全 文 ：·有效成分·
Biotransformation of artemisinin by fermentation
of Rhizopus chinensis andCunninghamella elegans
ZHAN Ji-xun
1, 2 , GUO Hong-zhu
1 , HAN Jian
1 , ZHANG Yuan-xing
2 , GUO De-an
1*
( 1. Sta te Key Labo ra tor y o f Na tural and Biomimetic Drug s, School of Pha rmaceutical Sciences, Peking Univ ersity ,
Beijing 100083, China; 2. State Key Labo ra to r y o f Bio reac to r Engineering , Ea st China Univ er sity
o f Science and Technolog y, Shanghai 200237, China)
Abstract: Object　 To study the microbial t ransfo rmation o f antimalarial drug a rtemisinin (Ⅰ ) .
Methods　 The enzymes secreted by fermenta tion of Rhizopus chinensis Sai to CICC 3043 and Cunninghamel-
la elegans Lendn. AS 3. 1207 in the potato medium were employed to transform artemisinin. Results　
Three products w ere obtained, among which deoxyartemisinin (Ⅱ ) w as also found in the controls wi thout
microo rganisms. The o ther two w ere identified as 3α-hydroxydeoxya rtemisinin (Ⅲ ) and 9β -hydrox-
ya rtemisinin (Ⅳ ) . Compound (Ⅳ ) was identified as a new one. Conclusion　 Artemisinin could be t rans-
formed by the tw o ti tle st rains o f microo rganisms. It i s easy to release one oxygen atom by breaking of
perexide bridge in potato medium and to form deoxyartemisinin, thus making i t lose it s antimalarial activi-
ty. The i ron ( Fe) in the po tato medium may have the above function.
Key words: bio t ransformation; a rtemisinin; Rhizopus chinensis saito; Cunninghamella elegans Lendn.
华根霉和雅致小克银汉霉对青蒿素的生物转化研究
占纪勋 1, 2 ,郭洪祝1 ,韩　健1 ,张元兴 2 ,果德安 1
( 1. 北京大学药学院 天然药物及仿生药物国家重点实验室 ,北京　 100083;　 2. 华东理工大学 生物反应器国家重点实验室 ,
上海　 200237)
摘　要: 目的　探讨微生物对青蒿素 (Ⅰ )的转化作用。 方法　通过华根霉和雅致小克银汉霉在土豆培养基中发酵
产酶 ,对青蒿素进行转化。 结果　两株菌对底物均有转化作用 ,分离出去氧青蒿素 deoxyar temisinin (Ⅱ ) , 3α-羟基
去氧青蒿素 3α-hydroxydeoxya r temisinin (Ⅲ )和 9β-羟基青蒿素 9β -hydroxya rtemisinin (Ⅳ )共 3个产物 ,其中Ⅳ为
一新化合物 ,同时振荡条件下底物在无菌培养基中也能发生微量转化得到产物Ⅱ 。 结论　青蒿素易被实验两株真
菌转化 ,同时过氧桥也易断裂而失去一个氧原子成为去氧青蒿素 ,丧失抗疟活性 ,起作用的可能是土豆培养基中的
铁元素。
关键词: 生物转化 ;青蒿素 ;华根霉 ;雅致小克银汉霉
中图分类号: R284. 1　　　文献标识码: A　　　文章编号: 0253 2670( 2002) 10 0869 04
　 　 Artemisinin Ⅰ , a lso called Qinghaosu, a
sesqui terpene lactone endoperoxide isolated f rom
the Chinese herbal plant Artemisia annua L. , i s an
impo rtant therapeutic agent combating multidrug-
resistant Plasmodium falciparum st rains
[1 ] . Some
biot ransforma tions o f artemisinin by micro organ-
isms and plant cells w ere repo rted
[2～ 6 ]
. Microbial
t ransfo rmation is defined as an enzymatic reaction
ca taly zed by the enzyme secreted in metabolic ac-
tivi ties o f microo rganisms, by w hich the st ructures
of specific subst rates w ere modified. Compa red
wi th chemical methods, i t has highly stereo and
chemo-selectivi ty and o ther advantag es, such as
mild reaction conditions, simple operation proce-
·869·中草药　 Chinese Traditiona l and He rbal Drug s　 2002年第 33卷第 10期
收稿日期: 2002-02-12作者简介:占纪勋 ( 1976-) ,男 ,江西人 ,在读博士 ,主要从事天然产物的分离及生物转化研究。 E-mail: zh anjixun@ sina. com
* 通讯作者　　 Tel: ( 010) 62901516　　 Fax: ( 010) 62092700
dures, less cost and low er pol lution. Some reac-
tions which can no t be fulfilled in chemical ap-
proach are facile processes by microbial t ransfor-
ma tion. Some microbial t ransfo rmations w ere es-
tablished as in v itro models for the predici ton of
mammalian drug metaboli tes [8, 9 ] . In this paper the
microbial transforma tion o f artemisinin by fermen-
tation of Rhizopus chinensis Sai to CICC 3043 and
Cunninghamella elegans Lendn. AS 3. 1207 is re-
ported.
