Quality Standard of Haleniae Herba

Xuemei ZHOU, Dong WANG, Han GAO, Jianping ZHANG, Li CUI

Inner Mongolia Autonomous Region Institute for Drug Control, Inner Mongolia Autonomous Region Key Laboratory for Standard Research of Chinese and Mongolian Medicine, Huhhot 010020, China

Abstract [Objectives] The research aimed to establish qualitative and quantitative detection methods of Haleniae Herba, and provide basis for improving its quality standard. [Methods] Using thin-layer chromatography (TLC), qualitative identification of Haleniae Herba was conducted. Using high-performance liquid chromatography, luteolin content of Haleniae Herba was determined. [Results] TLC identification method had good specificity, with clear spots and good degree of separation, and reference medicinal material of Haleniae Herba was prepared. Content determination results displayed that luteolin had good linear relationship in the range of 0.088-1.760 μg. The RSD values of precision, repeatability, stability and sample recovery of the instrument met the requirements. [Conclusions] The established method is simple and reproducible, and can be used for the quality control of Haleniae Herba.

Key words Haleniae Herba, Quality standard, Thin-layer chromatography, Content determination

1 Introduction

Haleniae Herba is the whole herb with flower of Gentianaceae plantHaleniacorniculata. It is harvested when the flowers bloom in summer and autumn. After removing impurities, it is dried in the shade. Its nature is bitter, sweet, flat, soft and greasy. It is mainly used to calm the "Xie Ri", clear away heat and close wound, and cure yellow eyes, bitter mouth, high fever, headache, yellow urine, "Xie Ri" heat, typhoid fever and pulse heat. Haleniae Herba was first recorded in theBiblicalCanonofMongolianMedicinewritten by Mongolian pharmacist Chambra Dorgi in the 19thcentury[1]. It was recorded that "H.corniculatais born in shady places such as forests. Its leaves are round and thick, and its stems are covered with round short hairs, and its flowers are light yellow and closed". This variety was included in the 1998 edition of Mongolian medicine volume of theDrugStandardsoftheMinistryofHealth[2]. The current quality standard of Haleniae Herba is relatively simple, only includes three items: character, microscopic identification and physical and chemical identification. In view of the lack of effective TLC identification items, there is no content determination item of chemical component based on efficacy. According to the requirements for quality control of traditional Chinese medicine in the general principles ofChinesePharmacopoeia(Volume IV, 2020 Edition)[3], the study carried out TLC identification and content determination in order to comprehensively control the quality of the medicinal material.

2 Instruments and reagents

2.1 InstrumentsJapan Shimadzu LC-20AT high-performance liquid chromatograph (with SPD-M20A type of diode array detector); Sartorius ME5 type of electronic balance; Mettler AE-100 electronic balance; KQ-500DE type of CNC ultrasonic cleaner.

2.2 ReagentsReference medicinal material of Haleniae Herba: it was self-made and identified as certified product by relevant experts. Luteolin control sample: it was bought from National Institutes for Food and Drug Control (lot No.111520-202107). Ursolic acid control sample: it was bought from National Institutes for Food and Drug Control (lot No.110742-201823). Methanol was chromatographically pure. Thin-layer plate: prefabricated silica gel G plate (Yantai Chemical Industry Research Institute). Other reagents were all AR, and water was high purity.

3 Methods and results

3.1 TLC identification1 g of sample powder was taken in conical bottle, and 10 mL of ethyl acetate was added. After ultrasonic treatment for 15 min, it was filtrated, and filtrate was taken as test solution. Taking toluene-ethyl acetate-formic acid (20∶4∶0.5) as developing agent, spots corresponding to ursolic acid appeared in the test sample, and Rf value was moderate. Moreover, the separation effect of main spots in the chromatogram of control medicinal materials was good (Fig.1A). Taking chloroform-acetone (9∶1) as developing agent, the ursolic acid spots in the chromatogram of the test sample were verified, and it was considered that there was ursolic acid in the test sample (Fig.1B). Finally, toluene-ethyl acetate-formic acid (20∶4∶0.5) was determined as the developing agent, and the effect was better. The effect of low temperature (6 ℃) and low humidity (20% of relative humidity) on thin-layer results was inspected. The results showed that low temperature and low humidity had little effect on the overall separation effect (Fig.2).

