全 文 ：·制剂与质量·
D eterm ination of marker com pounds in plan ts of Rhod iola L.
from d ifferen t habita ts by RP-HPLC
SHAO Yun2dong1, SU Yan2fang1, YU Hong2jian2, L I Fu2m ing3
(1. Co llege of Pharm aceu ticals and B io techno logy, T ian jin U niversity, T ian jin 300072, Ch ina; 2. J ianfeng N atural
P roduct R &D Co. , L td, T ian jin 300402, Ch ina; 3. Zhejiang J ianfeng Pharm acy Co. , L td, J inhua 321000, Ch ina)
Abstract: Object　To develop the reliab le R P2H PL C m ethods fo r the determ inat ion of sa lidro side, ty2
ro so l, ro savin, ro sin, and ro sarin in the p lan ts of R hod iola L. and to evaluate their species from differen t
hab ita ts. M ethods　M ethod É : m ethano l2w ater (015 mmo löL SD S in 1% acet ic acid aqueou s so lu t ion)
system fo r the analysis of sa lidro side; m ethod Ê : aceton it rile2w ater system fo r ro savin; m ethod Ë : aque2
ou s aceton it rile2pho spho ric gradien t system fo r sa lidro side, tyro so l, ro savin, ro sin, and ro sarin. Results　
T he con ten ts of sa lidro side in differen t species range from 01021% to 11420% , and tho se of ro savin in all
species are very lim ited o r undetected excep t in R hod iola rosea L. and R. sacha linensis. T he con ten ts of the
five m arker ingredien ts are sign if ican t ly species2 and hab ita t2dependen t. Conclusion 　T h ree R P2H PL C
m ethods are estab lished fo r quan t ita t ive analysis of the above five m arker ingredien ts in the m ean t im e, re2
spect ively. Evaluat ion of the quality of varied species of R hod iola L. show s that R. rosea grow ing in X in2
jiang U ygu r A u tonomou s R egion and R. sacha linensis grow ing in J ilin p rovince are the tw o bet ter species
con ta ined w ith abundan t above2m en tioned ingredien ts in Ch ina.
Key words: R hod iola L. ; sa lidro side; tyro so l; ro savin; ro sin; ro sarin; R P2H PL C
RP-HPLC 法测定不同品种和产地红景天中指标成分的含量
邵云东1, 苏艳芳1, 於洪建2, 李复明3Ξ
(11 天津大学药物科学与技术学院, 天津　300072; 21 天津尖峰天然产物公司, 天津　300402;
31 浙江尖峰药业有限公司, 浙江 金华　321000)
摘　要: 目的　建立快速、有效的反相高效液相 (R P2H PL C) 检测方法, 测定不同品种和产地红景天中红景天苷、红
景天芬、酪醇、红景天素、红景天任的含量, 以评价红景天的质量。方法　方法É : 甲醇2水 (015 mmo löL SD S 的 1%
醋酸水溶液) 检测红景天苷; 方法Ê : 乙腈2水检测红景天芬; 方法Ë : 乙腈2磷酸水溶液梯度洗脱同时检测 5 个有效
成分。结果　红景天苷的含量为 01021%～ 11420% ; 红景天芬除在蔷薇红景天和高山红景天中含量较高外, 在其他
品种中均非常低或低于检测限; 5 个有效成分的含量因品种或产地的不同而有很大的变化。结论　建立的 3 种R P2
H PL C 方法可同时测定红景天苷、红景天芬等 5 个有效成分。综合考虑不同品种中 5 个有效成分的含量, 以产自新
疆的蔷薇红景天和吉林的高山红景天为国内较好的品种。
关键词: 红景天属; 红景天苷; 酪醇; 红景天芬; 红景天素; 红景天任; 反相高效液相色谱
中图分类号: R 286102　　　文献标识码: B　　　文章编号: 0253 2670 (2004) 05 0505 04
1　 In troduction
T he R hod iola L. p lan t con ta in s abou t 90
species and their variou s species natu ra lly d isp lay a
circumpo lar d ist ribu t ion in moun ta inou s region s
w ith h igher la t itudes and elevat ion s of the N o r2 th2
ern H em isphere [1 ]. T radit ional fo lk m edicine,
p lan ts of R hod iola L. , especia lly R. rosea, u sed to
increase physica l endu rance, w o rk p roduct ivity,
longevity, resistance to h igh la t itude sickness, and
to trea t fa t igue, dep ression, nervou s system diso r2
ders, etc[2～ 5 ]. Since 1960, a variety of R hod iola
p roducts have been offered and con sum ed as adap2
togen s w ith variou s health2p romo ting effects.