1　 Results and discussion
Products Ⅱ -Ⅳ were isola ted from the fer-
mented broth of C. elegans AS 3. 1207 and tw o
products Ⅱ , Ⅲ f rom that of R. chinensis CICC
3043. But compoundⅡ was also obtained from the
controls in w hich the subst ra te was added wi thout
microo rganisms.
Compound Ⅱ was obtained as colo rless nee-
dles. TO FM S ofⅡ show ed a molecular w eight of
266 and the cha racteristic signals of a peroxide
bridge w ere absent in IR spectrum, which suggest-
ed an oxygen a tom lost. The
1
HNM R and
13
CNMR
were in good ag reement w ith those o f deox-
ya rtemisinin, thereforeⅡ was confi rmed to be de-
oxya rtemisinin[4 ] .
Compound Ⅲ was obtained as colo rless nee-
dles. The TOFM S o f Ⅲ suggested i ts molecular
w eight of 282. IR spect rum o fⅢ show ed a st rong
abso rption a t 3 495 cm- 1 and no characteristic sig-
nals of the peroxide bridge observed. Af ter a com-
parison o f the
1
HNMR and
13
CNMR spect ra wi th
those of 3α-hydroxydeoxyatemisinin, Ⅲ was iden-
ti fied as 3α-hydroxydeoxya temisinin. It was prev i-
ously repor ted to be a microbial t ransfo rmed prod-
uct o f ar temisinin by Penicill ium cbrysogenum
( ATCC 9480) [ 4] .
ProductⅣ was obtained as colo rless needles.
TO FM S ofⅣ show ed a mo lecular w eigh t of 298.
A st rong absorption at 3 491 cm- 1 and the signals
o f the pero xide bridge w ere observ ed, suggesting a
hydroxy group int roduced into the subst ra te
molecule. DEPT analysis show ed tha t the number
o f secondary carbon changed from four to three and
that of tertiary carbon increa sed from fiv e to six.
A new peak atδ73. 5 in the 13 CNM R spectrum was
found, which indica ted Ⅳ was a hydro xy lated
product o f subst rate and the hydroxy g roup must
be added at a secondary ca rbon. The hydroxy
g roup w as determined from CO SY, HSQC and
HMBC spect ra to be a t C-9 po sition. Compa ring
13 CNM R spect rum ofⅣ w ith that of ar temisinin,
i t w as found that C-10, C-8 shi fted downfield and
C-1, C-7, C-14 shif ted upfiled, which confi rmed
Fig 1.　 The struc-
ture of ar-
temisinin
Ⅳ to be a 9-hydroxy product.
In the 2D 1H-1 H NOESY spec-
trum , the co rrela tion of 9α-H to
1α-H and 7α-H strongly sug-
gested that the o rientation of 9-
OH as β configuration. There-
fo reⅣ was identi fied as 9β-hy-
droxya rtemisinin, w hich is a
new compound ( See Fig. 1) .
ProductⅡ was also isolated f rom the sub-
strate controls, w hich showed that it w as no t a
bio t ransfo rmed product but a product of chemical
reaction cataly zed by the iron ion exi ted in pota to
medium possibly. It could be inferred tha tⅢ was
a hydroxy la ted product o f deoxya rtemisininⅡ . A
two-step reaction including a chemical and a bio-
logical reaction resul ted in productⅢ .
