全 文 ：胀果甘草悬浮培养细胞合成甘草总黄酮
杨 英 , 何 峰 , 余龙江? , 陈雪红 ,
雷 晶 , 季家兴 , 付春华
( 华中科技大学生命科学与技术学院 , 湖北 武汉 430074 )
摘要 : 比较了胀果甘草 ( Glycyrrhiza inflata) 悬浮细胞在逐级放大摇瓶中的生长、黄酮产量以及营养消耗过
程 , 以便了解其放大规律。结果表明 , 在 250 和 500 mL 摇瓶中 , 细胞的最大生物量、黄酮产量以及最大比
生长速率没有显著性差异 , 但是在 1 L 的摇瓶中 , 这三种参数都显著地降低 , 分别比 250 mL 摇瓶中降低了
27% , 30 %和 27%。在逐级放大的摇瓶中 , 氮、磷、铵浓度都随着培养时间延长而逐渐降低 , 尽管在 1 L
的摇瓶中磷消耗得最慢 , 但三种摇瓶中磷在细胞生长对数期基本都被消耗尽了。此外 , 硝态氮在第 18 天
时基本被消耗完 , 而铵态氮在细胞收获时仍能维持在 100 mg?L。因此在反应器中培养时 , 主要的培养条件
还需进一步优化。
关键词 : 胀果甘草细胞 ; 生长 ; 黄酮产量 ; 营养消耗 ; 放大培养
中图分类号 : Q 945 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 04 - 444 - 03
Production of Flavonoids in Cell Suspension Culture of
Glycyrrhiza inflata (Leguminosae)
YANG Ying, HE Feng, YU Long-J iang
*
, CHEN Xue-Hong,
LEI J ing, J I Jia-Xing, FU Chun-Hua
( College of LifeScienceand Technology, Huazhong University of Scienceand Technology, Wuhan 430074 , China)
Abstract : The cell biomass, flavonoids production and nutrients consumption weremonitored to characterizethe culture of
progressive scale-up of Glycyrrhiza inflata . Themaximumbiomass, flavonoids production andthemaximumspecific growth
rate in the cultureof 250 and500 mL flasks remain similar, but were significantly higher than that of 1 000 mL flask . The
three parameters in theculture of 1 000 mL flaskwere27% , 30 % , and27% lower than that of 250 mL flask, respective-
ly . The concentrates of phosphate, nitrateandammoniumin progressive scale-upflasks decreased similarly . Phosphateand
nitrate were almost exhaustedon day 10 and18 in all flasks, respectively; whileammoniummaintainedabout 100 mg?L till
cells were harvested . The basic culture conditions needed to befurther optimized for higher flavonoids productionon abio-
reactor scale .
Key words: Glycyrrhiza inflata; Growth; Flavonoids; Nutrients consumption; Scale-up flask
Glycyrrhiza inflata Bat . is a traditional Chinese
medicine . One of the major compounds, flavonoids
have significant anti-oxidation activity, antitumor activ-
ity ( Kanazawa et al . 2003) , antihuman immunodefi-
ciency virus activity ( Hatano et al . 1988 ) and anti-
bacterial activity (Fukai et al . 2002) .
Thus, the G. inflata plant has fallen short of sup-
ply with an increasingdemand for flavonoids . Plant cell
culture is auseful method for theproductionof valuable
secondary metabolites .
云 南 植 物 研 究 2007 , 29 (4) : 444～446
Acta Botanica Yunnanica

? ?Correspondence author; E-mail : Yulj@ hust. edu. cn; Tel : 027 - 87792264; Fax: 027 - 87792265
Received date: 2006 - 12 - 05 , Accepted date: 2007 - 04 - 30
作者简介 : 杨英 ( 1977 - ) 女 , 博士 , 讲师 , 主要从事生物技术研究。
However, there were few studies on cell suspen-
sion culture of G. inflata, and its nutrients consump-
tion has not yet been systematically investigated . Cell
growth and flavonoids biosynthesis are sensitive to en-
vironmental conditions . Furthermore, scaling up the
culture fromflask to bioreactor isvery difficult . Conse-
quently, in the present study, the cell growth, fla-
vonoids accumulation and nutrient consumption were
investigated in scale-up flasks to understand the char-
acteristic of G. inflata cell , which may be beneficial
for scaling up in the bioreactor .
Materials and Methods
G. inflata cell were maintained in Murashige and Skoog
(MS) culturemedium containing 3% sucrose supplemented with
2 ,4 -dichlorophenoxyacetic acid (2 , 4 -D, 0. 5 mg?L) , naphtha-
lene acetic acid (NAA , 0 .5 mg?L) and6-benzyladenine (6-BA ,
0 . 5 mg?L) . For experiments, cells were cultured at 25℃ on a
rotatory shaker (120 r?min) with 5% inoculum in 250 , 500 mL
and 1 L flaskswith 70 , 140 and 280 mL medium (pH 5.8) , re-
spectively . The cells were harvested by filtration, and dried at
50℃ to constant dry weight ( DW ) . The maximum specific
growth rateswere calculated by theformula: (X2 - X1 )? ( t2 -
t1 ) , where X2 and X1 werethe DW of the cell at t2 and t1 days
during the exponential growth phase, respectively .
