20(S)-notoginse-
noside (9).9 The key differences were that the upfield shift of C-17
and C-21 from δC 54.7 and 25.8 in 9 to δC 50.6 and 22.8 in 10 and
the downfield shift of C-22 from δC 35.8 in 9 to δC 43.3 in 10.
These chemical shifts were also found among 20(R)- and 20(S)-
protopanaxatriol isomers.9 Thus, the structure of metabolite 10 was
elucidated as 20(R)-notoginsenoside-R2. The assignments made
from the 1H and 13C NMR data were confirmed by the HSQC and
HMBC spectra.
The molecular formula of 11 was determined to be C41H70O13
by HRESIMS ([M + Na]+, m/z 793.4764), which was consistent
with the disappearance of a glucose moiety signal at C-20 in the
1H and 13C NMR spectra compared with those of substrate 2. The
1H NMR spectrum showed an olefinic proton signal at δH 5.54 (t,
J ) 6.8 Hz), as well as two olefinic carbon signals at δC 139.2 and
125.2 in its 13C NMR spectrum, indicating that a double bond was
1
not present at C-24(25). The H and 13C NMR signals of Me-21
(δH 1.79, s; δC 12.7), Me-26 (δH 1.33, s; δC 29.4), and Me-27 (δH
1.37, s; δC 29.6) were determined, suggesting that a double bond
occurred at C-20(22) and a hydroxyl group was located at C-25.
This was confirmed by a HMBC experiment, showing correlations
of H-21 with C-20 (δC 139.2), C-22 (δC 125.2), and C-17 (δC 50.1),
of H-26 with C-25 (δC 69.1), C-24 (δC 43.9), and C-27 (δC 29.6),
and of H-27 with C-25 (δC 69.1), C-24 (δC 43.9), and C-26 (δC
29.4). The stereochemistry of the double bond at C-20(22) was
determined to be E since the signal of C-21 was observed at δC
12.7 in the 13C NMR spectrum, while in the case of a Z-type
The molecular formula of 10 was determined to be C41H70O13
1
structure, it would be expected at δC 25-30.10 The H and 13C
1
by HRESIMS ([M + Na]+, m/z 793.4658). Comparison of the H
NMR signals for the ring system and the glycosyl units of 11 were
very similar to those of 9. On the basis of these observations,
compound 11 was assigned as 3â,12â,25-trihydroxydammar-(E)-
20(22)-ene-6-O-â-D-xylopyranosyl-(1f2)-â-D-glucopyranoside.
and 13C NMR data of 10 (Table 1) with those of substrate 2
suggested that signals belonging to a glucose moiety at C-20 were
no longer present. The 1H NMR spectrum showed an olefinic proton
The molecular formula of compound 12 was determined as
C41H68O12 by HRESIMS ([M + Na]+, m/z 775.4606). Comparison
* Corresponding author. Tel: 86-21-50271516. Fax: 86-21-50272789.
E-mail: gda5958@163.com.
1
of its H and 13C NMR data with those of 2 suggested that the
† Shanghai Institute of Materia Medica.
‡ Shenyang Pharmaceutical University.
glucose moiety at C-20 was missing and a new double bond was
10.1021/np070053v CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/13/2007

1204 Journal of Natural Products, 2007, Vol. 70, No. 7
Notes
Table 1. 13C NMR Spectroscopic Data (100 MHz, C5D5N) of Compounds 10-12, 13, and 16
position
10
11
12
13
16
position
10
11
12
13
16
1
2
3
4
5
6
7
8
39.5
27.8
78.8a
40.2
61.4
79.4
45.0
41.2
50.2
39.7
32.3
70.9
49.0
51.8
31.4
26.7
50.6
17.4
17.7a
73.0
22.8
43.3
39.3
28.4
78.4a
39.9
61.0
79.1
44.8
41.0
50.3
39.4
32.2
72.2
50.1
50.5
31.9
27.5
50.1
17.0
17.4
139.2
12.7
125.2
39.5
28.8
78.8a
40.2
61.3
79.5
45.1
41.3
50.7
39.7
32.3
72.5
50.9
51.2
32.5
27.8
50.5
17.3
17.7
139.7
13.1
122.6
39.5
27.8
39.5
28.7
78.3
40.0
60.8
74.4
46.2
41.4
50.1
39.7
32.3
72.6
50.6
50.9
32.6
27.8
50.4
17.2
17.7
139.6
13.0
125.5
23
24
25
26
27
28
29
30
6-O-Glc
1′
2′
3′
4′
5′
6′
22.6
126.1
130.8
25.8
23.3
