Biotransformation of 2α,5α,10β,14β-Tetraacetoxy-4(20),11-taxadiene by Cell Suspension Cultures of Catharanthus roseus

• Abstract
• Introduction
• Materials and Methods
• General
• Plant tissue and cell culture
• Substrate
• Biotransformation
• Compound 2
• Compound 3
• Compound 4
• Compound 5
• Effects of stage of substrate addition on the transformation
• Results and Discussion
• Acknowledgements
• References

Abstract

Catharanthus roseus cell suspension cultures were employed for the biotransformation of 2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene (1), and four metabolites were obtained. Based on their physical and chemical data, the structures of the four metabolites were respectively elucidated as 10β-hydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (2), 5α-hydroxy-2α,10β, 14β-triacetoxy-4(20),11-taxadiene (3), 6α,10β-dihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (4), and 6α,9α,10β-trihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (5), among which 3 and 5 were characterized as new taxoids. The effects of the stages of substrate addition on the biotransformation were also investigated. The results revealed that the biotransformation rate for 1 reached 85.3 % and the yield of 2 70 % when 1 was administered during the mid-linear phase (9 - 12^th day) of the cell growth cycle. On the other hand, the yield for 4 reached the highest level of 11.8 % when 1 was added in the early linear phase (6^th day).

Introduction

The biochemical potential of plant cell cultures to produce specific secondary metabolites such as drugs, flavors, pigments and agrochemicals is of considerable interest in connection with their biotechnological utilization. Unfortunately, it has been reported that the accumulation of some secondary metabolites does not always occur in the cell cultures of higher plants [1]. However, there is evidence that such cultures retain an ability to specifically transform exogenous substrates administered to the cultured cells. Therefore, plant cell culture, as a bioreactor, is considered to be useful for transforming cheap and abundant compounds into valuable ones. The compound 2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene (1) is a taxane isolated from the callus cultures of Taxus spp. in high yield (ca. 1 - 2 % of dry weight) [2], [3], and might become one of the potential alternative resources of paclitaxel, one of the most effective anticancer drugs in clinical application for various cancers, or other structurally related bioactive compounds, such as taxinine and its derivatives which were reported to show high multi-drug-resistance reversal activity for some solid cancer cells [4], [5], [6], [7]. We previously reported that 1 was selectively hydroxylated at 9α-position by Ginkgo cell suspension cultures [8]. In the present paper, the specific hydroxylation and deacetylation of 1 by Catharanthus cell suspension cultures and the effects of the stage of substrate addition on the bioprocess are reported.

Materials and Methods

General

IR spectra were obtained on a Perkin-Elmer 983 spectrophotometer (KBr). Optical rotation values were measured by using a Perkin-Elmer 243B polarimeter. NMR spectra (¹H-NMR, ¹³C-NMR, DEPT, ¹H-¹H-COSY, HMQC, HMBC and NOESY) were recorded in CDCl₃ on Bruker DRX-500 or Varian INOVA-500 spectrometer (¹H-NMR, 500 MHz; ¹³C-NMR, 125 MHz) and chemical shifts were recorded in ppm using TMS as internal standard. FABMS spectra were measured on a KYKY-ZHP-5^# mass spectrometer in the positive mode. TLC analyses were performed on the precoated silica gel 60 F₂₅₄ plates (Merck). All chemicals were obtained from Beijing Chemical Factory.

