全 文 ：Isolation and Purification of Gonyautoxins from Two Strain of
Alexandrium minutum Halim
MIAO Yu-ping1 , 2 , CHEN Yi-min3 , ZHOU Hong-nong3 , and WEN Ren2＊
(1 .Key and Open Laboratory of Marine and Estuarine Fisheries , Ministry of Agriculture , East China Sea Fisheries Research
Institute , Chinese Academy of Fishery Sciences , Shanghai 200090 , China;2 .School of Pharmacy , Fudan University ,
Shanghai 200032 , China;3 .Institute of Fisheries Science , National Taiwan University , Taibei 10167 , China)
Abstract:Aim　To study isolation and purification of Gonyautoxins from two different strains of Alexandrium minutum
Halim.Methods　Ethanol extracts of the two culture strains of Alexandrium minutum Halim were fractionated by gel filtra-
tion and ion exchange chromatography to obtain gonyautoxins.The toxin profiles were tested by HPLC method.Results　
Both strains contain gonyautoxin-1 , gonyautoxin-2 , gonyautoxin-3 and gonyautoxin-4.The total gonyautoxin content of
Amtk4 strain is almost 10 times higher than that of Amtk2 strain.Conclusion　Alexandrium minutum Halim Amtk4 strain is
suitable for the preparation of Gonyautoxins.
Key words:Alexandrium minutum Halim;gonyautoxins;gel filtration chromatography;ion exchange chromatography
CLC number:R915.1;R931.711　　　Document code:A　　　Article ID:1003-1057(2004)2-103-03
Introduction
Marine algae have attracted much attention in the
search for lead compounds to develop new drugs in recent
years.Up to now , a variety of bioactive compounds have
been found from marine algae.Gonyautoxins(GTXs)is a
kind of paralytic shellfish toxins discovered in shellfish and
toxic algae.The toxins have particular importance as phar-
macological tools because of their unique action against so-
dium channels in the excitable membrane.Similar to tetro-
dotoxin(TTX), GTXs have good actions of analgesia , lo-
cal anesthesia and anticonvulsion;they can be considered
as lead compounds for developing new drug
[ 1] .
Figure 1　Structures of Gonyautoxins
Received date:2003-08-29.
＊Corresponding author:Tel 021-54237560 ,
E-mail rwen@shmu.edu.cn
　　Because of their complex structure , GTXS have not
yet been synthesized successfully.Up to the present time ,
GTXs have only been isolated from cultured toxic microal-
ga in foreign country.In China , the preparation of GTXs
has never been reported.Different strains of Alexandrium
minutum have different toxicant profile;this paper pre-
sents our study on the toxicant profile of two strains of Al-
exandrium minutum Halim.
Materials and Methods
Culture of Alexandrium minutum Halim Amtk2
strain
The two strains of Alexandrium minutum Halim were
isolated from purple clam culture pond at Tong-Kang in Tai-
wan recently.Cultures were maintained in 20 liter flasks
containing 15 literK medium incubated at 24℃and exposed
to a 14 h light 10 h dark cycle (2000-4000 lux)[ 2] .Cell
number of Alexandrium minutum in 1 mL aliquots from the
culture was counted under light microscope.
Gonyautoxins Extraction and Purification
The microalga cells were collected by filtration
through a glass fiber column (2 cm ×10 cm ID), then
extracted with 95% ethanol previously acidified to pH 2
with concentrated HCl.Chloroform was used for defatting
the ethanol extracts.Then it was concentrated to 4 mL by
vacuum-evaporation.Finally , the crude extract of toxin
was obtained.The toxicity of this crude preparation was
103Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)
determined by mouse bioassay
[ 3] .The toxin in the ex-
tracts was adsorbed to a column of Bio-Gel P-2 equilibrat-
ed with water.Furthermore , the column was first washed
with a sufficient volume of water , then eluted with 0.1
mol·L-1 acetic acid.The eluate of toxins adsorbed on
Bio-Gel P-2 was then vacuum-evaporated to obtain gon-
yautoxin mixture.The toxin mixture was purified by the
following step of ion exchange chromatography.The toxin
was exchanged into a column of Bio-Rex 70 equilibrated
with water and gradient eluted with 0.025 mol·L-1 acetic
acid (acetic acid concentration varied from 0 to 0.025
mol·L-1 in 120 min), using Hitachi L-4200 UV detector
on line detection.The fraction was collected every one
min.GTXs were analyzed by HPLC specially designed for
paralytic shellfish(PSP)toxins[ 4] .
GTXs standard and HPLC system
GTXs standard was obtained from National Marine
Institute of Canada.Their concentrations are GTX-1 ,
6.55μmol·L-1;GTX-2 , 1.52μmol·L-1;GTX-3 , 0.52
μmol·L-1 ;GTX-4 , 1.71 μmol·L-1.They were diluted
into 5 different concentrations to set up a standard curve.
The HPLC system is composed of a high pressure
pump (Hitachi L-6000)with a syringe loading sample
injector(Rheodyne 7125), Cosmosil 5C18-AR(4.6 mm×
200mm), Waters RP-18(4.6mm×250mm)HPLC col-
umn , a fluorescent monitor (Hitachi F-1000)equipped
with 150 W xenon lamp and a chromato-integrator(Hita-
chi D-2500)for calculation of peak area.
