全 文 ：·药剂与工艺·
A novel approach to deproteinization in isolation and
purif ication of lentinan fromLentinus edodes
WANG Wei-guo
1
, ZHAO Yong-liang
2
( 1. Depar tment o f Bio lo g y and Chemical Eng ineering , Nanyang Institute of Techno log y , Nanyang 473004, China;
2. Key Biotechnolog y Labo rato ry , Institute of Breeds and Resources, Chinese Ag ricultura l
Academy o f Science and Technolog y , Beijing 100081, China)
Abstract: Object　 Looking fo r a sui table approach to depro teinization in isolation and puri fica tion of
lentinan from Lent inus edodes . Methods　 The orthogonal test w as mainly used. Results　 A new approach
w as established fo r depro teinization in isolation and puri fica tion of a lentinan by o rthogonal test. The re-
sul ts indica ted that the majo r facto r affecting the deproteinization ra te ( DPR) was B ( 2-butanol) , the sub-
majo r facto r D ( ethanol ) ; while C ( methanol ) and E ( NaCl ) had li t tle inf luence. The influencing order fo r
DPR was B> D> A ( chlo rofo rm) > E> C. DPR va ried g rea tly when the amount of 2-butano l v aried, going
up slow ly fi rst , then going down rapidly. DPR decreased g radually as the concentration of ethanol in-
creased. DPR went up first , a nd then w ent dow n, w hen the content of chlo rofo rm increased. And DPR a l-
tered li t tle when the concentra tion o f methanol increased. The optimal condition fo r depro teinization w as
A2B2 C1D1 E2 , the best composi tion fo r the deproteinizing agent w as 40% of chlorofo rm , 10% of 2-butanol,
2% of methanol , 40% o f ethano l, 0. 01% o f NaCl. Further proven experiments showed tha t DPR was up
to 99. 98% under the best condi tion. The optimal tech nolog y of pret reatment of the dried L . edodes , such
as disti lling of L . edodes , concentra ting of boiled sample, precipi ta ting and deco lorizating o f rough
poly saccha rides, drying of rough polysaccha rides, which applied to massiv e production for indust ry, w as
also researched out through repea ted t rials. Conclusion　 The novel appro ach to depro teiniza tion has the
superiority of simple tech nolo gy , needless high-price apparatus, low er cost of pro cessing , and easily am-
pli fying. Thus, the technolo gy o f depro teiniza tion is of g reat impo rtance to the isolation and purification of
fungi and o ther poly saccha rides rich in proteins.
Key words: Lentinus edodes ( Berk. ) Sing. ; depro teiniza tion; polysaccharides; i sola tion; puri fica tion
一种在多糖分离纯化过程中新的脱蛋白方法
王卫国 1 ,赵永亮 2
( 1. 南阳理工学院 生物与化学工程系 ,河南 南阳　 473004; 2. 中国农业科学院品种资源研究所 生物技术重点实验室 ,北京
　 100081)
摘　要: 目的　寻找一种在多糖 (香菇多糖 )分离纯化过程中适宜的脱蛋白方法。方法　以正交试验法为主。结果　
通过正交试验法找到了一种在多糖 (香菇多糖 )分离纯化过程中新的脱蛋白方法。 5因素 3水平正交试验的结果表
明 ,影响脱蛋白率 ( DPR)的主要因素是 B(正丁醇 )、次主要因素是 D(乙醇 ) ,而因素 C(甲醇 )和 E(氯化钠 )的影响
则较小 ;影响 DPR的主次顺序为 B> D> A(氯仿 )> E> C。 DPR随着正丁醇浓度的增加而变化很大 ,开始缓慢上
升 ,然后迅速降低 ;随着乙醇浓度的增大 , DPR逐渐降低 ;而当氯仿浓度增大时 , DPR则开始上升 ,然后下降 ;当甲
醇浓度增大时 , DPR则变化很小。 脱蛋白的最佳条件是 A2B2 C1D1E2 ,即在氯仿 40% 、正丁醇 10% 、甲醇 2% 、乙醇
40% 、氯化钠 0. 01%的条件下 ,杂蛋白的去除率最高。 验证性试验结果表明 ,在此条件下 ,杂蛋白的去除率达
