Sibu MA,Yingrong ZHANG,Xiangling QU,Fengyun JIN*

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

Guoshangye,also known as Zishangye,Shizaozi,Yanguo,etc,is dry rhizome and pseudobulb ofPholidota yunnanensis Rolfein Orchidaceae,and it is used in tujia and miao’s folk frequently. Guoshangye 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 contents of β-sitosterol in 9 various batches of Guoshangye produced in different areas including shuitian town of Guiyang,Liuzhi special zone,Huishui,Kaili,Zunyi and so on,aiming at providing theoretical foundation for the determination of β-sitosterol in Guoshangye.

Materials and Methods

Experimental instruments

Following experimentalinstrumentswereused:WatersHPLC H-Class,Empower chromatographic work station,ultrasonic cleaning machine(HS20500D),scale(AB204-S),AB204-S type 1/100 000 electronic scale (Mettler-Toledo,Switzerland),digital thermostatic water bath(Changzhou Aohua),fuming cupboard,evaporation dish,and conical flask with cover.

Experimental regents

Following experimentalregents were used:β-sitosterol reference substance(National Institute for the Control of Pharmaceutical and Biological Products,batch number:110851-200403),methanol(MERCK,Germany), 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 Guoshangye 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 asPholidota yunnanensis Rolfein Orchidaceae.

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 elution program:0-15 min,methanol volume fraction 90% -93% ).The flow rate was of 0.3 ml/min,and the column temperature was of 30℃.An evaporative 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 ultrasonic treatment for 5 min,diluted to constant volume,and shaken to obtain the reference solution.

Preparation of reference solution

Preparation of test solutionAbout 0.5 g of Guoshangye powder(sieved with No.2 sieve)was added into a conical flask with cover,which was then added with 20 ml of 95% ethanol to conduct soaking for 20 min.The solution was subjected to weighing,ultrasonic extraction,cooling and weighing,and supplemented to an adequate weight.Filtration was performed to obtain subsequent filtrate,which was evaporated at 70℃.The dry matter was cooled naturally and dissolved in methanol,and the obtained solution was then transferred into a 5 ml volumetric flask,diluted to constant volume,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 injection and analyzed under above chromatographic conditions,and it was shown that the separation degrees between β-sitosterol and adjacentchromatographic peaks were greater than 1.5.The results were shown as following Fig.1.

Investigation of linear relation

The reference solution was injected in volumes of 1.8,2.1,2.4,2.7,3.0 and 3.3 ul,respectively.Linear regression was performed with the natural logarithmsXof injection masses(μg)as horizontal coordinates and the natural logarithmsYof peak area integral value as vertical coordinates.The regression equation wasY=0.140 8X+10.978 (r=0.999 1).The results showed that there was good linear relation 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 calculated to haveRSD(n=6)=1.53%,indicating 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 performed under above chromatographic conditions in an amount of 6 μl,obtaining a result ofRSD=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 hadRSD=0.54%,indicating 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 reference substance.Content determination 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 material,2 solutions were prepared in parallel.Under above chromatographic conditions,8 μl of No.1 sample was injected,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.

Table 1 Determination results of recovery test

Conclusions and Discussion

β-sitosterol has absorption in ultraviolet 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 advantages of high sensitively and simple and rapid operation.This paper provided reliable basis for the determination of β-sitosterol content in Guoshangye.We also verified the method,and found the standard curve had good linear 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 certain reference value for quality control of Guoshangye.

Table 2 Contents of β-sitosterol in Guoshangye from different habitats(n=9)

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