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Biotransformation of artemisinin by Catharanthus roseus and Ginkgo biloba cell suspension cultures

长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究(英文)



全 文 :·药材·
Biotransformation of artemisinin byCatharanthus roseus and
Ginkgo biloba cell suspension cultures
HAN Jian, DAI Jun-gui , CU I Ya-jun, ZHAN Ji-xun, GUO Hong-zhu, GUO De-an
( Schoo l o f Pharmaceutica l Sciences, Mode rn Research Center fo r TCM , Peking Univ er sity , Beijing 100083, China)
Abstract: Object  To investiga te the biot ransfo rmation of the antimalarial compound a rtemisinin
(Ⅰ ) by Catharanthus roseus and Ginkgo biloba cell suspension cultures. Methods  Plant tissue cul ture
technolog y was employed. The product was isola ted on si lica g el column ch romatog raphy and i ts st ructure
w as elucidated by spect ro scopic evidence. Results  One product w as obtained and i ts st ructure w as charac-
terized as 3α- hydroxydeoxya rtemisinin (Ⅱ ) . Conclusion  Bo th of C . roseus and G.biloba cell suspension
cultures can bioconver t a rtemisinin.
Key words: Catharanthus roseus ( L. ) G. Don; Ginkgo biloba L. ; bio t ransformation; artemisinin; cell
suspension culture
长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究
韩 健 ,戴均贵 ,崔亚君 ,占纪勋 ,郭洪祝 ,果德安⒇
(北京大学药学院 中医药现代研究中心 ,北京 100083)
摘 要: 目的 对抗疟药物青蒿素 (Ⅰ )进行了生物转化研究。 方法 利用长春花及银杏植物细胞悬浮培养细胞进
行生物转化。用硅胶柱色谱进行产物的分离 ,波谱方法鉴定产物的结构。结果 此两种植物悬浮细胞体系均能将青
蒿素转化成 3α-羟基去氧青蒿素 (Ⅱ )。 结论 此两种植物悬浮细胞体系均能有效转化青蒿素。
关键词: 长春花 ;银杏 ;生物转化 ;青蒿素 ;悬浮细胞
中图分类号: R282. 13   文献标识码: A   文章编号: 0253 2670( 2003) 02 0166 03
    Artemisinin ( Qinghaosu ) , a sesqui terpene
lactone, is an antimala rial ag ent isola ted f rom the
Chinese herbal medicine Artem isia annua L.
[1 ] .
Thereaf ter ar temisinin and its deriv ativ es have re-
ceived considerable at tention because of thei r activ-
ity against resistant st rains o f Plasmodium f alci-
parum and ef ficacy against cerebral mala ria. As an
antimalarial drug , the high reversion rate and poo r
solubili ty of artemisinin in wa ter, was limi ted i ts
use in clinics. A number of deriv ativ es of
a rtemisinin have been synthesized from dihy-
droartemisinin, and out of these, a rtemether, ar-
teether, a rtesunic and ar telinic acid are ei ther cur-
rently in use o r being evaluated fo r use[ 2] . At the
same time some bio transforma tion o f a rtemisinin
and i ts analogues, such as arteannuin B,
artemether and arteether etc. , has been ca rried out
during the past years[ 3~ 6] . M oreover , the cy to tox ic
effect o f some a rtemisinin deriv atives have been
studied
[7, 8 ]
. It can be concluded tha t a rtemisinin
and i ts deriv ativ es are g et ting to be the focus of
many investiga to rs. To da te, there is no repo rt on
the bio t ransformation of artemisinin by plant cell
suspension cultures. In the present paper, The
bio t ransfo rmation o f I by Catharanthus roseus ( L. )
G. Don and Ginkgo biliba L. cell suspension cul-
tures w as repo rted, respectiv ely.
