Study on TLC Differentiation for Chaige Changyuan Mixture

Yuanfeng YANG, Yuchuan LI, Yunxia HAN, Xunrong ZHOU

Abstract [Objectives] This study was conducted to establish TLC methods for Scutellariae Radix， Peucedani Radix and Puerariae Radix in Chaige Changyuan Mixture.

[Methods]The preparation methods of test solutions， the development conditions， the color development inspection method and other factors were investigated for thin-layer chromatography. The three main medicines of Scutellariae Radix， Peucedani Radix and Puerariae Radix were qualitatively identified.

[Results] The TLC spots of Scutellariae Radix， Peucedani Radix and Puerariae Radix could be identified well by TLC， and corresponding negative control had no interference.

[Conclusions]The method is simple， available and reproducible， and can be used for the quality control of Chaige Changyuan Mixture.

Key words Chaige Changyuan Mixture; TLC; Scutellariae Radix; Radix Puerariae; Peucedani Radix

Received： March 6， 2022  Accepted： May 23， 2022

Supported by First-class Discipline Construction Project in Guizhou Province （Chinese Pharmacy） （GNYL[2017]008）： Subsidiary Subject （GNYL[2017]008-7）; Scientific Research Project of Guizhou University of Traditional Chinese Medicine （3040-04020001406）.

Yuanfeng YANG （1985-）， female， P. R. China， PhD， associate chief pharmacist， devoted to research about material basis of hospital preparations and traditional Chinese medicine.

*Corresponding author. E-mail： 2398662779@qq.com.

Chaige Changyuan Decoction is composed of 14 traditional Chinese medicines such as Radix Bupleuri， Scutellariae Radix， Radix Puerariae and Peucedani Radix. It has the functions of dispelling cold， dispelling dampness and guiding stagnation. It can clear half exterior and half interior， prevent evil and disease， and is used for the symptom that external dampness invades the human body surface and stays on the human body surface， affecting the operation of the defensive qi on the body surface and the function of protecting the human body. Because the decoction is cumbersome to decoct， and the liquid medicine is inconvenient to carry， patients often have poor compliance for administration. In order to facilitate patients to take it， preparation researchers in the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine made it into a mixture that is closer to the decoction in functional characteristics. In the prescription， Radix Puerariae is the monarch medicine， which has the effects of expelling pathogenic factors from muscles for clearing heat， and relieving exterior syndrome. It mainly contains isoflavonoids， coumarins， puerarin， triterpenoid saponins and other chemical components[1]. Peucedani Radix has the effects of directing qi downward to resolve phlegm， dispersing wind and clearing heat， and its main active ingredient is coumarin[2]. Scutellariae Radix has the functions of clearing away heat and dampness， purging fire for removing toxin， and its active ingredients are mainly flavonoids[3]. At present， the quality standard of the relevant Chaige Changyuan Mixture has been only reported for the content detection of puerarin[4]， and there is no relevant report on thin layer identification. Because TLC has the advantages of convenience， rapidity， specificity and good reproducibility[5]， in this study， the thin layer chromatography identification of Scutellariae Radix， Radix Puerariae and Peucedani Radix in Chaige Changyuan Mixture was investigated， so as to establish reliable identification methods. This study provides a basis for the research on the quality control standard of Chaige Changyuan Mixture.

Materials and Methods

Experimental materials

Medicinal materials

The reference medicinal material of Scutellariae Radix （batch number 120955-201810）， the reference medicinal material of Peucedani Radix （batch number 120951-201706）， and the reference medicinal material of Radix Puerariae （batch number 121551-201805） were all provided by National Institutes for Food and Drug Control. The medicinal materials of Chaige Changyuan Mixture （batch numbers 20200301， 20200302， 20200303） were weighed according to the prescription amount， soaked in water for 30 min， and decocted for 3 times （10 times of water for the first time （deducting 2 times of the water absorption rate）， and 8 times of water for the second and third times）， 30 min each time. The filtrates of the three times were combined and cold-stored and stood for 12 h. The supernatant was drawn and concentrated under normal pressure to 800 ml， which was added with 200 g of xylitol and 0.5 g of ethylparaben （dissolved in a small amount of ethanol）， and with purified water to 1 000 ml， so as to obtain the preparation of Chaige Changyuan Decoction. The negative preparations lacking Scutellariae Radix， Peucedani Radix， and Radix Puerariae were all prepared by the R&D center of traditional Chinese medicine preparations of the hospital， and the rest of the reagents were of analytical grade.

