Phytochemical Analysis of Twelve Marker Analytes in Sogunjung-tang Using a High-Performance Liquid Chromatography Method

: Sogunjung-tang (SGJT) is a traditional herbal prescription that has been used in Korea for the treatment of abdominal pain since ancient times. In this study, an analytical method for the simultaneous quantiﬁcation of 12 marker analytes (gallic acid (GA), albiﬂorin (ALB), paeoniﬂorin (PAE), liquiritin apioside (LIAP), liquiritin (PIQ), benzoic acid (BA), coumarin (COU), liquiritigenin (LIQG), cinnamic acid (CINA), benzoylpaeoniﬂorin (BPAE), cinnamaldehyde (CINAD), and glycyrrhizinic acid (GLYA)) for quality evaluation of SGJT was developed based on high-performance liquid chromatography (HPLC) combined with a photodiode array detector. A Waters SunFire reverse-phased C 18 column was used for the chromatographic separation of the 12 marker analytes in SGJT using a two-mobile phases system consisting of 0.1% ( v / v ) aqueous formic acid and 0.1% ( v / v ) formic acid in acetonitrile. The developed analytical method was validated by assessment of linearity, limit of detection, limit of quantiﬁcation, recovery, and precision. Using the developed and validated HPLC method, the 12 marker analytes were determined to be present in 0.10–32.83 mg / g in SGJT.


Introduction
Traditional herbal prescriptions consist of two or more medicinal herbs; they are very diverse and contain many ingredients. Therefore, standardization is required for efficient quality control.
Sogunjung-tang (SGJT), also known as Xiaojianzhong-tang in Chinese and Shokenchu-to in Japanese, is an oriental medical prescription. According to Donguibogam (the Principles and Practices of Eastern Medicine), it is recorded that SGJT has been used to treat symptoms such as abdominal pain, wet dreams, and melalgia. SGJT consists of a combination of six herbal medicines: Paeoniae Radix, Cinnamomi Ramulus, Glycyrrhizae Radix et Rhizoma, Zingiberis Rhizoma Recens, Zizyphi Fructus, and Oryzae Gluten [1].
In studies on the biological activity of SGJT, Jung et al. [2] reported its inhibitory effects on Type I hypersensitivity and inflammatory reactions, and Kim et al. [3] reported its effects on cell proliferation and immune activity. In addition, Katami et al. [4] reported the results of genotoxicity studies such as bacterial reverse mutation and micronucleus tests.
Thus, a range of biological activities of SGJT and of many of the individual herbal medicine components constituting SGJT have been reported, and analytical methods for quality control based on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry have also been reported [2][3][4][5][6][7][8][9][10][11]. However, no standardization study for quality control of SGJT composed of combinations of these herbs has been reported.
In this study, five of the constituent medicinal herbs of SGJT, excluding saccharide-containing Oryzae Gluten, were mixed, extracted, and used for HPLC analysis. These extracts were used to develop and verify a method for the simultaneous analysis of 12 marker compounds for quality assessment of SGJT using HPLC separation combined with photodiode array detection for the simultaneous detection of the marker analytes. For the quality assessment of SGJT using the developed analysis, the following 12 marker analytes were assayed: GA, ALB, PAE, BA, and BPAE (Paeoniae Radix); COU, CINA, and CINAD (Cinnamomi Ramulus); and LIAP, LIQ, LIQG, and GLYA (Glycyrrhizae Radix et Rhizoma).

Plant Materials
Five of the raw medicines that constitute SGJT are shown in Table S1, and were purchased from a pharmaceutical manufacturer of herbal medicine, Kwangmyungdang Medicinal Herbs (KMH; Ulsan, Korea) in November 2017. Based on guidelines ("The Dispensatory on the Visual and Organoleptic Examination of Herbal Medicine"), the origins of these herbs were confirmed by Dr. Seung-Yeol Oh, president of KMH [13]. Voucher specimens (2017KE63-1 to 2017KE63-5) for each material have been deposited at the Herbal Medicine Research Division, Korea Institute of Oriental Medicine.

Preparation of Samples and Standard Solutions for Simultaneous Analysis by HPLC
To prepare sample solutions from the SGJT preparations for simultaneous quantification of the 12 marker analytes (GA, ALB, PAE, LIAP, LIQ, BA, COU, LIQG, CINA, BPAE, CINAD, and GLYA) by HPLC, each 10 mL of 70% methanol and distilled water was added to 100 mg of a lyophilized SGJT sample, and the mixture was extracted under ultrasonic conditions at room temperature for 60 min. Samples that were used for analysis of ALB, PAE, and GLYA were diluted tenfold with 70% methanol and distilled water before analysis.
A standard solution of each marker analyte was prepared in methanol at a concentration of 1.0 mg/mL and stored in a refrigerator (ca. 4 • C) until use. The sample and standard solutions were filtered through a 0.2 µm GHP membrane (Pall Life Sciences, Ann Arbor, MI, USA) before HPLC analysis.

HPLC Equipment and Operating Conditions for Simultaneous Quantification of the 12 Marker Analystes
For quality control assessments of SGJT by simultaneous analysis of major marker analytes, the analysis method used previously [16,17] was modified and applied. Briefly, the HPLC system used for quantitative analysis was a Shimadzu Prominence LC-20A series HPLC (Kyoto, Japan), coupled to two solvent delivery units (LC-20AT), online degasser (DGU-20A 3 ), a forced air circulation type column oven (CTO-20A), automatic sample injector (SIL-20A), and a photodiode array detector PDA (SPD-M20A); the system was controlled by LabSolution software (Version 5.53, SP3). Other detailed analysis parameters are shown in Table S2.