·891·中草药　 Chinese T raditional and Herba l D rugs　第 34卷第 10期 2003年 10月
收稿日期: 2002-12-12基金项目:河南省南阳市科委资助的攻关项目 ( No. 981074)作者简介:王卫国 ( 1963— ) ,男 ,河南省汝州人 ,硕士 ,副教授 ,发表论文 30余篇 ,主 (副主 )编出版论著 4部 ,主持完成并通过鉴定省级攻关项目 4项 ,参与完成并通过鉴定省级攻关项目 1项 ,完成国家发明专利 2项 ,现主持省市级科研项目 5项 ,研究方向为生物制药、生物技术。 Tel: ( 0377) 3121605 ( O)　 Fax: ( 0377) 3121404　 E-mail: w gwwang@ 163. net　w gww ang@ mai l. nyis t. n et
99. 98%。通过反复试验 ,还找到了适用于工业化生产的包括香菇原料的蒸煮、煮沸样品的浓缩、粗多糖的沉淀和脱
色、粗多糖的干燥等在内的原料预处理的最佳工艺。 结论　这种新的脱蛋白方法具有工艺简单、无需贵重设备、加
工成本低、容易放大等特点 ,特别适合于多糖类产品的精制或其他需要去除杂蛋白的单元操作。
关键词: 香菇 ;脱蛋白 ;多糖 ;分离 ;纯化
中图分类号: R284. 2; R286. 02　　　文献标识码: B　　　文章编号: 0253 2670( 2003) 10 0891 05
　　 It s w ell know n that 1970s is the time o f g ene
engineering in the secto r o f life science, 1980s the
time of protein engineering , 1990s and 21st centu-
ry the time of po lysaccharides engineering
[1 ]
. The
g rea t success has been achiev ed in the upst ream of
bioengineering such as having acquired many hy-
brid microbes. How ever, broad applications of
them to indust ry and people s life are very few.
The main reason affecting thei r application is that
w e have no t solv ed th e problems of dow nst ream of
bioengineering well. The problem o f depro teiniza-
tion is alw ays a crucial technolo gy in the aspects of
the genetic engineering, proteins engineering, es-
pecially for po lysaccharides engineering . Clinic
practice indicated tha t higher purity of polysaccha-
rides has g rea tly potentia l applica tion and ma rket.
There are many methods of isolation and purifica-
tion of pro teins; however, the ra re methods of de-
proteinization in isolation and puri fica tion of
poly saccha rides a re ef fective. The published meth-
ods such as th e improved gel elect ropho resis
method
[ 2]
described by Guard-Pet ter ( 1995) , Sub-
micron Magnetic Pa rticles method
[3 ]
repor ted by
Khng ( 1998 ) , solvent-f ree bio t ransfo rmations
[ 4]
described by D′Souza ( 1999) , the pa rticular emul-
sans method
[5 ]
inv ented by Gutnick ( 1980) , Enzy-
ma tic Transfo rmations
[6 ]
described by Millqvist-
Fureby ( 1998 ) , or the t raditional hot phenol
method
[ 7]
described by Westpha l are all dif ficul t to
be used in larg e scale, though they are fai rly ef fi-
cient. In the present paper, “ a novel approach to
depro teiniza tion in isola tion and puri fica tion of
lentinan” that can be applied to any sca le commer-
cially is repor ted. L . edodes was taken as experi-
menta l ma terials fo r the reason of the high nour-
ishment of L . edodes and the la rg ely clinic applica-
tion o f lentinan but rare repo rts about puri fica tion
o f lentinan around the w o rld.
1　Materials and methods
1. 1　 Materials: Lentinus edodes ( Berk. ) Sing. in-
cluding brindle L . edodes , nonbrindle L . edodes
( purchased from mush room market o f Nanyang,
Henan Province ) . Chlo ro fo rm ( AR) , 2-butanol
( AR) , methanol ( AR) , ethanol ( Food Grade) ,
NaCl ( AR) ( purchased from No. 1 Factory of
Tianjin Chemical Reagent ) .
1. 2　 Methods of pretreatment
1. 2. 1　 Grinding and decolo riza tion o f material.
