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Journal of Chinese Pharmaceutical Sciences 2004 , 13 (1) 53

Preparation of High2Purity Linolenic Acid from Oil of Lithospermum


Erythrorhizon by Urea Inclusion and Column Chromatography
3
HAN Xi2jiang , XU Ping , MENG Xiang2li , LIANG Zhi2hua , ZHANG Zong2shuang , and YANG Zhan2cheng
( Department of Applied Chemistry , Harbin Institute of Technology , Harbin Heilongjiang 150001 , China)

Abstract: Aim  To separate high purity linolenic acid from the oil of Lithospermum erythrorhizon growing in the
Northeast of China. Methods  Urea inclusion and column chromatography were used. Results  Unsaturated fatty acid was
separated , with a purity of 99130 wt % of linolenic acid. Conclusion  The experiment shows excellent reproducibility and
high feasibility for industrial production.
Key words : Lithospermum erythrorhizon ; linolenic acid ; urea inclusion ; column chromatography
CLC number : R917 ; R284. 2    Document code : A    Article ID : 100321057 (2004) 12053205

. cn
expected. In this study , with the aim of low cost , simple
Introduction

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operation and industrial feasibility , we separated and ex2

.a
tracted unsaturated fatty acids from the oil of Lithosper2

s
Lithospermum erythrorhizon is a traditional Chinese

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medicinal herb which has the functions of promoting coro2 mum erythrorhizon by urea inclusion and column chroma2

jc
[1 ]

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nary circulation , antivirus , detoxicating and antisepsis . tography. Preprocessing conditions of urea inclusion and
Lithospermum erythrorhizon oil has important medical val2

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separating techniques of column chromatography are also
ues. It has high content of unsaturated fatty acids , espe2

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discussed.

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ciallyα2 andγ2 linolenic acids , whose formulas are 9 ,12 ,
Experimental
152C17 H29 COOH and 6 ,9 ,122C17 H29 COOH , respectively.
α2 Andγ2 linolenic acids compose 43 % - 46 % of the un2 Reagents , instruments and measuring conditions
saturated fatty acid , of which γ2 linolenic acid occupies The oil of Lithospermum erythrorhizon was extracted
13 % - 14 % andα2linolenic acid 30 % - 32 %. Linolenic by CO2 supercritical extraction. Other reagents , such as
acid is an important active substance in the brain , which KOH , absolute ethanol , absolute methanol , urea , an2
didates vessels and resists arteriosclerosis. It is honored hydrous Na2 SO4 , ether , petroleum ether , silica gel , and
[2 ,3 ]
as“Brain Gold”as it promotes humans′IQ . AgNO3 are all analytically pure.
The main methods for separating and extracting un2 Instruments : 6890NΠ5973N gas chromatograph2mass
saturated fatty acids from vegetable oil at present are crys2 spectrometer (USA) , Varian CP 3800 gas chromatograph
tallization at low temperature , fractionation by adsorption , (USA) , LD2522 centrifuge ( Beijing) , refrigerator , wa2
ter2bath , and chromatographic column ( 500 mm ×50
[4 - 6 ]
CO2 supercritical extraction , urea inclusion , etc .
However , most researches are conducted in the laborato2 mm) .
ry , which are far beyond industrialization. What is worse , Chromatographic condition : carrier gas , N2 ; column
the linolenic acid separated and extracted is not so pure as head pressure , 8 psi ; sampling room temperature , 250 ℃;
detector , FID ( 250 ℃) ; chromatographic column , CPW
Received date : 2003201208. AX 52CB (220 ℃) ; sampling volume , 110μL.
Foundation item : Heilongjiang Science and Technology Bureau
Mass spectrometric condition : ionization power , EI ;
( GG01C405203)
3
ionization energy , 70eV ; ion source temperature , 230 ℃;
Corresponding author : Fax 86245126418750 ,
E2mail hanxijiang @263. net
quadrupole temperature , 150 ℃.
Biograph: Dean of Chemistry Laboratory Center and professor in the Reactions
Department of Chemistry of Harbin Institute of Technology. Hydrolysis  A certain amount of the oil of Litho2
Copyright © 2004 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn
54 Journal of Chinese Pharmaceutical Sciences 2004 , 13 (1)

spermum erythrorhizon was put into a mixture solution of Table 1  Experiment of optimum amount of KO H

