全 文 ：民族药果上叶中 β-谷甾醇的
含量测定
马四补 1，2，张应蓉 2，屈相玲 3，靳凤云 2* （1.中
央民族大学，北京 100081；2.贵阳中医学院 ，
贵州贵阳 550002；3. 贵阳中医学院第二附属
医院，贵州贵阳 550001）
摘 要 [目的]测定不同产地果上叶中 β-谷
甾醇的含量。 [方法]采用超高效液相色谱蒸发
光散射检测器（UPLC-ELSD）测定不同产地果
上叶中 β-谷甾醇的含量 。 色谱条件为：BEH
C18 柱，甲醇-水为流动相，蒸发光散射检测器
检测参数为漂移管温度 90℃ ， 载气 （N2）20
psi，流速为 0.3 ml/min，柱温 30 ℃。 [结果]不
同产地的果上叶中 β-谷甾醇的含量不同，3、4
批次含量较高。 该操作在常温下进行，灵敏度
高，操作简便、快速。 [结论] 该研究为果上叶
药材果上叶中 β-谷甾醇的含量测定提供了理
论依据。
关键词 β-谷甾醇；民族药；果上叶；测定
基金项目 贵州省中医药管理局中医药、民族
医药科学技术研究课题（QZYY2012-34）。
作者简介 马四补（1979-），男，贵州威宁人，
副教授，在读博士 ，主要从事民族医药研究 ，
E-mail：453175568@qq.com。*通讯作者，靳凤云，
教授，硕士研究生导师，研究方向为中药质量控
制与新药开发，E-mail：jinfengyun@163.com。
收稿日期 2015-07-22
修回日期 2015-10-23
Determination of β-sitosterol Content in
Ethnodrug Guoshangye
Sibu MA1,2, Yingrong ZHANG2, Xiangling QU3, Fengyun JIN2*
1. Minzu University of China, Beijing 100081, China;
2. Guiyang university of Traditional Chinese Medicine, Guiyang 550002, China;
3. The Second Affiliated Hospital of Guiyang University of Traditional Chinese Medicine, Guiyang 550001, China
Supported by Traditional Chinese Medicine and Ethnic Minority Medicine Technique
Research Subject of Guizhou Administrative Bureau of TCM (QZYY2012-34).
*Corresponding author. E-mail: jinfengyun@163.com
Received: July 22, 2015 Accepted: October 23, 2015A
Agricultural Science & Technology, 2015, 16(11): 2546-2548
Copyright訫 2015, Information Institute of HAAS. All rights reserved Storage and Processing
G uoshangye, also known asZishangye, Shizaozi, Yanguo,etc, is dry rhizome and pseu-
dobulb of Pholidota yunnanensis Rolfe
in Orchidaceae, and it is used in tujia
and miao’s folk frequently. Gu-
oshangye is recorded in “Guizhou
traditional Chinese medicinal material
and Ethnic Minority Medicine Quality
Standard” (2003 Edition), and has a
long history of application in Guizhou
individually for treating cough mainly[2].
In this paper, we determine the con-
tents of β-sitosterol in 9 various batch-
es of Guoshangye produced in differ-
ent areas including shuitian town of
Guiyang, Liuzhi special zone, Huishui,
Kaili, Zunyi and so on, aiming at pro-
viding theoretical foundation for the
determination of β-sitosterol in Gu-
oshangye.
Materials and Methods
Experimental instruments
Following experimental instru-
ments were used : Waters HPLC
H-Class, Empower chromatographic
work station, ultrasonic cleaning ma-
chine (HS20500D), scale (AB204-S),
AB204-S type 1/100 000 electronic
scale (Mettler-Toledo, Switzerland ),
digital thermostatic water bath
(Changzhou Aohua), fuming cup-
board, evaporation dish, and conical
flask with cover.
Experimental regents
Following experimental regents
were used: β-sitosterol reference sub-
stance (National Institute for the Con-
trol of Pharmaceutical and Biological
Products, batch number: 110851-
200403), methanol (MERCK, Ger-
many), chromatographic pure
methanol (Tianjin Kermel Chemical
Reagent Co., Ltd.), analytically pure
ethanol, Wahaha purified water
(Hangzhou Wahaha Group Co., Ltd.).
Experimental medicinal materials
Nine various batches of Gu-
oshangye were collected from different
habitats including shuitian town of
Guiyang, Liuzhi special zone, Huishui,
Kaili, Zunyi and so on, and identified
by Sun Qingwen associate professor
from Department of Pharmacognosy
of Guiyang university of Traditional
Chinese Medicine as Pholidota yun-
nanensis Rolfe in Orchidaceae.
Abstract [Objective] The contents of β-sitosterol in Guoshangye from different habi-
tats were determined. [Method] UPLC-ELSD was adopted to determine the contents
of β-sitosterol in Guoshangye from different habitats, in which BEH C18 column (2.1
mm×50 mm, 1.7 μm) was used with a mobile phase of methanol-water at a flow
rate was of 0.3 ml/min, and the column temperature was of 30 ℃; and an evapora-
tive light-scattering detector was used, the temperature in drift tube was of 90 ℃ ,
and the pressure of N2 was of 20 psi. [Conclusion] This research provided theoreti-
cal foundation for the determination of β-sitosterol in Ethnodrug Guoshangye.
