Quantitative Analysis of 18 Marker Components in the Traditional Korean Medicine, Cheongsangbangpung-Tang, Using High-Performance Liquid Chromatography Combined with Photodiode Array Detector

: Cheongsangbangpung-tang (CSBPT) is a traditional herbal medicine that has been used in many regions of Asia, including Korea, China, and Japan, for the treatment of purulent inﬂammation and eczema on the face. In this study, a method for the simultaneous analysis of 18 marker components, geniposide ( 1 ), coptisine chloride ( 2 ), prim - O -glucosylcimifugin ( 3 ), berberine chloride ( 4 ), liquiritin apioside ( 5 ), liquiritin ( 6 ), ferulic acid ( 7 ), narirutin ( 8 ), 5- O -methylvisammisoide ( 9 ), hesperidin ( 10 ), arctigenin ( 11 ), baicalin ( 12 ), oxypeucedanin hydrate ( 13 ), wogonoside ( 14 ), baicalein ( 15 ), arctiin ( 16 ), glycyrrhizin ( 17 ), and pulegone ( 18 ), was developed for quality control of CSBPT. The novel approach, which is based on high-performance liquid chromatography (HPLC) separation coupled with photodiode array detection, was veriﬁed by the assessment of linearity, limit of detection, limit of quantiﬁcation, accuracy, recovery, and precision. Analysis of CSBPT by using the established assay revealed that compounds 1 – 18 were present in concentrations of 0.27–18.31 mg/g.


Introduction
Oriental medical prescriptions consisting of a combination of two or more medicinal herbs and containing many components have been widely used in Asian countries, such as Korea, China, and Japan, to enhance human health and prevent various diseases. However, there is a limit to continuous use without standardization of these herbal medicine prescriptions. Therefore, standardization for their continuous efficacy and quality control must be performed.
Cheongsangbangpung-tang (CSBPT; "Qing-Shang-Fang-Feng-Tang" in Chinese and "Seijo-bofu-to" in Japanese) is an oriental medical prescription that has been used in the treatment of purulent inflammation including acne and boils on the face and head [1,2]. CSBPT was first recorded in Gogeumuigam of Gong Shin in the Ming Dynasty of China, and it has also been included in Dongeuibogam of Heo Jun in the Joseon Dynasty of Korea [1,2]. This prescription consists of a combination of 12 [2].
Studies have been reported on the efficacy of CSBPT with respect to its antibacterial effects on Propionibacterium acnes and Staphylococcus aureus [3][4][5], its effect on atopic allergy [6], skin regeneration, wrinkle improvement, whitening, and moisturizing effects [1]. Saruwatari et al. [7] assessed the safety of this prescription in a study on herbal drug interactions with cytochrome P450 (CYPs), N-acetyltransferase 2, and xanthine oxidase.
Recently, Yang et al. [8] conducted and reported a pilot study on the effectiveness and safety of chronic urticarial in combination with Xiao-Feng-San and Qing-Shang-Fang-Feng-Tang.

Plant Materials
Twelve crude plant materials, shown in Table S1, were purchased from a pharmaceutical manufacturer of herbal medicine (Kwangmyungdang Medicinal Herbs (KMH); Ulsan, Korea) in November 2017. The origins of these plant materials were confirmed by Dr. Seung-Yeol Oh, president of KMH, according to the guideline "The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine" [21]. Voucher specimens (2018KE69-1 to 2018KE63-12) of the crude plant material were deposited at the Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea).

Chemicals and Reagents
The standards used in this study were supplied from manufacturers specializing in . HPLC-grade acetonitrile, water, and trifluoroacetic acid (≥99.0%), and ACS regent-grade formic acid (≥98.0%), glacial acetic acid (≥100.0%), and phosphoric acid (85.0%) were used in the mobile phase, and they were purchased from JT Baker (Phillipsburg, NJ, USA), Merck KGaA (Darmstadt, Germany), and Fujifilm Wako Pure Chemical Co., Ltd. (Osaka, Japan), respectively. Methanol and dimethyl sulfoxide (99.7%) used to prepare the test and standard solutions were purchased from JT Baker (Phillipsburg, NJ, USA) and Merck KGaA (Darmstadt, Germany), respectively.

Preparation of Test Solution and Standard Solution for HPLC-PDA
To perform quantitative analysis on the 18 marker components of CSBPT using HPLC-PDA, a test solution was prepared at a concentration of 10 mg/mL using 70% methanol. The prepared solution was extracted using an ultra-sonicator at room temperature for 60 min and then filtered with a 0.2 µm polypropylene (GHP) membrane (Pall Life Sciences, Ann Arbor, MI, USA).
Each of the standard solution for compounds 1-18 was prepared at a concentration of 1000 µg/mL using either methanol or DMSO-methanol solution (1:1), and then stored at approximately 4 • C until use.

