- Research article
- Open Access
- Open Peer Review
Simultaneous quantification and antiatherosclerosis effect of the traditional Korean medicine, Hwangryunhaedok-tang
© Seo et al.; licensee BioMed Central. 2015
- Received: 15 July 2014
- Accepted: 24 March 2015
- Published: 8 April 2015
Hwangryunhaedok-tang (HHT) is a traditional herbal medicine that is used for the treatment of fever, inflammation, gastritis, and hypertension. In this study, we performed simultaneous determination of the five components, geniposide (1), baicalin (2), coptisine (3), palmatine (4), and berberine (5) in HHT by using a high-performance liquid chromatography–photodiode array (HPLC–PDA) analysis. We also evaluated the antioxidative activity of HHT and compounds 1–5 by measuring their effects on low-density lipoprotein (LDL) oxidation and antiproliferative abilities in vascular smooth muscle cells (VSMCs).
Five compounds were separated within 40 min by using a Gemini C18 column (temp. 35°C; two-component gradient elution; flow rate 1.0 mL/min; detector 240 and 277 nm). The activities of HHT and compounds 1–5 were tested with the radical scavengers 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 2,2-diphenyl-1-picrylhydrazyl, in thiobarbituric acid reactive substance assays, and in relative electrophoretic mobility assays using CuSO4-induced LDL oxidation systems. The antiproliferative effects of samples on platelet-derived growth factor (PDGF)-induced VSMC proliferation were studied by using a cell proliferation assay.
Regression analysis of the five major compounds showed good linearity (r2 ≥ 0.9997) in different concentration ranges. The recoveries of the five compounds were in the range 86.31–110.78%, with relative standard deviations below 2.1%; those of intra- and interday precision were 0.04–3.78% and 0.04–1.69%, respectively. HHT reduced the oxidation properties of LDL induced by CuSO4 and inhibited cell proliferation in PDGF-treated VSMCs. Among the five components, compound 2 could effectively suppress LDL oxidation and PDGF-induced VSMC proliferation.
The established HPLC–PDA method will help to improve quality control of HHT. The results demonstrate that HHT has antiatherosclerotic activity and that it functions by modulating LDL oxidation and VSMC proliferation. The effects of HHT may be attributed, at least I part, to compound 2.
- Simultaneous quantification
- Antiatherosclerosis effect
- Traditional Korean medicine
Traditional herbal formulas are generally composed of various herbs and have been used for the prevention of and therapy for a variety of diseases for thousands of years. Moreover, because herbal medicines tent to have a mild effect and few side effects, many people are becoming increasingly interested in traditional herbal medicines [1-4]. Hwangryunhaedok-tang (HHT) is a traditional Korean herbal medicine consisting of four medicinal herbs, Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex, and Gardeniae Fructus in 1:1:1:1 proportions and is called Orengedokuto in Japan and Hwanglianjiedu-tang in Chinese . HHT has been used clinically for the treatment of various symptoms including inflammatory diseases [6,7], gastrointestinal disorders , diabetes mellitus , brain injury [10-12], and acute liver injury [13,14]. Recently, the preventative effect of HHT on atherosclerosis was reported in models in vivo . However, the underlying antiatherosclerotic mechanism of HHT has not yet been thoroughly elucidated. In this study, we investigated the antioxidant effects of HHT on low-density lipoprotein (LDL) and antiproliferative effect on vascular smooth muscle cells (VSMCs), which are key atherosclerotic events [16,17]. Furthermore, chromatographic analysis was performed by using a high-performance liquid chromatography–photodiode array (HPLC–PDA) system to enable the simultaneous quantification of five major compounds, geniposide (1) in Gardeniae Fructus, baicalin (2) in Scutellariae Radix, and coptisine (3), palmatine (4), and berberine (5) in Coptidis Rhizoma and Phellodendri Cortex, for quality control of HHT.
The four crude herbs that make up HHT, Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex, and Gardeniae Fructus, were purchased from Omniherb (Yeongcheon, Korea) and HMAX (Jecheon, Korea). The origin of each herbal medicine was taxonomically confirmed by Prof. Je Hyun Lee, Dongguk University, Gyeongju, Korea. Voucher specimens (2008–KE20–1 through KE20–4) have been deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine.