1 Results and discussion
·166· 中草药  Chinese T raditional and Herbal Drug s 第 34卷第 2期 2003年 2月
⒇收稿日期: 2002-10-20作者简介:韩 健 ,女 ,辽宁省人 ,硕士 ,主要从事生物药学 ,天然产物的生物转化及活性研究。
* 通讯作者: Tel: 86-10-62091516  Fax: 86-10-62092700  E-mai l: gda@ mai l. bjmu. edu. cn
CompoundⅠ ( Fig . 1) w as administered to the
ten-day-old cell cult rues, and incubated fo r addi-
tional seven day s. The cells and medium were har-
vested and ex t racted as described in the experimen-
tal section. One product w as isolated f rom the
medium by chroma tog raphic methods, and i ts
st ructure w as identi fied as 3α-hydroxydeoxy-
a rtemisinin ( Fig. 1) on the basis of i ts chemical and
spect ral da ta. Mass spect ra l da ta o f productⅡ in-
dicated that there w as no change in the molecular
w eight (m /z [M ]
+
282) . The IR spectrum show ed
a broad peak at 3 490 cm
- 1 , which suggested the
presence o f a hydroxyl g roup, w hile no character-
istic signal o f a perox ide bridge ( 831, 881, 1 115
cm
- 1
) was observ ed. The
13
CNMR spectrum sh-
ow ed a new signal a t 69. 4. All of these data indi-
cated tha tⅡ was a hydroxyla ted deoxyartemisinin.
The 13 CNMR and 1HNM R data ofⅡ were in good
ag reement wi th those repo rted in li terature
[3 ]
. The
yields o fⅡ by C . roseus and G. biloba cul tured cells
w ere 10% and 13% , respectiv ely. Addi tional test
show ed that compoundⅠ added to the same medi-
um without cell cultures and incuba ted in the same
condition yielded no products, suggesting that Ⅱ
be an enzyma tic product. The fact thatⅠ was con-
ver ted toⅡ by bo th C. roseus and G.biloba cul tured
cells suggested that some plant cell suspension cul-
tures migh t po ssess simi lar enzyme systems to re-
sul t in the lo ss o f one of the peroxide oxygen atoms
to give th e epox ide and have the abi li ty o f hydroxy-
lation though the precise o rder of the pa thw ay is
stil l unknown. The bioactivi ty studies on the anti-
tumo r and antimala rial activities of the t ransfo rmed
productⅡ is under w ay.
Fig. 1  Structures of compoundⅠ andⅡ
This result should be of some value fo r the fu-
ture bio t ransfo rmation studies of ar temisinin. Al-
though there has been some chemical methods to
synthesize artemisinin and i ts derivativ es, the tox ic
risk and high cost of these methods limi t thei r
uses. Unti l now plants are sti ll the main source fo r
the production of a rtemisinin. But th e content of
artemisinin in natural resources or cul tured cells of
A. annua i s low , w hich made it urgent to develop
some ef fectiv e methods w ith biotechno logy and
biochemical engineering to improve the production
of ar temisinin.
2  Experiment
2. 1  General experimental condi tions. IR spect ra
w ere obtained on a Perkin-Elmer 983B spect ropho-
tometer ( KBr) . NM R spect ra ( 1HNM R and 13 CN-
MR) w ere recorded in CDCl3 on INOVA-500 spec-
trometer, and chemical shif ts w ere reco rded in δ
using TM S as internal standa rd. TOF mass spec-
tra was measured on a MALDI TO F mass spec-
trometer. All chemicals w ere obtained from Bei jing
Chemical Factory.
2. 2  Tissue and cell cul ture of C . roseus. The
seedlings of C. roseus ( identi fied by Prof. GUO De-
an ) were obtained from the Medicinal Plant Ga r-
den o f Peking Univ ersi ty Heal th Science Center.
Young leaves w ere used to initia te calli. The plant
materials were disinfected by immersing in 70%
ethano l fo r 30 seconds, followed by 0. 1% HgCl2
fo r ten minutes, w ashed fiv e times wi th sterili zed
w ater, then cut into small pieces ( about 0. 5 cm×
0. 5 cm ) and aseptical ly transferred to M urashig e
and Skoog s medium ( M S) supplemented w ith 1. 0
mg /L o f 2, 4-dichlorophenoxyacetic acid ( 2, 4-D)
and 7. 0 g /L o f aga r. The pH o f the medium was
adjusted to 5. 8 befo re being auto claved at 121℃
fo r 20 minutes. The calli w ere ini tiated f rom all of
explants w ithin four w eeks of culture in the dark-
ness at ( 25± 2)℃ . The calli cul tures w ere main-
tained on the medium o f the same composi tion and
the same culture condi tion by subculture every four
w eeks. Three-w eek-o ld f riable cal li were used fo r
initiation of suspension cul tures. M S medium sup-
plemented wi th 0. 5 mg /L of 6-benzyl-aminopurine
( 6-BA) , 0. 5 mg /L o f N AA and 0. 2 mg /L of 2, 4-
dichlo rophenoxyacetic acid ( 2, 4-D ) which w as
proved to be the best fo r g ood g row th of the cul-
·167·中草药  Chinese T raditional and Herbal Drug s 第 34卷第 2期 2003年 2月
tures in liquid medium. Cell cul tures w ere subcul-
tured every three w eeks a t the inocula tion of 5 g /L
o f dry w eight in 500 mL of Erlenmeyer f lasks wi th
150 mL o f fresh medium and incubated on a rota ry
shaker at 110 r /min in the darkness at ( 25± 2)℃ .