Instruments

DZKW-4 type electronic constant-temperature water bath （Shanghai Jinqiao Kexi Instrument Factory）; ZF-20D type dark chamber UV analyzer （Gongyi Yuhua Instrument Co.， Ltd.）; 98-1-B type electronic thermostat electric heating jacket （Tianjin Taisite Instrument Co.， Ltd.）; SK5010HP ultrasonic cleaner （200 W， frequency 53 kHz; Shanghai Kudos Ultrasonic Instrument Co.， Ltd.）; HCP-400A pulverizer （Zhejiang Yongkang Jinsui Machinery Factory）; JY 3001 pharmaceutical electronic balance （Shanghai Minqiao Precision Scientific Instrument Co.， Ltd.）; silica G-plate （Qingdao Marine Chemical Plant， for thin-layer chromatography）; polyamide film （produced by Luqiao Sijia Biochemical Clinker Factory， Taizhou City， Zhejiang Province， for thin layer analysis）.

Experimental methods

Thin layer identification of Scutellariae Radix

First， 20 ml of the product and 20 ml of negative preparation lacking Scutellariae Radix were added with 30 ml of methanol-ethyl acetate （1∶3） mixed solution， respectively. The two mixtures were heated with refluxing for 0.5 h. After cooling， the supernatants were evaporated to dryness， and the two parts of residues were dissolved with 2 ml of methanol as the test solution and negative control solution， respectively. Then， 1 g of the reference medicinal material of Scutellariae Radix was taken， and prepared by the same method to the reference medicinal material solution. According to the test of thin-layer chromatography （General Rule 0502）， 5 μl of the test solution， 5 μl of the negative control solution and 2 μl of the reference medicinal material solution were drawn， respectively， pointed on the same polyamide film， and developed with toluene-ethyl acetate-methanol-formic acid （10∶10∶3∶1∶2）. Finally， the polyamide film was taken out， air-dried and sprayed with 5% ferric chloride， and observed in sunlight[6].

Thin layer identification of Peucedani Radix

First， 20 ml of the product and 20 ml of negative preparation lacking Peucedani Radix were added with 20 ml of chloroform， respectively. The two mixtures were shaken and extracted twice， 20 ml each time. The chloroform extracts of each mixture were combined and evaporated to dryness. The two parts of residues were dissolved in 4 ml of methanol， respectively， as the test solution and negative control solution， respectively. Then， 1 g of the reference medicinal material of Peucedani Radix was taken， added with 20 ml of chloroform， and ultrasonically treated for 10 min. After filtration， the filtrate was evaporated to dryness， and 2 ml of methanol was added to the residue to dissolve it， so as to obtain the reference medicinal material solution[7]. According to the test of thin layer chromatography （general rule 0502）， 4 μl of each of the above three solutions was drawn， pointed on the same silica gel G plate， respectively， and developed with petroleum ether （60-90 ℃）-ethyl acetate （3∶1）. The silica gel G plate was taken out， air-dried， and sprayed with 10% sulfuric acid ethanol solution. Finally， it was blow-dried with hot air， and inspected under UV light （365 nm） immediately.

Thin layer identification of Radix Puerariae

First， 20 ml of the product was diluted with 10 ml of water， and added with water-saturated n-butanol and extracted twice with shaken， 40 ml each time. The n-butanol extracts were combined， and evaporated to dryness， and the residue was dissolved in 4 ml of methanol to obtain the test solution. Then， 20 ml of the negative preparation lacking Radix Puerariae was added with water-saturated n-butanol， and extracted twice with shaken， 20 ml each time. The n-butanol extracts were combined， and evaporated to dryness， and the residue was dissolved in 4 ml of methanol to obtain the negative control solution. Next， 2 g of the reference medicinal material of Radix Puerariae was taken， added with 20 ml of methanol， and stood for 2 h. After filtration， the filtrate was evaporated to dryness， and the residue was dissolved in 1 ml of methanol to obtain the reference medicinal material solution. According to the test of thin layer chromatography （general rule 0502）， 2 μl of each of the above three solutions was drawn， pointed on the same silica gel G plate， respectively， and developed with chloroform-methanol-water （20∶2.5∶0.25） as the developing agent. The silica gel G plate was taken out， air-dried， and sprayed with 10% sulfuric acid ethanol solution. Finally， it was heated at 105 ℃ until the color of the spots was clear， and observed under ultraviolet light （365 nm）.

