全 文 ：·制剂与质量·
Stud ies on ra ising y ield of effective con stituen t
in Trigonella f oenum -g raecum by cellulase
J IAN G B in2hu i1, ZHAO Yu2qing23 , ZH EN G D an13 3 , J IN G Yu2, HU X iao2m in1, ZH EN G L ong2x i1
(1. Co llege of R esources and C ivil Engineering, N o rtheast U niversity, Shenyang 110004, Ch ina;
2. L iaon ing Co llege of T radit ional Ch inese M edicine, Shenyang 110032, Ch ina)
Abstract: Object　To imp rove the yield of the effect ive con st ituen t in T rig onella f oenum 2g raecum L.
by cellu lase. M ethods　To take the yield of sapon in as index to confirm the op t im al enzym e and the best
enzymo lysis condit ion fo r imp roving the yield of sapon in. Results　T he yield of the effect ive con st ituen t in
experim en ta l group is abou t 316 t im es of that in con tro l group; the best experim en ta l condit ion fo r enzy2
mo lysis is: tempera tu re 50 ℃, pH 414, t im e 72 h. Conclusion 　By u sing cellu lase, the yield of effect ive
con st ituen t of T. f oenum 2g raecum — sapon in cou ld be increased.
Key words: T rig onella f oenum 2g raecum L. ; cellu lase; sapon in
纤维素酶提高胡芦巴有效成分收率的研究
姜彬慧1, 赵余庆23 , 郑　丹13 3 , 荆　宇2, 胡筱敏1, 郑龙熙1Ξ
(11 东北大学资源与土木工程学院, 辽宁　沈阳　110004; 21 辽宁中医学院, 辽宁　沈阳　110032)
摘　要: 目的　利用纤维素酶提高胡芦巴有效成分的收率。方法　以甾体皂苷收率为指标, 确定提高胡芦巴有效成
分收率的最佳酶及最佳酶解条件。结果　实验组有效成分的收率约是对照组的 316 倍; 酶解的最佳实验条件为: 温
度 50 ℃, pH 414, 反应 72 h。结论　利用纤维素酶可以提高胡芦巴有效成分——甾体皂苷的收率。
关键词: 胡芦巴; 纤维素酶; 甾体皂苷
中图分类号: R 286102　　　文献标识码: B　　　文章编号: 0253 2670 (2004) 06 0626 04
　　T he research of ex tract ing the effect ive con s2
t ituen t from the natu ra l m ateria ls depending on en2
zym e p repara t ion o r enzym atic engineering is a new
techno logy ju st arising after the m iddle of 1990’s.
Dom est ic and overseas researches indica te that the
b io logica l enzym e can be u t ilized to realize the ex2
t ract ion and iso la t ion of the effect ive con st i2 tuen t
from the Ch inese m edicinal m ateria ls[1～ 3 ]. In o rder
to imp rove the yield of the effect ive con st i2 tuen t
from the m edicinal m ateria ls, som e scho lars have
ever m ade som e research u sing th is techno lo2
gy [4, 5 ]. A t p resen t, the dom est ic and overseas
scho lars m ain ly concen tra te on the research of the
cellu lo se as fo r the app lica t ion of enzym e m ethod
to ex tract ion of t rad it ional Ch inese m edicine. Be2 cau se the cell w all of mo st Ch inese m edicinal m ate2ria ls is compo sed of cellu lo se, the effect ive con2st ituen ts are a lw ays w rapped w ith in the cell w all.Cellu lase can be u sed to b reak the Β2D 2gluco sechain, w h ich is in favo r of the ex tract ion of effec2t ive con st ituen ts.T rig onella f oenum 2g raecum L. (T FL ) is theseed of T rig onella L inn. , one L egum ino sae p lan t,w h ich is con sidered as the trad it ional u se fo r w ar2m ing the k idney to invigo ra te y ang fo r a long h is2to ry. T he app lica t ion of the dried m atu re seed ofT FL has already been reco rded in Ch inese P ha rm a2cop oeia as the typ ica l Ch inese m edicine. T he studyof modern pharm aco logy show s that T FL has thefunct ion s of reducing the sugar and fa t in the b lood
·626· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 6 期 2004 年 6 月
Ξ 收稿日期: 2003212211
作者简介: 姜彬慧 (1962—) , 女, 黑龙江人, 副教授, 博士研究生, 研究方向为资源与微生物技术。3 通讯作者　T el: (024) 862247253 3 东北大学 2003 届本科毕业生
as w ell as f igh t ing again st tumo r, in w h ich mo re
at ten t ion of dom est ic and overseas scho lars has
been at t racted. T he m ain chem ical con st ituen ts of
the p lan t are sapon in, stero iden in, f lavone and its
g lyco side, t riterpene, a lkalo id, coum arin, o rgan ic
acid, lipa, and o thers, among w h ich the con ten t of
d io sgen in and yamogen in is abou t 016% —1% bu t
on ly 0115% —0132% in o ther p lan ts from sam e
genu s[6 ].