Plant tissue and cell culture

Plant material of Catharanthus roseus (L.) G. Don (Apocynaceae) was collected from the Greenhouse of Peking University Health Science Center, and identified by Professor Dean Guo. The voucher specimen (No. 010 417) was deposited in the herbarium of Department of Natural Medicines, Peking University. The leaves of C. roseus were disinfected by immersion in 70 % ethanol for 30 s, followed by 0.1 % HgCl₂ for 10 min, and then washed with sterilized distilled water for 5 times. To initiate callus cultures, disinfected leaves were cut into small pieces (0.5 cm × 0.5 cm) and aseptically transferred to Murashige and Skoog’s medium (MS) [9] supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 7.0 g/L agar. The pH value was adjusted to 5.8 prior to autoclaving at 121 °C for 20 min. The cultures were maintained on the same medium at 25 ± 2 °C in the dark by subculturing every four weeks. Three-week-old friable callus cultures were used to initiate cell suspension cultures in MS medium supplemented with 0.5 mg/L 6-benzylaminopurine (6-BA), 0.5 mg/L naphthaleneacetic acid (NAA) and 0.2 mg/L 2,4-D. Cell cultures were subcultured every week within the first month after callus cultures were transferred to liquid medium. When growing stably, cell cultures were filtered aseptically through filter paper under vacuum and 15 g fresh cell cultures inoculated into each 500-mL flask with 150 mL medium every three weeks. All the cell suspension cultures were maintained on a rotary shaker at 110 rpm at 25 ± 2 °C in the dark. The cultures were harvested from shake flasks every three days to determine the pH value and the dry weight for the kinetic studies. The pH values of the sample flasks were directly measured before the cell cultures were filtered. The cell cultures from the sample shake flasks were filtered under vacuum and washed thoroughly with distilled water, and then dried at 50 °C to a constant weight to calculate the biomass.

Substrate

2α,5α,10β,14β-Tetraacetoxy-4(20),11-taxadiene (1) was isolated from callus cultures of T. yunnanensis, and characterized by chemical and spectral methods as described in the literature [2]. The substrate was dissolved in EtOH and diluted to 10.0 mg/mL before use.

Biotransformation

0.5 mL of the substrate solution was added into each flask with 15-day-old cell cultures, and 0.5 mL EtOH alone instead of substrate solution into each parallel flask as the control. After additional six days of incubation, the cell cultures were filtered under vacuum and washed with distilled water (3 × 20 mL). The filtrate was collected, extracted with EtOAc (3 × 150 mL) and the dried cultures were extracted in an ultrasonic bath (3 × 50 mL) at room temperature, then both the extracts were concentrated under vacuum at 40 °C, respectively. The residues were dissolved in acetone and analyzed by TLC, developed with acetone-petroleum ether (60 - 90 °C) (1 : 2.5) , and visualized by spraying with 10 % H₂SO₄ (in EtOH) followed by heating at 105 °C. The TLC results showed that several new spots with higher polarity appeared in the chromatogram of the medium extract compared with those of the culture extracts of substrate feeding and the controls. For preparative biotransformation, 100 mg of substrate were distributed into 20 flasks with 15-day-old cell cultures, and after six days of incubation, the culture media were collected, extracted and concentrated as described above. The obtained residue (160 mg) was separated on a silica gel (5∼40 mesh ) column (0.8 × 25 cm), eluted with acetone-petroleum ether (60 - 9 °C) (1 : 5, 1 : 4, 1 : 3, 1 : 2, 1 : 1, 60 mL for each gradient eluent) to yield 40 mg of 2 (between 40 and 50 mL), 3 mg of 3 (60 - 80 mL), 10 mg of 4 (120 - 140 mL) and 2 mg of 5 (260 - 270 mL).

