Structure elucidation of metabolites of swertiamarin produced by Aspergillus niger

Article abstract of DOI:10.1016/j.molstruc.2007.07.031

The in vitro metabolism of swertiamarin was carried out in preparative scale using the fungus Aspergillus niger and the metabolites were isolated by semi-preparative HPLC combined with liquid-liquid extraction. Two metabolites, erythrocentaurin and one new compound were obtained and identified by ¹H, ¹³C and 2D NMR and high resolution MS. The anti-inflammatory activity of the novel metabolite was tested and compared with that of swertiamarin in a mice model.

Full text of DOI:10.1016/j.molstruc.2007.07.031

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Journal of Molecular Structure 878 (2008) 22–25
www.elsevier.com/locate/molstruc
Structure elucidation of metabolites of swertiamarin produced
by Aspergillus niger
a
a
b
b,
*
Chang Jun , Zhao Xue-Ming , Liu Chang-Xiao , Zhang Tie-Jun
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
The National Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road,
Tianjin 300193, PR China
b
Received 15 March 2007; received in revised form 14 July 2007; accepted 26 July 2007
Available online 3 August 2007
Abstract
The in vitro metabolism of swertiamarin was carried out in preparative scale using the fungus Aspergillus niger and the metabolites
were isolated by semi-preparative HPLC combined with liquid-liquid extraction. Two metabolites, erythrocentaurin and one new com-
1
pound were obtained and identified by H, 13C and 2D NMR and high resolution MS. The anti-inflammatory activity of the novel
metabolite was tested and compared with that of swertiamarin in a mice model.
ꢀ 2007 Elsevier B.V. All rights reserved.
Keywords: Swertiamarin; Aspergillus niger; Erythrocentaurin; (Z)-5-Ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c]pyridine-1-one;
Anti-inflammatory
1. Introduction
isolated and identified as erythrocentaurin and (Z)-
5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,
4-c]pyridine-1-one, a novel compound, by NMR and high
resolution MS. The anti-inflammatory activity of the new
compound was investigated in an animal model.
Swertiamarin is a secoiridoid compound and a major
active constituent of Enicostemma axillare subsp [1], Genti-
ana campestris [2], Swertia mussotii Franch [3] and Swertia
franchetiana [4], and has been an important traditional
drug in China for many years. Its structure is shown in
Fig. 1. Swertiamarin has a wide range of therapeutic effects
[5–8]. And swertiamarin has been used as an efficient tradi-
tional Chinese drug for serum hepatitis. Swertiamarin,
however, has no or little oral therapeutic effect prior to
bacterial metabolism in the intestinal tract. The first step
in the metabolic pathway of swertiamarin following oral
administration is the hydrolysis of glucosyl group catalyzed
by b-D-glucosidase [9,10]. Aspergillus niger is a major
microorganism producing b-D-glucosidase [11,12] and has
been successfully applied to the biotransformation of some
drugs [13,14]. Herein, swertiamarin is metabolized by
A. niger in preparative scale, and the metabolites were
2. Experiment section
2.1. Chemicals, microorganism and animal
Swertiamarin was extracted and purified from Chinese
herb Swertia mussotii Franch (authenticated by professor
ZHANG at Tianjin Institute of Pharmaceutical Research,
China; its Chinese name was zangyinchen) in our labora-
tory and to approximately 80% purity. Methanol and ace-
tonitrile were HPLC grade (Honeywell International Inc.,
USA). Other chemicals used here were analytical grade
or better and purchased from Tianjin agent Co., Ltd.,
China.
Aspergillus niger CICC 2169 was supplied by China
Center of Industry Culture Collection and maintained in
slants at 4 ꢁC and subcultured every two months. The
*
Corresponding author. Tel./fax: +86 22 23006848.
E-mail address: changjun8772@126.com (C. Jun).
0022-2860/$ - see front matter ꢀ 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2007.07.031

