Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae)

Article abstract of DOI:10.1080/14786419.2011.589055

A new sesquiterpene glycoside, artemisinic acid 3 - O - D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3 - hydroxyartemisinic acid (2, 36.69%) and 3 - hydroxyartemisinic acid -D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC⁵⁰ value of 0.56molmL¹.

Full text of DOI:10.1080/14786419.2011.589055

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Three sesquiterpene compounds
biosynthesised from artemisinic acid
using suspension-cultured cells of
Averrhoa carambola (Oxalidaceae)
Li Yang a , Jianhua Zhu a , Liyan Song b , Xiaojian Shi a , Xingyi Li a
& Rongmin Yu a
a Department of Natural Product Chemistry , Jinan University ,
Guangzhou 510632 , P.R. China
b Department of Pharmacology , Jinan University , Guangzhou
510632 , P.R. China
Published online: 20 Oct 2011.
To cite this article: Li Yang , Jianhua Zhu , Liyan Song , Xiaojian Shi , Xingyi Li & Rongmin Yu (2012)
Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured
cells of Averrhoa carambola (Oxalidaceae), Natural Product Research: Formerly Natural Product
Letters, 26:15, 1388-1394, DOI: 10.1080/14786419.2011.589055
To link to this article: http://dx.doi.org/10.1080/14786419.2011.589055
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Natural Product Research
Vol. 26, No. 15, August 2012, 1388–1394
Three sesquiterpene compounds biosynthesised from artemisinic acid using
suspension-cultured cells of Averrhoa carambola (Oxalidaceae)
a
a
a
Li Yanga, Jianhua Zhua, Liyan Songb , Xiaojian Shi , Xingyi Li and Rongmin Yu
*
*
aDepartment of Natural Product Chemistry, Jinan University, Guangzhou 510632, P.R. China;
bDepartment of Pharmacology, Jinan University, Guangzhou 510632, P.R. China
(Received 7 November 2010; final version received 17 April 2011)
A new sesquiterpene glycoside, artemisinic acid 3-ꢀ-O-ꢀ-D-glucopyranoside
(3, 31.24%) and other two biotransformation products, 3-ꢀ-hydroxyartemisinic
acid (2, 36.69%) and 3-ꢀ-hydroxyartemisinic acid ꢀ-D-glucopyranosyl ester
(4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the
cultured cells of Averrhoa carambola. The three biotransformation products were
obtained for the first time by using the suspension-cultured cells of A. carambola
as a new biocatalyst system, and their structures were identified on the basis of the
physico-chemical properties, NMR and mass spectral analyses. The results
indicate that the cultured cells of A. carambola have the abilities to hydroxylate
and glycosylate sesquiterpene compounds in a regio- and stereoselective manner.
Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated
against K562 and HeLa cell lines. Compound 4 showed strong activity against
HeLa cell line, with the IC50 value of 0.56 mmol mLꢀ1
.
Keywords: Averrhoa carambola; suspension-cultured cells; biotransformation;
artemisinic acid; regio- and stereoselective hydroxylation; glycosylation
1. Introduction
Artemisinin is a sesquiterpene lactone with a peroxide bridge, which is the functional
moiety against both chloroquine-sensitive and multidrug-resistant strains of Plasmodium
falciparum. Modern pharmacological research also showed that artemisinin possessed very
good anti-hepatitis and anti-cancer effects besides anti-malarial activity (Efferth, Dunstan,
Sauerbrey, Miyachi, & Chitambar, 2001; Romero et al., 2005). Artemisinic acid (1) was
proposed to be one of the intermediates in biosynthesis pathway leading to artemisinin in
Artemisia annua (Zhang et al., 2008; Zhu et al., 2010). The content of 1 was much higher
than that of artemisinin in the leaves of A. annua (Gupta et al., 1996) and it also showed
good anti-tumour activity (Zhou, S.L. Wan, & Y.P. Wan, 2006). Compound 1 has a
chemical structure (cadinane-type sesquiterpene) similar to that of artemisinin. Therefore,
the utilisation of 1 as a starting material is of practical importance for the synthesis of
artemisinin analogues.
Over the past few decades, biotransformation has been extensively studied because it is
considered an important method for converting inexpensive and plentiful substances into
expensive and scarce ones. Recently, plant cell cultures as an important biotransformation
system have been widely used because of their biochemical potential to produce specific
*Corresponding authors. Email: tsly@jnu.edu.cn; tyrm@jnu.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.589055
http://www.tandfonline.com