Fou r d ifferen t enzym es ( including cellu lase,
g lucoam ylase, Β2glucanase N S44053, cellob iase
N S37040) w ere u sed to carry ou t the enzymo lysis
on T FL and also confirm the best enzym e and best
enzymo lysis condit ion of imp roving the yield of
sapon in from T FL.
1　M a ter ia ls and appara tus
111　M ateria ls: T FL is p rovided by the R esearch
Cen ter of N ew M edicine of L iaon ing Co llege of
T radit ional Ch inese M edicine. T he samp le is kep t
w ith con stan t w eigh t by fire dry and kep t in the
drying appara tu s after grinding and screen ing. T he
standard sapon in is pu rchased from the Ch inese B i2
o logica l P roducts A u then t ica t ion Sta t ion; Cellu2
lase com es from Shanghai L izhu2Dongfeng B io logi2
cal T echno logy Co. , L td. ; Glucoam ylas, Β2gluca2
nase, and cellob iase are all p rovided by Beijing
N ovey Company. T he o ther reagen ts are pu rely
analyzed.
112　A pparatu s: JC101—A D igita l E lectro therm ic
B last ing D ry Box from Shanghai J iacheng A ppara2
tu s & Equ ipm en ts Facto ry; 721—T ype Spectro2
pho tom eter from Shanghai A nalyt ica l In st rum en t
Facto ry; D K—97—É E lectric Con stan t T empera2
tu re W ater Bath from T ian jin C ity T aist A ppara tu s
Co. , L td.
2　M ethods and results
211　P repara t ion of experim en ta l reagen t: C it ric
acid buffer so lu t ion is p repared acco rd ing to the
file [7 ]: Enzym e so lu t ion: Cellu lase so lu t ion, to
w eigh 50 m g cellu lase in accu racy and disso lve it in
10 mL m easu ring flask, keep the concen tra t ion at
5 m gömL. T he concen tra t ion of o ther enzym ic
p repara t ion so lu t ion is 5%.
Standard so lu t ion: To accu ra tely w eigh 110
m g sapon in standard and disso lve it in 5 mL m ethyl
a lcoho l w ith con stan t vo lum e, then p reserve it in
the refrigera to r a t 4 ℃ ( the concen tra t ion of the
sapon in is 012 m gömL ).
T FL so lu t ion fo r experim en t: To w eigh 5 g
T FL and separa tely p lace it in the 250 mL m easu r2
ing flask (20 m gömL ) , then add cit ric acid to bal2
ance so lu t ion, m ake a con stan t vo lum e after 10
m inu tes superson ic o scilla t ion s, then sto re it in the
refrigera to r a t 4 ℃ fo r standby.
212　T he draw ing of standard cu rve of sapon in:
A cco rd ing to the m ethod in the litera tu re [8 ] , to
take the con ten t of sapon in (C , Λg) to the co rre2
sponding ab so rbency A in o rder to p rocess linear
regression; sapon in regression linear equat ion:
C = 79193 A - 01520 1; r= 01997 5; and the co rre2
sponding ab so rbency A w ith in (0—60 Λg) po ssess
good linear dependence.
213　T he op t im um enzym e fo r hydro lyzed T FL :
In con tro l group to w eigh 20 m gömL T FL p re2pa2
ra t ion T FL so lu t ion fo r experim en t 215 mL , and
215 mL dist illed w ater, pH 414, 50 ℃, w ater bath
48 h; and 1 mL w ater sa tu ra ted no rm al bu tyl a lco2
ho l in to the 1 mL liqu id, then after shak ing,
standing, dem ix ing, cen trifugat ion, pu t superna2
tan t liqu id in evapo ra t ing dish; repeat the above
opera t ion fou r t im es, to ta lly co llect 5 mL w ater
sa tu ra ted no rm al bu tyl a lcoho l in evapo ra t ing dish,
af ter 100 ℃ vo la t ilizing on the bain2m arie u se
fo rm aldehyde to disso lve the sapon in st ick ing to
the 25 mL evapo rat ing dish, w ash it severa l t im es
and vo la t ilize the w ater bath at 60 ℃, take 1 mL
sapon in so lu t ion in the test tube w ith 1 mL m ark s,
acco rd ing to the m ethod of test ing the tran sparency
at the w avelength of 560 nm , then accoun t yield of
enzym atic sapon in th rough the regression equat ion
and the fo llow ing fo rm u la.