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23
2.4. Structure determination
High resolution mass spectrometry was performed on a
Bruker Apex II FI-ICR mass spectrometer with ESI (Agli-
ent Corp., Palo Alto, USA) as the ion source. Positive ion
mode was employed.
The purified metabolites were dissolved in DMSO-d6
(for metabolite 1) or CDCl3 (for metabolite 2) for NMR
1
analysis. The H, 13C and 2D NMR were performed on a
Bruker AV400 (Bruker BioSpin Group, Faellanden, Swit-
zerland). Chemical shifts were presented as d values relative
to TMS as internal standard. Melting point was recorded
on a Yanaco MP-3 micro melting point apparatus (Yanaco
Corporation, Kyoto, Japan) and uncorrected. The UV
spectrum was obtained with a Hitachi U-2000A spectro-
photometer (Hitachi Ltd., Tokyo, Japan). IR spectrum
was performed on a FT-IR spectrophotometer (Perkin-
Elmer, Waltham, USA) in KBr disks.
Fig. 1. The structures of swertiamarin and metabolites.
compositions of medium for biotransformation were as fol-
lows: 8 g/L glucose, 5 g/L peptone, 5 g/L yeast extract, 5 g/
L KH2PO4, 5 g/L NaCl, 1 g/L MgSO4Æ7H2O and 1 g/L
MnSO4Æ4H2O with pH 6.5.
Male Wistar rats (180–250 g) were purchased from the
animal breeding center of Tianjin Medical University,
Tianjin, China.
2.5. Anti-inflammatory activity
2.2. Biotransformation in preparative scale
For determination of the effects on carrageenan-induced
paw edema, the following protocol was employed: 15 min
after the oral administration of either test sample or dosing
vehicle, each mouse was injected with 25 lL of freshly pre-
pared suspension of carrageenan (20 mg/mL) in physiolog-
ical saline (154 mmol/L NaCl) into subplantar tissue of the
right hind paw. As the control, 25 lL saline solution was
injected into that of left hind paw. Paw edema was mea-
sured at 100, 200, 250 and 300 min following induction
of inflammation. The difference in footpad thickness
between the right and left foot was measured with a pair
of dial thickness gauge calipers (Ozaki Co., Tokyo, Japan).
Mean values of treated groups were compared with mean
values of the control group and analyzed using statistical
methods.
The preparative scale metabolism by A. niger CICC
2169 was carried out with 40 shake flasks (250 mL) each
containing 50 mL of medium. After inoculation with
spores of A. niger CICC 2169 at 28 ꢁC and 160 rpm for 2
days, swertiamarin dissolved in methanol was added at a
final concentration of 0.5 mg/mL. Following 5 days of
growth, the broth was filtered and stored at 4 ꢁC.
2.3. Isolation of the metabolites
The culture was concentrated into about 200 mL under
reduced pressure at 60 ꢁC and extracted three times with
equal volume of petroleum (60–90 ꢁC). The medium was
further extracted three times with equal volume of chloro-
form. The pooled chloroform layer was dried over sodium
sulfate and evaporated into dryness in vacuum. The resi-
due was then reconstituted in 15% acetonitrile in water
and the solution was subjected to semi-preparative HPLC
separation. The semi-preparative HPLC was performed
on a Hewlett-Packard 1100 system (Surplus Lab Inc.,
Muskegon, USA) consisting of a binary pump and
UV2300 detector and a C18, 5 l, 250 · 10 mm column.
The mobile phase consisted of A (0.04% acetic acid in ace-
tonitrile, v/v) and B (0.04% acetic acid in water, v/v). The
gradient elution procedure was as following: 0–10 min,
15% A; 10–30 min, 15%–24% A; 30–35 min, 24%–100%
A; 35–45 min, 100% A; 45–50 min, 100%–15% A. The
pooled eluate was concentrated under reduced pressure
and lyophilized. The purity of the isolated metabolites
was measured by both HPLC and TLC. The HPLC
was carried out with two different mobile phases, 10% ace-
tonitrile in water and 15% acetonitrile in water, respec-
tively. The mobile phase of TLC was mixture of methyl,
acetic ether and acetic acid (methyl:acetic ether:acetic
acid = 9:4:1, v/v/v).
Data obtained from animal experiments were expressed
as means standard error ( SEM). Statistical differences
between the treatments and the control were tested by
one-way analysis of variance (ANOVA) and Student-
Newman-Keuls post hoc test. A value of p < 0.05 was
considered to be significant.
3. Results and discussion
3.1. Structure elucidation
After the pooled chloroform layer was evaporated into
dryness in vacuum, 1.5 g of residue was obtained. The
residue was dissolved in 10 mL of 15% acetonitrile in water
and filtered, and then the filtrate was subjected to semi-pre-
parative HPLC separation. The pooled peaks from semi-
preparative HPLC was concentrated under pressure and
lyophilized. Finally, 275 mg of metabolite 1 (98%) and
117 mg of metabolite 2 (96%) were obtained.
The data of metabolite 2 was as following: Colorless
needles. MS m/z: 177.0451 ([M+H]+); 1H NMR (400