W ith dram at ica lly increase of dem and fo r R hod iola2
·505·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 5 期 2004 年 5 月
Ξ 收稿日期: 2003210215
based phytom edicines in the la te 1980s, and be2
cau se of their sign if ican t species2dependen t varia2
t ion in phytochem istry and pharm aco logy, the
standard iza t ion of R hod iola p roducts o r ex tracts is
of great impo rtance. Scien t if ic invest iga t ion reveal2
ed that tw o group s of chem ical con st ituen ts w ere
respon sib le fo r the un ique pharm aco logica l p roper2
t ies of R hod iola p roducts: one is phenylp ropano ids
including ro savin, ro sin, and ro sarin [6, 7 ] , and the
o ther is phenylethano l derivat ives including
salidro side and tyro so l. T herefo re, the R hod iola
ex tracts o r p repara t ion s are u sually standard ized
fo r bo th ro savin and salidro side to guaran tee their
quality and effect iveness.
　　Ch ina has abundan t resou rces of p lan ts of
R hod iola L. , and nearly 80 species of them are dis2
t ribu ted in Ch ina. T he analysis of R hod iola p rod2
ucts is repo rted on ly fo r sa lidro side, and the stan2
dard iza t ion by determ inat ion of ro savin has no t
been repo rted in Ch ina. M o reover, none of the re2
po rted H PL C m ethods, the detect ion of sa lidro2
side, and tyro so l in R hod iola p roducts[8, 9 ] w as sat2
isfacto ry. T hu s, in ou r ongo ing effo rt to so lve the
above2m en tioned p rob lem s, th ree H PL C m ethods
w ere developed fo r the quan t ita t ive analysis of the
five m arker compounds, such as sa lidro side,
ro savin, tyro so l, ro sin, and ro sarin in one run, re2
spect ively.
2　Exper im en t
211　P repara t ion of stock so lu t ion s of reference
Salidro side and ro savin (Ch rom aD ex ) , 2 m g
fo r each w ere accu ra tely w eighed, and disso lved in
tw o 10 mL vo lum etric f lask s w ith m ethano l as
stock so lu t ion fo r m ethod É and Ê , respect ively.
Salidro side, tyro so l, ro savin, ro sin, and ro sa2
rin, 1 m g fo r each, w ere accu ra tely w eighed,
m ixed together, and disso lved in a vo lum etric f lask
w ith m ethano l as stock so lu t ion fo r m ethod Ë .
212　Calib ra t ion cu rve
T he stock so lu t ion s w ere dilu ted 2, 4, 8, 12,
16 t im es in m ethano l, respect ively. U sing A nastar
ch rom atography data system , all these standard
w ere in jected to genera te a five2po in t ca lib ra t ion
cu rve fo r the th ree m ethods respect ively. Standard
cu rve, as show n in T ab le 1, w as linear w ith R 2 no t
less than 01998 6.
213　Samp le p repara t ion
T he p lan t m ateria ls of R hod iola L. w ere co l2
lected from w ild locat ion in Ch ina. A ll the samp les
w ere iden t if ied by Yu Hong2jian, a sen io r engineer
from T ian jin TCM Facto ry. T h ree ex tract ing
m ethods including u lt rason ic ex tract ion, Soxh let
appara tu s ex tract ion, and room tempera2 tu re ex2
t ract ion w ere compared. R esu lts show ed that u l2
t rason ic ex tract ion w as a simp le, fast, and eff i2
cien t m ethod. T he roo ts of each species (abou t 200
g) w ere finely pow dered, 015 g of each samp les
w as accu ra tely w eighed, u lt rason ica lly ex tracted
w ith 30 mL of 60% aqueou s ethano l fo r 20 m in
th ree t im es, ex tract ion so lu t ion s w ere cen trifuged
at 5 000 röm in fo r 15 m in, super2 natan ts w ere
com b ined and adju sted to a 100 mL vo lum etric
f lask w ith 60% aqueou s ethano l. A ll samp le so lu2
t ion s w ere filtered th rough a 0145 Λm A crodisc sy2
ringe filter befo re being sub jected to H PL C analy2
sis. Each samp le so lu t ion (10 ΛL ) w as in jected fo r
H PL C analysis.