Compound 2

10β-Hydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene, colorless needles. [α]_D ²⁵: + 30.7° (c 0.012, MeOH); IR: υ_max ^(KBr): = 3456, 2940, 1726, 1620, 1373, 1236, 1023 cm^-1; ¹H-NMR δ = 5.35 (1H, dd, J = 7.5 Hz, 2.5 Hz, H-2), 5.28 (1H, t, J = 2.5 Hz, H-5), 5.26 (1H, brs, H-20a), 5.11 (1H, dd, J = 5.5 Hz, 11.5 Hz, H-10), 4.99 (1H, dd, J = 9.0 Hz, 4.5 Hz, H-14), 4.86 (1H, brs, H-20b), 2.92 (1H, d, J = 7.5 Hz, H-3), 2.80 (1H, dd, J = 18.5 Hz, 9.0 Hz, H-13β), 2.40 (1H, m, H-13), 2.35 (1H, dd, J = 12.0 Hz, 15.0 Hz, H-9β), 2.18 (3H, s, 14-OCOCH₃ ), 2.05 (3H, s,5-OCOCH₃ ), 2.02 (3H, s, 2-OCOCH₃ ), 1.98 (3H, 18-CH₃), 1.90 (1H, m, H-7), 1.88 (1H, d, J = 2.5 Hz, H-1), 1.72 (3H, s, 16-CH₃), 1.67 (1H, dd, J = 14.5 Hz, 5.5 Hz, H-9α), 1.21 (1H, m, H-7), 1.18 (3H, s, 17-CH₃), 0.84 (3H, s, 19-CH₃); ¹³C-NMR δ = 170.0 (2-OCOCH₃, 5-OCOCH₃), 169.8 (14-OCOCH₃), 142.3 (C-4), 138.5 (C-11), 132.5 (C-12), 116.8 (C-20), 78.3 (C-5), 70.7 (C-2), 70.6 (C-14), 67.3 (C-10), 59.0 (C-1), 47.0 (C-9), 41.8 (C-3), 39.6 (C-15), 39.4 (C-13), 37.4 (C-8), 33.8 (C-7), 32.1 (C-17), 28.9 (C-6), 25.3 (C-16), 22.5 (C-19), 21.8 (C-18), 21.5 (14-OCOCH₃), 21.4 (2-OCOCH₃), 21.0 (5-OCOCH₃); FABMS (NBA): m/z = 485 (M + Na, 17), 341 (1), 281 (7), 135 (100).

Compound 3

5α-Dihydroxy-2α,10β,14β-triacetoxy-4(20),11-taxadiene, white powder. [α]_D ²⁰: + 35.2° (c 0.162, CHCl₃); IR υ_max ^(KBr): = 3432, 2940, 1726, 1636, 1373, 1236, 1023 cm^-1; ¹H-NMR δ = 6.11 (1H, dd, J = 5.5 Hz, 12.0 Hz, H-10), 5.34 (1H, dd, J = 2.5 Hz, 6.0 Hz, H-2), 5.10 (1H, brs, H-20a), 5.03 (1H, dd, J = 5.0 Hz, 10.0 Hz, H-14), 4.77 (1H, brs, H-20b), 4.19 (1H, brs, H-5), 3.21 (1H, d, J = 6.5 Hz, H-3), 2.77 (1H, dd, J = 9.0 Hz, 18.5 Hz, H-13α), 2.35 (1H, dd, J = 5.0 Hz, 18.5 Hz, H-13β), 2.32 (1H, dd, J = 12.0 Hz, 14.5 Hz, H-9β), 2.12 (1H, m, H-7), 2.08 (3H, s, OCOCH₃ ), 2.05 (6H, s, OCOCH₃ , CH_{3 -} 18) , 2.02 (3H, s, OCOCH₃ ), 1.84 (1H, d, J = 1.5 Hz, H-1), 1.72 (2H, m, H-6), 1.65 (3H, s, CH₃ - 16),1.59 (1H, dd, J = 5.5 Hz, 15.0 Hz, H-9α), 1.25 (1H, m, H-7), 1.11 (3H, s,CH₃ - 17), 0.80 (3H, s, CH₃ - 19); ¹³C-NMR: δ = 170.1(OCOCH₃ × 3), 147.5 (C-4), 135.8 (C-11), 134.4 (C-12), 113.5 (C-20), 76.2 (C-5), 70.8 (C-2), 70.5 (C-14), 70.3 (C-10), 58.9 (C-1), 43.7 (C-3), 39.9 (C-9, C15), 39.2 (C-13), 37.2 (C-8), 33.0 (C-7), 31.8 (C-17), 30.7 (C-6), 25.3 (C-16), 22.1 (C-19), 21.4 (OCOCH₃, C-18), 20.7 (OCOCH₃ × 2); FABMS (NBA): m/z = 485 (M + Na, 17), 462 (4), 403 (4), 343 (22), 299 (4), 283 (26), 265 (28), 136 (75), 91 (100).