43.9
69.1
29.4
29.6
31.4
16.3
16.5
27.4
124.9
130.8
25.7
17.6
31.7
19.1 (18.7)b
45.6 (45.8)b
69.7
29.9
30.2
23.7
44.3
69.5
29.7
30.0
32.2
17.6
16.9
78.7a
40.2
61.3
79.4
45.0
41.1
50.1
39.7
32.1
17.7a
31.7
31.7
16.7
17.1
16.7
16.8
16.7
16.9 (17.1)b
9
10
11
12
13
14
15
16
17
18
19
20
21
22
103.5
79.9
78.1
71.8
79.4
63.0
103.2
80.0
77.6
71.4
79.5
62.6
103.1
80.2
78.0
71.7
79.9
62.9
103.5
80.3
78.0
71.8
79.8
63.0
101.8
78.5
79.4
72.4
78.4
63.1
71.0 (70.9)b
48.3 (48.9)b
51.7
31.3
26.9 (26.7)b
54.7 (50.8)b
17.4
Xyl(or Rha)
1′′
2′′
3′′
4′′
5′′
6′′
105.0
75.9
78.8a
71.0
67.3
104.6
75.5
78.4a
70.9
66.9
104.5
75.9
78.8a
71.3
67.3
104.9
75.8
78.7a
71.2
67.3
101.9
72.3
72.6
74.2
69.5
18.8
17.6
73.3
27.2 (22.8)b
36.5 (44.0)b
a Values in vertical column may be interchanged. b Chemical shifts in parentheses due to the 20(R) epimer of 13.
apparent. The 1H NMR spectrum showed two olefinic proton signals
at δH 5.48 (t, J ) 6.8 Hz) and 5.21 (t, J ) 7.2 Hz), which was
consistent with the four olefinic carbon signals observed at δC 139.7,
130.8, 124.9, and 122.6 in the 13C NMR spectrum. The location of
the double bond was supported by a HMBC experiment, which
exhibited correlations of H-21 (with C-20, C-22, and C-17), H-26
(with C-24, C-25, and C-27), and H-27 (with C-24, C-25, and C-26).
The geometrical isomerism of the double bond at C-20(22) was
C-24 (δC 44.3), and C-26 (δC 29.7) confirmed the location of the
double bond at C-20(22) and the hydroxylation at C-25. Thus, the
structure of metabolite 16 was concluded to be 3â,12â,25-
trihydroxydammar-(E)-20(22)-ene-6-O-R-L-rhamnopyranosyl-(1f2)-
â-D-glucopyranoside.
The pH of the broth was determinted to be around 3.4 after
growing a culture of A. coerulea for 6 days without substrate, which
indicated that this organism could decrease the pH of the medium.
Therefore, autoclaved culture controls of 2 and 6 were performed
in parallel in order to determine whether some of the metabolites
obtained were artifacts of acid-catalyzed hydrolysis rather than being
produced by enzymatic conversion. The HPLC results obtained
showed that no metabolites were present. This suggested that this
organism could decrease the pH value of the medium but did not
cause any acid-catalyzed transformations.
Thus, the present results show that microbial transformation of
three 20(S)-protopanaxatriol-type saponins (1-3) by A. coerulea
yielded metabolites with C-20 deglycosylation, side-chain oxida-
tion-reduction, and C-20 hydroxyl group isomerization. The
deglycosylation of the terminal glycosyl at C-6 was also found in
the metabolites of 2 and 3 (4-8).
The three substrates 1-3, with various glycosyl substituents at
C-6, were converted into the same metabolites (4-8) and the same
side-chain-modified metabolites (9-18) by A. coerulea. This
suggests that enzymes secreted by A. coerulea are capable of
transforming the 20(S)-protopanaxatriol-type saponins with high
yield and regioselectivity but with low selectivity for the glycoside
type and linkage at C-6. In our preliminary screening, we also
investigated two 20(S)-protopanaxadiol-type saponins, ginsenosides-
Rb1 and -Rd, but found that A. coerulea was not capable of
transforming these two substrates.
1
determined to be E.10 The H and 13C NMR signals for the ring
system and the glycosyl units of 12 were very similar to those of
9. Therefore, 12 was concluded to be 3â,12â-dihydroxydammarane-
(E)-20(22),24-diene-6-O-â-D-xylopyranosyl-(1f2)-â-D-glucopyra-
noside.
