Cytotoxic biotransformed products from andrographolide by Rhizopus stolonifer ATCC 12939

Article abstract of DOI:10.1016/j.molcatb.2009.11.002

Biotransformation of andrographolide (1) by Rhizopus stolonifer ATCC 12939 was investigated. Ten bioconversion products were isolated and identified. Their structures were elucidated by high resolution mass spectroscopy (HR-MS), extensive NMR techniques,

Full text of DOI:10.1016/j.molcatb.2009.11.002

Journal of Molecular Catalysis B: Enzymatic 62 (2010) 242–247
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Cytotoxic biotransformed products from andrographolide
by Rhizopus stolonifer ATCC 12939
Xiangjiu He∗, Xiaobin Zeng, Hui Hu, Yixuan Wu
College of Pharmacy, Wuhan University, 185 Donghu Rd, Hubei, Wuhan 430072, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2009
Received in revised form 3 November 2009
Accepted 3 November 2009
Available online 10 November 2009
Biotransformation of andrographolide (1) by Rhizopus stolonifer ATCC 12939 was investigated. Ten
bioconversion products were isolated and identified. Their structures were elucidated by high res-
olution mass spectroscopy (HR-MS), extensive NMR techniques, including 1H NMR, 13C NMR, DEPT,
1H–1H correlation spectroscopy (COSY), two dimensional nuclear Overhauser effect correlation spec-
troscopy (NOESY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple
bond coherence (HMBC). Their structures were identified to be 12(R),13(R)-12-hydroxyandrographolide
(2), 12(S),13(S)-12-hydroxyandrographolide (3), isoandrographolide (4), 3-dehydro-isoandrographolide
(5), 14-deoxy-11,12-didehydroandrographolide (6), 3-oxo-14-deoxy-11,12-didehydroandrographolide
(7), 3-dehydroandrographolide (8), 14-deoxyandrographolide (9), 3-dehydro-14-deoxyandrographolide
(10) and 3-dehydro-14-deoxyandrographolide-19-oic acid (11). Among them, compounds 5 and 11 are
novel compounds. The biosynthetic pathways of andrographolide by R. stolonifer were proposed. Most of
the products showed potential antiproliferative activities against human breast cancer (MCF-7), human
colon cancer (HCT-116) and human leukemia (HL-60) cell lines, and their structure–activity relationships
(SAR) were discussed in detail.
Keywords:
Andrographolide
Microbial transformation
Rhizopus stolonifer
Oxidative products
Antiproliferative activities
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
atoma HepG2 cells via alteration of reactive oxygen species
(ROS) [11], induce apoptosis via caspase-8-dependent pathway in
Andrographolide was the major active constituent of
Andrographis paniculata Nees, famous traditional Chinese
human cancer cells [12], enhance 5-fluorouracil-induced apoptosis
via caspase-8-dependent mitochondrial pathway involving p53
participation in hepatocellular carcinoma (SMMC-7721) cells [13].
The importance of microbial biotechnology in the production
of steroid drugs and hormones was realized for the first time in
1952 when Murray and Peterson patented the process of 11␣-
hydroxylation of progesterone by a Rhizopus species. Since then,
microbial transformations have provided adequate tools for the
large scale productions of natural or modified steroid analogues.
Microbial transformation of natural products are currently favored
when compared to their natural counterparts due to some thera-
peutic advantages, such as an increased potency, longer half-lives
in the blood stream, simpler delivery methods and reduced side
effects. The preferential use of whole cells over enzymes as bio-
catalysts for the production of these pharmaceutical derivatives
mostly results from the costs of the latter enzyme isolation, purifi-
cation and stabilization. Furthermore, the use of microbial models
to mimic mammalian metabolism is well known [14,15].
a
and Ayurvedic medicine for the treatment of gastric disor-
ders, infectious diseases and common colds. It chemically
designated as 2(3H)-furanone,3-[2-[decahydro-6-hydroxy-5-
(hydroxymethyl)-5,8a-dimethyl-2-methylene-1-napthalenyl]
ethylidene] dihydro-4-hydroxy (Fig. 1), which exhibited several
anti-inflammatory properties, including inhibition of NF-␬B acti-
vation through covalent modification of reduced Cysteine 62 of
p50 [1], inhibition of intercellular adhesion molecule-1 expression
in monocytes activated by tumor necrosis factor-␣ [2], suppres-
sion of cyclooxygenase-2 (COX-2) expression in neutrophils and
microglial cells [3], and IFN-␥ and IL-2 production [4,5]. Andro-
grapholide exerted immunomodulatory effects by interfering with
NFAT activation and ERK1 and ERK5 phosphorylation in T-cells
[6]. It also had hepatoprotective [7], and anti-HIV [8] activities.
