全 文 ：天然产物研究与开发 Nat Prod Res Dev 2012，24:1343-1347
文章编号:1001-6880(2012)10-1343-05
Received December 26，2011;Accepted April 18，2012
Funding:The Knowledge Innovation Programs of the Chinese Academy
of Sciences (NO． KSCX2-EW-J-26)
* Corresponding author :Tel:86-971-6132750;E-mail:hufz@ nwipb． ac．
cn
青藏高原红景天属植物药材中五种有效成分 HPLC分析
迟晓峰1，2，矫晓丽1，2，董 琦1，肖远灿1，胡风祖1*
1中国科学院西北高原生物研究所，西宁 810008;2 中国科学院研究生院，北京 100049
摘 要:为了比较红景天属不同红景天药材的有效成分的差异，采用 HPLC-DAD法建立了同时测定青藏高原 14
种红景天属药材中红景天苷、酪醇、没食子酸、儿茶素、表儿茶素含量的方法．色谱柱为 Phenomenex Luna C18(250
× 4． 6 mm，5 μm) ;以甲醇-水为流动相，梯度洗脱;流速为 1． 0 mL /min;检测波长为 276 nm;柱温为 30 ℃。结果
表明，在五种化合物在选定的条件下能得到较好的分离，线性关系良好。方法回收率在 97． 36%以上，RSD小于
2． 31%。该方法简单快速，可为红景天属药材的质量控制提供科学依据。
关键词:HPLC，有效成分，红景天
中图分类号:R284. 2 文献标识码:A
Determination of Five Bioactive Compounds in Medicinal Plants
Belonging to Rhodiola L． Genus in Qinghai-Tibetan Plateau by HPLC
CHI Xiao-feng1，2，JIAO Xiao-li1，2，DONG Qi1，XIAO Yuan-can1，HU Feng-zu1*
1 Northwest Institute of Plateau Biology of Chinese Academy of Sciences，Xining 810008 China;
2Graduate school of Chinese Academy of sciences，Beijing 100049，China
Abstract:In order to evaluate the differences and similarities between the primary constituents in Rhodiola L． genus，an
improved liquid chromatography method coupled with diode array detector was developed for the simultaneous determina-
tion of Salidroside，tyrosol，gallic acid，catechin and epicatechin in fourteen medicinal plants belonging to the Rhodiola
L． genus in Qinghai-Tibetan Plateau． A column of Phenomenex Luna C18(250 × 4． 6 mm，5μm)was used． The mobile
phase consisted of acetonitrile and water with gradient elution． The flow rate was 1． 0 mL /min with UV detection wave
length at 276 nm． The column temperature was 30 ℃ ． The experimental results show that the five bioactive compounds
were separated perfectly under the conditions of determination． Their recoveries are over 97． 36% and the RSDs are less
than 2． 31% ． this method was sample，accurate and sensitive，so it can be used for the quality control of Rhodiola．:
Key words:HPLC，Bioactive compounds，Rhodiola
Introduction
Rhodiola rosea，also known as“golden root”or“rose-
root”belongs to the plant family Crassulaceae，grows at
elevated altitudes in the Arctic and in mountainous re-
gions throughout Europe and Asia［1］． The genus Rhodi-
ola L．(Crassulaceae)consists of nearly 200 species in
which at least 20 species are commonly used in tradi-
tional medical practice in Eastern Europe and Asia es-
pecially in traditional Tibetan medicine in China［2-4］．
R． rosea has been most intensively studied in Russia
and Scandinavia，where it has been purported to stimu-
late the nervous system，decrease depression，enhance
work performance，eliminate fatigue，and prevent high
altitude sickness［5-10］．
In China，Rhodiola crenulata H． Ohba is the only au-
thorized herb according to Chinese Pharmacopoeia［11］．
However，in recent years，increased commercial de-
mands of the source had seriously threatened its surviv-
al． In view of this，to find suitable substitute in the
Qinghai-Tibet Plateau becomes extremely crucial．
Rhodiola rosea contains flavonoids，monoterpenes，trit-
erpenes，phenolic acids，and phenylethanol derivatives
such as salidroside and tyrosol specific to this plant．
Significant antioxidant activities have been documented
for the extracts of various Rhodiola species，which have
been attributed to a variety of antioxidant compounds
including gallic acid，catechin and epocatechin［12］．