Compound 4

6α,10β-Dihydroxy-2α,5α,14β- triacetoxy-4(20),11-taxadiene, white powder. [α]_D ²⁵: + 47.1° (c 0.0072, MeOH); IRυ _max ^(KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023 cm^-1; ¹H-NMR δ = 5.41 (1H, s, H-20a), 5.36 (1H, dd, J = 2.0 Hz, 6.0 Hz, H-2), 5.06 (1H, dd, J = 6.0 Hz, 12.0 Hz, H-10), 5.04 (1H, brs, H-5), 5.01 (1H, dd, J = 9.5 Hz, 5.5 Hz, H-14), 4.97 (1H, s, H-20b), 3.89 (1H, m, H-6), 2.95 (1H, d, J = 6.0 Hz, H-3), 2.80 (1H, dd, J = 9.0 Hz, 19.0 Hz, H-13β), 2.42 (1H, dd, J = 3.5 Hz, 19.0 Hz, H-13α), 2.39 (1H, m, H-9β), 2.20 (3H, s, 18-CH₃), 2.06 (3H, s, OCOCH₃ ), 2.02 (3H, s, OCOCH₃ ), 1.98 (1H, dd, J = 4.5 Hz, 14.5 Hz, H-7β), 1.94 (3H, s, OCOCH₃ ), 1.91 (1H, d, J = 2.5 Hz, H-1), 1.72 (3H, s, 16-CH₃), 1.67 (1H, dd, J = 5.5 Hz, 15.0 Hz, H-9α), 1.48 (1H, d, J = 14.5 Hz, H-7α), 1.19 (3H, s, 17-CH₃), 1.04 (3H, s, 19-CH₃); ¹³C-NMR: δ = 169.9 (OCOCH₃ × 2), 169.5 (OCOCH₃), 138.6 (C-4), 138.5 (C-11), 132.3 (C-12), 120.4 (C-20), 81.6 (C-5), 70.6 (C-2), 70.2 (C-6, C-14), 67.3 (C-10), 58.8 (C-1), 47.4 (C-9), 41.5 (C-3), 41.3 (C-7), 39.6 (C-15), 39.4 (C-8), 37.5 (C-13), 32.1 (C-17), 25.4 (C-16, C-19), 21.5 (C-18, OCOCH₃), 21.4 (OCOCH₃), 21.0 (OCOCH₃); FABMS (NBA): m/z = 501 (M + Na, 28), 401 (4), 341 (4), 299 (8), 281 (12), 154 (94), 136 (100).

Compound 5

6α,9α,10β-Trihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene, colorless needles. [α]_D ²⁵: + 42.3° (c 0.018, MeOH); IRυ_max ^(KBr): = 3432, 2940, 1726, 1636, 1373, 1236, 1023 cm^-1; ¹H-NMR: δ = 5.46 (1H, s, H-20a), 5.37 (1H, dd, J = 2.5 Hz, 6.0 Hz, H-2), 5.08 (1H, brs, H-5), 5.01 (1H, s, H-H-20b), 4.98 (1H, dd, J = 5.1 Hz, 9.3 Hz, H-14), 4.75 (1H, d, J = 9.5 Hz, H-10), 4.01 (1H, d, J = 9.5 Hz, H-9), 3.93 (1H, m, H-6), 2.96 (1H, d, J = 6.0 Hz, H-3), 2.82 (1H, dd, J = 8.6 Hz, 18.6 Hz, H-13α), 2.44 (1H, dd, J = 5.1 Hz, 18.6 Hz, H-13β), 2.20 (3H, s, 18-CH₃), 2.06 (3H, s, OCOCH₃ ), 2.02 (3H, s, OCOCH₃ ), 2.00 (1H, m, H-7), 1.96 (3H, s, OCOCH₃ ) , 1.90 (1H, d, J = 2.0 Hz, H-1), 1.64 (3H, s, 16-CH₃), 1.62 (1H, m, H-7), 1.24 (3H, s, 17-CH₃), 1.20 (3H, s, 19-CH₃); ¹³C-NMR: δ = 170.0 (OCOCH₃), 169.8 (OCOCH₃), 169.4 (OCOCH₃), 138.2 (C-4), 136.7 (C-11), 134.8 (C-12), 121.1 (C-20), 82.0 (C-5), 79.3 (C-9), 72.1 (C-10), 70.5 (C-14), 69.8 (C-6, C-2), 58.7 (C-1), 44.1 (C-8), 43.6 (C-3), 39.5 (C-15), 37.4 (C-13), 34.2 (C-7), 31.8 (C-16), 26.0 (C-17), 21.6 (C-18, C-19), 21.3 (OCOCH₃), 21.3 (OCOCH₃), 20.8 (OCOCH₃); FABMS (NBA): m/z = 517 (M + Na, 7), 154 (100), 136 (82).