Key words β-sitosterol; Ethnodrug; Guoshanye; Determination
DOI:10.16175/j.cnki.1009-4229.2015.11.041
Agricultural Science & Technology2015
Table 1 Determination results of recovery test
Sample weight∥g Content insample∥mg
The added
amount∥mg
Measured
amount∥mg
Recovery
rate∥%
Average recovery
rate∥% RSD∥%
0.250 4 0.132 7 0.128 0.259 7 99.22 97.69 2.14
0.250 3 0.132 6 0.254 7 95.39
0.250 4 0.132 7 0.261 3 100.47
0.250 2 0.132 6 0.256 4 96.72
0.250 2 0.132 6 0.256 3 96.64
Experimental methods
Chromatographic conditions [3 -7]
America Waters BEH C18 column (2.1
mm×50 mm,1.7 μm) was used with a
mobile phase of methanol-water to
perform gradient elution (gradient elu-
tion program: 0-15 min, methanol vol-
ume fraction 90%-93%). The flow rate
was of 0.3 ml/min, and the column
temperature was of 30℃. An evapora-
tive light-scattering detector was used.
The temperature in drift tube was of 90
℃ , and the pressure of N2 was of 20
psi.
β-sitosterol reference substance
(1.00 mg) was weighed accurately and
put in a 10 ml volumetric flask, which
was then added with methanol, and
the solution was subjected to ultrason-
ic treatment for 5 min, diluted to con-
stant volume, and shaken to obtain the
reference solution.
Preparation of reference solution
Preparation of test solution About
0.5 g of Guoshangye powder (sieved
with No.2 sieve) was added into a con-
ical flask with cover, which was then
added with 20 ml of 95% ethanol to
conduct soaking for 20 min. The solu-
tion was subjected to weighing, ultra-
sonic extraction, cooling and weighing,
and supplemented to an adequate
weight. Filtration was performed to ob-
tain subsequent filtrate, which was e-
vaporated at 70 ℃ . The dry matter
was cooled naturally and dissolved in
methanol, and the obtained solution
was then transferred into a 5 ml volu-
metric flask, diluted to constant vol-
ume, and shaken. Filtration was finally
performed with a 0.2 um microfiltration
membrane to obtain the test solution.
Results and Analysis
System suitability test
The reference solution and test
solution were subjected to sample in-
jection and analyzed under above
chromatographic conditions, and it
was shown that the separation de-
grees between β-sitosterol and adja-
cent chromatographic peaks were
greater than 1.5. The results were
shown as following Fig.1.
Investigation of linear relation
The reference solution was inject-
ed in volumes of 1.8, 2.1, 2.4, 2.7, 3.0
and 3.3 ul, respectively. Linear re-
gression was performed with the natu-
ral logarithms X of injection masses
(μg) as horizontal coordinates and the
natural logarithms Y of peak area inte-
gral value as vertical coordinates. The
regression equation was Y= 0.140 8X+
10.978 (r =0.999 1). The results
showed that there was good linear re-
lation between the peak area logarithm
of β-sitosterol and reference injection
volume logarithm in the range of 0.18-
0.33 μg.
Precision test
Two ul of the reference solution
was injected continuously for 6 times,
peak areas was determined , and
β - sitosterol peak areas were calculat-
ed to have RSD (n=6)=1.53%, indi-
cating that the instruments had good
precision.
Reproducibility test
A sample powder of the same
batch (No. 6) was prepared into to 5
test solutions in parallel according to
2.3, and sample injection was per-
formed under above chromatographic
conditions in an amount of 6 μl, ob-
taining a result of RSD=1.87%.
Stability test
The test solution prepared from
No. 6 sample was determined at 0, 2,
4, 6, 8 and 12 h since preparation, and
it was calculated that the β-sitosterol
peak areas had RSD= 0.54%, indicat-
ing that the content of the test solution
was stable in 12 h.
Recovery test
Five parts of No.6 sample were
accurately weighed, each of which
was about 0.5 g, and accurately added
with 0.128 mg of the β-sitosterol refer-
ence substance. Content determina-
tion was performed according to above
content determination method, and the
results were shown in Table 1.
Determination of sample content
Guoshangye materials collected
from different habitats were prepared
into test solutions according to 2.3,
and for each batch of medicinal mate-
rial, 2 solutions were prepared in par-
allel. Under above chromatographic
conditions, 8 μl of No. 1 sample was
a, Solvent blank; b, β-sitosterol reference substance; c, Guoshangye sample.
Fig.1 Chromatograms of β-sitosterol reference substance and Guoshangye sample
2547
Agricultural Science & Technology 2015
Responsible editor: Yingzhi GUANG Responsible proofreader: Xiaoyan WU
Table 2 Contents of β-sitosterol in Guoshangye from different habitats (n=9)
Batch Weighed sample amount∥g Content of β-sitosterol∥mg/g
1 0.500 0 0.30
2 0.500 1 0.44
3 0.500 0 0.64
4 0.500 1 0.65
5 0.500 2 0.43
6 0.500 0 0.53
7 0.500 2 0.39
8 0.500 1 0.46
9 0.500 0 0.56injected, and the injection volumes of
other batches were of 6 μl. Areas of
chromatographic peaks were recorded
for calculating β-sitosterol contents in
Guoshangye from different habitats,
and the results were shown in Table 2.
Conclusions and Discus-
sion
β-sitosterol has absorption in ul-
traviolet terminal [8]. In the study, the
content of β-sitosterol in Guoshangye
was determined by UPLC-ELSD
method, which was performed under
room temperature, with the advan-
tages of high sensitively and simple
and rapid operation. This paper provid-
ed reliable basis for the determination
of β-sitosterol content in Guoshangye.
We also verified the method, and
found the standard curve had good lin-
ear relation in the range of 0.018 -
0.033 μg. The results showed that the
contents of β-sitosterol in Guoshangye
from different habitats were different,
and the third and fourth batches had
higher contents. This paper has cer-
tain reference value for quality control
of Guoshangye.
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Fig.2 linear curve of β-sitosterol
2548