Based on this，in this paper Salidroside，tyrosol，gallic
acid，catechin and epicatechin were selected as the typ-
ical and pharmacologically components to evaluate the
quality of several medicinal plants belonging to the
Rhodiola L． Genus from Qinghai-Tibetan Plateau． In
this paper a fast and precise HPLC-DAD method was
developed for simultaneous determination of total Sali-
droside，tyrosol，gallic acid，catechin and epicatechin in
several medicinal plants belonging to the Rhodiola L．
Genus from Qinghai-Tibetan Plateau aiming to identify
distributional differences and explore the potential re-
sources of the Rhodiola．
Experimental
Chemicals and materials
The reference standard of Salidroside，tyrosol，gallic
acid，catechin and epicatechin were purchased from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing，China)．
Methanol was of HPLC grade and obtained from Merck
(Germany) ，while all other chemicals used were of an-
alytical grade． Water was purified on a Milli-Q system
(Millipore，Bedford，MA)and used throughout the study．
Thirteen samples of different species of Rhodiola were
selected for analysis (Table 1)． Samples used in this
study were collected directly at different parts of Qing-
hai-Tibetan Plateau． At each sampling site the sample
was acquired from different positions more than 1m a-
part，and at least five individual plants were collected
randomly to ensure representative sampling． The
samples were identified by Professor Chen shi-long in
Northwest Institute of Plateau Biology of Chinese Acad-
emy of Sciences．
Table 1 Description of the Rhodiola samples
No Name Origin Altitude(m)
HJT-1 R． chrysanthemifolia Langkazi Tibet 4400
HIT-2 R． Coccinea Changdu Tibet 3960
HJT-3 R． kirilowii Changdu Tibet 4270
HJT-4 R． tieghemii Nangqian Qinghai 4110
HJT-5 R． alsia Linzhi Tibet 4220
HJT-6 R． sexifolia Qushui Tibet 3560
HJT-7 R． yunnanensis Linzhi Tibet 3470
HJT-8 R． himalensis Leiwuqu Tibet 4080
HJT-9 R． fastigiata Huashixia Qinghai 4320
HJT-10 R． bupleuroides Naqu Tibet 4550
HJT-11 R． algida Qilian Qinghai 4010
HJT-12 R． kirilowii Hualong Qinghai 3380
HJT-13 Rhodiola crenula Lhasa Tibet 3870
Apparatus and chromatographic conditions
Five bioactive compounds in fourteen species of Rhodi-
ola were analyzed by HPLC． The HPLC system consis-
ted of a Waters (Milford，MA，USA)515 pumps cou-
pled to a Waters 2996 diode array detector． Chromato-
graphic separation of five bioactive compounds was a-
chieved on a Phenomenex Luna C18(250 × 4． 6 mm，5
μm)． The temperature of the column was maintained at
30 °C． The mobile phase consisted of solvent A (meth-
anol)and B (deionized water)． A gradient elution pro-
gram was used as follows:0-5 min 10% -20% A (v /v，
linear gradient) ，5-40 min 20% -70% A (v /v，linear
gradient)with the flow rate of 1． 0 mL min-1 ．
Fig. 1 Typical HPLC chromatograms of (A)mixed standards and (B)Rhodiola crenula． Gallic acid (1) ，salidroside
(2) ，tyrosol (3) ，epicatechin (4)and catechin (5)．
4431 Nat Prod Res Dev Vol. 24
Preparation of standard solutions
The standard of five bioactive contents were accurately
weighed and then dissolved in methanol to produce
stock solutions，which were diluted to appropriate con-
centration for the construction of calibration curves． All
standard and sample solutions were kept at 4 °C in the
refrigerator．
Preparation of sample solutions
The radix of plant material was powdered and sieved
through a 0． 315 mm sieve． An accurately weighed sam-