Note: 1. Test sample No.1; 2. Control medicinal material of Haleniae Herba; 3. Control sample of ursolic acid; 4. Test sample No.2. A. Toluene-ethyl acetate-formic acid (20∶4∶0.5) as developing agent; B. Chloroform-acetone (9∶1) as developing agent.Fig.1 TLC photo of Haleniae Herba

Note: 1. Test sample No.1; 2. Control medicinal material of Haleniae Herba; 3. Control sample of ursolic acid; 4. Test sample No.2.Fig.2 TLC photo of Haleniae Herba (low temperature and low humidity)

3.2 Selection of chromatographic conditions(i) Chromatographic column. Octadecyl silane bonded silica gel as filler, Shimadzu C18250 mm×4.6 mm 5 μm column and phenomenex C18250 mm×4.6 mm 5 μm column were used in the study.

(ii) Mobile phase. Combining the determination method for content of Lamiophlomis Herba in theChinesePharmacopoeia(Volume I, 2020 Edition), methanol-0.2% phosphoric acid solution (46∶54) was taken as mobile phase. The results showed that there was good separation effect and moderate retention time. So, methanol-0.2% phosphoric acid solution (46∶54) was determined as mobile phase.

(iii) Column temperature. In the experiment, 30 and 35 ℃ of column temperature was compared. The result showed that the retention time was slightly different, but the resolution and the number of theoretical plates did not change. So, column temperature was determined as 30 ℃ in the experiment.

(iv) Detection wavelength. The spectral scanning of luteolin was conducted from 190-800 nm by diode array detector (Fig.3). The results showed that luteolin had the maximum absorption peak at 348.90 nm. Combining the determination method for content of Lamiophlomis Herba in theChinesePharmacopoeia(Volume I, 2020 Edition), the determination wavelength was selected as 350 nm.

Fig.3 Luteolin spectrum

(v) Number of theoretical plates. The determination results showed that the theoretical plate number of luteolin peak was more than 5 000, which could achieve the separation from adjacent peaks, and met the requirements ofR>1.5 in theChinesePharmacopoeia. Therefore, this standard stipulated that the number of theoretical plates calculated according to luteolin peak should not be lower than 5 000. The chromatograms of the control sample and the test sample obtained under the above chromatographic conditions were shown in Fig.4-5.

Fig.4 Chromatogram of luteolin control sample

Fig.5 Chromatogram of Haleniae Herba test sample

3.3 Selection of preparation method of test solution

3.3.1Selection of extraction solvent. Combining the determination method for content of Lamiophlomis Herba in theChinesePharmacopoeia(Volume I, 2020 Edition), methanol was taken as extraction solvent, and better separation effect was obtained. So, methanol was selected as extraction solvent.

3.3.2Selection of extraction method. Methanol heating reflux and ultrasonic extraction were used to compare, and there was little difference in the results. Because ultrasonic extraction was simpler and easier, methanol ultrasonic extraction was selected.

3.3.3Investigation on extraction efficiency. 5 copies of sample powder (No.3 sieve) were taken, and each was 0.5 g. 25 mL of methanol was precisely added, and the plug was inserted for weighing. The ultrasonic treatment (350 W of power, 40 kHz of frequency) was conducted for 15, 30, 60 and 90 min. It was taken out for cooling, and then it was weighed again. The lost weight was made up with methanol, and it was shaken evenly. After filtrating, the filtrate was taken. Determined by above chromatographic conditions, the contents of luteolin in different ultrasonic extraction times were 1.68, 1.87, 1.83 and 1.84 mg/g, respectively. After ultrasonic treatment for 30 min, the content of luteolin did not increase, so the ultrasonic time was determined as 30 min.

