Fujun SUN Juan BAO Shimei ZHAO Zonghua ZHENG Pengfei XIA

Abstract [Objectives] This study was conducted to establish a method for determining the contents of Chaenomeles cathayensis （Hemsl.） Schneid.， and analyze the changes in effective components of C. cathayensis grown in Guizhou， so as to provide data support for the production and quality control of C. cathayensis. [Methods] The contents of ursolic acid and oleanolic acid in C. cathayensis in different areas of Guizhou were determined. The HPLC method was adopted under following conditions： chromatographic column waters C18 column （4.6 mm×150 mm， 5.0μm）; mobile phase： methanol-0.1 mol/L ammonium acetate （85∶15）; column temperature： 25 ℃; detection wavelength： 257 nm; flow rate 1.0 ml/ min. [Results] Through methodological investigations， HPLC could be used to detect the contents of the two terpenoids oleanolic acid and ursolic acid in C. cathayensis from Guizhou. The content of oleanolic acid ranged from 0.076% to 0.144%， and the content of ursolic acid ranged from 0.201% to 0.439%. [Conclusions] A method for determining the contents of C. cathayensis was established using the HPLC method with oleanolic acid and ursolic acid as the index components. The method is accurate， reliable， and simple and easy to implement， and can serve as a reference and support for quality evaluation and standard improvement of C. cathayensis.

Key words Chaenomeles cathayensis; HPLC; Oleanolic acid; Ursolic acid

Chaenomeles cathayensis （Hemsl.） Schneid. is a plant of Rosaceae[1]， which is cultivated throughout Guizhou[2]. The fruit is used as a medicine， which is used by Guizhou folks in many ways. It has the effects of calming the liver and relaxing muscles， and reducing dampness in the stomach and is used for treating arthritis with fixed pain caused by dampness， pain in waist and knee joints， vomiting and diarrhea， twitch， and beriberi[3]. It is a local Chinese medicinal material in Guizhou， which was included in the 2003 edition of Quality Standards for Chinese Medicinal Materials and Ethnic Medicines in Guizhou Province. In recent years， as the medical and health benefits of Chaenomeles plants have been further explored， modern medicine has proved that Chaenomeles plants are rich in organic acids and a variety of nutrients[4]， among which organic acids have biologically activity such as anti-tumor， anti-bacterial and anti-thrombotic effects[5]. Oleanolic acid and ursolic acid are the main chemical components in the organic acids of the genus Chaenomeles[6]， which have anti-tumor， liver protection， antibacterial and anti-inflammatory effects[7]. In this study， oleanolic acid and ursolic acid were selected as the control indicators to explore the method for determining oleanolic acid and ursolic acid contents of C. cathayensis. The contents of oleanolic acid and ursolic acid in C. cathayensis from different regions under the same harvesting conditions and time were determined by HPLC， establishing a scientific accurate detection method. This study provides data support for the quality research of C. cathayensis.

Materials and Methods

Instruments and Materials

Instruments

Waters e2695 high performance liquid chromatograph （quaternary pump， 2489UV/Vis ultraviolet detector， Empower workstation， Waters Technology Co.， Ltd.）; METTLER TOLEDO balance; KQ3200DE CNC ultrasonic cleaner （Kunshan Ultrasonic Instruments Co.， Ltd.）.

Materials

Methanol （chromatographically pure）; ultrapure water; oleanolic acid （batch number： 110709-201808， content 91.1%）; ursolic acid （batch number： 110742-201823， content 99.9%）; reference substances， all provided by the National Institutes for Food and Drug Control; samples， collected from the development zone of Anshun City in Guizhou Province， Minzhu Town of Panzhou City， and Sifang Beach of Luyin Lake in Duyun City， all identified by Professor Wei from College of Pharmacy， Guizhou University of Traditional Chinese Medicine as ripe fruit of C. cathayensis （Hemsl.） Schneid.

Experimental methods

Chromatographic conditions

Column： waters C18 column （4.6 mm×150 mm， 5.0 μm）; mobile phase： methanol-0.1 mol/L ammonium acetate （85∶15）; column temperature： 25 ℃; detection wavelength： 257 nm; flow rate 1.0 ml/min; injection volume： 10 μl.

Solution preparation

Preparation of control solution

A certain amount of oleanolic acid reference substance （18.97 mg） and ursolic acid reference substance （12.03 mg） were weighed accurately and added in a 25 ml measuring flask， respectively. Methanol was added into each measuring flask to constant volume， giving a stock solution. Then， 2 ml of oleanolic acid reference substance stock solution and 3 ml of ursolic acid reference substance stock solution were accurately measured into the same 10 ml measuring flask， followed by dilution with methanol to constant volume （concentration： oleanolic acid 138.25 μg/ml， ursolic acid 144.22 μg/ml）.

Preparation of test solution

About 2 g of C. cathayensis powder （passed through No.3 sieve） was accurately weighed and added into a 25 ml volumetric flask， followed by the addition of 20 ml of methanol. The sample was ultrasonically extracted （power 500 W， frequency 40 kHz） for 30 min， taken out and cooled. The extract was diluted with methanol to constant weight， shaken well， and filtered through a 0.45 μm microporous membrane， obtaining the filtrate as the test solution.