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1389
secondary metabolites that could confer useful elements such as Favours, pigments and
agrochemicals (Gren, Wojcieszynska, Guzik, Perkosz, & Hupert-Kocurek, 2010; Ishihara,
Hiroki, Toshifumi, & Nobuyoshi, 2003; Liu et al., 2010).
There are some research papers about the biotransformation of artemisinic acid (1)
(Elmarakby, EI-Feraly, EIsohly, Croom, & Hufford, 1988; Kawamoto, Asada, Sekine, &
Furuya, 1998). More recently, we reported a few biotransformation products of
artemisinic acid, which were obtained using two biocatalyst systems (the cell suspension
cultures of Catharanthus roseus and Panax quinquefolium crown galls; Zhu et al., 2010).
The suspension culture system of Averrhoa carambola L. was established by our
research group, and the results of the primary experiment showed that artemisinin
derivatives could be biotransformed by this culture system. In this article, artemisinic acid
(1), a typical sesquiterpene, was used as a substrate in cultured cells of A. carambola in
order to expand the range of artemisinin derivatives, to find out new reaction types of
biotransformation in plant culture cells, and to investigate the bioconversion capability of
this new system on important sesquiterpene compounds.
2. Results and discussion
The results demonstrated that artemisinic acid (1) was converted into three products (2–4)
by the suspension-cultured cells of A. carambola.
The HR–ESI–MS spectrum of 3 included a pseudomolecular ion [M þ Na]þ peak at
m/z 435.19874, consistent with a molecular formula of C21H32O8 (Calcd for C21H32NaO8
,
435.19894). The molecular weight of 3 was 162 more than that of substrate 2, indicating
that compound 3 might be the glycosylation product of hydroxyl-artemisinic acid. In the
1H-NMR spectra of 3, an anomeric proton signal was observed at ꢁ 4.37 (1H, d,
J ¼ 7.6 Hz), indicating that the configuration of sugar was ꢀ type. This result was further
confirmed by the study of acid hydrolysis of 3. The sugar generated by acid hydrolysis was
compared with the standard of ꢀ-D-glucose by paper chromatography, and found that the
Rf value of both were the same. In addition, the component sugar in 3 was indicated to be
ꢀ-glucopyranosyl on the basis of the patterns of the carbon and proton signals caused by
the sugar moiety in NMR spectra. There were 21 carbon signals in the 13C-NMR spectra.
On comparison of the data of 1 and 3, the carbon signal at C-15 in 3 was shifted upfield
(ꢁ 23.6 ! 21.2), and one carbon signal was shifted markedly downfield (ꢁ 26.3 ! 77.7).
These data suggested that the upfield shift of the C-15 signal was caused by a ꢂ-effect due
to hydroxylation of 1 at C-3. Correlations were observed in the HMBC spectrum between
the proton signal at ꢁ 4.37 (Glc-1-H) and the carbon resonance at ꢁ 77.7 (C-3), suggesting
that glucose was connected to C3-OH. To elucidate the stereochemistry of C3-OH in 3, the
NOESY spectrum was determined. NOEs were observed between H-3 (ꢁ 3.86) and Me-15
(ꢁ 1.76), H-3 and H-2ꢃ (ꢁ 1.66). However, no NOE was observed between H-3 and H-10
(ꢁ 2.44). These data could only be accounted for by the assignment that the configuration
of H-3 in 3 was ꢃ (ꢀ-OH) type. To further confirm the configuration of H-3, acid
hydrolysis of 3 was done. TLC showed that the aglycone of 3 had the same Rf value as that
of 2, indicating that the aglycone of 3 is 3-ꢀ-hydroxyartemisinic acid. All the information
indicated that the ꢀ-D-glucopyranosyl residue was attached to 3-ꢀ-OH of 1. Thus, 3 was
identified as artemisinic acid 3-ꢀ-O-ꢀ-D-glucopyranoside, which was a new compound.
The structures of 2 and 4 were determined to be 3-ꢀ-hydroxyartemisinic acid (2) and
3-ꢀ-hydroxyartemisinic acid ꢀ-D-glucopyranosyl ester (4), respectively, according to the
spectra of ESI–MS, 1H-NMR and 13C-NMR and by comparing the data with those from
the literature (Zhu et al., 2010).