Results and Disscution
The mouse assay results of both extracts showed typi-
cal PSP poisoning features.Analyzed by HPLC specially
designed for PSP toxins showed that both strains contain
gonyautoxin-1 , gonyautoxin-2 , gonyautoxin-3 , and gonyau-
toxin-4;the retention times of GTX-4 , GTX-1 , GTX-3 ,
and GTX-2 are 6.2 , 7.1 , 11.5 , and 15.6 min , respective-
ly , just the same as those of the GTXs standard.The HPLC
chromatograms of Gonyautoxins are shown in Figure 2.
Figure 2 　HPLC chromatograms of gonyautoxins of the two
strains of Alexandrium minutum The HPLC Column:cosmosil
5C18-AR(4.6 mm×250 mm)
Figure 3 　 HPLC chromatogram of gonyautoxins prepared
HPLC column:Waters RP-18(4.6 mm×250 mm)
Table 1　Toxin content of the two strains of Alexandrium minutum(×10-10mol cell)
Strain GTX-4 GTX-1 GTX-3 GTX-2 Total
Amtk2 (4.42±0.19)E-05 (1.36±0.09)E-04 (1.35±0.11)E-06 (1.66±0.08)E-06 (1.83±0.11)E-04
Amtk4 (5.25±0.18)E-04 (1.19±0.17)E-03 (3.78±0.13)E-05 (4.38±0.13)E-05 (1.79±0.19)E-03
104 Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)
Figure 4　Toxin composition of the two strains of Alexandrium
minutum
The toxin composition of the two strains of Alexandrium
minutum is shown in Table 1 and Figure 3.
Table 1 indicates that the total toxin content of
Amtk2 is (1.83±0.05)×10-14 mol cell , and that of
Amtk4 strain is (1.79±0.10)×10-13 mol cell.This
means that Amtk4 strain contains gonyautoxins almost 10
times that of Amtk2 strain.Amtk4 strain is much more
suitable than Amtk2 strain for the preparation of Gonyau-
toxins.HPLC chromatogram of Gonyautoxins prepared is
shown in Figure 3 (the HPLC column was changed for
good separation , so the whole separation time is different
from that shown in Figure 2).
GTX-4 andGTX-1 are a pair of isomers , and GTX-3
and GTX-2 are another pair of isomers.From Figure 4 we
know that the contents of GTX-4 and GTX-1 are much
higher than those of GTX-3 and GTX-2 in both strains.In
Amtk2 strain , the content of (GTX-4 +GTX-1) is
98.35% in total , while that of(GTX-3+GTX-2)is only
1.65% in total.Similarly , in the Amtk4 strain , the con-
tent of(GTX-4+GTX-1)is 95.45% in total , and that
of(GTX-3+GTX-2)is only 4.55% in total.The de-
tailed data of toxin composition of the two Alexandrium
minutum strains are shown in Table 2.
Table 2　Toxin composition of the two strains of Alexandrium
minutum(%)
Strain GTX-4 GTX-1 GTX-3 GTX-2
Amtk2 24.19 74.16 0.74 0.91
Amtk4 29.20 66.21 2.11 2.44
Acknowledgement
The authors thank professor M.A.Quilliam , the
Institute for Marine Bioscience , National Research Coun-
cil in Canada and professor Yoshiteru Oshima , Graduate
School of Pharmaceutical Sciences , Tohoku University in
Japan for their quantitative check of gonyautoxins.
References
[ 1] Song JJ , Miao QW.Toxicology of marine organism [ M] .Bei-
jing:Beijing Science and Technology Press , 1996.221.
[ 2] Anderson DM , White AW , Baden DG.Factors significant to
marine dinoflagellate culture [ A] .In:Keller MD, Guillard RRL.
ed.Toxic dinoflagellates [ M] .New York:Elsevier, 1985.113-
116.
[ 3] Hellrich K.Official methods of analysis of the association of
official chemists [ M] .15th ed.Arlington:Virginia , 1990.Chap.
35 , 881-882.
[ 4] Oshima Y.Postcolumn derivativization liquid chromatographic
method for paralytic shellfish toxins [ J] .J AOAC Int , 1995 , 78:
528-532.
从两株微小亚历山大藻中分离纯化膝沟藻毒素
缪宇平1 ,2 , 陈逸民3 , 周宏农3 , 闻　韧2
(1.中国水产科学研究院东海水产研究所 农业部海洋与河口渔业重点开放实验室 , 上海 200090;
2.复旦大学药学院 ,上海 200032;3.台湾大学理学院 ,台北 10617)
摘要:目的　从两株微小亚历山大藻(Alexandrium minutum Halim)中分离纯化膝沟藻毒素(Gonyautoxins)。方法　用胶滤层
析 、离子交换层析等方法从人工培养的两株微小亚历山大藻酸酒精萃取物中分离纯化膝沟藻毒素 , 再用 HPLC 法对毒素进行
定性定量测定。结果　从两株藻株培养液中均分离得到膝沟藻毒素 GTX-4 , GTX-1 , GTX-3 , GTX-2 ,其中 Amtk4藻株的毒素含量
为Amtk2 的近 10倍。结论　微小亚历山大藻Amtk4 藻株适于制备膝沟藻毒素。
关键词:微小亚历山大藻;膝沟藻毒素;胶滤层析;离子交换层析
105Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)