System suitability test

Oleanolic acid and ursolic acid reference solutions， test solution and methanol solution were injected into a liquid chromatograph， and the chromatograms were recorded. It can be seen that there was no interference in the blank， and the retention time of oleanolic acid and ursolic acid was the same in the chromatograms of the reference substances and the test substance. The separation of oleanolic acid and ursolic acid reached 1.51， and the chromatographic conditions met the experimental requirements. The results are shown in Fig. 1 and Fig. 2.

Linear relationship investigation

From the control solution under "Preparation of control solution"， 4， 8， 12， 16， 20 and 30 μl were taken and injected into the liquid chromatograph under the chromatographic method in "Chromatographic conditions"， and the chromatograms were recorded. The peak area A was used to calculate the concentration （μg/ml） by linear regression， and the linear regression equations were： oleanolic acid： y=494 147x+7 610.3 （R2=0.999 3） and ursolic acid： y=426 881x-1 635 （R2=0.999 7）. The results showed that oleanolic acid and ursolic acid had a good linear relationship with peak area in the ranges of 55.30-414.75 and 57.74-432.66 μg/ml， respectively.

Precision test

The mixed reference solution under "Preparation of control solution" was taken and injected for 6 times consecutively under the chromatographic conditions of "Chromatographic conditions" to determine the peak areas of oleanolic acid and ursolic acid. The RSD of the peak area of oleanolic acid was 0.6%， and the RSD of ursolic acid peak area was 0.7%. The precision of the instrument was good.

Stability test

The same sample was taken at 0， 4， 8， 12， 16， and 24 h and injected under "chromatographic conditions" to determine the peak areas of oleanolic acid and ursolic acid. The RSD values of the sample peak areas were 0.7% and 0.7%， respectively， indicating that the sample had good stability in 24 h.

Repeatability test

According to the method under "Preparation of test product"， 6 test product solutions were prepared in parallel with medicinal materials produced in the development zone of Anshun， and the contents of oleanolic acid and ursolic acid were determined under "chromatographic conditions". The average content of oleanolic acid was 0.143%， and the RSD value was 1.5%; and the average content of ursolic acid was 0.441%， and the RSD value was 1.4%. It showed that the method had good repeatability.

Recovery test

Six samples from the development zone of Anshun City， Guizhou （crushed， passed through No.3 sieve）， each 1 g， were accurately weighed， and added in a 25 ml volumetric flask， respectively. Then， 2 ml of the oleanolic acid reference solution （concentration： 0.691 3 mg/ml） and 10 ml of ursolic acid reference substance solution （concentration： 0.480 7 mg/ml） were accurately added， and the test substance solutions were prepared according to the method under "Preparation of test solution"， and determined for component contents.

Results and Analysis

Results of recovery test

The average recoveries of oleanolic acid and ursolic acid were calculated to be 99.2% and 99.2%， respectively， indicating that the method had high accuracy. The results are shown in Table 1.

Sample determination

Moisture determination

About 3 g of each medicinal powder sample was taken and measured according to the 2015 edition of Chinese Pharmacopoeia （General Rule 0832， Method 2）， and the determined moisture contents of the C. cathayensis medicinal materials are shown in Table 2.

Content determination

The C. cathayensis medicinal powders from different regions of Guizhou Province were taken and prepared according to the method under "Solution preparation" into test solutions， which was determined for component contents. The total amounts of oleanolic acid and ursolic acid in the medicinal materials were calculated based on dried products. The results are shown in Table 2.

Conclusions and Discussion

C. cathayensis is a traditional medicinal material in Guizhou. In the 2003 edition of Quality Standards for Chinese Medicinal Materials and Ethnic Medicines in Guizhou Province， the contents of relevant components of the C. cathayensis medicinal material were not determined， and there are certain limitations in evaluating the quality of the medicinal materials. Our research team analyzed the active ingredients of the medicinal material and believed that organic acids are the material basis for the curative effect of the medicinal material. Combined with related reports， we confirmed that the pentacyclic triterpenoid organic acids oleanolic acid and ursolic acid with significant biological activity could be used as indicators. Oleanolic acid and ursolic acid are isomers with similar polarities， which brings certain difficulties to chromatographic separation. We compared methanol-0.2% acetic acid solution （82.5∶17.5）， methanol-water-glacial acetic acid- triethylamine （265∶35∶0.1∶0∶05）， acetonitrile-methanol-0.5% ammonium acetate solution （67∶12∶21） and other mobile phases， but the ideal separation effect was not achieved. Using methanol-0.1 mo/L ammonium acetate （85∶15） as the mobile phase achieved better separation results， and the samples were negative without interference， so we used methanol-0.1 mo/L ammonium acetate （85∶15） as the chromatographic condition. We also explored and verified the extraction conditions of samples， and a method for determining the contents of C. cathayensis medicinal materials was established. Five regions where the output of C. cathayensis is relatively large were selected and the contents in the medicinal materials were determined using the method. The results showed that the contents of oleanolic acid and ursolic acid in C. cathayensis from different producing areas in Guizhou Province were quite different. The oleanolic acid content was between 0.076% and 0.144%， and the ursolic acid content was between 0.201% and 0.439%， showing that the content difference was nearly 1 time. The factors affecting the quality of medicinal materials and the formulation of quality control limits for medicinal materials need to be studied and discussed in depth. The developed HPLC method for determining the contents of C. cathayensis based on the efficacy and composition characteristics of the medicinal material is accurate， reliable， simple and quick， and can be used as a content determination method for quality control of C. cathayensis.

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