Andrographolide is also a potential cancer therapeutic agent [9],
which could induce cell cycle arrest and mitochondrial-mediated
apoptosis in human leukemic HL-60 cells [10] and human hep-
In the continuing research, the metabolites of andrographolide
in rats [16–18] and human being [19–21] had been carried out in
our research group, and series of sulfate and dehydrate metabo-
lites of andrographolide were isolated and identified. Meanwhile,
the microbial conversions of andrographolide were screened and
∗
Corresponding author.
E-mail address: hexiangjiu@whu.edu.cn (X. He).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.11.002

X. He et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 242–247
243
Fig. 1. Biotransformation pathways of andrographolide (1) by R. stolonifer.
several fungi were selected for further studies. The present work
was an attempt to get the microbial transformation products of
andrographolide by Rhizopus stolonifer ATCC 12939 and provide
some structure–bioactivity relationships against human tumor cell
proliferation of andrographolide.
of an ␣,␤-unsaturated lactone. The high resolution ESI-MS gave the
ion peak [M−H]− at m/z 347.1835 (calcd 347.1858), according to its
molecular formula of C20H28O5.
In the 1H NMR of compound 5, there was a pair of olefinic
protons at ı 4.85 (H-17a, br. s) and 4.55 (H-17b, br. s), which
were exocyclic methylene protons of Andrographis diterpenoids.
The lowest field proton signal at ı 6.55 (H-12, t, J = 7.1 Hz) was des-
ignated to the ␤-olefinic proton of ␣,␤-unsaturated lactone. The
proton signal linked to oxygenated carbon of H-3 at ı 3.38 in andro-
grapholide was disappeared. In the lowest field of its 13C NMR,
there was a new carbonyl carbon signal at ı 213.1. The carbon
of C-12 (151.0) was shifted to lower field about 2 ppm compared
to andrographolide (1), which was appeared at ı 149.3. In the
HMBC spectrum, the carbon signal at ı 213.1 correlated with the
signals at ı 2.80 (H-2a, dt, J = 15.1, 3.8 Hz), 2.40 (H-2b, m), 0.91
(20-CH3, s) and 1.60 (H-5), suggested C-3 was a carbonyl carbon.
The carbonyl carbon of lactone at ı 171.5 correlated with the sig-
nals at ı 6.55 (H-12) and 4.76 (H-14, dd, J = 6.2, 3.0 Hz), indicated
the carbon–carbon double bond of the ␣,␤-unsaturated lactone
located at C-12(13). In the NOESY spectrum, the signal of ı 6.55
(H-12) had NOE correlations with 4.76 (H-14). Compared the spec-
troscopic data with those of compound 4 and the literature [23],
the carbon–carbon double bond at C-12(13) was determined as Z-
form, instead of E-form in andrographolide. When compared the
signals of compound 5 with those of andrographolide reported in
the literature [23], the proton signals of H-11 to H-15 and the car-
bon signals of C-11 to C-15 were almost identical. Therefore, the
C-14 was determined as S-configuration, which the configuration
of C-14 in the literature was established from single-crystal X-ray
analysis. Based on above analysis, compound 5 was elucidated to
be 3-dehydro-isoandrographolide, which was a novel compound.
The full assignments of proton and carbon signals were given in
Table 1.
2. Results
Thirty-two fungi were screened for the biotransformation of
andrographolide and R. stolonifer ATCC 12939 was chosen for fur-
ther study. After 96 h incubation, ten biotransformation products
(Fig. 1) were isolated and identified from the broth of R. stolonifer
in potato medium. Some of them have considerable in vitro
antitumor activities compared to the substrate–andrographolide
(1).
2.1. Identification of biotransformation products
Ten metabolites of andrographolide were identified by chemical
and spectroscopic ways. Among them, two were novel compounds
and were elucidated to be 3-dehydro-isoandrographolide (5)
and 3-dehydro-14-deoxyandrographolide-19-oic acid (11).
Others were known compounds, which were identified as
12(R),13(R)-12-hydroxyandrographolide (2) [22], 12(S),13(S)-
12-hydroxyandrographolide (3) [22], isoandrographolide (4)
[23], 14-deoxy-11,12-didehydroandrographolide (6) [18], 3-
oxo-14-deoxy-11,12-didehydroandrographolide
(7)
[22,24],
3-dehydroandrographolide (8) [25], 14-deoxyandrographolide
(9) [18], and 3-dehydro-14-deoxyandrographolide (10) [22],
respectively.