Dried L . edodes was smashed into the size of mi llet
( d = 1 — 1. 2 mm ) in a pulv erizer XM— 7
( Xuchang Mechanic Facto ry ) . The pulverized
dried L . edodes powder w as deco lorized by 95%
ethano l ( pow der-ethanol= 1∶ 2) .
1. 2. 2　 Boi ling and fi lt rating o f material. The de-
colo rized mushroom powder w as boi led for 4 hours
at 96℃ w ith the ra tio of L . edodes to wa ter 1. 0∶
12. 0 first , then distilled at 96℃ fo r 4 hours w ith
the ratio o f L . edodes to wa ter 1. 0∶ 8. 0. Af ter
each disti llation, i t w as pressed and fi ltered w ith
heat-resistant cloth , respectiv ely.
1. 2. 3　 Concentration of boiled sample. Put the
fi rst and second distilled liquid into a boi ler, and
then heated i t at 94℃ until the amount of the con-
centra ted liquid decreased to 20% o f i t s ini tial v o l-
ume. This process takes about 3— 5 hours.
1. 2. 4　 Precipi ta tion and decolo riza tion of rough
po lysaccharides. Added 95% ethano l to the con-
centra te ti ll the final concentration o f ethanol of
the precipi ta to r is up to 70% . It should be stir red
continuously while adding the ethanol. Af ter agita-
tion, the sample w as kept sti ll for 8— 10 hours to
let rough lentinan precipi tate.
1. 2. 5　 Dry of rough po lysaccharides. The precipi-
ta ted rough po lysaccharide was condensed under
cool condition ( 4℃ — 10℃ ) and dried a t 65℃
fo r 2— 4 hours o r pressed wi th squeezer at 20℃
— 30℃ .
1. 3　 Deproteinization of rough po lysaccharides
·892· 中草药　 Chinese T raditional and Herba l D rugs　第 34卷第 10期 2003年 10月
1. 3. 1　 Selection o f the agents o f depro teiniza tion.
The st rateg y of deproteinization w as determined by
using cheap organic solvents commercially and con-
venient ly rather than using other methods as de-
scribed in the int roduction. In this experiment ,
chloroform ( A) , 2-butano l ( B) , methanol ( C) ,
ethanol ( D ) , NaCl ( E ) were chosen from the
view points of commerce, application and ef fective-
ness.
1. 3. 2　 Techno log y of depro teinization. The tech-
nolog y of deprotenization w as designed as follow s.
　　 Rough polysaccha ride sample dis til led w ater wa ter-
　 soluble solution ch loroform+ 2-bu tanol
sti rring 10- 20 min
　 colloidal mix ture meth anol+ ethanol+ NaCl
s ti rring 5- 10 min
1. 3. 3　 Decision of optimal composition of depro-
teiniza tion agents. The content of each agent fo r
depro teiniza tion was determined th rough or tho-
gonal test ( O T) . In this resea rch, the major fac-
to rs af fecting depro teinization and levels of each
facto r w ere set as Table 1. Th e experimental
scheme w as show n in Table 2.
Table 1　 Factors and levels
Lev els
Facto rs /%
A B C D E
1 30 5 2 40 0. 005
2 40 10 4 50 0. 01
3 50 20 6 60 0. 02
1. 3. 4　 Determination o f the DPR. The DPR was
determined atλ= 280 nm with ult rav iolet spect ro-
photometer 756 MC ( The First Analytical Appara-
tus Facto ry of Shanghai, China) . The pro tein con-
tent w as tested in rough po ly- saccharides that
w ere not depro teinized as 100% .
2　 Results and discussion
2. 1　 Pret rea tment of the dried L . edodes. It i s
v ery impo rtant to break the dried L . edodes to
pieces wi th the size o f mil let ( d= 1— 1. 2 mm) . If
i t w asn t sha ttered wi th a kibbler o r it w asn t
small enough, too much non-sta rch poly saccharid-
es wi ll remain in the material and w on t be ex t ract-
ed ef fectiv ely. If i t is g round too much, i t w ill be
dif ficul t to press and fil ter the sample. Thus, in
order to ex tract polysaccha rides f rom L . edodes
completely, th e ma terial must be shat tered proper-
ly.