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L. Yang et al.
100
80
60
40
20
0
0
1
2
3
4
5
6
Co-culture time (day)
Figure 1. Time-course of biotransformation of artemisinic acid (1) by the cultured cells of
A. carambola. Yields of 1 (^), 2 (g), 3 (m) and 4 (g) are plotted.
H
H
H
H
H
HO
R1
hydroxylation
glycosylation
H
O
HO
HO
R2
O
O
3 R1 = OGlc, R2 = OH
1
2
4 R1 = OH, R2 = OGlc
Figure 2. Biotransformation of artemisinic acid (1) by the suspension-cultured cells of A. carambola.
To investigate the biotransformation pathway, a time course in the conversion of 1 was
followed. As shown in Figure 1, the concentration of substrate 1 decreases sharply in 1 day
(100% ! 38%), indicating that transformation happened on the 1st day. At the same time,
compound 2 reached its maximum transformation rate (36.69%); after its climax, the
concentration of 2 decreased gradually, indicating that 2 may act as one of the interme-
diates. Compound 3 reached its maximum transformation rate (31.24%) on the 2nd day,
and then remained stable until the 4th day. The amount of 4 increased tenuously,
suggesting that 4 may be the end product of this process. All products were found in the
cultures, and no product was detected in the medium.
The proposed biosynthesis pathways of 2, 3 and 4 are shown in Figure 2. Artemisinic
acid was first regio- and stereoselectively hydroxylated in the allyl group to yield product 2
and then was glycosylated to yield products 3 and 4, respectively.
From Figure 1, we can see that hydroxylated product (2) and glycosylated product (3)
were the prominent products, both of which achieve their maximum transformation rate in
a short reaction time (36.69%, 1 d; 31.24%, 2 d), and compound 4 was obtained in a lower
yield. This result indicated that plant culture cells of A. carambola have the abilities to
regio- and stereoselectively hydroxylate and glycosylate sesquiterpenes. Compound 4 was
reported to be the product of artemisinic acid when the cultured cells of A. annua were
used as biocatalysis (Kawamoto et al. 1998). However, there are no reports on the

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1391
biotransformation of artemisinic acid (1) to clarify the biotransformation pathways of 1–3.
For the first time, this article describes the biosynthesis pathway of compound 3.
The anti-tumour activity of compounds 3 and 4 was investigated against K562 and
HeLa cell lines. The IC50 values of 3, 4 and cisplatin as a positive control against HeLa cell
line were 2.25 ꢁ 0.13, 0.56 ꢁ 0.01 and 5.46 ꢁ 0.37 mmol Lꢀ1, and those of 3, 4 and
adriamycin (ADR) as a positive control against K562 cell line were 4.10 ꢁ 0.61, 1.30 ꢁ 0.50
and 0.27 ꢁ 0.12 mmol Lꢀ1 against K562 cell line, respectively. These data demon-
strated that ꢀ-D-glucopyranosyl ester (4) exhibited stronger anti-tumour activity than
3-ꢀ-O-ꢀ-D-glucopyranoside (3) against the K562 and HeLa cell lines, and the HeLa cell
line was more sensitive to those products than the K562 cell line.
3. Experimental
3.1. General
The melting points were measured on a X-5 melting point apparatus. Optical rotations
were obtained on a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on a
Bruker DRX-400 spectrometer, the chemical shifts (ꢁ) were given in ppm relative to TMS
as an internal standard and coupling constants were given in Hz. ESI–MS data were
obtained with 4000 Q TRAP LC/MS/MS by direct inlet using MeOH as solvent. HR–ESI–
MS spectrum was obtained on a Micromass Q-TOF and an Agilent 6210 LC/MSD TOF
mass spectrometers. HPLC was performed on a Agilent 1200 liquid chromatograph system
(Palo Alto, CA, USA), equipped with vacuum degasser, quaternary gradient pump, auto-
sampler and DAD, connected to a Agilent ChemStation software. A Pheomenex synergi
C18 column (5 mm, ’4.6 ꢂ 250 mm2) and a Pheomenex ODS C18 guard column
(4.6 ꢂ 12.5 mm2, mm) were used. Silica gel (100–200 and 200–300 meshes) used for
column chromatography and silica GF254 (10–40 mm) for TLC were supplied by the
Qingdao Marine Chemical Factory, China, and ODS from YMC Co. Ltd., Japan.
3.2. Substrate
Artemisinic acid (1) was extracted and isolated from A. annua by our research group. The
structure of 1 was determined by MS and NMR, and its purity was estimated to be greater
than 98% by the HPLC analysis.
3.3. Plant cell cultures
Culture cells of A. carambola were sub-cultured on MS liquid medium (Murashige and
Skoog medium containing 3% sucrose without agar) supplemented with 2.0 mg Lꢀ1
6-benzylaminopurine (6-BA) and 2.0 mg Lꢀ1 2,4-dichlorophenoxyacetic acid (2,4-D) at
25ꢃC in the dark at 2-week intervals.
3.4. Biotransformation of artemisinic acid (1)
The substrate (1, 100 mg) was administered to 20 flasks which contained suspension-
culture cells of A. carambola. Co-culture proceeded for 2 days at 25ꢃC on a rotary shaker
(110 rpm) in the dark. After incubation, the cultures and medium were separated by
filtration with suction. The dried cultures were extracted with methanol for four times by
ultrasound-assisted extraction. Each of MeOH fractions was concentrated and partitioned
between H2O (30 mL) and EtOAc (40 mL ꢂ 3). EtOAc fractions were combined and
evaporated to dryness in vacuo. The residue was chromatographed on silica gel columns
using petroleum ether–ethyl acetate as solvent systems. The eluting fractions were further