Compound 5 was obtained as white amorphous powder and was
positive for the Legal and Kedde reactions, suggesting the presence

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X. He et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 242–247
Table 1
1H and 13C NMR data of compounds 5 and 11.
Position
Compound 5
Compound 11
ıC
ıC
ıH (H, multiple, J in Hz)
ıH (H, multiple, J in Hz)
1
2
39.5
1.79 (H, m)
1.38 (H, m)
39.2
1.81 (H, br. d, 12.1)
1.22 (H, br. t, 12.1)
36.3
2.80 (H, dt, 15.1, 3.8)
2.40 (H, m)
36.2
2.76 (H, m)
2.35 (H, m)
3
4
5
213.1
55.0
57.1
–
–
212.0
60.1
56.5
–
–
1.60 (H, o)
1.26 (H, br. d, 9.6)
6
24.8
1.76 (H, m)
1.68 (H, m)
25.0
2.06 (H, m)
1.89 (H, br. d, 11.3)
7
38.8
2.38 (H, m)
2.03 (H, br. d, 12.1)
38.6
2.40 (H, br. d, 11.3)
2.13 (H, m)
8
9
10
148.9
58.0
40.0
–
148.7
57.6
40.0
–
2.00 (H, m)
–
1.75 (H, br. d, 8.2)
–
11
12
24.5
2.85 (2H, t, 7.1)
23.6
1.71 (H, br. t, 9.8)
1.53 (H, o)
151.0
6.55 (H, t, 7.1)
25.7
2.60 (H, m)
2.21 (H, o)
13
14
129.2
69.8
–
131.2
147.3
–
4.76 (H, dd, 6.2, 3.0)
7.23 (H, br. s)
15
75.1
4.36 (H, dd, 10.1, 6.2)
4.06 (H, dd, 10.1, 3.0)
72.5
4.70 (2H, s)
16
17
171.5
108.4
–
175.2
108.5
–
4.85 (H, br. s)
4.55 (H, br. s)
4.86 (H, br. s)
4.67 (H, br. s)
18
19
22.4
64.8
1.30 (3H, s)
23.3
1.53 (3H, s)
4.20 (H, d, 11.1)
4.56 (H, d, 11.1)
180.2
20
15.5
0.91 (3H, s)
13.5
1.03 (3H, s)
Notes: (1) All spectra were recorded on Bruker AV-400, 400 MHz for 1H and 100 MHz for 13C in CD3OD. (2) The carbon signals were assigned unambiguously on 1H NMR, 13
C
NMR, COSY, TOCSY, NOESY, HMQC, and HMBC spectra. (3) “m” indicates multiples, “o” indicates overlapped.
Compound 11 was obtained as colorless needle crystals (in
methanol) and also an ␣,␤-unsaturated lactone judged from its
positive for the Legal and Kedde reactions. The molecular for-
mula C20H26O5 was drawn from its HR-ESI-MS, which gave the ion
peak [M−H]− at m/z 345.1733. In the 1H NMR, an olefinic proton
of ␣,␤-unsaturated lactone appeared at ı 7.23 (H-14, br. s). The
spectroscopic data of compound 11 were very similar to those of
compound 10. There was also a pair of olefinic protons at ı 4.86 (H-
17a, br. s) and 4.67 (H-17b, br. s) of the exocyclic methylene protons
for Andrographis diterpenoids. There were 7 sp2 hybrid carbon sig-
nals in the 13C NMR, which included three carbonyl carbons at ı
212.0 (C-3), 180.2 (C-19) and 175.2 (C-16). In the HMBC spectrum,
the carbon signal at ı 212.0 correlated with the signals at ı 2.76 (H-
2a, m), 2.35 (H-2b, m), 1.53 (18-CH3, s) and 1.26 (H-5), suggested
C-3 was a carbonyl carbon. The carboxylic carbon at ı 180.2 (C-19)
had long range correlation with ı 1.53 (18-CH3, s), suggesting the
hydroxymethyl group at C-19 of andrographolide was oxygenated
to a carboxylic acid. In the NOESY spectrum, the ␤-orientation sig-
nal at 1.26 (H-5, br.d., J = 9.6 Hz) has correlation with the signal at
1.53 (18-CH3, s), suggested the 18-CH3 was ␤-orientation and the
carboxylic group linked at C-4 was ␣-orientation. The signal of lac-
tone carbonyl carbon at ı 175.5 correlated with ı 7.23 (H-14), 2.60
(H-12a) and 2.21 (H-12b), suggested the ␣,␤-unsaturated bond of
lactone located at C-13(14). Based on above evidences, compound
11 was elucidated to be 3-dehydro-14-deoxyandrographolide-19-
oic acid, which was a novel compound as far as known. The full
assignments of proton and carbon were given in Table 1.