Product Ⅳ was a hydro xy l product of
artemisininⅠ owing to the enzyme secreted by C .
elegans AS 3. 1207. The biological activi ties of Ⅳ
are current ly under investiga tion ( See Fig. 2) .
In conclusion, microbial t ransfo rmation is a
useful too l in new drug research. By using various
kinds of enzymes o f microo rganisms and some
chemical methods, i t i s possible to t ransform dif-
ferent na tural products, modi fy thei r st ructures o r
activ e si tes and obtain new bioactive compounds
fo r new drug development.
2　 Experimental
2. 1　 General experimental procedures: Melting
points w ere determined on a micromelting point ap-
paratus and uncor rected. 1D and 2D NMR spect ra
w ere reco rded in CD3 Cl on an INOVA-500 inst ru-
ment a t 500 M Hz by using TM S as internal stand-
ard . IR spect rum was run on a Perkin -Elmer 983
·870· 中草药　 Chinese Traditiona l and He rbal Drug s　 2002年第 33卷第 10期
Fig 2.　 The possible biotransformat ion pathways of artemisinin byRhizopus chinensis andCunninghamella elegans
FT-IR and recorded in KBr pellets. Optical ro ta-
tions w ere measured on Perkin-Elmer 243 spec-
trometer using MeOHas so lv ent. TO FM S was ob-
tained w ith a Perkin-Elmer Q STAR mass spec-
trometer. TLC analyses w ere perfo rmed on si lica
g el G. Separa tion and purification w ere carried out
by column ch romatog raphy on si lica gel. The si lica
g el w as obtained from Qingdao Haiyang Chemical
Group Co. , P. R. China.
2. 2　 Microorganisms: R. chinensis CICC 3043
w as obtained from China Center o f Indust rial Cul-
ture Collection and C. elegans AS 3. 1207 from
China General Microbiological Cul ture Collection
Center.
2. 3　 Medium: All the experiments of culture and
biot ransforma tion w ere ca rried out in po tato medi-
um.
2. 4　 Cultura l and general biot ransforma tion pro-
cedure: Microo rganisms used in this study w ere in-
ocula ted in to 250 mL Erlenmeyer f lasks containing
50 mL o f po tato medium from PDA ( po tato dex-
trose aga r ) slants. The fermenta tion flasks were
placed on ro ta ry shakers, opera ting at 180 r /min at
28℃ . Artemisinin w as prepared as a 20 mg /mL
solution in acetone. Af ter 48 h of incubation, 0. 3
m L o f subst rate so lution was added into the fer-
menta tion bro th. The cultures w ere maintained
under the identical condi tions fo r further four
days. Cul tural cont rols consisted of fermenta tion
blanks in w hich micro organisms w ere g row n wi th-
out subst rate. Substrate controls consisted o f ster-
i le medium containing the same amount of sub-
strate and incuba ted under the same condi tions.
Af ter addi tional four day s, the t ransformed
broth and the controls w ere fil tered and the fi l-
t rates were ex t racted wi th the same vo lume of E-
tOAc fo r three times. The solvent w as evapo rated
to dryness under reduced pressure and the residue
w as disslov ed in acetone. TLC analyses w ere ca r-
ried out on silica gel plates using petroleum ether
( 60℃～ 90℃ ) -acetone ( 5∶ 2) as the developing
sy stem, and visualiza tion of pla tes w as performed
by using 10% H2 SO4-EtO H as spray reagent. The
results show ed bo th st rains could bio t ransfo rm the
subst rate.
Prepa ra tive scale biot ransforma tion experi-
ments by R . chinensis CICC 3043 and C. elegans
AS 3. 1207 w ere carried out in tw enty 1 L Erlen-
meyer flasks containing 250 mL of po ta to medium
respectiv ely. Artemisinin, 200 mg , w as used in
each bio t ransfo rmation system. Al l operations
w ere perfo rmed under the condi tions described
above.
2. 5　 Isolation and cha racteriza tion of bio t rans-
fo rmed products: yellow residue, 600 mg , w as ob-
tained from the fermented broth o f R. chinensis CI-
CC 3043 and 650 mg from that o f C . elegans AS
3. 1207. The residues were cho romatog raphed on
silica g el co lumns. Bo th columns w ere eluted w ith
per tro leum ether ( 60℃～ 90℃ ) -acetone ( 10∶ 1) .