KOH2H2 O2C2 H5 OH. Protected by N2 , it was refluxed at Experiment No. Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ

60 ℃ in a water2bath till the solution turned into even yel2 KOHΠg 2 4 6 8 10

low. Then the solution was cooled at room temperature. TΠmin 45 37 32 30 28

pH value was adjusted to 3 - 4 with dilute hydrochloric Mixed fatty acid Πg 17. 5 18 17 17 16

acid , which laminated the solution. The yellow mixed fat2 Linolenic acidΠwt %(methyl ester) 29. 5 33. 1 36. 6 38. 2 37. 9

ty acids were separated , and anhydrous Na2 SO4 was used


Table 2  Experiment of optimum saponification temperature
as a dehydrating agent .
Ⅵ Ⅶ Ⅷ Ⅸ Ⅹ
Methyl2esterification  Mixed fatty acids obtained
Experiment No.

Temperature Π℃ 40 50 60 70 80
from the above step were added to a mixture solution of
TΠmin 70 50 30 30 25
concentrated H2 SO4 2CH3 OH. Protected by N2 , it was re2
Mixed fatty acid Πg 17. 2 18. 3 17. 2 16. 1 17. 1
fluxed at 60 ℃ in a water2bath till the reaction ended.
Linolenic acid Πwt %(methyl ester) 36. 5 35. 5 37. 8 37. 1 36. 8
The solution was laminated by adding distilled water after

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(20 g of Lithospermum Erythrorhizon Oil , 8g of KOH)

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being cooled. The upper yellow solution was separated ,

c
which was methyl esters of mixed fatty acids , and an2

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From Table 1 it can be seen that the amount of KOH

s
hydrous Na2 SO4 was used as a dehydrating agent .
not only influenced the reaction rate , but also the purity

p
Urea inclusion  Urea , methanol and mixed methyl

c
of linolenic acid. The low purity of linolenic acid in Ⅰ-

j
ester were put into a three2neck flask. Protected by N2 , it

.
Ⅲ suggested incomplete reaction. In order to ensure reac2

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was refluxed at 60 ℃ in a water2bath till the solution be2 tion rate and complete reaction and save material mean2

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came clear. The product was cooled at room temperature while , 8 g ( 0114 mol ) of KOH were chosen per 20 g of

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for 1 h. It was placed in refrigerator for 12 h refrigeration Lithospermum erythrorhizon oil .
and 12 h freezing. Then it was placed at room tempera2 Table 2 shows that the reaction temperature mainly
ture for 1 h. The organic phase was extracted with ether , influenced the reaction rate , but not the quality of the
the solid phase was separated , and enriched methyl esters products. Generally speaking , the optimum temperature
of unsaturated fatty acids were yielded. Then it was would be 60 ℃.
hydrolyzed again to obtain unsaturated fatty acids. In addition , water and ethanol are solvents in
Column chromatography  Treated with 10 wt % hydrolysis , which seldom influenced the reaction. As an2
AgNO3 solution , silica gel was baked in oven for 18 h at alyzed above , the condition of hydrolysis would be : oil of
Lithospermum erythrorhizon ( g) ∶KOH ( g) ∶ water ( mL ) ∶
120 ℃. Then it was soaked in petroleum ether for 6 h , as
ethanol ( mL ) = 5 ∶2∶5∶20. The reaction best took place
fixed phase , before it was stored in the column. One
lucifugally at 60 ℃ in water2bath , protected by N2 .
gram mixed fatty acids was added in column , and then
Methyl2esterification
eluting agent was gradually dropped ( 80 wt % petroleum
Mixed fatty acids being urea included directly may
ether , 18 wt % ether , 2 wt % glacial acetic acid ) , with
be oxidized. Due to high polarity of the fatty acids , urea
the effluence being monitored by gas chromatography.
would adsorb them and affect yield. Methyl esters of fatty
Results and Discussion acids are relatively more stable and less polar , which is
beneficial to the separation of each component . There2
Hydrolysis
fore , fatty acids are methyl2esterificated first .
The main factor influencing hydrolysis is saponifica2
Effects of amounts of methanol and concentrated
tion condition. Optimum designs are listed in Tables 1
H2 SO4 on the reaction are shown in Tables 3 and 4.
and 2.