2.2. Antiproliferative activities against human tumor cells in vitro
The antiproliferative activities against human tumor cell lines
of the bioconversion products were evaluated. Most bioconver-
sion products showed considerable cytotoxic activities against
human breast cancer (MCF-7), human colon cancer (HCT-116) and
leukemia (HL-60) cell lines, and the results were shown in Table 2.
3. Discussion
Most biotransformation products of andrographolide by R.
stolonifer were oxygenated or dehydrated products. Nucleophilic
reaction could be occurred easily at the ␤-carbon since the dou-
ble bond at C-12(13) of andrographolide was an ␣,␤-unsaturated
lactone, and compounds 2–3 were a pair of hydrated products
of andrographolide at C-12(13) through a nucleophilic reaction.
After hydration of the double bond at C-12(13), the configuration
of the double bond could be converted to Z-form through dehy-
dration (compounds 4 and 5). The hydroxyl group at C-14 could
undergo dehydration easily and formed conjugated double bonds
(Compounds 6, 7, 9–11). Compound 6 was a double dehydration
product of andrographolide, which was the main microbial con-
version product of andrographolide by R. stolonifer. On other side,
the lactone ring is very stable, which the result is similar to those of
andrographolide metabolites in vivo by either rats or human being
[16–22]. From above analysis, the proposed biosynthetic path-
ways of microbial conversion products of andrographolide were

X. He et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 242–247
245
Table 2
4.3. Culture and biotransformation procedures
IC50 values for the inhibition of human hepatoma and leukemia cells of andro-
grapholide (1) and its metabolites 2–11 (mean SD, n = 8).
Screening scale biotransformations of andrographolide were
carried out in 250 mL Erlenmeyer flasks, which contained 60 mL of
potato medium. Microorganisms were transferred into the flasks
from the slants. The flasks were placed on rotary shakers, operat-
ing at 180 rpm at 28 ◦C. The substrate was dissolved in methanol
with a concentration of 10 mg/mL. After 48 h of culture, 0.5 mL
of the solution was added into the fermentation flasks and these
flasks were maintained under the same conditions for additional 4
days. Culture controls consisted of fermentation blanks in which
microorganisms were grown without substrate but adding the
same amount of methanol. When the fermentation finished, the
broths were filtered and the filtrates were extracted with the same
volume of ethyl acetate for three times. The cells were refluxed with
methanol. The extracts were evaporated to dryness under reduced
pressure and the residues were dissolved in methanol. The solu-
tions were spotted on silica gel plates which were developed by
chloroform–methanol system, and visualized by spraying with 10%
H2SO4 solution, followed by heating at 110 ◦C for 10 min. TLC anal-
yses revealed that R. stolonifer ATCC 12939 could biotransform the
substrate.
Compounds
IC50 (␮mol/L)
MCF-7
HCT-116
6.0 0.8
53.1 6.2
45.6 5.7
1.8 0.2
HL-60
7.5 1.2
17.6 2.1
28.3 1.9
23.1 1.9
41.5 6.0
1
2
3
4
9.5 1.3
45.2 5.1
56.3 7.2
2.1 0.4
5
6
7
8
9
10
11
53.1 6.1
15.1 1.8
95.2 10.1
71.5 8.0
10.1 1.6
172.1 15.0
190.7 20.0
66.3 5.8
6.8 1.0
188.0 21.0
56.2 6.3
8.6 1.1
10.5 1.6
163.1 15.2
105.6 12.0
15.3 2.1
167.6 18.1
131.0 16.7
88.3 10.2
151.7 16.8
shown in Fig. 1. Compounds 6 and 9 were also the andrographolide
metabolite in rats, which suggested microbial conversion could be
regarded as reference for in vivo metabolite study [18].
Most metabolites showed obvious in vitro antitumor activi-
ties. Compound 4, isoandrographolide, showed the most potential
antiproliferative activities against MCF-7 and HCT-116 cell lines,
with IC50 values were 2.1 0.4 and 1.8 0.2 ␮mol/L, respectively.