The procedure o f the cultiva tion o f G. biloba cells
w as perfo rmed as described previously
[ 9]
.
2. 3  Bio transforma tion of Ⅰ . The suspension
cells w ere cul tured in 500 mL of flask wi th 150 mL
liquid medium. One milliliter of the stock sub-
st ra te solution( 10 mg /mL) wa s added to one f lask
w ith suspension cell cul tures, and one addi tional
f lask wi thout substrate as the contro l. Af ter addi-
tional sev en day s of incubation, the cell cul trues
w ere fil tered under v acuum and w ashed th ree times
w ith distil led w ater. The fi lt rate w as co llected and
ex t racted three times by equiv alent v olume of
EtO Ac, and all the ex t racted so lutions w ere con-
centrated under v acuum a t 40℃ . The residues
w ere disso lv ed in acetone and analy zed by TLC de-
veloped w ith acetone-petro leum ether ( 1∶ 2. 5) ,
and detected by spraying w ith 10% EtO H ( in
H2 SO4 ) fo llow ed by heating a t 105℃ . The TLC
resul ts show ed that one new spot appeared in the
chroma tog ram of the medium ex tract. Fo r prepara-
tiv e bio transforma tion experiment , 1 mL o f sub-
st ra te solution was added to each f lask on the 10th
cultural day , and the to tal amount of Ⅰ adminis-
tered in C . roseus and G.biloba was 300 mg , 150
mg, respectively. Af ter additional sev en days of
incuba tion, al l the media w ere co llected, ex t racted
and concentrated as described above. The obtained
residue w as separated by silica gel ch romatog raphy
( silica gel G, 200— 300 meshes) , eluting wi th ace-
tone-petroleum ether ( 60℃— 90℃ ) ( 1∶ 5— 1∶ 1)
to yield 30 mg and 20 mg of Ⅱ by C . roseus and
G.biloba cell suspension cultures, respectively.
2. 4  Identi fica tion. 3α-Hydroxydeoxya rtemisinin
Ⅱ : white needles; 1HNMR ( CDCl3 , 500 MHz) δ
0. 93 ( 3H, d, J= 6 Hz, M e-14) , 1. 17 ( 3H, d J= 7
Hz, Me-13) , 1. 56 ( 3H, s, M e-15) , 1. 81 ( 1H,
m, H-8) , 1. 92 ( 1H, m, H-8) , 1. 97 ( 1H, m , H-
2) , 2. 07 ( 1H, dt, J= 12. 9, 4. 5 Hz, H-7) , 3. 18
( 1H, dq, J= 4. 5, 7. 2 Hz, H-11) , 3. 60 ( 1H, s,
H-3) , 5. 62 ( 1H, s, H-5) .
13
CNMR ( CDCl3 , 500
Hz): δ12. 6 ( 13-C) , 18. 4 ( 14-C) , 20. 5 ( 15-C) ,
23. 5 ( 8-C) , 30. 3 ( 2-C) , 32. 7 ( 11-C) , 33. 4 ( 9-
C) , 35. 1 ( 10-C) , 40. 6 ( 1-C) , 42. 0 ( 7-C) , 69. 4
( 3-C) , 82. 9 ( 6-C) , 98. 9 ( 5-C ) , 108. 9 ( 4-C) ,
171. 3 ( 12-C) .
Acknowledgements: We thank the Na tional Outstand-
ing Youth Founda tion by N SF of China and T rans-Centur y
T raining Prog r am Foundation fo r the Ta lents by the Min-
istry of Educa tion fo r financial suppor t.
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