Copyright © 2004 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn
Journal of Chinese Pharmaceutical Sciences 2004 , 13 (1) 55

Table 3  Experiment of optimum amount of methanol increase of double bonds , which increases the volume of
Experiment No. A B C D E the molecules and makes them difficult to form urea inclu2
Methanol ΠmL 60 70 80 90 100 sion complexes. In experiments , contact chances of urea
Methyl ester Πg 15. 5 13. 5 15. 0 15. 6 15. 7 and methyl esters should be increased as much as possible
Linolenic acid Πwt % 33. 2 34. 2 38. 1 38. 2 37. 9 by stirring , which meanwhile increases inclusion proba2
(Mixed fatty acid Π20 g ,Concentrated H2 SO4Π016 (mL) bility of urea to methyl esters of different bond lengths and
saturation degrees.
Table 4   Experiment of optimum amount of concentrated Experimental data of optimum amount of urea are
H2 SO4 shown in Table 5. Lack of urea leads to incomplete urea
Experiment No. F G H I J inclusion ; while excessive urea will adsorb methyl esters
Concentrated H2 SO4Πml 0. 2 0. 4 0. 6 0. 8 1. 0 of various fatty acids , which affects yield. In order to
TΠmin 84 73 42 43 41 save material and not to affect the purity of linolenic acid ,
Linolenic acid Πwt % 3 49. 8 50. 3 54. 1 49. 5 47. 2 20 g of urea , namely , urea ( g) ∶
methyl esters ( g) = 2∶
1

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( Fatty acid Π20 g , Methanol Π80 mL) 3 was applied in the reaction. Purity of unsaturated fatty ac2

c
It is the purity after urea conclusion.

.
ids was achieved at 9512 wt % after urea inclusion ( lino2

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lenic acid 5515 wt %) .

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Table 3 shows incomplete methyl2esterification reac2

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Effects of inclusion temperature on the results are
tion when methanol was not sufficient . Amount of concen2

c p
shown in Table 6. A long time is needed under a low

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trated H2 SO4 prominently influenced the reaction time.

.
temperature , which would decrease the purity of linolenic
More time was needed with low amount of KOH , and ef2

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acid ; too high a temperature is unfavorable to the forma2
fect of catalysis was not obvious ; thus purity of linolenic

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tion of inclusion complexes , which also results in low pu2
acid was relatively low. As shown in Table 4 , too much

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rity of linolenic acid. Generally , we choose 60 ℃, at
concentrated H2 SO4 accelerated the reaction , but by2
which purity of unsaturated fatty acids reached 9314 wt %
products were produced at the same time and the purity of
( linolenic acid 56 wt %) .
linolenic acid also decreased. The optimal conditions of
methyl2esterification are : mixed fatty acids ( g) ∶methanol
Table 5  Experiment of optimum amount of urea
concentrated H2 SO4 ( mL ) = 100 ∶
(mL ) ∶ 400 ∶3. The re2
Experiment No. 1 2 3 4 5
action occurred at 60 - 64 ℃ in a water2bath , protected
Urea Πg 10 20 30 40 50
by N2 .
Polyene fatty acid Πwt % 90. 1 95. 2 94. 8 93 90. 2
Urea inclusion
Linolenic acid Πwt % 43. 2 55. 5 50. 2 48. 7 40. 1
The mechanism of urea inclusion is traditionally ex2
plained as follows : separation of mixed fatty acids by urea
Table 6  Experiment of optimum inclusion temperature
inclusion occurs in terms of different saturation degrees
and carbon2chain lengths of the fatty acids Experiment No. 1 2 3 4 5
[7 ,8 ]
. Stable
Inclusion Temperature Π℃ 40 50 60 70 80
crystalline inclusion complexes are formed from urea and
TΠmin
saturated and mono2unsaturated components of methyl es2 120 100 60 40 30