Table 1　Cal ibration data for each reference
compound in method É —Ë
M ethods Compounds R 2 Regression equationÉ salidro side 0. 999 1 Y = 1. 56×104XÊ ro savin 0. 999 5 Y = 2. 58×104XË salidro side 0. 999 2 Y = 1. 44×104X
tyro so l 0. 999 1 Y = 3. 67×104X
ro savin 0. 999 3 Y = 2. 31×104X
ro sin 0. 998 6 Y = 2. 42×104X
ro sarin 0. 998 5 Y = 2. 41×104X
　　Y reflects peak area, X reflects amount of compound in ΛgömL
214　Stab ility test
Each samp le so lu t ion (10 mL ) w as in jected in2
to H PL C in each m ethod after 1, 3, 5, 7, 12 h, re2
spect ively. R esu lts show ed that a ll the rela t ive
standard devia t ion (R SD ) w ere less than 3100%
and indica ted the samp le so lu t ion s w ere stab le
w ith in 12 hou rs.
215　R ecovery test
T he recovery w as determ ined by adding
know n amoun ts of each reference compound to the
p lan t samp le p rio r to the ex tract ion, and all the
samp les w ere dealt w ith the sam e m ethod as de2
scribed in Sect ion 213. T he determ ined concen2
t ra t ion of each ingredien t w as divided by the added
concen tra t ion, and the recoveries w ere calcu la ted
in percen tages. T he added concen tra t ion s w ere
based on the m ean concen tra t ion of these ingredi2
en ts in th ree un sp iked samp les. R SD w as less than
3101% , and average recovery ra t io w as no t less
than 9510%.
3　Ana lytica l m ethods
　　H PL C analysis w as perfo rm ed on a Sh im adzu
·605· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 5 期 2004 年 5 月
L C—10A T vp system equ ipped w ith Sh im adzu
SPD—10A vp detecto r. Fo r the m ethods É andÊ , a Phenom enex C18 co lum n (250 mm ×416 mm ,
5 Λm ) w as u sed, and fo r m ethod Ë a Sh im adzu
V P—OD S co lum n (150 mm ×416 mm , 5 Λm ) w as
u sed.
M ob ile phase in m ethod É : M ethano l2w ater
so lu t ion (015 mmo löL SD S 1% acet ic acid w ater
so lu t ion ) (25∶75) ; f low rate: 018 mL öm in; de2
tect ion w avelength: 225 nm.
M ob ile phase in m ethod Ê : A ceton it rile2w a2
ter (25∶75) ; f low rate: 110 mL öm in; detect ion w avelength: 254 nm.M ob ile phase in m ethod Ë : So lu t ion A :012% aqueou s pho spho ric acid, so lu t ion B: A ce2ton it rile, the so lven t gradien t w as A , in it ia l 96%20 m in 70% ; 25 m in 96% ; 30 m in 96% (run t im e30 m in ) ; detect ion w avelength: 1—12 m in 225nm , 12—30 m in 254 nm ; flow rate: 018 mL öm in.Each samp le (10 ΛL ) w as in jected. A ll separa t ionw as at room temperatu re. Peak s w ere assigned bysp ik ing the samp les w ith reference compounds.T he resu lts can be seen in T ab le 2 and F ig. 1—3.