Validation of the HPLC Analytical Method
According to the International Conference on Harmonisation guidance for Q2B validation of analytical procedures [18], the developed HPLC analytical method was verified by testing parameters such as linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and precision. The linearity was assessed by the coefficient of determination (r 2 ) in the calibration curve of each marker analyte, and LOD and LOQ were calculated using the equations where σ is the standard deviation of the y-intercept and S is the slope of the calibration curve. Accuracy verification was performed through extraction recovery tests using the standard addition method, and was calculated using the equation The precision was assessed by determining the relative standard deviation (RSD) values of repeatability, and intraday and interday precisions. Repeatability was evaluated based on the RSD values of the retention time and peak area of each marker analyte measured repeatedly six times. Furthermore, intraday and interday precisions were evaluated as RSD values of results measured for samples on the same day and on three consecutive days, respectively.
The suitability of the system for the simultaneous analysis was confirmed by assessing parameters such as capacity factor (k ), selectivity factor (α), resolution (Rs), number of theoretical plates (N), and tailing factor (Tf ) values [19].

Determination of Marker Components of SGJT
To identify the marker components of SGJT, we investigated the major components of the constituent herbal medicine (GA, ALB, PAE, BA, and BPAE from P. lactiflora; COU, CINA, and CINAD from C. cassia; LIAP, LIQ, LIQG, and GLYA from G. uralensis; 6-GIN from Z. officinale; and SPI from Z. jujube) using the described HPLC system together with a distilled water-acetonitrile mobile-phase system containing formic acid ( Figure S1). As a result, 12 analytes were selected as marker components for quality control of SGJT. The chemical structures of the selected marker analytes are shown in Figure S2.

Optimization of HPLC Chromatographic Conditions
A range of analysis parameters, including types of reversed-phase C 18 column according to the manufacturer (SunFire (Waters, Milford, MA, USA), Gemini (Phenomenex, Torrance, CA, USA), Capcellpak UG120 (Shiseido, Japan), OptimaPak (RStech Corp., Korea)) and column temperature (30, 35, 40, and 45 • C) were varied to achieve efficient separation of the 12 marker analytes that were extracted from the SGJT sample. The optimal analysis was achieved with a SunFire C 18 column (4.6 × 250 mm, 5 µm), a column temperature of 40 • C, and two mobile-phase systems of distilled water-acetonitrile, both containing 0.1% (v/v) formic acid. Under the optimized analysis conditions, 12 marker analytes were baseline separated and eluted within 45 min with Rs values ≥ 3.45 ( Figure 1 and Table S3).

Validation of Developed HPLC Analytical Method
In the developed HPLC analytical assay, system suitability factors for the 12 marker analytes, namely, k , α, N, Rs, and Tf, were calculated to be 1.18-14.07, 1.02-4.46, 17782-1488677, 3.45-79.88, and 0.96-1.29, respectively (Table S3). As shown in Table 1, the calibration curve for each marker component used for quantitative analysis was calculated as the peak area (y) versus the concentration (x) in six different concentration ranges using mixed standard solutions. The r 2 values were 0.9991-1.0000, which indicated good linearity of the calibration curves. The LOD and LOQ concentration ranges calculated by the equations given in Section 2.6 were 0.04-0.73 µg/mL and 0.12-2.22 µg/mL, respectively. These results are summarized in Table 1. The extraction recovery for each component, which was used to evaluate the accuracy of the analytical method, was 95.74-103.30%, and the RSD (%) values were <3.00 ( Table 2). The RSD values for the repeatability, and intraday and interday precisions of each marker analyte, were all within 1.59%, showing good precision results ( Table 3). The above validation results confirm that the simultaneous analysis assay developed for quality control of SGJT is appropriate.   Table 3. Precision of the analytical method for the 12 marker analytes in SGJT.

Quantification of the 12 Marker Analytes in SGJT Samples
Simultaneous quantification was performed for the 12 marker analytes (GA, ALB, PAE, LIAP, LIQ, BA, COU, LIQG, CINA, BPAE, CINAD, and GLYA) selected for quality control of SGJT using the verified HPLC analytical assay. As shown in Table 1, simultaneous determinations of these analytes were conducted at 230 nm (ALB, PAE, BA, and BPAE), 254 nm (GLYA), 270 nm (GA), 275 nm (LIAP, LIQ, COU, LIQG, and CINA), and 286 nm (CINAD), based on the λ max of UV spectra of each component. The peak of each component was confirmed by comparison with the UV spectrum and retention time of the corresponding reference standard compound. The amounts of the 12 marker analytes in the lyophilized SGJT sample were measured to be 0.10-32.83 mg/g by using the developed and validated assay; among these components, PAE, the main component of Paeoniae Radix, was found in the highest concentration (32.83 mg/g) ( Table 4).

Conclusions
In the present study, 12 marker analytes were selected for quality control of SGJT using a simple and common HPLC system, with a simultaneous quantitative analysis method. The developed analysis assay was verified by assessing several parameters, including linearity, LOD, LOQ, accuracy, and precision, and was successfully applied to sample analysis. The developed and validated HPLC analysis assay is expected to be used to obtain data for the quality control and evaluation of SGJT and to form the basis for the analysis of other traditional Korea medicines in the future.

Conflicts of Interest:
The authors declare no conflict of interest.