The antiproliferative activities against the selected cell lines were
decreased significantly after the ␤-hydroxyl group has been oxi-
dized to the keto group (compounds 5, 7, 8, 10 and 11). Hydration
at 12(13)-en of andrographolide (compounds 2 and 3) could slightly
decrease the antiproliferative activities against MCF-7, HCT-116
and HL-60 cell lines. Epimerization of 12(13)-en of andrographolide
(compound 4) could increase the target bioactivity of MCF-7 and
HCT-60, while had an adverse result against HL-60 cell. Dehydra-
tion of the hydroxyl at C-14 almost had no effect to the target
bioactivities. These SAR results provided useful clues in the process
of andrographolide development.
Preparative scale biotransformation of andrographolide by R.
stolonifer was carried out in 1000 mL Erlenmeyer flasks containing
250 mL of potato medium, operating at 180 rpm at 28 ◦C. A total of
1500 mg of substrate was transformed. Other procedures were the
same as screening scale biotransformation.
4.4. Extraction and isolation
Brown residue (15.3 g) was obtained from the fermented broth
and cells of R. stolonifer. The residues were resolved in 2000 mL dis-
tilled water and extracted with the same volume of ethyl acetate
and water-saturated n-butanol for three times respectively. From
TLC analysis, the n-butanol fraction did not contain any metabolite
of andrographolide. The ethyl acetate fraction (3.3 g) was subjected
to an open silica gel column (30 mm × 350 mm) and eluted with
chloroform/methanol gradiently (chloroform, 50:1, 20:1, 10:1, 5:1,
2:1, and methanol, v/v). From chloroform/methanol 50:1 elution,
compound 6 (210.3 mg, 14.0% yield) and 9 (31.3 mg, 2.1% yield)
were obtained through recrystallization using acetone. The residue
of the chloroform/methanol 50:1 elution was applied to an ODS col-
umn (20 mm × 250 mm) and eluted with methanol/water, and the
methanol/water (7:3) fraction was subjected to a semi-preparative
RP-HPLC (Zorbax Rx-C18, 5 ␮m, 9.4 mm × 250 mm, Agilent) and
eluted with methanol/water (80:20, v/v) to obtain compounds
10 (17.0 mg, 1.1% yield) and 7 (11.2 mg, 0.7% yield). The chloro-
form/methanol 20:1 elution was subjected to a Sephadex LH-20
column (20 mm × 550 mm) and eluted with chloroform/methanol
4:6, and the second fraction was purified by the semi-preparative
RP-HPLC and eluted with methanol/water (65:35, v/v), and got
compounds 5 (18.1 mg, 1.2% yield), 4 (128.4 mg, 8.5% yield), and
2 (36.0 mg, 2.4% yield), respectively. The fourth fraction of the
Sephadex LH-20 column was subjected to the semi-preparative
RP-HPLC and eluted with methanol/water (70:30, v/v), and got
compounds 3 (28.0 mg, 1.9% yield) and 8 (25.7 mg, 1.7% yield).
The chloroform/methanol 10:1 elution was applied to an ODS col-
umn (20 mm × 250 mm) and eluted with methanol/water, and the
methanol/water 60/40 fraction got compound 11 (8.5 mg, 0.6%
yield) after recrystallization using methanol.
4. Experimental
4.1. General experimental procedures
IR spectra were determined on a Bruker IFS 55 spectrometer
(Bruker Inc., Fällanden, Switzerland) in KBr pellets. UV spectra
were measured on a Cary 5000 UV–Vis spectrophotometer (Var-
ian Inc., Palo Alto, CA). ESI-MS spectra were recorded on a Bruker
Esquire 2000 mass spectrometer. High resolution ESI-MS spectra
were recorded on a Bruker mass spectrometer (Bruker Inc., Fällan-
den, Switzerland). NMR spectra were measured on a Bruker AV-400
spectrometer (Bruker Inc., Fällanden, Switzerland). Analytical and
semi-preparative HPLC were carried on Waters 600 instruments
equipped with RI and PDA detectors (Waters Corp., Milford, MA).
Silica gels for column chromatography were products of Qing-
dao Marine Chemical Factory (Qingdao, China). Normal-phase and
reverse-phase preparative thin-layer chromatography were prod-
ucts of Merck (Darmstadt, Germany).
Andrographolide (1) was isolated from Andrographis paniculata
Nees, and its purity was above 98% determined by HPLC method.
4.2. Microorganisms and culture
All microbials, including R. stolonifer ATCC 12939 was purchased
from the American Type Culture Collection (ATCC, Rockville, MD).