Chemicals and reagents
Apparatus and conditions
A Shimadzu LC-20A HPLC system (Shimadzu, Kyoto, Japan) consisting of a system controller (CBM-20A), a solvent delivery unit (LC-20AT), an on-line degasser (DGU-20A3), a column oven (CTO-20A), a sample autoinjector (SIL-20 AC), and a photodiode array (PDA) detector (SPD-M20A). The data were processed by LCsolution software (version 1.24, Shimadzu, Kyoto, Japan). The analytical column used for the separation of the five components was a Phenomenex Gemini C18 (250 × 4.6 mm; particle size 5 μm, Torrance, CA, USA). The mobile phases consisted of solvent A (10%, v/v, acetonitrile in 0.2% SDS with phosphoric acid 200 μL/L) and solvent B (acetonitrile). The gradient conditions of the two mobile phases were: 10 → 40% B in 20 min, then 40 → 50% B in 20 min, then 50 → 100% B in 10 min, then 100 → 10% B in 5 min; the re-equilibrium time was 15 min. Column temperature was maintained at 35°C. The analysis was carried out at a flow rate of 1.0 mL/min, with PDA detection at 240 nm for iridoid and alkaloids and 277 nm for flavonoid compounds. The injection volume was 10 μL.
Preparation of standard solutions
Each stock solution of reference compounds 1–5 was accurately weighed and dissolved in methanol at a concentration of 1,000 μg/mL. All the stock solutions were kept at 4°C in a refrigerator until use and diluted to the appropriate concentration range to establish calibration curves.
Preparation of sample solutions
Composition of HHT
Calibration curves, range, limits of detection (LODs), and of quantification (LOQs)
Each calibration curve was established by plotting peak areas versus the concentration of standard solutions. The concentration ranges were 7.81–500.00 μg/mL for compounds 1 and 2, 1.56–50.00 μg/mL for compounds 3 and 5, and 4.69–300.00 μg/mL for compound 4. To assess LOD and LOQ values, stock solutions of all reference compounds were diluted with methanol. The LOD and LOQ values were determined as signal-to-noise (S/N) ratios of 3 and 10, respectively.
Precision and accuracy
Intra- and interday precisions were determined by using a standard addition method to prepare spiked samples, employing both standards and controls. Precisions are presented as the relative standard deviation (RSD) for intra- and interday. The repeatability of the developed method was evaluated by measuring six replicates of the mixed standard solutions. The RSD values of peak areas and retention times of each compound were used to evaluate the repeatability of the developed HPLC method. The test for recovery, which was carried out to evaluate the accuracy of the methods, was performed by adding three different concentrations (low, medium, and high) of five reference standards to 200 mg of HHT sample. This test was conducted in triplicate and evaluated by using the independently prepared calibration curves.
Determination of antioxidant activity
ABTS radical scavenging activity
where Acontrol is the absorbance of the negative control and Asample is the absorbance of the sample. RC50 values (the concentration required for 50% reduction of ABTS radical) were calculated from the concentration of sample required to reduce the absorbance by 50%.
DPPH radical scavenging activity
Radical scavenging activity of samples was determined by using DPPH as a free radical by the method described Moreno et al.  with some modifications. Briefly, 100 μL of various concentrations of sample was added to 100 μL of DPPH solution (0.15 mM in ethanol) in a 96-well plate. After 30 min incubation in the dark at room temperature, the absorbance was measured at 517 nm. Activity of scavenging (%) was calculated by using the above formula.
Determination of LDL oxidation
Oxidation of LDL by CuSO4
We examined the oxidation of LDL by CuSO4 by using a previously described method . LDL samples (500 μg protein/mL, Biomedical Technologies, Stoughton, MA, USA) were prepared at 37°C in a medium containing 10 mM phosphate buffer (pH 7.4) and various concentrations of samples. After 5 min, the oxidation was initiated by the addition of CuSO4 (25 μM). After 6 h oxidation, lipid peroxidation and electrophoretic mobility of LDLs were measured as described below.
Determination of thiobarbituric acid reactive substance (TBARS)
Lipid peroxidation of LDLs was estimated by determinng the level of malondialdehyde (MDA) generated by using a TBARS assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s protocols . After oxidation, 50 μg of LDLs was mixed with 200 μL of thiobarbituric acid (TBA) and incubated at 100°C for 30 min. Upon completion of the reaction, the absorbance at 535 nm was measured by using a microplate reader.