·871·中草药　 Chinese Traditiona l and He rbal Drug s　 2002年第 33卷第 10期
ProductⅡ , 3 mg , and Ⅲ , 66 mg , w ere obtained
from R. chinensis CICC 3043, whileⅡ , 2 mg , Ⅲ ,
47 mg , andⅣ , 70 mg from C . elegans AS 3. 1207.
9β -hydroxyartemisininⅣ , C15 H22O6 , colo rless
needles, mp 194℃～ 196℃ , [α]20D + 50. 0 ( c0. 20,
EtO H) Positiv e TOFM S m /z 299 [M+ 1 ]
+
, 316
[M+ NH4 ]
+ , 321 [M+ Na ]
+ , 337 [M+ K ]
+ , 614
[M+ NH4 ]
+ , 619 [2M+ Na ]
+ , 635 [ 2M+ K ]
+ ;
IRν( K Br)max ( cm- 1 ): 3 491 ( O H) , 1 741 ( C= O) , 835,
883 and 1 113 ( the peroxide bridge )
1
HNMR and
13
CNMR data see Table 1.
Table 1.　 1HNMR and 13CNMR spectral data
of compoundⅣ
Posi tion δC　　 δD　　　 DEPT
　　 1 　　 42. 9 1. 52 m CH
2 24. 7 1. 52 m, 2. 00 m CH2
3 35. 7 2. 07 m, 2. 43 m CH2
4 105. 5 C
5 93. 4 5. 93 s CH
6 78. 8 C
7 42. 2 1. 91 dq ( 14. 0, 5. 0) CH
8 32. 1 1. 18 m, 2. 12 m CH2
9 73. 5 3. 29 m CH
10 44. 4 1. 39 m CH
11 32. 5 3. 38 m CH
12 171. 6 C
13 12. 6 1. 23 d ( 7. 0) CH3
14 15. 5 1. 12 d ( 6. 0 CH3
15 25. 2 1. 46 s CH3
　　 In addition, subst rate, 80 mg , w as added into
four 1 L flasks containing 250 mL of blank medi-
um. They w ere maintained on the ro ta ry shakers
under identical condi tions. Finally product Ⅱ , 1
mg , w as iso lated f rom the medium.
Acknowledgement: We thank th e National Outstanding
Youth Foundation by NSF o f 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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蝉翼藤茎化学成分研究 (Ⅱ )
杨学东 ,徐丽珍 ,杨世林
(中国医学科学院 中国协和医科大学药用植物研究所 ,北京　 100094)
摘　要: 目的　对蝉翼藤 Securidaca inappendiculata的化学成分进行研究。 方法　利用硅胶柱色谱和中压液相色
谱方法对蝉翼藤茎的 95%乙醇提取物进行分离 ;采用 UV, IR, MS, 1D和 2DNMR等技术对所得化合物进行结构
研究。 结果　分离鉴定了 6个化合物 ,分别为: 4, 4′-二甲基 -1, 7-庚二酸 (Ⅰ ) ,肌醇 (Ⅱ ) ,豆甾醇 (Ⅲ ) ,维太菊苷
(Ⅳ ) ,鼠李糖 (Ⅴ )和蔗糖 (Ⅵ )。结论　Ⅰ 为首次从该植物中分离得到的化合物 ,其他化合物均为首次从本属植物中
分离得到。
关键词: 蝉翼藤 ;远志科 ; 4, 4′-二甲基 -1, 7-庚二酸
中图分类号: R284. 1　　　文献标识码: A　　　文章编号: 0253 2670( 2002) 10 0872 03
·872· 中草药　 Chinese Traditiona l and He rbal Drug s　 2002年第 33卷第 10期
收稿日期: 2001-12-25作者简介:杨学东 ,男 ,博士 ,现在中国医学科学院、中国协和医科大学药用植物研究所新药研究中心从事天然产物化学研究和中药新药开发。
Tel: ( 010) 62899705　　 E-mai l: xuedong y@ 263. n et