All culture and biotransformation experiments were performed in
potato medium. Potato medium was prepared by the following pro-
cedure: 200 g of mincing husked potato, added 800 mL water, was
boiled for half an hour. Then the solution was filtered and the filtrate
was added with water and 20 g glucose to 1 L.
12(R), 13(R)-12-hydroxyandrographolide (2): white powder,
C
20H32O6. Legal and Kedde Reactions: positive. IR ꢀmax (KBr) cm−1
:
3443, 2932, 1755, 1532, 1061, 891, 786. High resolution ESI-MS
(negative) m/z: 367.2137 [M−H]− (calcd for C20H31O6, 367.2121).
1H NMR (400 MHz, CD3OD): ı 4.97 (1H, br. t, J = 3.3 Hz, H-14), 4.94
(1H, t, J = 8.1 Hz, H-15a), 4.90 (1H, br. s, H-17a), 4.81 (1H, br. s,

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X. He et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 242–247
H-17b), 4.62 (1H, dd, J = 8.1, 2.6 Hz, H-15b), 4.51 (1H, d, J = 9.2 Hz,
H-12), 4.30 (1H, d, J = 11.2 Hz, H-19a), 3.72 (1H, m, H-3), 3.60 (1H,
(negative) m/z: 347.1840 [M−H]− (calcd for C20H27O5, 347.1858).
1H NMR (400 MHz, CD3OD): ı 6.81 (1H, dd, J = 7.1, 5.4 Hz, H-12),
5.01 (1H, d, J = 5.6 Hz, H-14), 4.81 (1H, br. s, H-17a), 4.66 (1H, br.
s, H-17b), 4.61 (1H, d, J = 11.2 Hz, H-19a), 4.40 (H, br. d, J = 11.2 Hz,
H-15a), 4.25 (1H, d, J = 11.2 Hz, H-19b), 4.11 (H, br. d, J = 11.2 Hz, H-
15b), 1.25 (3H, s, 18-CH3), 0.92 (3H, s, 20-CH3). 13C NMR (100 MHz,
CD3OD): ı 212.9 (C-3), 172.1 (C-16), 149.3 (C-12), 149.0 (C-8), 129.4
(C-13), 109.1 (C-17), 75.8 (C-15), 66.3 (C-14), 64.5 (C-19), 57.8 (C-9),
56.5 (C-5), 43.5 (C-4), 40.5 (C-10), 39.2 (C-1), 39.0 (C-7), 36.0 (C-2),
25.3 (C-11), 25.2 (C-6), 23.0 (C-18), 15.3 (C-20).
14-Deoxyandrographolide (9): colorless needle crystals (in ace-
tone), C20H30O4. Legal and Kedde Reactions: positive. IR ꢀmax (KBr)
cm−1: 3433, 2947, 1744, 1643, 1055, 957, 761. ESI-MS (negative)
m/z: 333 [M−H]−. 1H NMR (400 MHz, CD3OD): ı 7.23 (1H, s, H-
14), 4.92 (1H, br. s, H-17a), 4.86 (1H, br. s, H-17b), 4.38 (2H, m,
H-15), 4.23 (1H, d, J = 11.2 Hz, H-19a), 3.41 (H, m, H-3), 3.25 (1H, d,
J = 11.2 Hz, H-19b), 1.22 (3H, s, 18-CH3), 0.72 (3H, s, 20-CH3). 13C
NMR (100 MHz, CD3OD): ı 175.2 (C-16), 148.5 (C-8), 147.2 (C-14),
131.2 (C-13), 108.7 (C-17), 80.6 (C-3), 72.5 (C-15), 64.7 (C-19), 57.6
(C-9), 56.1 (C-5), 43.4 (C-4), 39.8 (C-10), 38.7 (C-7), 38.1 (C-1), 29.0
(C-2), 25.5 (C-12), 25.2 (C-6), 23.5 (C-11), 23.1 (C-18), 15.3 (C-20).
3-Dehydro-14-deoxyandrographolide (10): white powder,
d, J = 11.2 Hz, H-19b), 1.33 (3H, s, 18-CH3), 0.69 (3H, s, 20-CH3). 13
C
NMR (100 MHz, CD3OD): ı 177.3 (C-16), 148.5 (C-8), 107.2 (C-17),
80.3 (C-3), 76.0 (C-15), 73.0 (C-14), 70.5 (C-12), 64.0 (C-19), 55.9 (C-
5), 54.2 (C-9), 54.1 (C-13), 43.1 (C-4), 39.6 (C-10), 38.6 (C-7), 38.2
(C-1), 31.2 (C-11), 29.0 (C-2), 24.9 (C-6), 23.6 (C-18), 15.2 (C-20).