1392
L. Yang et al.
separated by silica gel columns using chloroform–methanol as solvent systems to afford
products 2, 3 and 4.
3.4.1. Artemisinic acid (1)
Colourless square crystal, m.p.: 130–131ꢃC. ESI–MS: 233 [M ꢀ H]ꢀ, 257 [M þ Na]þ.
1H-NMR (400 MHz, chloroform-d): ꢁ 0.89 (3H, d, J ¼ 6 Hz, Me-14), 1.59 (3H, s, Me-15),
2.6 (1H, br s, H-6), 2.71 (1H, m, 7-H), 4.98 (1H, s, H-5), 5.55 (1H, s, H-13ꢃ) and 6.45 (1H,
s, H-13ꢀ); 13C-NMR (100 MHz, chloroform-d): ꢁ 172.6 (C-12), 142.6 (C-11), 134.9 (C-4),
126.5 (C-13), 120.1 (C-5), 42.0 (C-7), 41.3 (C-1), 37.8 (C-6), 35.2 (C-9), 27.5 (C-10), 26.3
(C-3), 25.9 (C-8), 25.5 (C-2), 23.6 (C-15) and 19.7 (C-14).
3.4.2. 3-ꢀ-Hydroxyartemisinic acid (2)
1
Colourless needles (CHCl3); ESI–MS m/z: 249 [M ꢀ H]ꢀ and 273 [M þ Na]þ; H-NMR
(400 MHz, acetone-d6): ꢁ 0.97 (3H, d, J ¼ 6.4 Hz, Me-14), 1.74 (3H, s, Me-15), 2.27 (1H,
dd, J ¼ 14, 2.0 Hz, H-2), 3.94 (1H, d, J ¼ 5.6 Hz, H-3ꢃ), 5.16 (1H, s, H-5), 5.54 (1H, s,
H-13ꢃ) and 6.34 (1H, s, H-13ꢀ); 13C-NMR (100 MHz, acetone-d6): ꢁ 168.3 (C-12), 144.5
(C-11), 137.2 (C-4), 124.5 (C-13), 123.6 (C-5), 67.2 (C-3), 43.1 (C-7), 41.9 (C-1), 39.5 (C-6),
36.5 (C-9), 35.5 (C-2), 29.4 (C-10), 26.8 (C-8), 21.1 (C-15) and 20.6 (C-14).
3.4.3. Artemisinic acid 3-ꢀ-O-ꢀ- D-glucopyranoside (3)
15
Amorphous solid; m.p. 231–232ꢃC; ½ꢃꢄD ¼ þ7.0ꢃ(C ¼ 0.27, MeOH); HR–ESI–MS m/z:
435.19874 [M þ Na]þ (Calcd for C21H32NaO8, 435.19894); 1H-NMR (400 MHz, methanol-
d4): ꢁ 0.97 (3H, d, J ¼ 13.0, 13.0, 13.0 and 3.0 Hz, H-9ꢃ), 1.09 (3H, d, J ¼ 6.5 Hz, Me-14),
1.38 (1H, m, H-1), 1.66 (1H, m, H-2ꢃ), 1.76 (3H, s, Me-15), 2.44 (1H, m, H-10), 3.1–3.4
(6H, m, H-20, 30, 40, 50 and 60), 3.86 (1H, d, J ¼ 5.6 Hz, H-3ꢃ), 4.37 (1H, d, J ¼ 8.0 Hz, H-10),
5.18 (1H, s, H-5), 5.45 (1H, s, H-13ꢃ) and 6.25 (1H, s, H-13ꢀ); 13C-NMR(100 MHz,
methanol-d4): ꢁ 171.6 (C-12), 144.8 (C-11), 136.3 (C-4), 125.5 (C-13), 124.4 (C-5), 107.0
(C-10), 78.6 (C-50), 78.1 (C-30), 77.7 (C-3), 75.8 (C-20), 72.1 (C-40), 63.3 (C-60), 43.5 (C-7),
42.3 (C-1), 39.9 (C-6), 36.9 (C-9), 34.8 (C-10), 29.5 (C-8), 27.3 (C-2), 21.2 (C-15) and
20.6 (C-14).
3.4.4. 3-ꢀ-Hydroxyartemisinic acid ꢀ-D-glucopyranosyl ester (4)
1
Amorphous solid; ESI–MS m/z: 411 [M–H]ꢀ and 435 [M þ Na]þ; H-NMR (400 MHz,
pyridine-d5): ꢁ 0.97 (1H, dddd, J ¼ 13.0, 13.0, 13.0 and 3.0 Hz, H-9ꢃ), 1.06 (3H, d,
J ¼ 6.5 Hz, Me-14), 1.35 (1H, m, H-1), 1.46 (1H, dddd, J ¼ 13.0, 13.0, 13.0 and 3.0 Hz,
H-8ꢀ), 1.64 (1H, m, H-2ꢃ), 1.72 (1H, dd, J ¼ 13.0 and 2.0 Hz, H-9ꢀ), 1.89 (3H, s, Me-15),
2.25 (1H, m, H-10), 2.33 (1H, dd, J ¼ 14.0 and 2.5 Hz, H-2ꢀ), 2.87 (1H, br s, H-6), 2.99
(1H, ddd, J ¼ 13.0, 3.0 and 3.0 Hz, H-7), 4.03 (1H, ddd, J ¼ 9.5, 4.0 and 2.5 Hz, H-50), 4.07
(1H, dd, J ¼ 6.5 and 5.5 Hz, H-3), 4.30–4.5 (5H, m, H-20, 30, 40and 60), 5.29 (1H, s, H-5),
5.55 (1H, s, H-13ꢃ), 6.45 (1H, d, J ¼ 8.0 Hz, H-10) and 6.62 (1H, s, H-13ꢀ); 13C-NMR
(100 MHz, pyridine-d5): ꢁ 167.2 (C-12), 144.5 (C-11), 138.1 (C-4), 126.6 (C-13), 123.8 (C-5),
97.3 (C-10), 80.4 (C-50), 79.5 (C-30), 75.3 (C-20), 71.9 (C-40), 67.7 (C-3), 63.0 (C-60), 43.7
(C-7), 42.3 (C-1), 40.1 (C-6), 36.9 (C-9), 36.2 (C-2), 29.8 (C-10), 27.4 (C-8), 22.4 (C-15) and
21.5 (C-14).