Nitrate in themediumwas determined according to Hecht &
Mohr (1990) . Salicylic acid-H2 SO4 (5 % ) was put into theme-
dium, 20 min later, 8 % NaOH was added in . Then the absor-
bency was determined at 410 nm at 25℃ . Ammonium was as-
sayedas reported by Moore & Stein (1948) . After 2 mL dilute
medium, 3 mL ninhydrin reagent and 0 .1 mL 1 % ascorbic acid
were incubated together in 100℃ water for 15 min, and slowly
cooled inair . The absorbencywas determined at 580 nm . Addit-
ionally, phosphate was determined by colorimetry at 660 nmwhen
1 mL dilute medium and 3 mL phosphate reagent (2 . 5% ammo-
niummolybdate∶10% ascorbic acid∶water= 1∶1∶2 ) was placed
in 45℃ water for 20 min (Li , 1998) .
Theflavonoids were extracted with 30 volumes of ethanol?
water (70?30 , v?v) by sonicationfor 1 h at 25℃ . After centrifu-
gation at 10 000 rpm for 6 min, the supernatant was extracted
three timeswith EtOAc, then with 95% ethanol . The combina-
tion of flavonoids in cells and mediumwas determined by color-
imetry accordingto Zhang et al . (2001 ) . Rutin was used as a
standard sample .
Results and Discussion
Cultured with a cycle of 22 days, the maximum
biomass, flavonoids production and the maximum spe-
cific growth rateof the cells all decreased with progres-
sive scale-up flasks, and by 27% , 30% and 27% in
1 L flasks compared to that in 250 mL flasks, respec-
tively (Table 1) . But the differences were insignificant
between 250 and 500 mL flasks ( P > 0 .05 ) , which
was agreed with theobservation in Catharanthus roseus
cell suspension culture ( Zheng et al . 1998 ) . Addit-
ionally, a little foamwas observedon thesurfaceof the
Table 1 Compare of the maximum biomass, flavonoids production and
maximum specific growth rate in cell suspension culture of G. inflata in
progressive scale-up flasks . Datawith different superscript letters are sig-
nificantly different (one-way ANOVA followed by a multiple comparison;
a, b: P < 0 .05; B: P < 0 .01 , n= 3) .
Flasks
Maximum
biomass
( g DW?L )
Flavonoids
production
( mg?L )
Maximum specific
growth rate
( g DW?L?d)
250 ?mL 14 Y. 5±0 ?. 61a 80 . 35±4 .2a 0 +. 66±0 .03a
500 ?mL 13 Y. 3±0 ?. 82a 73 . 63±4 .1a 0 +. 61±0 .04a
1 ?L 10 U. 6±0 ?. 75B 56 .45±1 .4B 0 ). 48±0 .04b
Fig . 1 Time courses of phosphate, nitrate and ammonium consumption in cell suspension cultureof G. inflata grown in MS medium
■ , □ and ● represent for 250 , 500 mL and 1 L flasks, respectively . Each data indicated the means of three independent experiments .
5444 期 YANG Ying et al .: Production of Flavonoids in Cell Suspension Culture of Glycyrrhiza inflata . . .
1 L flasks in which the cells appeared brown and less
healthy in the later stage of cultivation . Zheng et al .
(1998) found that the oxygen mass transfer (KL a) in
10 L bioreactor was significantly higher than that in
flasks . Ten Hoopen ( 1994 ) reported that shear stress
was themain reasonmaking the cells brown .Thus, the
decreased biomass of brown cell and flavonoids produc-
tion in 1 L flasks might be due to shear stress and the
decreased dissolved oxygen .
The trends of phosphate, nitrate and ammonium
consumption in progressive scale-up flasks were very
similar that the nutrients all decreased sharply ( Fig .
1) . At the initial several days, the phosphate con-
sumptions in 250 , 500 mL and 1 L flasks were almost
alike, but later it was the slowest in 1 L flasks . The
phosphate in all flasks was almost exhausted on day 10
at the logarithm phase, which was observed in many
plants cell cultures (Shin et al . 2003 ) . Most plant
cells utilize ammoniumfirstly and nitrate later (Shin et
al . 2003) . In the study, the ammoniumconsumption
was faster than nitrate at the beginning of the cultiva-
tion . Low concentrations of ammoniumstimulatenitrate
uptake and of nitrate inhibite ammonium uptake
(Dortch, 1990) . Thus, the nitrate was almost runout
of on day 18 , while the ammonium still maintained
about 100 mg?L when the cells wereharvested . Conse-
quently, themain culture conditions needed to be fur-
ther optimized for higher flavonoids production on a
bioreactor scale .
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