12(S), 13(S)-12-hydroxyandrographolide (3): white powder,
C
20H32O6. Legal and Kedde Reactions: positive. IR ꢀmax (KBr) cm−1
:
3451, 2920, 1750, 1520, 1053, 931, 803. High resolution ESI-MS
(negative) m/z: 367.2130 [M−H]− (calcd for C20H31O6, 367.2121).
1H NMR (400 MHz, CD3OD): ı 5.10 (1H, br. s, H-17a), 5.03 (1H,
t, J = 2.1 Hz, H-14), 4.92 (1H, br. s, H-17b), 4.66 (1H, d, J = 7.1 Hz,
H-12), 4.54 (1H, br. d, J = 9.5 Hz, H-15a), 4.51 (1H, br. d, J = 9.5 Hz,
H-15b), 4.42 (1H, d, J = 11.0 Hz, H-19a), 3.62 (1H, m, H-3), 3.51 (1H,
d, J = 11.0 Hz, H-19b), 1.38 (3H, s, 18-CH3), 0.72 (3H, s, 20-CH3). 13
C
NMR (100 MHz, CD3OD): ı 178.3 (C-16), 148.5 (C-8), 107.5 (C-17),
80.4 (C-3), 75.4 (C-15), 69.6 (C-14), 66.0 (C-12), 65.0 (C-19), 56.0 (C-
5), 52.8 (C-9), 51.5 (C-13), 43.2 (C-4), 38.8 (C-7), 38.7 (C-10), 38.2
(C-1), 31.0 (C-11), 29.0 (C-2), 24.9 (C-6), 23.6 (C-18), 15.4 (C-20).
Isoandrographolide (4): colorless amorphous powder, C20H30O5.
Legal and Kedde Reactions: positive. IR ꢀmax (KBr) cm−1: 3441,
2932, 1742, 1613, 1043, 950, 821. ESI-MS (negative) m/z: 367
[M−H]−. 1H NMR (400 MHz, CD3OD): ı 7.03 (1H, s, H-12), 5.03 (1H,
br. s, H-17a), 4.86 (1H, br. s, H-17b), 4.72 (2H, s, H-15), 4.60 (1H,
t, J = 3.6 Hz, H-14), 4.22 (1H, d, J = 11.2 Hz, H-19a), 3.41 (1H, m, H-
3), 3.23 (1H, d, J = 11.2 Hz, H-19b), 1.20 (3H, s, 18-CH3), 0.71 (3H,
s, 20-CH3). 13C NMR (100 MHz, CD3OD): ı 171.8 (C-16), 151.2 (C-
12), 149.1 (C-8), 129.4 (C-13), 108.6 (C-17), 81.0 (C-3), 75.0 (C-15),
69.9 (C-14), 65.0 (C-19), 57.9 (C-9), 56.5 (C-5), 43.7 (C-4), 40.1 (C-
10), 39.1 (C-7), 38.1 (C-1), 29.0 (C-2), 25.3 (C-6), 24.8 (C-11), 23.4
(C-18), 15.6 (C-20).
C
20H28O4. Legal and Kedde Reactions: positive. IR ꢀmax (KBr)
cm−1: 3424, 2940, 1748, 1720, 1651, 1039, 921, 805. ESI-MS
(negative) m/z: 331 [M−H]−. 1H NMR (400 MHz, CD3OD): ı 7.20
(1H, s, H-14), 4.86 (1H, br. s, H-17a), 4.80 (1H, br. s, H-17b),
4.50 (1H, d, J = 12.1 Hz, H-19a), 4.41 (2H, m, H-15), 4.18 (1H, d,
J = 12.1 Hz, H-19b), 1.25 (3H, s, 18-CH3), 0.92 (3H, s, 20-CH3). 13C
NMR (100 MHz, CD3OD): ı 213.0 (C-3), 175.3 (C-16), 149.0 (C-8),
147.2 (C-14), 131.3 (C-13), 108.6 (C-17), 72.6 (C-15), 64.7 (C-19),
57.6 (C-9), 56.6 (C-5), 43.6 (C-4), 40.4 (C-10), 39.3 (C-1), 39.1 (C-7),
36.2 (C-2), 25.5 (C-12), 25.2 (C-6), 23.4 (C-11), 23.0 (C-18), 15.5
(C-20).