Effects of stage of substrate addition on the transformation

On the 0, 3^rd, 6^th, 9^th, 12^th, 15^th, 18^th culture day, 0.5 mL substrate solution was added to each 500 mL flask with 150 mL medium by 3 replicates. On the 21^st day, all the media in each flask were collected, extracted and concentrated as described above. The residues were dissolved in HPLC mobile phase solution to yield 2.0 mL of sample solution, and the amounts of residual substrate 1, as well as compounds 2 and 4 were analyzed by HPLC. HPLC analyses were performed by using a Zorbax C₁₈ column (25 cm × 4.6 mm I.D., 5 μm), eluted with methanol-acetonitrile-water (50 : 15 : 35, v/v/v) at a flow rate of 1.0 mL/min and detected at 227 nm. By using the substrate and obtained compounds 2 and 4 as standard samples, the regression equations of 1, 2 and 4 were determined to be: Y = 625 889 X + 154 506 (r = 0.9997), Y = 643 254 X + 18 322 (r = 0.9993), Y = 703 244 X - 52 361 (r = 0.9999), respectively, where Y refers to peak area, X the injection amount (in μg), and r the correlation coefficient.

Results and Discussion

Compound 1 was administered to the 15-day-old cell cultures, and four more polar metabolites were obtained after additional six days of incubation. On the basis of chemical and physical data, their structures were identified as 10β-hydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (2), 5α-hydroxy-2α,10β,14β-triacetoxy-4(20),11-taxadiene (3), 6α,10β-dihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (4), and 6α,9α,10β-trihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene (5), respectively, among which 3 and 5 are two new taxoid compounds. However, 2 and 4 have also been obtained as microbial transformation products by Hu and co-workers [10], while this is the first report that they were obtained from biotransformation system by plant cell cultures. The biotransformation reaction is illustrated in Scheme 1.

The FAB mass spectrum of 3 showed a quasi molecular ion peak [M + Na]⁺ at m/z 485, consistent with the molecular formula of C₂₆H₃₈O₇, suggesting that the loss of an acetyl group as compared with 1. The ¹H-NMR spectrum of 3 was similar to that of 1 except that the signal corresponding to H-5β (δ = 5.29, brs) shifted to higher field at δ = 4.19 (brs), also suggesting the presence of a hydroxy group rather than an acetoxy group at the C-5 position. This conclusion was further supported by the appearance of a carbon resonance at δ = 78.5 instead of δ = 76.2 in its ¹³C-NMR spectrum. Therefore, compound 3 was elucidated to be the 5-deacetyl derivative of 1, and all the ¹H-NMR and ¹³C-NMR signals of 3 were assigned according to its ¹H-¹H COSY, HMQC and HMBC spectra (see Material and Methods).

The FAB mass spectrum of 5 showed a quasi molecular ion peak [M + Na]⁺ at m/z 517, consistent with the molecular formula of C₂₆H₃₈O₈. The ¹H-NMR spectrum of 5 was similar to that of 4 except that signals corresponding to H-9α (δ = 1.67, dd, J = 5.5, 15.0 Hz) or H-9β (δ = 2.39, m) in 4 had disappeared. At lower field, an additional oxygen-bearing methine signal was observed at δ = 4.01 (d, J = 9.5 Hz), which suggested the existence of a hydroxy group at the C-9 position. As a result, the signal of H-10α (δ = 5.06, dd, J = 6.0, 12.0 Hz) shifted to higher field (δ = 4.75, d, J = 9.5 Hz), and the coupling constant showed that the introduced hydroxy group is at the α-position. This was confirmed by the signal of C-9, which was significantly shifted to the lower field at δ = 79.3 in ¹³C-NMR spectrum, and was also confirmed by comparing with the NMR data of the reported compound 9α-hydroxy-2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene [8]. So the structure of 5 was identified as 6α,9α,10β-trihydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene, and all the ¹H-NMR and ¹³C-NMR signals of 5 were assigned according to its ¹H-¹H COSY, HMQC and HMBC spectra (see Material and Methods).