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24
C. Jun et al. / Journal of Molecular Structure 878 (2008) 22–25
MHz, CDCl3) d: 4.57 (2H, t, J = 6.0 Hz, H-3), 3.09 (2H, t,
J = 6.0 Hz, H-4), 6.79 (1H, dd, J = 17.6, 11.2 Hz, H-6),
5.59 (1H, dd, J = 0.7, 17.6Hz, Ha-7), 5.81 (1H, dd,
J = 0.7, 11.2 Hz, Hb-7), 9.18 (1H, s, H-8), 8.85 (1H, s,
H-11); 13C NMR (100 MHz, CDCl3) d: 163.6 (C-1), 66.6
(C-3), 24.2 (C-4), 132.6 (C-5), 127.8 (C-6), 120.5 (C-7),
151.3 (C-8), 121.5 (C-9), 141.0 (C-10), 150.7 (C-11). The
data of metabolite 2 was the same as that of erythrocentau-
rin [14]. So metabolite 2 was identified as erythrocentaurin,
a known compound having effect of inhibition of the
enzyme system of cells of skin [15].
(3470 cm-1) and one conjugated lactone (1720 cm-1) were
observed. The data of UV showed that the
kmax = 279.2 nm which means that one conjugated struc-
ture exists in the metabolite 1.
There were no proton and carbon signals arising from
glucopyranosyl unit were observed in the NMR data of
metabolite 1, which suggested that the glucosyl group
was hydrolyzed. Examination of the 13C NMR properties
of metabolite 1 and gentianine [10] revealed that some sim-
ilarities between them except that the d values of C-6, C-8
and C-12. Thus fragment A was obtained (Fig. 2). The data
of COSY correlation of H-11 to H-12 (Fig. 3) indicated
that there is a single bond between C-11 and C-12, which
was ascertained by the expected upfield shift for C-12
(13.97 ppm) and significant downfield shift for C-11
(128.84 ppm). The NOESY correlations of H-11 and H-
12 indicated the Trans-sterochemistry of H-11. So the frag-
ment B was obtained (Fig. 2). The double bond between C-
11 and C-5 was confirmed by the long-range correlation of
H-11 to C-6, C-10, C-5 and C-12 (Fig. 3). The C-8 to C-9
linkage was ascertained by the correlation of H-8 to C-6,
C-9, C-10 and C-1 in the HMBC spectrum (Fig. 3). Based
on the correlation of H-6 to C-8, C-10, C-11, C-5 and C-12,
C-6 was arranged to be linked to C-5. The nitrogen was
arranged between C-6 and C-8, and the hydroxyl group
was linked to C-8, which was confirmed by the shifts for
C-8 (87.2 ppm) and C-6 (57.2 ppm). The R-sterochemistry
of OH-8 was established by the NOESY correlations of
The data of metabolite 1 was as following: less polar
than parent compound (by TLC analysis); Amorphous
24
powder; m.p. 140–142 ꢁC; ½aꢀD ꢁ 6:7ꢂ; UV (MeOH) kmax
:
279.2 nm; IR (CHCl3) vmax: 3470, 1720, 1625, 1560, 1470,
1380 cm-1; ESIMS m/z 196.0976 ([M+H]+); element analy-
sis: Anal. C 61.58%, H 6.66%, N 7.14%, O 24.62%, calcd
for C10H13O3N, C 61.53%, H 6.71%, N 7.18%, O 24.59%.
The 1D and 2D NMR data were shown in Table 1 and
Fig. 3, respectively.
On the base of the element analysis (C 61.58%, H 6.66%,
O 24.62%, N 7.14%), the molecular formula was estab-
lished as C10H13O3N, which is consistent to the result of
high resolution ESIMS ([M+H]+ at m/z 196.0976). The
13C NMR spectrum (Table 1) exhibited 10 signals contain-
ing one carbonyl (C-1), one olefinic carbon (C-11), three
quaternary carbons (C-5, C-9 and C-10), one methyl car-
bon (C-12), one methine carbon (C-8) and three methylene
1
carbons(C-3, C-4 and C-6). H NMR spectrum gave the
signals of one olefinic proton at d 5.52 (H-11), one methyl
proton at d 1.77 (H-12), three methylene protons at d 4.33
(H-3), 2.62 (H-4) and 4.46 (H-6), and one methine proton
at d 5.52 (H-8). In the data of IR, one hydroxy group
Table 1
The dH, dC and corresponding fragments of metabolite 1
Carbon dH (DMSO-d6
dC (DMSO-
d6100 MHz)
Corresponding
fragments
Fig. 2. Structures of fragment A and fragment B.
400 MHz)
1
163.6
65.45
22.66
131.1
57.2
Carbonyl carbon
(CO)
Methylene carbon
(CH2)
Methylene carbons
(CH2)
Quaternary carbon
(C)
3
4
5
6
4.33 (m)
2.62 (m)
4.46 (d, J = 14.2)
Methylene carbons
(CH2)
4.57 (d, J = 14.2)
8
5.52 (s)
87.2
128.4
145.97
128.84
13.97
Methine carbon
(CH)
Quaternary carbon
(C)
Quaternary carbon
(C)
Olefinic carbon
(CH)
9
10
11
12
NH
6.11 (q, J = 7.1)
1.77 (d, J = 14.2)
3.72 (d, J = 7.2)
Fig. 3. The selected COSY and HMBC correlations of metabolite 1. The
data of HMBC were collected in a 1024 · 64 matrix with 2 transient per t1
increment. The multiple-bond delay was adjusted to a coupling constant
of 4 Hz; the recycle period was 1.0 s. Other parameters were as follows:
Methyl carbon
(CH3)
n
1JCH = 34.5 ms; JCH = 80 ms; f1 = 400.123 Hz; f2 = 100.622 Hz.

