全 文 ：第 23 卷　第 1 期　　　　　　　　　　　　　植　　　物　　　研　　　究 2003年　1 月
Vol.23　No.1　　　　　　　　　　　BULLETIN OF BOTANICAL RESEARCH Jan., 　2003
无载体固定化黄花蒿细胞生产青蒿素
胡风庆1　陈长兰1　王关林2
(1.辽宁大学生命科学系 , 沈阳　110036)
(2.辽宁师范大学生物工程研究所 , 大连　116029)
摘　要　疟疾是一种严重危害人类健康的流行病 ,主要由疟原虫经蚊虫叮咬引起 。目前 ,在临床
上疟原虫对治疗疟疾的药物(如氯奎等)有较强的耐药性 ,并表现出明显的交叉耐药性。来自黄花
蒿的青蒿素具有极其明显的抗疟活性 ,成为临床首选的药物 ,因此青蒿素的获取成为关键。本研
究采用无载体固定化法培养黄花蒿生产青蒿素 ,初步研究了无载体固定化细胞的生长特性。检测
发现 ,利用该方法生产的青蒿素是常规细胞培养法的 9倍 ,因此该方法有望成为青蒿素生产的首
选方法。
关键词　疟疾;青蒿素;黄花蒿;无载体固定化
SELF-IMMOBILIZED AGGREAGE CULTURE
OF ARTEMISIA ANNUA L.FOR IMPROVED ARTEMISININ
HU Feng-Qing 1　CHEN Chang-Lan1　WANG Guan-Lin2
(1.Department of Life and Science , Liaoning Univ ersity , shenyang 110036)
(2.I nsititute of Biotechnology , Liaoning Normal University , dalian 116029)
Abstract　Malaria is a kind of serious pandemic w hich is caused by plasmodia through mosqui to s
sting and bite.At present , drugs of curing malaria are:chloroquine , ethylamine pyrimidine et al ,
but the most serious question is anti-drug character of plasmodia on those drugs , particular to chlo ro-
quine.Furthermore , the anti-chloroquine plasmodia st rain have cross -anti character in clinical.
Artemisinin f rom Artem isia annua L.has evidently anti-malaria activity and become the best drug
in clinical.At present , the key question is that how to acquire artemisinin.For this reason w e utilize
self-immobilized agg regate culture of Artemisia annua L.to produce Artemisinin , and primarily
studied character of self-immobilized culture of Artem isia annua L.cell.Through determining , it
find that this method can acquire 9-times quanti ty artemisinin mo re than normal method.Therefore
this method may be possible became the best method of artemisinin produce.
Key words　malaria;artemisinin;Artemisia annua L.;self-immobilized aggregate
　　Malaria , also named dabaizi , is a kind of seri-
ous pandemic w hich is caused by plasmodia through
mosqui to s sting and bite.The numbers which die
of malaria are more than AIDS in every year.Today
malaria is a kind of most popular , most serious epi-
demic disease.It still exists and does g reat harm to
第一作者简介:胡风庆(1971-),男 ,讲师 ,沈阳药科大学在读博士研究生 ,主要从事植物分子生物学与植物天然产物的研究与开发。
收稿日期:2002-03-26
human health[ 1 ,2] .
At present , drugs of curing malaria are:chloro-
quine , ethy lamine pyrimidine et al , but the most se-
rious question is ant i-drug character of plasmodia
on those drugs , particular to chloroquine.Further-
more , the anti-chlo roquine plasmodia st rain have
cross-ant i character , so the w ork of searching for
new -type anti-malaria drug is going on and w ish
to relieve the harm of malaria to human health[ 1 , 3] .