In order to shed light on the effects of the stages of substrate addition on the biotransformation process and to determine the optimal addition stages for the production of different products, the kinetics of cell growth and pH values as well as the residual amount of 1 and the yields of 2 and 4 at the different addition stages for 1 were investigated. The results (Fig.[1]) showed that the cell suspension cultures of C. roseus grew rapidly, and the whole growth period included three phases: 1) linear phase (0 - 15^th day), 2) stationary phase (15 - 18^th day), and 3) death phase (18 - 21^st day). The pH values remained relatively stable between 5.0 and 6.0 throughout the growth period. As shown in Fig. [2], when 1 was added during the mid-linear phase (9 - 12^th day), the biotransformation rate of 1 reached the highest level of 85 %, and the yield of 3 70 %, which suggested that this phase is the best for the production of 2. However, when 1 was added on the 6^th day, the yield of 4 reached its point of 11.8 %. Obviously, the specific reaction of 1 incubated with C. roseus cell cultures could be considered as 10-deacetylation due to the fact that product 2 possesses the highest yield.

In order to shed light on the process of 1 to 2 and 3 described above and to show that it was an enzymatic one, an additional experiment was performed. Compound 1 was added to the media with the pH value adjusted to exactly the same range (5.0 - 6.0) and was incubated without cells under the same conditions. Under the above conditions, no product was generated, which suggested the process of 1 to 2 and 3 to be enzymatic.

In conclusion, we reported here that 2α,5α,10β,14β-tetraacetoxy-4(20),11-taxadiene can be specifically hydroxylated at the 6α and 9α positions as well as deacetylated at C-10 by C. roseus cell cultures and this provided a powerful pathway to prepare bioactive taxoids or the intermediates for the semisynthesis of other bioactive agents. Future study may focus on isolating and purifying the responsible enzymes from the cell cultures, and then biocatalyze efficiently the substrate to the desired products by purified enzymes instead of whole cells.

Acknowledgements

We wish to thank The National Outstanding Youth Foundation by NSF of China and Trans-Century Training Program Foundation for the Talents by the Ministry of Education for financial support.

References

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Dean Guo, PhD

The State Key Laboratory of Natural and Biomimetic Drugs

School of Pharmaceutical Sciences, Peking University

Xueyuan Road 38

Beijing 100083

P.R.China

Email: gda@ bjmu.edu.cn

Phone: +86-10-62091516

Fax: +86-10-62092700

References

• 1 Suga T, Hirata T. Biotransformation of exogenous substrates by plant cell cultures.  Phytochemistry. 1990;  29 2393-406 (and references cited therein)
  CrossrefPubMedSearch in Google Scholar
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• 3 Wu Y, Zhu W, Lu J, Hu Q, Li X. Selection and culture of high-yield sinenxans cell lines of Taxus spp. distributed in China.  Chinese Pharmaceutical Journal. 1998;  33 15-8
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  PubMedSearch in Google Scholar
• 5 Sako M, Suzuki H, Yamamoto N, Hirota K. Highly increased cellular accumulation of vincristine, a useful hydrophobic antitumor-drug, in multidrug-resistant solid cancer cells induced by a simply reduced taxinine.  Bioorganic & Medicinal Chemistry Letters. 1999;  9 3403-6
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Dean Guo, PhD

The State Key Laboratory of Natural and Biomimetic Drugs

School of Pharmaceutical Sciences, Peking University

Xueyuan Road 38

Beijing 100083

P.R.China

Email: gda@ bjmu.edu.cn

Phone: +86-10-62091516

Fax: +86-10-62092700