Metabolite 13 was assigned a molecular formula of C41H72O14,
as determined by HRESIMS ([M + Na]+, m/z 811.4877). When
compared with 2, it was found that the glucose moiety at C-20
was missing and a hydroxyl group was introduced into the substrate
molecule. In its NMR spectrum, the olefinic signals were absent,
which suggested that 13 possesses a C8-saturated side chain with
1
two hydroxyl groups. The H NMR signals of the C-21 (δH 1.43,
overlap), C-26 (δH 1.43, overlap), and C-27 (δH 1.43, overlap)
methyl groups were overlapping, indicating that the hydroxyl groups
1
are located at C-20 and C-25. The HMBC correlations of the H
NMR signal at δH 1.43 with C-17 (δC 54.7), C-20 (δC 73.3), C-22
(δC 36.5), C-24 (δC 45.6), and C-25 (δC 69.7) confirmed the
hydroxylation positions to be at C-20 and C-25. Consequently, the
structure of metabolite 13 was determined as 3â,12â,20,25-
tetrahydroxydammarane-6-O-â-D-xylopyranosyl-(1f2)-â-D-glu-
copyranoside. Metabolite 13 was isolated as a mixture of C-20
1
stereoisomers, and the additional signals in the H and 13C NMR
spectra were completely assigned.
The molecular formula of metabolite 16 was established as
C42H72O13 by HRESIMS ([M + Na]+, m/z 807.4812). Comparison
of the 1H and 13C NMR data of 16 with those of substrate 3
indicated that the glucose moiety at C-20 was absent. The 1H NMR
spectrum showed an olefinic proton signal at δH 5.52 (t, J )
6.8 Hz), together with the two olefinic carbon signals at δC 139.6
and 125.5 in the 13C NMR spectrum, indicating that a double bond
was no longer at C-24(25). In the 13C NMR spectrum, the C-21
signal was observed at δ 12.7, which suggested that the double
bond is at C-20(22), and the stereochemistry was determined to be
E.10 The HMBC correlations of H-21 with C-20 (δC 139.6), C-22
(δC 125.5), and C-17 (δC 50.4), of H-26 with C-25 (δC 69.5), C-24
(δC 44.3), and C-27 (δC 30.0), and of H-27 with C-25 (δC 69.5),
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured on a Perkin-Elmer 341 polarimeter. IR spectra were measured
on a Perkin-Elmer 577 spectrometer. The H and 13C NMR spectra
1
were carried out on a Bruker AM-400 spectrometer at 400 and
100 MHz in pyridine-d5, and chemical shifts are expressed in δ (ppm)
relative to tetramethylsilane (TMS). ESIMS and HRESIMS were
recorded on a Finnigan LCQDECA instrument. Silica gel (200-300 mesh;
Qingdao Haiyang Chemical Co. Ltd., Qingdao, People’s Republic of
China) was used for open column chromatography. Reversed-phase
preparative HPLC was performed on an Agilent 1100 Series HPLC
system (Agilent Technologies, Deutschland, Waldbronn, Germany)

Notes
Journal of Natural Products, 2007, Vol. 70, No. 7 1205
Figure 1. Structures of ginsenoside-Rg1 (1), notoginsenoside-R1 (2), ginsenoside-Re (3), and metabolites 4-18.
connected to a UV detector, using a C18 column (250 mm × 10.0 mm,
5 µm; Deerfield, IL) eluted with MeOH-H2O at a flow rate of
1.5 mL/min.
Chemicals and Materials. Ginsenoside-Rg1 (1), notoginsenoside-
R1 (2), and ginsenoside-Re (3) were purchased from Kunming Zhong-
zheng Bio-technology Co. Ltd (Kunming, People’s Republic of China),
and the purity was above 98% by HPLC analysis. All solvents were
AR grade (Sinopharm Chemical Reagent Co. Ltd., People’s Republic
of China).
flasks each containing 400 mL of potato broth medium. The fungus
was incubated for 2 days before 25 mg of substrate 1 (1 mL, 95%
ethanol) was fed to each flask. Incubation conditions and the extraction
process were the same as described above and afforded a crude extract
(2.52 g). The extract was subjected to column chromatography on MCI
gel (100 g). The column was eluted with H2O (600 mL), methanol
(800 mL), and acetone (500 mL). The fraction that eluted with methanol
(827 mg) was chromatographed on a silica gel column (60 g), with
CHCl3-MeOH (10:1-7:1) as solvent, which yielded fractions A-D.