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3.5. Hydrolisation of compound 3
The solution of 3 (1 mg) in 0.5 mL of HCl (1 mol Lꢀ1) was heated at 100ꢃC for 2 h and then
evaporated. Compared with standard glucose and compound 2, the reaction mixture was
detected using TLC methods.
3.6. Time-course of biotransformation
The cultures of A. carambola (10 g) were transferred to a 500-mL Erlenmeyer flask
containing 200 mL medium and cultured by continuous shaking for 11 days at 25ꢃC.
Substrate 1 (5 mg per flask) was added to the cultures and incubated at 25ꢃC in a rotary
shaker (110 rpm). At 1-day interval, three of the flasks were taken out and the cultures and
medium were separated by filtration. The extraction and analytic procedures were the
same as those described above. The yield of the products was determined on the basis of
the peak area from HPLC and expressed as a relative percentage to the total amount of
whole reaction products. A binary gradient elution system consisted of water (A) and
methanol (B), and separation was achieved using the following gradient programme:
0–5 min 40–65% B; 5–10 min 65–70% B; 10–15 min 70–85% B; 15–20 min 85–100% B;
20–25 min 100% B, and finally, reconditioning the column with 40% B isocratic for 5 min.
The flow rate was 0.8 mL minꢀ1 and the system operated at 30ꢃC. The detection
wavelength was set at 230 nm.
3.7. MTT cell proliferation assay
The inhibitory effect of products 3 and 4 on the growth of K562 and HeLa cells was
evaluated in vitro by MTT[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium-
bromide] assay as described in the literature (Sabo et al., 2005). Cisplatin and adriamycin
(ADR) were used as positive controls. The concentrations of hydroxyl products and
positive controls on the selected cell lines were in the range 0.0041–1.0000 mmol mLꢀ1. (The
values of IC50 are given as means ꢁ standard deviation of three independent experiments.)
Acknowledgements
This study was supported by a grant from National Natural Science Foundation of China
(no. 81073004). The authors thank Dongbo Yu of University of Texas Southwestern Medical
Center, USA, for proof-reading our manuscript.
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