3.3.4Preparation of test solution. (i) Preparation of control solution. An appropriate amount of luteolin control sample was accurately weighed, and methanol was added to prepare 40 μg/mL of control solution.(ii) Preparation of test solution. 0.5 g of sample powder (No.3 sieve) was taken in a conical flask with a stopper, and 25 mL of methanol was accurately added. After inserting the stopper, it was weighed. Ultrasonic treatment was conducted for 30 min. After cooling, it was weighed again, and the lost weight was made up with methanol. After shaking evenly, it was filtrated, and the filtrate was the test solution.

3.3.5Peak purity check. 10 μL of control solution and 10 μL of test solution were precisely taken in the liquid chromatograph, and the diode array detector was used to verify the purity of the luteolin peak. The test results were shown in Fig.6. The results showed that the luteolin peak in the tested sample was a single component.

Note: A. Profile chart; B. Purity chart; C. 3-point chart.Fig.6 Purity test of luteolin peak

3.3.6Linear test. 1.103 mg of luteolin control sample was taken in 25 mL of graduated flask. Methanol was added to dissolve and dilute it to the graduate. 2, 5, 10, 20, 30 and 40 μL of control solution was precisely taken in the liquid chromatograph, and it was measured according to above chromatographic conditions. The injection volume was regressed by peak area, and the results showed that luteolin had good linear relationship in the range of 88.24-1 764.80 ng. Regression equation wasY=4 437.9X-30 637,r=0.999 9.

3.3.7Precision. Repeatability test: 6 copies of same test sample (No.1) were taken, and each was 0.5 g. They were prepared into test solution according to above preparing method, and then they were measured by above hromatographic conditions, with 10 μL of injection volume. The results were shown in Table 1.

Table 1 Repeatability test results of luteolin

3.3.8Accuracy. 6 copies of same test sample (No.1, 1.80 mg/g) were taken, and each was 0.25 g. After precise weighing, it was set in conical bottle with stopper. 0.6 mg of control sample and 25 mL of methanol were precisely added, and it was measured according to above method. The results were shown in Table 2.

Table 2 Sample adding recovery test of luteolin

3.3.9Range. According to the accuracy test method, 6 samples in low and high concentration range were taken respectively, about 0.1 and 0.5 g (No.1, 1.80 mg/g). After precise weighing, 0.25 and 1.0 mg of luteolin control samples were added in turn (the concentration was about 70% and 3 times of the limit). According to the method under accuracy item, the recovery rate andRSDof 6 samples at high and low concentrations were calculated. The results were shown in Table 3.

Table 3 Scope investigation results

Seen from Table 3, at two points equivalent to about 70% and 3 times of the content limit, the requirements of precision, accuracy and linearity were met.

3.3.10Durability test. Chromatographic columns of different manufacturers and models were changed. According to the determined chromatographic conditions, No.1 and No.2 samples in the repeatability experiment were taken for determination. The results were shown in Table 4.

Table 4 Durability test of different chromatographic columns

3.3.11Stability test. The same test solution was determined at 0, 2, 4, 8, 12 and 24 h respectively. Peak area integral was 1 739 069, 1 727 327, 1 728 957, 1 726 082, 1 738 109, 1 764 620, andRSDwas 0.83%. It was clear that the peak area integral of luteolin was basically stable within 24 h.

3.4 Content determination of test sample and determination of content limitThe sample powder was processed and determined according to the above method. The determination results of two batches of samples were shown in Table 5.

Table 5 Determination results of luteolin content in samples

It was clear that luteolin content in Haleniae Herba was more than 1.7 mg/g. There may be differences in content due to different origin of medicinal materials and different harvest time. It was tentatively determined that it was calculated as a dry product, and the content of luteolin (C15H10O6) should not be less than 1.5 mg/g (0.15%).

4 Discussion

Haleniae Herba is a common variety in Mongolian medicine, with remarkable curative effect in clinical use. With the development of the times, its quality standard has seriously lagged behind other medicinal varieties. Therefore, it is of great significance to study and improve its quality standards. In this study, the preparation and revision of the quality standard of Haleniae Herba were deeply studied from the aspects of TLC identification and content determination. The results showed that it was feasible to control the quality of Haleniae Herba by TLC identification and content determination.