2. 2　 Distilla tion o f L . edodes . Fo r bo th f resh and
dried ma terials, the operation o f disti llation is the
fi rst and key step during the whole process of len-
tinan preparation. The suitable boiling condition
decides whether or not enough non-starch poly-
saccharides of L . edodes can be ex t racted. It is
crucial to choo se the optimal disti lling tempera-
ture and propo rtion o f the ma terial to w ater. The
experimental results indica ted that the optimum
distilling temperature is 96℃ , the propo rtion of
the material to w ater is 1∶ 20, the proper period of
boi ling is 4— 6 hours. If the steaming tem- pera-
ture is beyond 100℃ , it wi ll no t only increase the
co st of operation, but also lead to the lentinan
deg radation. If the tempera ture is too low , the liq-
uid can t be boiled, and the ef fect of steaming is
lessened. To decrease the remaining poly saccha-
rides tho roughly in th e material , steaming i t again
and i t should be car ried out a fter the first treat-
ment a t the same condi tion.
2. 3　 Concentra tion o f boiled sample. The concen-
trating is another impo rtant step to save the con-
sumption of precipitate agent. There are many
w ays o f concentrating the sample to choo se f rom.
In this experiment , w e culled the boiling w ay in
the light o f lessening the investment of production
equipment. The practice show ed tha t the boiled
liquid volume w as decreased to 20% — 25% of i ts
initiated vo lume at 94℃ fo r 3— 5 hours. It i s still
fairly ef fectiv e.
2. 4　 Precipi ta tion and deco lorization o f rough
po lysaccharides. The concentrated sample is thick
and da rk. In order to g et pure prepa ra tion, de-
co lorization has to be conducted. Much pigment in
rough po lysaccharides can be dissolved in ethanol.
Some flo cculent w hi te sediment appeared instantly
along wi th adding ethanol into the concentrate.
The mo re ethanol was added, the mo re flo cculent
w hi te sticky sediment appear red. Fo r the high effi-
·893·中草药　 Chinese T raditional and Herba l D rugs　第 34卷第 10期 2003年 10月
ciency of sedimentation and deco lorization, ethanol
w as added ti ll the final concentration of ethanol
reached to 65% — 70% . The so lution should be
sti rred wi th putting ethano l in. The sample wa s
kept sti ll fo r 8— 10 hours to get mo re f locculent.
This step w as repeated to deplete the pigment
more. Rough po lysaccharides 98. 5% have been
obtained and pigment ( 95% ) has been eliminated
finally.
2. 5　 Drying of rough po lysaccharides. The best
method of drying bio-products is lyophi li zation o r
f reeze-drying. In o rder to keep the continui ty of
w hole operation process and reduce the co st , the
w ay o f pressing or squeezing w as chosen. By this
w ay , the w hole period of drying can be decreased
w ithin 30 minutes.
2. 6　 Optimum composi tion of depro teiniza tion a-
gent. The resul ts of orthogonal test for DPR with
fiv e factors, three lev els are show n in Table 2.
　　 The values of the maximum di fference R in
Table 2 indicated tha t the majo r facto r af fecting
DPR is B ( 2-butanol ) , the sub-ma jo r facto r is D
( ethanol ) ; the minor facto r is C ( methanol ) , and
the sub-mino r facto r is E (NaCl ) . The inf luencing
o rder fo r DPR is B> D> A> E> C.
　　 It can be seen tha t the DPR va ried g reat ly
w hen the amount o f 2-butanol v aried, g oing up
slow ly fi rst , then going down rapidly f rom the size
o f numerical v alue and i ts changing tendency of
K 1 , K 2 , and K 3 , o f each factor in Table 2. The
DPR decreased g radual ly as the concentra tion of
ethanol increa sed. The DPR went up fi rst, and
then w ent dow n, w hen the concentration o f chlo ro-
form scaled up. The DPR altered li tt le w hen the
concentra tion o f methano l increased.
　　 The optimal combina tion for depro teiniza tion
is A2 B2 C1D1 E2 acco rding to the max imum value a-
mong K 1 , K 2 , and K 3 , of each facto r in Table 2.
