Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound
Yu Kim, Soo-Jin Jeong, Chang-Seob Seo, Hye-Sun Lim, Eunjin Sohn, Jiyeon Yun, Bu-Yeo Kim
Ukgansan (UGS), a traditional herbal formula composing seven medicinal herbal plants, has been applied in Asian countries for treating neurosis, insomnia, and irritability. Here, the current study performed a simultaneous determination of the seven marker compounds (liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I) using high-performance liquid chromatography (HPLC), to establish quality control of UGS. A 70% ethanol extract of
... and a mixture of the seven compounds were separated using a C-18 analytical column on a gradient solvent system of 1.0% (v/v) aqueous acetic acid and acetonitrile. Data were recorded at a UV wavelength of 250 nm for glycyrrhizin; 276 nm for liquiritin apioside, liquiritin, and atractylenolide I; and 325 nm for ferulic acid, decursin, and decursinol angelate. The results exhibited high linearity (correlation coefficient (r 2 ) ≥ 0.9998) and proper precision (0.38-3.36%), accuracy (95.12-105.12%), and recovery (95.99-104.94%) for the seven marker compounds. The amount of the seven marker compounds at the concentrations from 0.190 to 16.431 mg/g. In addition, the current study evaluated the antioxidant effects of UGS by measuring their scavenging activities against the 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2 -diphenyl-1-picrylhydrazyl (DPPH) radicals using in vitro cell-free systems and observed its antioxidant activity. Among the seven components of the UGS extract, ferulic acid dramatically enhanced the scavenging of ABTS and DPPH radicals compared with other compounds. The concentrations of ferulic acid required for a 50% reduction (RC 50 ) in ABTS and DPPH radicals were 16.22 µM and 41.21 µM, respectively. Furthermore, UGS extract exerted the neuroprotective effect and blocked the inflammatory response in neuronal hippocampal cells and microglia, respectively. Overall, the established method of HPLC will be valuable for improving the quality control of UGS extract, and ferulic acid may be useful as a potential antioxidant agent. Atractylodes japonica, Poria cocos, Bupleurum falcatum, Angelica gigas, Cnidium officinale, and Glycyrrhiza uralensis. UGS has been utilized to manage various diseases such as neurosis, insomnia, and irritability in children and is a herbal medicine that is approved by the Ministry of Health, Labor and Welfare of Japan. Modern pharmacological and clinical studies have suggested that UGS has the potential to improve insomnia [1,2], borderline personality disorder , neuroleptic-induced tardive dyskinesia , drug-induced parkinsonism , cognitive impairment , and the behavioral and psychological symptoms of dementia (BPSD)    . It is well known that the occurrence of these diseases is closely related to oxidative stress    . Thus, antioxidant activity should be considered, in addition to the efficacy of therapeutic drugs. The quality of herbal formulas depends on environmental factors, such as harvesting, storing, processing, and formulating methods. Because of their multiple components and the effect of the environment, quality assessment of the major components of herbal formulas is required for investigating their efficacy and safety  . Although the identification and simultaneous determination of the components of UGS using high-performance liquid chromatography (HPLC) and HPLC-Q-TOF-MS have been reported [14, 15] , there are few studies on simultaneous determination of the compounds in UGS using HPLC. Therefore, the current study conducted a simultaneous determination of the marker compounds of UGS for the quality control of UGS using a photodiode array HPLC detector (denoted the HPLC-PDA method), as it is the most popular analytical method. The HPLC-PDA method is a convenient and rapid analytical method to separate and identify the multiple compounds in herbal formulas [16, 17] . In this study, simultaneous analysis of seven compounds (liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I) in a UGS extract was performed, and method validation was carried out using the HPLC-PDA method. Moreover, the antioxidant activities of the seven marker compounds were determined using in vitro radical scavenging assays and the biological effects in neuronal cell lines HT22 hippocampal cells and BV-2 microglia. A variety of drugs have shown the neuroprotective activity against damage-induced HT22 cells and the inhibitory effects on neuroinflammation in lipopolysaccharide-stimulated BV-2 cells    .