ple (200 mg)was put into a conical flask and then ex-
tracted respectively with 40 mL of 50% aqueous meth-
anol by sonication for 40 min at 40° C． The solutions
were filtered through a 0． 45 μm nylon membrane filter
before subjecting 10 μL aliquots to HPLC analysis． De-
terminations were performed after three separate extrac-
tions of each sample，and each extract was injected in
triplicate．
Results and Discussion
Method development
Several chromatographic columns including Phenome-
nex Luna-C18 column (250 × 4． 6 mm，5 μm) ，Phe-
nomenex Gemini-C18 column (250 × 4． 6 mm，5 μm) ，
Waters Symmetry-C18column (150 × 3． 9 mm，5 μm)
were tested，and the current column proved to be the
best in this application． A variety of mobile phases were
investigated to optimize the chromatographic conditions
for the analysis of the five bioactive components． The
methanol-deionized water system was chosen as mobile
phases because of its better separation，resolution and
shorter duration for the five contents than others． A gra-
dient elution method was developed for the separation
because of the differences in polarity，solubility and
other characteristics of the five components． The gradi-
ent program was optimized as followed:10% -20%
(methanol)in 0-5 min，20% -70% (methanol)in 5-
40 min with the flow rate of 1． 0 mL min-1 ． All the com-
ponents can be separated within 20 min under this con-
dition．
Validation of developed method
Linearity and Limit of Detection
Standard stock solutions containing the five analytes
were prepared and diluted to appropriate concentrations
for plotting the calibration curves． The regression curves
were obtained from six concentration levels and then
the calibration curves were constructed by plotting the
peak areas versus the concentration of each analytes．
The limit of detection (LOD)and the limit of quantifi-
cation (LDQ) ，defined as the lowest concentration that
could be measured with accuracy and precision． The
minimum concentration，which could be calculated at
S /N = 3 and 10，respectively． The calculated results are
summarized in Table 2． All the analytes showed good
linearity (R ＞ 0． 999)in the investigated ranges． The
LOD and LOQ of the eleven analytes were 0． 02-0. 12
and 0． 06-0． 35 μg /mL，respectively．
Table 2 Linear regression data，LOD and LOQ of five compounds
Compound Regression equationa Linear range (μg /mL) r2 LOD (μg /mL) LOQ (μg /mL)
Gallic acid Y = 2． 04X -0． 005 3． 91-391． 2 0． 9991 0． 02 0． 07
Salidroside Y = 2． 33X-0． 0062 1． 70-170． 0 0． 9998 0． 02 0． 06
Tyrosol Y = 3． 01X -0． 0032 5． 02-100． 4 0． 9992 0． 04 0． 13
Epicatechin Y = 3． 0X + 0． 003 3． 50-70． 0 0． 9993 0． 12 0． 35
Catechin Y = 3． 0X + 0． 0018 1． 51-30． 20 0． 9996 0． 07 0． 22
a Y is the peak area，X is the concentration injected．
Accuracy and Precision
Recovery test using the addition of known amounts of
standard mixture solution of the five bioactive compo-
nents to the marked sample was applied to determine
the accuracy of the method． These mixtures were ana-
lyzed by the proposed method and the experiment was
performed in triplicate and recoveries (%) ，RSD (%)
were calculated． The intra-day and inter-day precisions
were investigated by determining a mixed standard so-
lution in six replicates during a single day and by du-
5431
Vol. 24 CHI Xiao-feng，et al:Determination of Five Bioactive Compounds in Medicinal
Plants Belonging to Rhodiola L． Genus in Qinghai-Tibetan Plateau by HPLC
plicating the experiments on 3 consecutive days． All the data were shown in Table 3．