Fraction B was purified by HPLC (MeOH-H2O, 85:15) to yield
metabolite 4 (14.9 mg). Fraction C was purified using HPLC (MeOH-
H2O, 80:20) to yield metabolites 5 (8.4 mg), 6 (10.6 mg), and 7
(6.3 mg). A compound obtained from fraction D was identified as
Microorganisms and Culture Conditions. All microorganisms
screened in our experiments were obtained from the China General
Microbiology Culture Center, People’s Republic of China. Stock
cultures of the fungi were stored on potato dextrose agar slants at
4 °C. Seed cultures were obtained by transferring fungi from stock
cultures to potato dextrose broth. Screening scale biotransformation
was carried out in 250 mL Erlenmeyer flasks containing 100 mL of
medium. The flasks were placed on a rotary shaker operating at
160 rpm at 26 °C. After 2 days of incubation, the substrates (0.2 mL,
10 mg/mL, 95% ethanol) were added into each flask, and these flasks
were maintained under fermentation conditions for 4 days. Then the
cultures were pooled and filtered. The filtrates were extracted three
times with equal volumes of n-BuOH, and the extractions were
evaporated in vacuo and analyzed by HPLC. An autoclaved culture
control was carried out in 250 mL Erlenmeyer flasks containing
100 mL of liquid medium. On growing the culture of A. coerulea for
6 days without substrate, the pH of the broth was checked. Then, the
incubations were autoclaved and the substrates ( 0.2 mL, 10 mg/mL,
95% ethanol) were added. Incubation was carried out for another 4
days, and the n-BuOH extract of the broth was analyzed by HPLC.
Culture controls consisted of fermentation blanks in which fungi were
grown without substrate but fed with the same amount of 95% ethanol.
Substrate controls were composed of sterile medium to which the
substrate was added and incubated without fungi.
1
8 (11.5 mg), by H and 13C NMR spectroscopic analysis.
Biotransformation of Notoginsenoside-R1 (2) and Isolation of
Metabolites. Biotransformation of 2 by the procedure described above
yielded a methanol fraction (868 mg), which was chromatographed on
silica gel (65 g). Elution of the column with CHCl3-MeOH (20:1-
7:1) afforded fractions A′-G′. Fractions B′ and E′ were subjected to
HPLC eluted with MeOH-H2O (80:20) to yield metabolites 4
(12.7 mg) and 12 (21.1 mg), respectively. HPLC (MeOH-H2O, 70:
30) of fraction F′ afforded 9 (20.8 mg), 10 (15.6 mg), and 11 (7.3 mg).
Fraction C′, upon HPLC (MeOH-H2O, 80:20), gave 5 (4.0 mg), 6
(8.6 mg), and 7 (5.5 mg). Further chromatography on silica gel of
fractions D′ and G′ yielded the metabolites 8 (4.0 mg) and 13
(11.0 mg), respectively.
20(R)-Notoginsenoside-R2 (10): fine needles (MeOH); mp 239-
240 °C; [R]2D0 +17 (c 0.17, MeOH); IR (KBr) νmax 3405, 1637,
1047 cm-1; 1H NMR (pyridine-d5, 400 MHz) δ 0.87, 1.01, 1.24, 1.40,
1.48, 1.65, 1.70, 2.10 (3H each, all s, H3-30, 19, 18, 21, 29, 27, 26,
28), 4.97 (1H, d, J ) 7.2 Hz, H-1′), 5.80 (1H, d, J ) 7.2 Hz, H-1′′);
13C NMR (pyridine-d5, 100 MHz), see Table 1; HRESIMS m/z 793.4658
[M + Na]+ (calcd for C41H70O13Na, 793.4714).
Biotransformation of 1 and Isolation of Metabolites. The prepara-
tive scale biotransformation of 1 was carried out in twenty 1000 mL
3â,12â,25-Trihydroxydammar-(E)-20(22)-ene-6-O-â-D-xylopyra-
nosyl-(1f2)-â-D-glucopyranoside (11): white, amorphous powder;

1206 Journal of Natural Products, 2007, Vol. 70, No. 7
Notes
[R]D20 +12 (c 0.12, MeOH); IR (KBr) νmax 3405, 1639, 1049 cm-1; 1H
NMR (pyridine-d5, 400 MHz) δ 0.79, 0.96, 1.19, 1.33, 1.37, 1.48, 1.79,
2.07 (3H each, all s, H3-30, 19, 18, 26, 27, 29, 21, 28), 4.93 (1H, d,
J ) 6.8 Hz, H-1′), 5.75 (1H, d, J ) 7.2 Hz, H-1′′); 13C NMR (pyridine-
d5, 100 MHz), see Table 1; HRESIMS m/z 793.4764 [M + Na]+ (calcd
for C41H70O13Na, 793.4714).