That is, the best composi tion fo r the depro-
teiniza tion agent is 40% of chloroform , 10% of 2-
butanol, 2% o f methano l, 40% o f ethanol , and
0. 01% o f NaCl. Further proved experiments indi-
cated tha t the DPR was up to 99. 98% under the
best condition. The obvious advantag e fo r this ap-
proach is that all ag ents used are easily removed
from the products compared wi th o ther methods.
Table 2　 DPR with f ive factors, three levels by OT
No.
Factors
A B C D E
DPR/%
1 1 1 1 1 1 　 80. 5
2 1 1 1 1 2 82. 7
3 1 1 1 1 3 81. 3
4 1 2 2 2 1 88. 1
5 1 2 2 2 2 92. 9
6 1 2 2 2 3 89. 7
7 1 3 3 3 1 50. 9
8 1 3 3 3 2 55. 8
9 1 3 3 3 3 53. 2
10 2 1 2 3 1 70. 8
11 2 1 2 3 2 74. 2
12 2 1 2 3 3 73. 2
13 2 2 3 1 1 98. 5
14 2 2 3 1 2 99. 3
15 2 2 3 1 3 99. 0
16 2 3 1 2 1 63. 3
17 2 3 1 2 2 66. 6
18 2 3 1 2 3 64. 7
19 3 1 3 2 1 75. 2
20 3 1 3 2 2 79. 8
21 3 1 3 2 3 76. 4
22 3 2 1 3 1 83. 3
23 3 2 1 3 2 88. 2
24 3 2 1 3 3 84. 9
25 3 3 2 1 1 67. 7
26 3 3 2 1 2 70. 6
27 3 3 2 1 3 68. 1
K 1 　 675. 1 　 694. 1 　 695. 5 　 747. 7 　 678. 3
K 2 709. 6 823. 9 695. 1 696. 7 710. 1
K 3 694. 2 560. 9 688. 1 634. 5 690. 5
R 34. 5 263 7. 4 113. 2 31. 8
　　 As described in the int roduction, higher puri-
ty o f po lysaccharides has g reat ly potential clinic
application. And proteins a re majo r residues of
po lysaccharides in the prepara tion of fungi polysac-
charides as edible mushrooms are rich in proteins.
It wi ll influence the therapeutical ef fect o f polysac-
charides heav ily i f the residue w as no t removed.
　　 Further studies need to be ca rried on wh ether
the new approach fo r depro teinization searched out
could be adapted for o thers materials and polysac-
charides.
　　 Acknow ledgements: Prof. Bonnie and Mr. M ichael
being with Nanyang Institute o f Techno log y presently r e-
vised o f the pape r from USA.
References:
[1 ]　 Wurtman, Judi th J, Shear J L, et al . Th erapeutic carboh y-
drate blends useful for aiding premens trual syndrome [ P].
US: 5760014, 1998-06-02.
·894· 中草药　 Chinese T raditional and Herba l D rugs　第 34卷第 10期 2003年 10月
[2 ]　 Guard-Pet ter J, Lak shmi B, Carlson R, et al . Characteri za-
tion of lipopolysacch arid e h eterogen eity in Sal-monella en teri-
tidis by an imp roved gel elect rophoresis m ethod [ J] . Apple
Environ Microbiol , 1995, 61: 2845-2851.
[3 ]　 Khng H P, Cunli f fe D, Davies S, et al . Th e s ynthesi s of sub-
micron magnetic particles and th ei r u se for preparativ e pu ri fi-
cation of p ro teins [ J] . B iotech Bioeng , 1998, 60: 419-424.
[4 ]　 D′Sou za S M, Alexander C, Carr S W , et al . Di rected nucle-
ation at a calcite-templated polymer su rface [ J]. N ature,
1999, 398: 312-316.
[5 ]　 Gutnick , David L, Rosenberg , et al . Production of alpha-
emuls an s [P] . U S: 4234689, 1980-11-18.
[6 ]　 Mil lqvis t-Fureby A, Gi ll I S, V ulf son E N. Enzymatic t rans-
formations in supersaturated solutions: I. A gen eral s tudy
w ith glycosidas es [ J ]. Biotech B ioen g , 1998, 60: 190-196.