Table 3 Precision，repeatability，stability and recovery of 5 compounds
Compound
Precision (RSD，%)
Intra － day (n = 6) Inter － day (n = 3)
Repeatability
(RSD，%，n = 6)
Stability
(RSD，%，n = 7)
Recovery (%，n = 3)
Mean RSD，%
Gallic acid 0． 21 1． 78 1． 47 1． 23 97． 36 1． 31
Salidroside 0． 34 1． 21 2． 35 2． 23 102． 3 2． 16
Tyrosol 0． 46 2． 13 1． 99 2． 12 99． 11 2． 31
Epicatechin 0． 71 2． 06 1． 68 1． 18 98． 94 1． 72
Catechin 0． 57 1． 49 2． 35 2． 03 101． 1 2． 16
Repeatability and stability
To further evaluate the repeatability of the developed
method，each extract was analyzed in six replicates．
Stability of sample solution was analyzed at 0，2，4，8，
12，24 and 48 h within 2 days at room temperature，re-
spectively． No significant change was observed in the
five components indicating the solution was stable for at
least 48 h under room temperature．
Sample analysis
The developed method was applied to the simultaneous
quantification of Salidroside，tyrosol，gallic acid，cate-
chin and epicatechin in Rhodiola of different species．
The five contents in samples were identified by compa-
ring the retention time and the UV-Vis spectra of stand-
ards to those in samples． The typical chromatograms are
shown in Fig． 1 and the contents of each sample were
calculated and shown in Table 4．
Table 4 Contents (mg/g)of five bioactive compounds in thirteen different species of Rhodiola
No． Salidroside Tyrosol Gallic acid Catechin Epicatechin
1 4． 9 0． 49 3． 85 2． 18 0． 45
2 4． 43 0． 88 6． 72 1． 33 0． 12
3 1． 95 0． 79 8． 33 3． 14 0． 33
4 3． 2 0． 44 3． 36 2． 19 0． 24
5 3． 8 0． 75 4． 96 0． 98 0． 67
6 4． 1 0． 38 6． 87 0． 77 0． 34
7 5． 04 0． 42 3． 76 NDa ND
8 ND ND 15． 27 ND 0． 45
9 13． 7 1． 48 4． 67 2． 12 0． 12
10 8． 3 0． 19 11． 88 3． 44 0． 33
11 33． 3 8． 18 4． 82 2． 78 0． 45
12 4． 43 0． 56 5． 06 1． 94 0． 66
13 12． 70 6． 77 4． 55 3． 44 0． 77
aND:not detected
Table 4 shows that considerable differences were found
for the five components within the 13 species of Rhodi-
ola L． Salidroside (0-33． 33 mg /g)and tyrosol (0-
8. 18 mg /g)were found in all the samples except sam-
ple 8． The highest content of Salidroside was found in
sample 11 and the content was extremely higher than
others，while the high content of tyrosol was also found
in the high Salidroside samples． Gallic acid (3． 36-
15. 27 mg /g)was found in all the 12 samples with the
highest content found in sample 8 which lack of sali-
droside and tyrosol． Among the analyzed compounds，
salidroside and gallic acid were the two major com-
pounds while epicatechin (0-0． 77 mg /g)was the least
component． Catechin (0-3． 44 mg /g) ，also an impor-
6431 Nat Prod Res Dev Vol. 24
tant component was not detected in sample 7 and 8．
Conclusion
A method has been established for estimation of five
components in fourteen different species of Rhodiola．
Chemical polymorphism was found in the fourteen spe-
cies belonging to the Rhodiola L． Genus in the Qinghai-
Tibetan Plateau．
References
1 Saratikov SA，Krasnov EA，Rhodiola rosea is a Valuable Me-
dicinal Plant (Golden root)． Monograph Tomsk State Univer-
sity Press，Tomsk，1987． 252．
2 Bao W，et al． People’s Military Medical Press，China，2003．
1-12．
3 Northwest institute of plateau biology of Chinese Academy of
Sciences，Tibetan Medicine． Qinghai peoples’Press，China，
1991． 432-434．
4 Richard PB，Patricia LG，Zakir Ramazanov． Rhodiola rosea a
Phytomedical Overview． Herbal Gram，2002，56:40-52．