[R]D20 -14 (c 0.12, MeOH); IR (KBr) νmax 3417, 1637, 1049 cm-1; 1H
NMR (pyridine-d5, 400 MHz) δ 0.97, 0.99, 1.26, 1.37, 1.80, 2.01 (3H
each, all s, H3-30, 19, 18, 27, 29, 21, 28), 1.36 (6H, s, H3-26, 29), 1.80
(3H, d, J ) 4.4 Hz, H3-6′′), 5.28 (1H, d, J ) 6.8 Hz, H-1′), 6.51 (1H,
brs, H-1′′); 13C NMR (pyridine-d5, 100 MHz), see Table 1; HRESIMS
m/z 807.4812 [M + Na]+ (calcd for C42H72O13Na, 807.4871).
3â,12â-Dihydroxydammarane-(E)-20(22),24-diene-6-O-â-D-xy-
lopyranosyl-(1f2)-â-D-glucopyranoside (12): white, amorphous
powder; [R]2D0 +14 (c 0.17, MeOH); IR (KBr) νmax 3415, 1637,
1047 cm-1; 1H NMR (pyridine-d5, 400 MHz) δ 0.83, 1.22, 1.23, 1.49,
1.58, 1.65, 1.82, 2.10 (3H each, all s, H3-30, 18, 19, 29, 27, 26, 21,
28), 4.96 (1H, d, J ) 7.2 Hz, H-1′), 5.78 (1H, d, J ) 7.2 Hz, H-1′′);
13C NMR (pyridine-d5, 100 MHz), see Table 1; HRESIMS m/z 775.4606
[M + Na]+ (calcd for C41H68O12Na, 775.4658).
3â,12â,20,25-Tetrahydroxydammarane-6-O-â-D-xylopyranosyl-
(1f2)-â-D-glucopyranoside (13): white, amorphous powder; [R]2D0
+14 (c 0.17, MeOH); IR (KBr) νmax 3415, 1641, 1047 cm-1; 1H NMR
(pyridine-d5, 400 MHz) δ 0.82 (3H, s, H3-30), 0.98 (3H, s, H3-19),
1.23 (3H, s, H3-18), 1.43 (9H, overlap, H3-21, 26, 27), 1.49 (3H, s,
H3-29), 2.11 (3H, s, H3-28), 4.97 (1H, d, J ) 7.2 Hz, H-1′), 5.79 (1H,
d, J ) 7.2 Hz, H-1′′); 13C NMR (pyridine-d5, 100 MHz), see Table 1;
HRESIMS m/z 811.4877 [M + Na]+ (calcd for C41H72O14Na, 811.4820).
Biotransformation of Ginsenoside-Re (3) and Isolation of Me-
tabolites. Biotransformation of 3 by the procedure described above
yielded a methanol fraction (892 mg), which was further chromato-
graphed on silica gel (65 g). Elution of the column with CHCl3-MeOH
(20:1-7:1) gave fractions A′′-E′′. Fraction A′′ was purified by HPLC
(MeCN-H2O, 60:40) and yielded metabolite 4 (10.8 mg). HPLC
(MeOH-H2O, 80:20) of fraction B′′ afforded 5 (1.7 mg), 6 (7.8 mg),
and 7 (5.0 mg). Fraction D′′ was subjected to HPLC (MeCN-H2O,
50:50) to yield 17 (21.5 mg). HPLC (MeOH-H2O, 75:25) of fraction
E′′ afforded 14 (30.0 mg), 15 (13.8 mg), and 16 (3.8 mg). Fractions
C′′ and F′′ were subjected to further chromatography on silica gel and
yielded metabolites 8 (7.0 mg) and 18 (11.3 mg).
Acknowledgment. This work was supported by Shanghai Com-
mission of Science and Technology (0511021024) and National
Supporting Program for TCM from the Ministry of Science and
Technology of China (2006BAI08B03-03).
Supporting Information Available: Table listing microorganisms
used for preliminary screening. This information is available free of
charge via the Internet at http://pubs.acs.org.
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3â,12â,25-Trihydroxydammar-(E)-20(22)-ene-6-O-R-L-rhamnopy-
ranosyl-(1f2)-â-D-glucopyranoside (16): white, amorphous powder;
NP070053V