Relative electrophoretic mobility (REM) assay
The electrophoretic mobility of LDLs was measured by using agarose gel (0.8% agarose in TAE buffer) electrophoresis and Coomassie Brilliant Blue R-250 staining. Electrophoresis was performed at 100 V for 30 min. REM was defined as the ratio of the distances migrated from the origin by oxLDL versus native LDL .
Vascular smooth muscle cell (VSMC) proliferation assay
Rat embryonic thoracic aorta smooth muscle-derived A7r5 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as a monolayer culture at 37°C in a humidified atmosphere of 5% CO2, 95% air in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Inc., NY, USA), containing 10% v/v fetal bovine serum (FBS, Gibco Inc., Grand Island, NY, USA) and 1% penicillin–streptomycin (P/S). Upon the attainment of 70–80% confluency, the cells were incubated in serum-free DMEM containing 0.5% bovine serum albumin for 20–24 h. Cytotoxicity and proliferation assays were performed by using the Cell Counting Kit-8 (CCK-8) as described by the manufacturer (Dojindo Laboratory, Kumamoto, Japan). Briefly, cells were seeded onto 96-well plates and grown in a final volume of 100 μL media per well. After treatment as indicated in the text for 24 h, 10 μL of kit reagent was added and the sample was incubated for an additional 3 h. Absorbance was measured at a wavelength of 450 nm by using a microplate reader.
Statistical evaluation of the results was performed by using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test by using GraphPad InStat 3.05 software (GraphPad Software Inc, San Diego, CA, USA).
Optimization of chromatographic conditions
Regression equation, linearity, LOD, and LOQ
Regression equation, linear range, correlation coefficient, LODs, and LOQs for marker compounds (n = 3)
Linear range (μg/mL)
Regression equation a
Correlation coefficient (r2)
LOD b (μg/mL)
LOQ c (μg/mL)
7.81 − 500.00
y = 14575.90x + 29400.74
7.81 − 250.00
y = 41028.20x + 12271.19
1.56 − 50.00
y = 45048.93x + 3766.28
4.69 − 300.00
y = 37568.06x + 15349.20
1.56 − 50.00
y = 43158.92x + 4420.01
Accuracy and precision
Recoveries for the assay of the five investigated compounds in HHT
Spiked amount (μg/mL)
Detected amount (μg/mL)
Precision of the analytical results (n = 5)
Spiked Conc. (μg/mL)
Detected Conc. (μg/mL)
Detected Conc. (μg/mL)
HHT sample analysis
Amounts of the five marker compounds in the HHT sample by HPLC (n = 3)
Antioxidant activity of HHT and its components
Compounds 1–5 were also tested in ABTS and DPPH assays to determine their contribution to the antioxidant property of HHT. In the ABTS assay, compounds 2–4 displayed RC50 values of 11.28 ± 0.21, 35.72 ± 0.54, and 163.17 ± 2.64 μM, respectively (Figure 3B). The scavenging activity of compound 5 was 42% at 250 μg/mL concentration. In the DPPH assay, compound 2 had an estimated RC50 value of 17.76 ± 0.15 μM (Figure 3D).
Effect of HHT and its components on Cu2+-mediated oxidation of LDL
Effect of HHT and its components on PDGF-induced VSMC proliferation
A simple, reliable, and accurate HPLC–PDA method was developed and validated for simultaneous separation and determination of compounds 1–5 in the traditional Korean herbal medicine, HHT. The developed method showed good linearity, precision, and accuracy and is therefore a suitable method with which to assess the quality of HHT and its components for quality control purposes. In this study, we have shown that HHT can reduce the oxidation of LDL and inhibit PDGF-induced VSMC proliferation, which are key atherosclerotic events. Compound 2, as one of the components in HHT, also exhibits an antioxidant effect on LDL and an antiproliferative effect on VSMCs. Although further studies are needed, these observations suggest that HHT acts, to inhibit LDL oxidation and suppress PDGF-induced VSMC proliferation, at least in part, through the effect of compound 2.
This research was supported by a grant (no. K13030) from the Korea Institute of Oriental Medicine.
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