In 1972 , Chinese scientists ex tracted
Artemisinin from Artem isia annua L , which has
anti-malaria activity[ 4] .Because of i ts definitive ef-
fect , quick action , low or no side effect , safety and
trust iness and sui table price , the drug is w elcomed
more and mo re.At present , artemsis is acquired
from artificial plant , because the plant of produce-
artemisinin is generally distributed in Asia , except
Asian region the o ther countries and areas have no t
distribution[ 5] .There are also some questions that
are not solved by using other methods to acquire
artemisinin , which impede its use and spread.At
the same time , the requirement of artemisinin from
patients of all the w orld can not be at all satisfied on-
ly through plant Artemisia annua L.and every
year a large mount of artemisinin are reqi red in clini-
cal[ 6 ～ 8] .In order to solve artemisinin source ques-
tion , dif ferent domains of scientists utilize dif ferent
methods , among of w hich plant cell culture method
has more advantage than other methods , that makes
i t become a key w ay of broadening artemisinin
source and become a hot point of research today[ 1 ,9] .
As secondary metabolic product in the process
of Artemisia annua L .g row th , Artemisinin s pro-
duction is of ten associated wi th tissue dif ferentia-
tion.In many cases , t issue differentiation is needed
for the biosynthesis of Artemisinin.In dispersed cell
culture of Artemisia annua L ., the necessary lack
of significant cellar org anization o r tissue dif ferentia-
tion usually results in low productivity of
Artemisinin.
Plant cell has the ability to agg regate sponta-
neously into macroscopic clumps displaying some lev-
el of cellar/ t issue differentiation , named self -im-
mobilizat ion , which eliminates the need of artificial
immobilization supports and could be exploited fo r
large-scale phytochemical synthesis.
Utilized t rait of plant cell , we studied self-im-
mobilized culture of Artemisia annua L.cell.
1　Materials and methods
1.1　Induction of Artem isia annua L .Callus
The callus was induced from the explants of the
leaf of Artemisia annua L.The explants were
w ashed 3 times w ith the distilled water , immersed
for 60 sec in 70% ethanol(v/v), surface-sterilized
in HgCl2(0.1%, v/v)for 5 min and finally rinsed
wi th sterile w ater.The explants cut into segments
of 1 cm2 were placed on B5 agar medium containing
2%sucrose , 0.2 mg/L BA , 0.5mg/L 2 , 4-D and
0.7% agar , then incubated at 25℃ in the light.
Callus fo rmation would be observed after 3 w eeks.
The callus w ere then excised and cultured under the
same conditions as above.Subculturing w as carried
out at 15day interval.
1.2 　Ini tiation of the self -immobilized agg regate
culture
Approx .5g fresh w t of actively g row ing callus
w as transferred to 250 mL Erlenmeyer f lask con-
taining 50 mL liquid medium which w as composed
of the same components as the agar medium.Flask
w as shaken at 115 rpm and 8 hours in the light at 25
℃.After 1 week , the medium was decanted , leav-
ing only small cellular agg regate in the flask.The
flask w as then supplemented wi th 50 mL fresh
medium and incubation w as continued.This proce-
dure w as repeated every 3 days.After 3 w eeks , few
dispersed cells were observed in the medium and the
medium remained almost clear.Once the self-im-
mobilized agg regate culture w as established , i t w as
subcultured every 10 day s by decanting all the medi-
um from the flask and adding fresh medium.Dis-
persed cells in the decanted medium were also col-
lected and cultured fo r comparison.
1.3　Analysis of artemisinin
The preparation and analysis of the intercellular
and ex tracellular artemisinin content in the samples
refer to the methods of Nair[ 10] and Sipahimalani[ 11] .
The major step is:1)After cell culture , cell is sepa-
rated from medium;2)cell is toasted at 75℃unt il
the w eight is no longer change;3)af ter dry cell is
78 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　23 卷
g rinded , use M eCN(50%)to ext ract , concentrate
and f ilter , the concentrate liquid is ready to deter-
mine;4)Use hexane to ext ract artemisinin in the
medium , then distillate in o rder to eliminate hex-
ane , distillating product is dissolved by M eCN ,
which is ready to determine.