HPLC Equipment and Chromatographic Separation Conditions
The simultaneous analysis of the 18 marker compounds for quality evaluation of CS-BPT was performed by using a modification of the previous methods [24,25]. A Shimadzu Prominence LC-20A series (Kyoto, Japan) HPLC system was used that combines a PDA for detection of the analyte and LabSolution software (Version 5.53, SP3, Kyoto, Japan) for data acquisition and processing. Other detailed analysis data are presented in Table S2.
Accuracy was assessed by conducting extraction recovery tests using the standard addition method.
The precision of the assay was evaluated by plotting the relative standard deviation (RSD) of each parameter, such as repeatability and intra-and inter-day precisions. In other words, the repeatability was evaluated by the RSD value of the retention time and peak area of each marker component after six repeated measurements of a standard solution.
Intra-and inter-day precisions were assessed by calculating the RSD value based on the data measured on one day and on three consecutive days, respectively.
System suitability in the established assay was verified by confirming parameters such as the capacity factor (k ), selectivity factor (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf ) values [27].

Selection of Marker Components for Quality Assessment of CSBPT
The major components that were identified in each herbal medicine are described in Section 3.1 and were also identified in the CSBPT sample. Thus, the 18 compounds were detected in the CSBPT sample among a total 31 components, as shown in Figure S2. These 18 compounds were selected as suitable marker components for the quality control of CSBPT; the chemical structures of the components are shown in Figure S3.

Method Validation of the Developed HPLC Analytical Method
For quality control of CSBPT based on compounds 1-18, the optimized analysis method was verified by evaluating factors such as system suitability, linearity, LOD, LOQ, and precision (repeatability, and intra-and inter-day precisions). The values of the system suitability factors k′, α, N, Rs, and Tf were calculated to be 3.23-13.80, 1.02-1.18, 24,7354.40-1,161,300.13, 3.85-36.85, and 1.01-1.22, respectively (Table S3). The r 2 values of compounds 1-18 calculated from the corresponding calibration curves were ≥0.9996, which confirmed the excellent linearity of the analysis assay. The LOD and LOQ values of compounds 1-18 were 0.07-0.36 μg/mL and 0.21-1.10 μg/mL, respectively. Regression equation, r 2 , LOD, and LOQ data are presented in Table 1. The extraction recovery of compounds 1-18 for evaluation of the accuracy of the developed assay was 97.03-104.04%, and the RSD (%) value was <3.00 ( Table 2). The RSD values of repeatability, and intra-and inter-day precisions of compounds 1-18 were 0.01-3.09%, showing good precision results (Table 3). These data confirmed that the developed HPLC assay was suitable for the quality control of CSBPT.

Method Validation of the Developed HPLC Analytical Method
For quality control of CSBPT based on compounds 1-18, the optimized analysis method was verified by evaluating factors such as system suitability, linearity, LOD, LOQ, and precision (repeatability, and intra-and inter-day precisions). The values of the system suitability factors k , α, N, Rs, and Tf were calculated to be 3.23-13.80, 1.02-1.18, 247,354.40-1,161,300.13, 3.85-36.85, and 1.01-1.22, respectively (Table S3). The r 2 values of compounds 1-18 calculated from the corresponding calibration curves were ≥0.9996, which confirmed the excellent linearity of the analysis assay. The LOD and LOQ values of compounds 1-18 were 0.07-0.36 µg/mL and 0.21-1.10 µg/mL, respectively. Regression equation, r 2 , LOD, and LOQ data are presented in Table 1. The extraction recovery of compounds 1-18 for evaluation of the accuracy of the developed assay was 97.03-104.04%, and the RSD (%) value was <3.00 ( Table 2). The RSD values of repeatability, and intra-and inter-day precisions of compounds 1-18 were 0.01-3.09%, showing good precision results (Table 3). These data confirmed that the developed HPLC assay was suitable for the quality control of CSBPT.   Table 3. Precision validation of compounds 1-18 in the developed analytical assay.

Quantification of Compounds 1-18 in CSBPT Samples
By using the developed and verified HPLC analysis method, quantification of the selected 18 marker components was performed for quality assessment of CSBPT samples. Quantification of compounds 1-18 was achieved by detection at the maximum absorption wavelength in the UV spectrum of each component. The amount of compounds 1-18 in the freeze-dried CSBPT sample was found to be 0.27-18.31 mg/g; among these analytes, compound 12, the major constituent of Scutellariae Radix, was identified as the most abundant (17.82-18.31 mg/g; Table 4).

Conclusions
We developed a simultaneous analysis method for selected compounds 1-18 that enables the quality assessment of CSBPT based on a common and widely-used HPLC-PDA system. The developed assay was validated by evaluating linearity, LOD, LOQ, recovery, and precision, and it was successfully applied to the analysis of CSBPT samples. This HPLC-PDA analytical assay can be used as a framework to obtain basic data for quality assessment of CSBPT and other traditional Korean medicines in the future.  Table S1: Composition of CSBPT, Table S2: Chromatographic conditions for simultaneous quantification of compounds 1-18 in CSBPT, Table S3: System suitability of compounds 1-18.

Conflicts of Interest:
The authors have declared no conflict of interest.