Saponin yield= (25 CV ×10- 3öW )×100%
Fou r p ieces of 215 mL T FL p repara t ion: T FL
so lu t ion fo r experim en t w ith 20 m gömL concen2
t ra t ion w ere m easu red in con tro l group by separa2
tely adding 215 mL and 5 m gömL cellu lase; pH
414, 50 ℃, w ater bath 48 h; 5% Β2glucanase, pH
518, 50 ℃ w ater bath 48 h; 5% Β2glucanase Ê ;
·726·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 6 期 2004 年 6 月
pH 518, 50 ℃, 48 h; and 5% glucoam ylase, pH
414, 25 ℃, w ater bath 48 h, acco rd ing to the
m ethod of test ing the tran sparency at the w ave2
length of 560 nm and accoun t the ra te of enzym atic
sapon in u sing fo rm u la (É ). T he comparison of d i2
fferen t resu lts of enzym e hydro lyzed sapon in yield
can be seen in T ab le 1. T he resu lt is cellu lase >
glucoam ylase> cellob iase> Β2glucanase É > con tro l
group. T he best is the group of celluase, the h igh
yield of sapon in is abou t 316 t im es than that in the
con tro l group.
Table 1　Sapon in y ield compar ison of d ifferen t
enzyme hydrolyzed TFL
N o. M ethods Sapon in yield ö%
1 　　con tro l group 0. 564
2 　　cellu lase 2. 041
3 　　glucoam ylase 1. 245
4 　　Β2glucanase 0. 699
5 　　cellob iase 0. 915
214　Choo se fo r op t im um condit ion fo r cellu lase
hydro lyzed T FL : T ake T FL , and 5 m gömL the
cellu lase in to triangu lar bo t t le, in d ifferen t t im e
(4, 24, 48, 72, 96, 108 h) , d ifferen t tempera tu re
(20 ℃, 37 ℃, 45 ℃, 50 ℃, 55 ℃, 65 ℃) , d ifferen t
pH (410, 412, 414, 416, 418) and differen t T FL
concen tra t ion s. A ccoun t the yield of sapon in acco r2
ding to the m ethod in 213 after the react ion term i2
nat ion.
21411 　 T he determ inat ion of the best react ion
t im e of cellu lase hydro lyzed T FL : U nder the con2
dit ion of 50 ℃ ho t w ater w ith pH 414, heat ing at
d ifferen t t im es, the rela t ion cu rve of react ion t im e
of cellu lase hydro lyzed T FL and the yield of cellu2
lase hydro lyzed sapon in is indica ted in F ig. 1.
W ith the t im e runn ing, the cellu lase hydro lyzed
T FL funct ion w as increased by and by, the func2
t ion w as imp roved step by step in 4—72 h as w ell,
un t il 72 h la ter it reached the peak, then low ered
dow n. It suggests tha t the mo st adap t ive t im e fo r
cellu lase hydro lyzed T FL be 72 hou rs. T h is
sounds that cellu lase can develop the largest func2
t ion under the mo st su itab le condit ion s.
21412 　 T he determ inat ion of the best react ion
temperatu re of cellu lase hydro lyzed T FL : U nder
the condit ion of pH 414, ho t w ater w ith heat ing at
F ig. 1　Effect on sapon in y ield in d ifferen t
enzyme hydrolyz ing time
72 h, in differen t tempera tu res, the rela t ion cu rve
of react ion temperatu re of cellu lase hydro lyzed
T FL and the yield of cellu lase hydro lyzed sapon in
is indica ted in F ig. 2. T he cellu lase hydro lyzed
T FL funct ion began from 40 ℃, it nonoccu rrenced
in 20 ℃—40 ℃. T he funct ion w as imp roved step
by step from 45 ℃ as w ell, un t il 50 ℃ it reached
the peak, then low ered dow n. It suggests tha t the
mo st adap t ive temperatu re fo r cellu lase hydro lyzed
T FL be 50 ℃. T h is sounds that cellu lase can de2
velop the largest funct ion under the mo st su itab le
condit ion s.