[7 ]　 Wes tph al O, Crow el l P, Burnet t P. Determination of th e car-
bohyd rate com posi ti on of w ood pulps b y gas ch romat- ograph y
of the alditol acetates [ J] . Anal Chem , 1967, 39 ( 1 ): 121-
124.
交泰丸方药 4种提取方法的比较研究
张学兰 1 ,李正杰 2
( 1. 山东中医药大学 ,山东 济南　 250014; 2. 河北以岭医药集团 ,河北 石家庄　 050091)
摘　要: 目的　优选交泰丸方药的提取工艺。方法　以小檗碱、肉桂酸、总生物碱 、挥发油、干浸膏为指标 ,在药材粒
度、溶剂量、煎提温度、滤过、浓缩等条件相同的前提下 ,对半仿生提取法 ( SBE法 )、水提取法 ( W E法 )、半仿生提取
醇沉法 ( SBAE法 )、水提取醇沉法 ( WAE法 )进行比较研究。 结果　 5个指标综合评价 Y值为: SBE法> WE法>
SBAE法 > W AE法。 结论　交泰丸方药的提取法以 SBE法为佳。
关键词: 交泰丸 ;提取方法 ;小檗碱 ;肉桂酸 ;总生物碱
中图分类号: R284. 02; R286. 02　　　文献标识码: B　　　文章编号: 0253 2670( 2003) 10 0895 04
Comparison of four extracting methods for Jiaotai Pill
*
ZHANG Xue-lan
1 , L I Zheng-jie
2
( 1. Shandong Univ er sity o f TCM , Jinan 250014, China; 2. Hebei Yiling
Pha rmaceutical Group, Shijia zhuang 050091, China)
Abstract: Object　 To evalua te the ex t racting techno logy o f Jiao tai Pi ll components. Methods　 Four
methods— the semi-bionic ex t raction ( SBE) , the w ater ex t raction ( WE) , the semi-bionic ex t raction by
precipi tation wi th alcohol ( SBAE) , and th e w ater ex t raction by precipitation w ith alcohol ( WAE) w ere
used to ex t ract Jiao tai Pill component , wi th berberine, cinnamic acid, to tal alkaloids, vo la tile oil and dried
ex t ract taken as the markers and to study the four methods under the same condi tions of drug g ranularity,
solvent amount , decocting temperature, fi lt ration, concentra tion, etc. Results　 The comprehensiv e values
Y were SBE> WE> SBAE> WAE. Conclusion　 SBE method is bet ter than the o ther three methods in the
ex t raction of Jiao tai Pi ll components.
Key words: Jiaotai Pill; ex tracting methods; berberine; cinnamic acid; to tal alkaloids
* Jiao tai Pil l is a Chinese prescription consisted of Rhizoma Copt idis andCortex Cinnamomi . It has the
function of co ordination betw een the hear t and the kidney.
　　交泰丸由黄连、肉桂组成 ,具有交通心肾的功
能。临床用于失眠、心律失常、口腔疾病等取得较好
效果 [1 ]。 为进一步探讨该方药采用半仿生提取
( SBE)法提取是否较目前普遍应用的提取方法为
佳 ,本实验根据 SBE法理论 [2 ] ,在优选出 SBE法提
取条件 [3 ]、药材组合方式及醇沉较佳浓度的基础上 ,
以小檗碱、肉桂酸、总生物碱、挥发油、干浸膏量为指
标 ,对该方药作 4种提取方法的比较研究。
1　仪器与药品
Beckman高效液相色谱仪 (美国贝克曼公司 ) ,
·895·中草药　 Chinese T raditional and Herba l D rugs　第 34卷第 10期 2003年 10月
收稿日期: 2002-12-18基金项目:山东省自然基金资助项目 ( Q00C05)作者简介:张学兰 ( 1963— ) ,女 ,山东蓬莱人 ,副教授 ,硕士生导师 ,主要从事中药制剂及炮制研究。
Tel: ( 0531) 8836297　 E-mai l: zh angxl2440@ 163. com