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C. Jun et al. / Journal of Molecular Structure 878 (2008) 22–25
25
Table 2
Anti-inflammatory effects of swertiamairn and metabolite 1 against carrgeenan-induced paw edema in mice at five different dose levels (n = 6)
Dose(mg/kg)
Swelling thickness (·10ꢁ2 mm) SEM (inhibition %)
100 min
200 min
250 min
300 min
Cka
Sb
58.5 4.1
65.8 5.4
68.9 4.6
73.2 5.2
10
30
50
80
100
54.8 3.2(6.3)
50.2 3.8(14.2)
49.6 3.5(15.2)
48.9 3.3(16.4)
50.4 4.1(13.8)
63.2 4.1(4.0)
61.6 3.3(6.4)
60.5 4.1(8.1)
45.4 3.6(31.0)*
56.8 4.3(13.7)
65.6 4.4(4.8)
63.7 4.5(7.5)
62.4 4.2(9.4)
51.2 4.2(25.7)*
60.7 5.3(11.9)
70.4 5.3(3.8)
68.5 4.7(6.4)
67.9 4.3(7.2)
55.8 5.2(23.8)**
68.8 5.6(6.0)
M1c
10
30
50
80
100
55.3 5.2(5.5)
42.3 3.6 (27)*
54.6 3.2(6.7)
55.2 4.4(5.6)
57.5 4.2(1.7)
63.7 6.3(3.2)
46.7 5.3(29.0)**
62.5 4.1(5.0)
60.3 5.4(8.4)
61.8 3.8 (6.1)
65.8 5.5(4.5)
69.9 6.2(4.5)
45.7 5.6(33.7)***
51.7 5.2(25.0)*
52.5 6.2(23.8)*
65.7 5.5(4.6)
46.3 5.8(36.7)***
52.8 5.6(27.9)*
55.8 6.5(23.8)*
68.9 5.8(5.9)
*P < 0.05; **P < 0.01; ***P < 0.001 significant from control.
a
CK, control group.
S, swertiamarin.
b
c
M1, (Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c]pyridine-1-one.
H-8 with H-6. Thus metabolite 1 was identified as (Z)-
5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,
4-c]pyridine-1-one, consistent with the degree of unsatura-
tion calculated from the molecular formula.
Acknowledgement
We thank the National Natural Science Foundation of
China (NSFC-20536040 and NSFC-30630075).
3.2. Anti-inflammatory activity
References
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In order to test the anti-inflammatory activity, metabolite
1 and swertiamarin were orally administered and their effects
on the edema of mice were evaluated. As expected, the sub-
plantar injection of carrageenan increased the volume of
the injected paw (edema) during the period of 6 h. Both com-
pounds possess anti-inflammatory activity (Table 2). Swert-
iamarin significantly reduced edema at 80 mg/kg. And
metabolite 1 has an extremely significant effect on easing
edema at 30, 50 and 80 mg/kg, respectively. Compared with
swertiamarin, metabolite 1 has a higher anti-inflammatory
action. But at both high dose (100 mg/kg) and low dose
(10 mg/kg), the anti-inflammatory activities of the swertiam-
arin and metabolite 1 dramatically decreased.
[8] S.K. Bhattacharya, P.K. Reddy, S. Ghosal, A.K. Singh, P.V. Sharma,
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Aspergillus niger CICC 2169 was successfully applied to
the biotransformation of swertiamarin. Two active metab-
olites were isolated and identified from the broth after 5
days of fermentation. One of metabolites was proven to
be a novel compound with higher anti-inflammatory activ-
ity than the parent compound.
In conclusion, A. niger CICC 2169 is a successful and
valuable model for further research of development of new
drugs.
[15] X.W. Yang, M.Y. Hao, Metabolite analysis for chemical
constituents of tranditional Chinese medicines, Beijing, 2003,
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