Polyene fatty acid Πwt % 85. 3 90. 1 93. 4 92. 7 91. 4


ters of mixed fatty acids at a low temperature , achieving
Linolenic acid Πwt %
the aim of separating those components from other unsat2 38. 4 43. 5 56 47. 5 39. 1

urated methyl esters. Urea is a crystal of tetragonal lat2


tice , which has no free space for other molecules. Howev2 The inclusion complexes were extracted with ether
er , when it is dissolved in organic solvents as metha2 after 12 h refrigeration and 12 h freezing ; thus methyl es2
[9 ]
nol , urea inclusion complexes of hexagonal lattice are ters of unsaturated fatty acids were enriched.
produced through strong hydrogen bonds ( tested by In all , the optimum condition of urea inclusion
XRD) . Unsaturated fatty acid molecules bend with the would be : urea ( g) ∶
methyl esters ( g) = 2∶
1. Inclusion
Copyright © 2004 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn
56 Journal of Chinese Pharmaceutical Sciences 2004 , 13 (1)

took place at 60 ℃for 1 h , protected by N2 . Ether is ex2


tractant.
Gas chromatographic analysis of methyl esters after
urea inclusion is shown in Figure 1. Purity of unsaturated
methyl esters was above 95 wt % , among which linolic
acid was 1116 wt % , α2 , and γ2 linolenic acids 50 - 55
wt % , and 182carbon tetraene acid 3317 wt %. However , Figure 2  GC of linolenic acid after column chromatography
urea inclusion could not separateα2 , andγ2 linolenic ac2
ids from other unsaturated fatty acids , which can only Fatty acids after column chromatography contain
separate saturated and mono2unsaturated from those con2 59135 wt %α2linolenic acid , and 39195 wt %γ2linolenic
taining 2 or more double bonds. Therefore , column chro2 acid , total purity of linolenic acid achieving 99130 wt %.
matography was chosen to purifyα2 , and γ2 linolenic ac2
ids further. Conclusion

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From the above the discussion , we easily achieve the

c . c
optimum reaction conditions and amount of every reactant :

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Amount of the reactants in hydrolysis should be : oil

s
of Lithospermum erythrorhizon (g) ∶KOH (g) ∶
water (mL) ∶

c p
ethanol ( mL ) = 5∶
2∶ 20. It takes place at 60 ℃ in wa2
5∶

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ter2bath , protected by N2 . pH = 3 - 4.

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Amount of the reactants in esterification should be :

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Figure 1  GC spectrum of polyene fatty acid methyl ester after
mixed fatty acids ( g ) ∶methanol ( mL ) ∶concentrated