3-Dehydro-isoandrographolide (5): white amorphous powder,
C
20H28O5. Legal and Kedde Reactions: positive. IR ꢀmax (KBr) cm−1
:
3-Dehydro-14-deoxyandrographolide-19-oic acid (11): colorless
needle crystals (in methanol), C20H26O5. Legal and Kedde Reac-
tions: positive. IR ꢀmax (KBr) cm−1: 3425, 2936, 1753, 1725, 1643,
1068, 1021, 842. High resolution ESI-MS (negative) m/z: 345.1733
[M−H]− (calcd for C20H27O5, 347.1702). 1H and 13C NMR see
Table 1.
3432, 2928, 1751, 1720, 1650, 1021, 975, 831. High resolution
ESI-MS (negative) m/z: 347.1835 [M−H]− (calcd for C20H27O5,
347.1858). 1H and 13C NMR see Table 1.
14-Deoxy-11,12-didehydroandrographolide (6): colorless plate
(in acetone), C20H28O4. Legal and Kedde Reactions: positive. IR ꢀmax
(KBr) cm−1: 3442, 2928, 1751, 1656, 1543, 1062, 945, 821. ESI-MS
(negative) m/z: 331 [M−H]−. 1H NMR (400 MHz, CD3OD): ı 7.40
(1H, br. t, J = 6.2 Hz, H-14), 6.80 (1H, dd, J = 15.6, 9.8 Hz, H-11), 6.13
(1H, d, J = 15.6 Hz, H-12), 4.71 (1H, d, J = 2.3 Hz, H-17a), 4.50 (1H,
d, J = 2.3 Hz, H-17b), 4.43 (2H, m, H-15), 4.25 (1H, d, J = 11.5 Hz, H-
19a), 3.38 (1H, m, H-3), 3.21 (1H, d, J = 11.5 Hz, H-19b), 1.26 (3H, s,
18-CH3), 0.75 (3H, s, 20-CH3). 13C NMR (100 MHz, CD3OD): ı 174.8
(C-16), 150.1 (C-8), 146.6 (C-14), 136.5 (C-11), 129.6 (C-13), 122.5
(C-12), 109.1 (C-17), 81.2 (C-3), 71.6 (C-15), 65.0 (C-19), 62.8 (C-9),
55.8 (C-5), 43.8 (C-4), 39.6 (C-10), 38.5 (C-1), 37.8 (C-7), 28.9 (C-2),
24.4 (C-6), 23.3 (C-18), 15.8 (C-20).
3-Oxo-14-deoxy-11,12-didehydroandrographolide (7): colorless
powder, C20H26O4. Legal and Kedde Reactions: positive. IR ꢀmax
(KBr) cm−1: 3432, 2923, 1747, 1712, 1661, 1530, 1041, 930, 850.
ESI-MS (negative) m/z: 329 [M−H]−. 1H NMR (400 MHz, CD3OD): ı
7.42 (1H, br. t, J = 6.3 Hz, H-14), 6.72 (1H, dd, J = 15.2, 9.3 Hz, H-11),
6.10 (1H, d, J = 15.2 Hz, H-12), 4.78 (1H, d, J = 2.1 Hz, H-17a), 4.61
(1H, d, J = 2.1 Hz, H-17b), 4.52 (1H, d, J = 12.1 Hz, H-19a), 4.50 (2H,
m, H-15), 4.11 (1H, d, J = 12.1 Hz, H-19b), 1.28 (3H, s, 18-CH3), 0.93
(3H, s, 20-CH3). 13C NMR (100 MHz, CD3OD): ı 213.3 (C-3), 172.5
(C-16), 148.7 (C-8), 145.8 (C-14), 135.3 (C-11), 128.9 (C-13), 122.4
(C-12), 109.1 (C-17), 71.0 (C-15), 64.7 (C-19), 61.0 (C-9), 56.7 (C-5),
55.2 (C-4), 39.5 (C-1), 39.0 (C-10), 38.0 (C-7), 36.3 (C-2), 24.5 (C-6),
21.9 (C-18), 15.5 (C-20).
4.5. Cell culture and assay for cytotoxic activity
Human breast cancer (MCF-7), human colon cancer (HCT-116)
and leukemia (HL-60) were used for bioactivity evaluation of
andrographolide and its metabolites based on established protocols
[26].
Acknowledgements
This research was partly supported by the Start Fund of Wuhan
University and the starting scientific research fund for returned
overseas students of Ministry of Education of China.
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