F ig. 2　Effect on sapon in y ield in d ifferen t enzyme
hydrolyz ing temperature
21413　T he determ inat ion of the best react ion pH
of cellu lase hydro lyzed T FL : U nder the condit ion
of 50 ℃, ho t w ater heated fo r 72 h, a t d ifferen t
pH values, the rela t ion cu rve of react ion pH of cel2
lu lase hydro lyzed T FL and the yield of cellu lase
hydro lyzed sapon in is indica ted in F ig. 3.
It show ed in F ig. 3 that, cellu lase hydro2
lyzed T FL funct ion w as increased by and by w ith
pH runn ing. W hen pH = 414, it reached the peak,
then low ered dow n. So the op t im um adap t ive pH
fo r cellu lase hydro lyzed T FL is 414, w h ich ino scu2
la tes w ith the mo st adap t ive pH of cellu lase. T h is
sounds like that cellu lase can develop the st rongest
funct ion under the mo st su itab le pH condit ion s.
·826· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 6 期 2004 年 6 月
F ig. 3　Effect on sapon in y ield in d ifferen t pH
21414　 Influence of enzym atic react ion cau sed by
the change of samp le concen tra t ion, and the deter2
m inat ion of M ichaelis2M en ten equat ion: A fter 72 h
under the condit ion of 50 ℃, pH 414, and the hy2
dro lyt ic act ion by 5 m gömL cellu lase, the resu lt
fo r the react ion velocity of variou s samp le w ith dif2
feren t concen tra t ion s is show n in T ab le 2.
Table 2　Change of reaction speed of d ifferen t
sample af ter enzyme hydrolyz ing
Samp leöm g [S ]ö(m g·mL - 1) 1[S ] V ö(m g·d- 1) V - 1ö(d·m g- 1)
　5 5 0. 20 0. 011 1 90. 09
100 10 0. 10 0. 015 1 66. 22
150 15 0. 07 0. 042 0 23. 81
200 20 0. 05 0. 045 4 22. 03
250 25 0. 04 0. 044 4 22. 52
　　U nder the condit ion of con stan t react ion tem 2
pera tu re, pH and enzym atic concen tra t ion, the p ri2
m ary speed of enzym atic react ion increased fo llo2
w ing the increase of samp les’ concen tra t ion [S ].
W h ile as samp le concen tra t ion s reached a certa in
lim it, the react ion speed becam e con stan t, con si2
dering as the largest react ion speed. T he rela t ion
betw een the speed and concen tra t ion of sub stra2
tum can be show n by M ichaelis2M en ten equat ion.
V =
V m ax·[S ]
K m + [S ]
K m: M 2constan t, V m ax: the mo st reaction speed, [S ]:
samp le concen tra t ion. w hen V = V m axö2, K m = [S ]
K m is one characterist ic2con stan t of enzym e. It
is one m ethod to research enzym e by m easu ring
K m. If the M ichaelis2M en ten equat ion changes in to
doub le recip rocal equat ion, one p ictu re is m ade by
1öV to 1ö[S ], each po in t is linked together and
m ake the nodal increm en t is m ade to be - 1öK m on
the la tera l ax is in o rder to calcu la te K m value.
1
V =
K m
V m ax
× 1[S ]+
1
V m ax
T he M ichaelis2M en ten con stan t of hydro lysis
T FL by cellu lase is K m = 200 m gömL ; 1öV m ax =
215, V m ax = 0104 m göd. So the rela t ion sh ip equa2
t ion betw een the react ion speed of hydro lysis T FL
by cellu lase and concen tra t ion of samp le as below :
V = 0104·[S ]200+ [S ]
3　D iscussion
T he resu lt of such research has indica ted that
the impo rtan t funct ion of cellu lase hydro lyt ic T FL
is enzym e’s funct ion of b reak ing w all of p lan t cell,
becau se comparing w ith the con tro l group , the
h igh yield of sapon in is abou t 316 t im es than that
in the con tro l group. T he resu lt of the research is
the sam e as the p redecesso r’s resu lt, and the argu2
m en t is a lso iden t if ied. So the research po in ts ou t:
du ring the period of ex tract ing effect ive m edi2 cine
elem en t from the p lan t, f irst ly it can be hydro2
lyzed by cellu lase acco rd ingly the release of the ef2
fect ive con st ituen t has been increased, the yield of
the effect ive con st ituen t has been imp rov2 ed, fo r
imp roving the techno logy of ex tract ing the effec2
t ive con st ituen t, ra ising the yield of the trad it ional
Ch inese m edicine the above m ethod has po ssessed a
very impo rtan t app lied co st.
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·926·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 6 期 2004 年 6 月