Artemisinin is analysised by gas-liquid chro-
matog raphy method using 2000mm ×3mm stainless
steel column , 3%OV -17 , 60 ～ 80 CHRO-
MORBW.Gasification temperature is 265℃, col-
umn temperature is 250℃, rate of flow of N2 is
40mL/min.
1.4　Microscopy observat ion of agg regate
Af ter 3 weeks , morphology and condition of
self-immobilized agg regate cells of Artemisia an-
nua L.were observed under an invert microscope
every 3 days.
2　Results and discussion
2.1 　The characteristics of self -immobilized ag-
g regate culture
The self -immobilized aggregate culture of
Artemisia annua L.was composed of light yellow ,
spherical and compact ag gregates wi th well-round-
ed surfaces , somewhat like the plant nodules.The
sizes of self -immobilized agg regates were mainly
from 2 mm to 3mm , accounting for 80%of the total
biomass.No aggregate was larger than 5 mm.Un-
der optimal culture conditions , there were virtually
no dispersed cells or cell debirs in the flasks , so the
culture liquid w as almost clear.
The cells wi thin agg regate are tight ly packed
and a distinct radical pattern of cell g row th is evi-
dent , which show s a thin sect ion of agg regates
viewed under an invert microscope.It can be also
observed that many of large agg regates contain cavi-
ties at their centers.In addition , the self-immobi-
lized aggregates of Artem isia annua L .could be
continuously subcultured and maintained their mor-
phological integ rity during the culture.
Self-immobilized agg regate culture is as a dis-
tinct culture sy stem as the dispersed cell culture.
The fo rmation of the aggregates is supposed to rep-
resent a morphpogenetic pathw ay similar to that of
embryogenesis.
F ig.1　Growth curve of Artemisia annua L.
2.2　Kinetics of grow th and artemisinin product ion
The g row th curve of the self-immobilized ag-
gregates is similar to that of dispersed cells
(Figure1).The maximum dry w eight obtained from
the agg regate culture w as 11.4g/L on day 30 , 2.28
times as the value of the ini tial inoculum and corre-
sponding to a double times of 20 day s.In contrast ,
the dispersed cells have a low er maximum biomass
(10.2g/L , on day 40), representing a 2.04 times
increase of biomass w ith the double time of 40 days.
The artemisinin product ion of the self-immo-
bilized aggregate culture w as found to be partially
grow th-related.The maximum artemisinin content
attained w as 0.038% dry weight , nearly 9 times
the corresponding value of the dispersed cell culture ,
which w as show n not to be grow th-related.Yield
of per 50mL sample of self-immobilized agg regate
culture on day 40 is no t only higher than yield of
dispersed cell culture , but also Artemisinin yield in
medium is higher than int racellular artemisinin
yield , which help to ext ract directly artemisinin
f rom medium and simplify technology of production
(Table 1).
In this wo rk , the improvement of artemisinin
production w as obtained wi thout undergoing st rain
selection , optimal media and culture condition or us-
ing elicitior and precursors.Therefore , further in-
creases in artemisinin yield may be possible af ter fur-
ther experiments and researches , and also it w ill be a
realization that large - scale production of
artemisinin in reaction equipments.
791 期　　　　　　　　　　　　　　胡风庆等:无载体固定化黄花蒿细胞生产青蒿素
Table 1　Artemisisnin yield of self-immobilized aggregate culture
and dispersed cell culture on day 40(mg/50mL sample)
Total artemisinin yield Int racellular artemisinin yield Artemisinin yield in medium
Self-imm obilized aggregate
Culture(percentage)
3.76
(100)
2.43
(63.1)
1.33
(36.9)
Dispersed cell culture
(percentage)
0.42
(100)
0.31
(72.2)
0.10
(23.8)
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80 　　　　　　植　　物　　研　　究　　　　　　　　　　　　　　　　　　23 卷