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urea conclusion ( a. Oleic acid ; b.γ2Linolenic acid ; c.α2Linolenic
acid ;d. 182carbon tetraene acid) H2 SO4 ( mL ) = 100∶
400∶
3. It takes place at 60 - 64 ℃in
water2bath , protected by N2 .
Column chromatography Amount of the reactants in urea inclusion should be :
Column chromatography is an effective way to sepa2 urea ( g) ∶
methyl esters ( g) = 2 ∶1. Inclusion takes place
rate and purify various fatty acids with different numbers at 60 ℃ for 1 h , protected by N2 . Cool the products at
of double bonds. Silica gel treated by AgNO3 solution is room temperature for 1 h. Place it in refrigerator for 12 h
has an affinity to π elec2
+
used as stationary phase. Ag refrigeration and 12 h freezing. Ether is extractant .
trons in double bonds. The force will be stronger with an Urea inclusion can separate saturated and mono2un2
increase of double bonds , which makes samples stay in saturated from those containing 2 or more double bonds ,
column for a longer time. Therefore , fatty acids with dif2 but not linolic acid , linolenic acids , and 182carbon tet2
ferent numbers of double bonds are separated. raene acid. The yield of unsaturated fatty acids after urea
Gas chromatographic analysis of fatty acids is shown inclusion is 75 wt % , with purity above 90 wt %.
in Figure 2 , where the 11134 min peak and the 12146 Treated with 10 wt % AgNO3 solution , silica gel is
min peak are methyl ester of γ2andα2linolenic acids , re2
baked in oven for 18 h at 120 ℃. It is soaked in petro2
spectively. Column chromatography can separate linolenic leum ether for 6 h as stationary phase. Eluting agent is a
acids in the oil of Lithospermum erythrorhizon , but it can2 mixture of 80 wt % petroleum ether , 18 wt % ether and 2
not separate γ2and α2linolenic acids , which are isomers. wt % glacial acetic acid. Elute the sample at 40 dΠmin to
γ2andα2linolenic acids have great physiological activities , separate the unsaturated fatty acids. Gas chromatography
which are used to manufacture drugs for cardiovascular shows that purity of linolenic acids is 99130 wt %.
disease. Usually , there is no need to separate them. Oth2 The experiment shows excellent reproducibility and
er special techniques are required to do that . the results are stable. We surely think that it is feasible
Copyright © 2004 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn
Journal of Chinese Pharmaceutical Sciences 2004 , 13 (1) 57

for industrial production. unsaturated fatty acids [J ] . Chin Oils Fats , 1999 , 24 ( 2 ) :
32237.
Acknowledgements [5 ] Zheng MY. Studies on continuous extraction of EPA and DHA
from fish oil by using supercritical carbon dioxide [J ] . Food
Authors acknowledgeHeilongjiang Science and
Sci , 2002 , 23 (4) : 76280.
Technology Bureau for financial support ( GG01C4052
[6 ] Median AR. Concentration of stearidonic , eicosapentaenoic and
03) .
docosahexaenoic acids from cod liver oil and the marine Mi2
croalga isochrysi Gatabana [J ] . J Am Oil Chem Soc , 1995 ,
References
72 :5752580.
[1 ] Xue L , Meng Q , Lu DW. Structured kinetic model of Litho2 [ 7 ] Schlenk H. Urea inclusion compounds of fatty acids [J ] . Prog
spermum erythrorhizon cell suspension culture [J ] . Chem Eng Chem Fats Other Lipids . 1953 , 2 :2432247.
Chin Univ , 1999 , 13 (2) :1412143. [8 ] Weng XC , Dong XW , Ren GP. Application of urea inclusion
[2 ] Shao Q , Zhang H , Bian J . Studies on a new functional oil : in the separation of fatty acids [J ] . Chin Oils Fats , 1994 , 19
conjugated linoleic oil [J ] . Food Sci , 2002 , 23 (2) :1642168. (6) :40243.
[3 ] Winkler K, Steinhart H. Identification of conjugated isomers of [ 9 ] Kim Sanguk , Lee Wonyoung , Yeo Sangdo , et al. Super carbon

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linolenic acid and arachidonic acid in cheese [J ] . J Sep Sci ,

c
dioxide extraction of perilla oil [J ] . Foods Bio , 1996 , 5 ( 4) :

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2001 , 24 :6632665. 3002302.

.a c
[4 ] Huang HQ , Bao SX. Separation and purification of polyvalent

jc p s
.
尿素包合与柱层析相结合从紫草油中分离高纯度亚麻酸

w w w 韩喜江 , 徐 平 , 孟祥丽 , 梁志华 , 张宗双 , 杨占成

( 哈尔滨工业大学 应用化学系 , 哈尔滨 150001)

摘要 : 目的  从中国东北地区产的紫草油中提取高浓度亚麻酸 。方法  采用尿素包合法和柱层析法 。结果  分离得到


了不饱和脂肪酸 ,其中亚麻酸含量为 99130 wt % 。结论  实验重现性好 ,工业化生产的可行性较高 。

关键词 : 紫草油 ; 亚麻酸 ; 尿素包合 ; 柱层析

Copyright © 2004 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University http://www.jcps.ac.cn

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