5 Shevtsov VA，Zholus BI，Shervarly VI，et al． A randomized
trial of two different doses of a SHR-5 extract versus placebo
and control of capacity for mental work． Phytomedicine，
2003，10:95-105．
6 Kanupriya，Prasad D，Sai RM，et al． Cytoprotective and an-
tioxidant activity of Rhodiola imbricata against tert-butyl hy-
droperoxide induced oxidative injury in U-937 human macro-
phages． Mol Cell Biol，2005，275:1-6．
7 Lia TL，Xua GH，Wu LL，et al． Pharmacological studies on
the sedative and hypnotic effect of salidroside from the Chi-
nese medicinal plant Rhodiola sachalinensis． Phytomedicine，
2007，14:601-604．
8 Qu ZQ，et al． Pretreatment with Rhodiola Rosea extract re-
duces cognitive impairment induced by intracerebroventricu-
lar streptozotocin in rats:implication of anti-oxidative and
neuroprotective effects． Biomed Environ Sci，2009，22:318-
326．
9 Chen X，et al． Salidroside attenuates glutamate-induced apop-
totic cell death in primary cultured hippocampal neurons of
rats． Brain Res，2008，1238:189-198．
10 Samuel ES，et al． Protection of human cultured cells against oxi-
dative stress by Rhodiola rosea without activation of antioxidant
defenses． Free Radical Biol Med，2009，47:577-584．
11 Pharmacopoeia Commission of PRC，Pharmacopoeia of the
People’s Republic of China，vol． I． Beijin:Chemical Industry
Press，2005． 106．
12 Ohsugi M，et al． Active-oxygen scavenging activity of tradi-
tional nourishing-tonic herbal medicines and active constitu-
ents of Rhodiola sacra． J Ethnopharm，1999，1:
櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵櫵
111-119．
(上接第 1443 页)
3 Yamamoto C，Nagai H，Takahashi K，et al． Cortical represen-
tation of taste-modifying action of miracle fruit in humans．
Neurolmage，2006，33:1145-1151．
4 Chen CC，Liu IM，et al． Improvement of insulin resistance by
miracle fruit (Synsepalum dulcificum)in fructose-rich chow-
fed rats． Phytotherapy Research，2006，20:987-992．
5 Chen P(陈萍) ，Yang TS(杨通顺) ，Wang H(王欢)． Study
on extraction methods of mysterious fruit genomic DNA．
Guangdong Agr Sci(广东农业科学) ，2008，3:26-28．
6 Zhao SR(赵素容) ，Lu YW(卢兖伟) ，Wu CZ(吴祖泽)．
Advances in research on hypoglycemic constituents of natural
products． Bull Acad Mil Med Sci(军事医学科学院院刊) ，
2005，29:280-283．
7 Ma Y(马燕) ，Zhang J(张晶) ，Wang Y(王亚) ，et al． Ex-
perimental study on hypoglycemic effect of astragalus hypo-
glycemic granule． Chin J Exp Tradit Med Form(中国实验方
剂学杂志) ，2011，17:157-160．
8 Zheng GH(郑光海) ，Piao HS(朴惠顺)． Hypoglycemic
effects of the ethyl acetate extract of Potentilla supina on al-
loxan diabetic mice． W Chin J Pharm Sci(华西药学杂志) ，
2010，25:416-417．
9 Ning XF(宁雪飞) ，Chen LL(陈亮亮) ，Tang HS(唐海
淑)． Distribution，Extraction and Activities of the Proteins
from Peganum harmala L． ． Nat Prod Res Dev(天然产物研
究与开发) ，2009，21:800-805．
10 Hui RH(回瑞华) ，Hou DY(侯冬岩) ，Li XC(李学成) ，et
al． Determination of protein in fagopyrum． Food Sci(食品科
学) ，2004，25:230-232．
11 Zhao YY(赵英永) ，Dai Y(戴云) ，Cui XM(崔秀明) ，et al．
Determination of protein contents of Radix Aconiti Kus-
nezoffii using coomassie brillant blue G-250 dye binding． J
Yunnan Nationalities University(云南民族大学学报) ，
2006，15:235-237．
12 Groop L，Groop PH，Stenman S，et al． Comparison of pharma-
cokinetics，metabolic effects and mechanisms of action of gly-
buride and glipizide during long term treatment． Diabetes
Care，1987，10(6) :71-78．
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Vol. 24 CHI Xiao-feng，et al:Determination of Five Bioactive Compounds in Medicinal
Plants Belonging to Rhodiola L． Genus in Qinghai-Tibetan Plateau by HPLC