Results and Analysis

Identification results and analysis

The results of Scutellariae Radix thin-layer identification showed that in the chromatograms of the test substance， there were spots of the same color at the positions corresponding to the chromatogram of the control medicinal material， and the negative control had no interference， as shown in Fig. 1. Validated by the three batches of samples， the method has good repeatability and is feasible.

The results of Peucedani Radix thin-layer identification showed that in the chromatograms of the test substance， there were fluorescent spots of the same color at the positions corresponding to the chromatogram of the control medicinal material， and the negative control had no interference， as shown in Fig. 2. Validated by the three batches of samples， the method has good repeatability and is feasible.

The results of Radix Puerariae thin-layer identification showed that in the chromatograms of the test substance， there were fluorescent spots of the same color at the positions corresponding to the chromatogram of the control medicinal material， and the negative control had no interference， as shown in Fig. 3. Validated by the three batches of samples， the method has good repeatability and is feasible.

Screening of extraction solvents for test solution preparation

Because the preparation is composed of 14 traditional Chinese medicines with complex components， there were many substances dissolved in the test solution， including both the components to be determined and other interfering components. Therefore， when preparing the test solutions， according to different components contained in the preparation and the different physicochemical properties of the components to be identified[8]， selecting a suitable solvent to extract the components to be tested could eliminate the interference of other components. In the preparation of the test solution for identification of Radix Puerariae in Chaige Changyuan Mixture， the pre-experiment adopts the preparation method of test solution contained in the 2015 edition of Chinese Pharmacopoeia under the Radix Puerariae item： using methanol as the extraction solvent.   First， 10 ml of the mixture was measured and evaporated to dryness in a water bath， and the residue was added with 20 ml of methanol， obtaining a solution， which was stood for 120 min. Then， the solution was filtered， and the filtrate was evaporated to dryness. Then， the residue was added with 1 ml of methanol to dissolve it， obtaining the test solution. However， the TLC with this test solution had too many spots and negative interference. Because the main components of Radix Puerariae were isoflavonoids， water-saturated n-butanol was selected to extract isoflavonoids， and the results showed that the spots were clear and the negative control had no interference. Therefore， water-saturated n-butanol was selected as the extraction solvent for the preparation of the test solution of Radix Puerariae.

Effects of developing conditions （mobile phase ratio） on thin layer chromatography

In the thin-layer identification of Radix Puerariae， because the main component of Radix Puerariae are isoflavonoids， the solubility in water is greater than that of flavonoids. When the developing ratio of chloroform-methanol-water of 7∶2.5∶0.25 was used in the experiment， it was found that due to too large polarity， the chromatographic spots overlapped， and the separation effect was unsatisfactory. Therefore， according to the polarity of isoflavonoids being slightly larger than that of flavonoids， the developing system was slightly adjusted to avoid overlapping spots. After repeated experiments， the development system was selected to be chloroform-methanol-water （20∶2.5∶0.25）.

Choice of colorimetric inspection method of thin layer chromatography

According to the screening test of the test solution preparation method and the results of the systematic screening test of the developing system， when the test solutions of the two medicinal materials of Radix Puerariae and Peucedani Radix were developed， air-dried， and then directly inspected under an ultraviolet light （365 nm）， Radix Puerariae showed abundant chromatographic spots， which might easily cause some interference in the negative control; and the chromatographic spots of Peucedani Radix were vague and difficult to identify. When Radix Puerariae was developed by 10% sulfuric acid ethanol at -105 ℃ and then inspected under ultraviolet light （365 nm）， the results showed that the chromatographic spots were few and clear， and the negative control had no interference. Through spraying with 10% sulfuric acid ethanol-hot air and immediately inspection under 365 nm ultraviolet light， the results showed that the chromatographic spots were clear and easy to identify. Therefore， the thin-layer identification methods of Radix Puerariae and Peucedani Radix in this study were finally established.

Conclusions

The TLC identification methods of Scutellariae Radix， Peucedani Radix and Radix Puerariae in Chaige Changyuan Mixture established in this study are simple and effective， with clear spots， good repeatability and strong specificity， and can be used as the quality control of Changge Changyuan Mixture.

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