Table 2　Analysis of d ifferen t spec ies of Rhod iola raw mater ia ls in Ch ina and extracts
from Tian j in J ianfeng Natura l Product R&D Co. , L td. (values in gö100 g)
　　Species H ab itats(P rovinces) T im e Salidro side T yro so l Ro savin Ro sin Ro sarin
　R hod iola rosea X in jiang June 2002 1. 232 0. 132 2 1. 621 0. 311 0. 214
　R. sacha linensis J ilin Ju ly 2001 0. 351 0. 084 5 0. 212 0. 101 0. 084
　R. sacha linensis Yunnan June 2001 0. 241 0. 001 4 0. 014 - -
　R. crenu la te Yunnan Ju ly 2001 1. 420 0. 231 1 0. 032 0. 008 -
　R. crenu la te Sichuan Sep. 2001 0. 411 0. 054 1 - - -
　R. crenu la te Q inghai Sep. 2001 1. 024 0. 084 2 - - -
　R. k irilow ii Q inghai Sep. 2001 0. 667 0. 023 4 - - -
　R. f astig ia ta Yunnan Ju ly 2001 0. 021 - - - -
　R. a lterna T ibet Sep. 2001 0. 321 0. 021 4 0. 012 - -
　R. rosea ex tract X in jiang 011223 3. 584 0. 524 1 6. 745 1. 412 1. 110
　R. crcnu la ta ex tract Yunnan 020102 4. 521 0. 642 1 0. 122 0. 021 -
　R. coccinea ex tract Q inghai 020203 4. 661 0. 682 3 - -
F ig. 1　Chromatogram of sa l idroside (method É )
F ig. 2　Chromatogram of rosav in (method Ê )
4　Results and d iscussion
　　T he developed m ethod Ë perm it ted the first
sim u ltaneou s detect ion of bo th “ sa lidro sides ”
(sa lidro side and tyro so l) and“ro savin s”( ro savin,
ro sin, and ro sarin ). Yet the R P2H PL C condit ion
w as som ew hat comp lica ted becau se of the gradien t
mob ile phase, changing detect ion w avelength and
rela t ively longer runn ing t im e. T hu s, m ethod É
fo r sa lidro side and m ethod Ê fo r ro savin have also 12salidro side　22tyro so l　32ro savin　42ro savin　52ro sinF ig. 3　Chromatogram of f ive reference substances(A) , R. rosea raw mater ia l (B) , andseparated by method Ëbeen developed in o rder to m eet the dem and fo r thedetect ion of d ifferen t types of m arker ingredien ts.T hese th ree m ethods are p roved to be rap id, sen si2t ive, and reliab le H PL C m ethods fo r the evaluat ionof R hod iola species and their rela ted ex tract o rp roducts.T he resu lts of the determ inat ion of the m arkeringredien ts of d ifferen t R hod iola species and them arket ex tracts availab le in Ch ina show ed thattheir con ten ts are sign if ican t species2dependen t andalso influenced by the w ild locat ion even fo r thesam e species. In conclu sion, R. rosea from the
·705·中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 5 期 2004 年 5 月
X in jiang U ygu r A u tonomou s R egion and R. sacha2
linensis from J ilin P rovince are tw o species w ith
h igher con ten ts of the m arker ingradien ts.
References:
[ 1 ]　D eng G C, D u N S, Sun D S. T he curren t R&D situation of
R hod iola L. [J ]1 Ch in J M od A pp l P harm (中国现代应用药
学) , 1994, 11 (4) : 472511
[ 2 ]　M arkus G, Yurdanur Y. A nalysis of the m arker compounds of
R hod iola rosea L. by RP2H PL C [J ]1 Chem P harm B u ll,
2001, 49 (4) : 46524671
[ 3 ]　Kiryanov A A. D eterm in ing b io logically active componen ts of
R hod iola rosea rh izom es [J ]1 K h im P rir W oed inen ii, 1991
(3) : 32023241
[ 4 ]　Satsyperova I F. B io logically active substances in rh izom es o r
R hod iola rosea L. [J ]1 In trod uced in S t. P etersbu rg , R astitel’ ny e R esu rsy , 1993, 29 (2) : 262311[ 5 ]　M aslow a L V. Pharm aco logical active compounds in R hod iola[J ]1 E xp C lin P harm , 1994, 57 (1) : 612631[ 6 ]　Germ ano C, Ram azanov Z. A rtic R oot R hod iola rosea—T heP ow erf u l N ew G inseng A lterna tive [M ]1 N ew Yo rk: Kens2ington P ress, 19991[ 7 ]　Bocharova O A. T he effect of R hod iola rosea ex tract on inci2dence rate of superficial b ladder carcinom a relap ses [J ]1 U rolN ef rol, 1995 (2) : 462471[ 8 ]　M ing H Q , X ia G Q , Zhang R J. T he research p rogress ofR hod iola L. [L ]1 Ch in T rad it H erb D rug s (中草药) , 1988,19 (5) : 372431[ 9 ]　W an S, You X T , W an T P. A nalysis of salidro side in R hod i2ola L. by RP2H PL C [J ]1 A cta P harm S in (药学学报) , 1992,27 (11) : 84928521
超声循环提取灵芝中三萜类化合物的研究
黄书铭1, 杨新林13 , 张自强1, 徐建兰2朱鹤荪3Ξ
(11 北京理工大学生命科学与技术学院, 北京　100081; 21 无锡市三联高科技开发有限公司, 江苏 无锡　214023;
31 北京理工大学 材料科学研究中心, 北京　100081)
摘　要: 目的　研究超声循环技术在灵芝中三萜类化合物提取中的应用。方法　在常规提取方法的基础上, 增加超
声循环的处理步骤。结果　通过试验对比, 超声循环提取所需各种溶剂用量减少, 提取时间缩短, 目的产物提取率
提高了 40%。与常规方法提取得到的目的产物之间存在着良好的相关性。结论　超声循环技术用于灵芝中三萜类
化合物的提取具有良好的应用前景。
关键词: 灵芝; 三萜类化合物; 超声循环技术; 高效液相色谱
中图分类号: R 28412; R 286102　　　文献标识码: B　　　文章编号: 0253 2670 (2004) 05 0508 03
Study on ultra son ic c ircula tion techn ique to extraction of tr iterpeno ids
from Ganoderm a lucidum
HUAN G Shu2m ing1, YAN G X in2lin1, ZHAN G Zi2qiang1, XU J ian2lan2, ZHU H e2sun3
(1. Schoo l of L ife Science and T echno logy; 2. San lian H i2T ech L im ited Company, W uxi 214023, Ch ina;
3. R esearch Center of M ateria l Science, Beijing Inst itu te of T echno logy, Beijing 100081, Ch ina)
Abstract: Object　To study the app lica t ion of u lt rason ic circu la t ion techn ique fo r the ex tract ion of
t riterpeno ids from Ganod erm a lucid um (L eyss. ex F r. ) Karst. M ethods　O n the basis of conven t ional
m ethod, the p rocessing step s of u lt rason ic circu la t ion trea tm en t w ere added. Results　T he comparat ive
experim en ts show ed that less amoun ts of variou s so lven ts and sho rter ex tract ion t im e w ere needed fo r u l2
t rason ic circu la t ion ex tract ion, w ith the p roduct ra t io of abou t 40 percen t h igher than that of conven t ional
m ethod. Fu rthermo re, a good con sistency of the target p roduct analyzed by H PL C w as found betw een tw o
differen t ex tract ion m ethods. Conclusion 　T he u lt rason ic circu la t ion techn ique has a po ten t ia l app lica t ion
to the ex tract ion of t riterpeno ids from G. lucid um .
Key words: Ganod erm a lucid um (L eyss. ex F r. ) Karst. ; t riterpeno ids; u lt rason ic circu la t ion tech2
n ique; H PL C
　　灵芝在我国已有两千多年的药用历史。近年来,
从灵芝的子实体、孢子和菌丝体所提取的三萜类化
合物组份 ( t riterpeno id componen ts) 已被证实具有
抗肿瘤、免疫调节等作用[1～ 4 ]。灵芝三萜类化合物的
·805· 中草药　Ch inese T radit ional and H erbal D rugs　第 35 卷第 5 期 2004 年 5 月
Ξ 收稿日期: 2003210208
基金项目: 国家重点科技项目 (攻关)计划
作者简介: 黄书铭 (1963—) , 男, 安徽省六安市人, 蚌埠高等专科学校副教授, 北京理工大学在职博士生。E2m ail: sm h519@bit. edu. cn3 通讯作者　T el: (010) 68911949　Fax: (010) 68911040　E2m ail: x lyang@bit. edu. cn