Simultaneous quantification and antiatherosclerosis effect of the traditional Korean medicine, Hwangryunhaedok-tang

Background 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). Methods 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. Results 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. Conclusions 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.


Background
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][2][3][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 [5]. HHT has been used clinically for the treatment of various symptoms including inflammatory diseases [6,7], gastrointestinal disorders [8], diabetes mellitus [9], brain injury [10][11][12], and acute liver injury [13,14]. Recently, the preventative effect of HHT on atherosclerosis was reported in models in vivo [15]. 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 highperformance 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.

Plant materials
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.
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 C 18 (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
A decoction of HHT was prepared in our laboratory from a mixture of chopped crude herbs. HHT was prepared as described in Table 1 and extracted with distilled water at 100°C for 2 h under pressure (98 kPa) using an electric extractor (COSMOS-660; Kyungseo Machine Co., Incheon, Korea). The extract was evaporated to dryness and freezedried (17.1% yield). Lyophilized HHT extract (250 mg) was dissolved in distilled water (25 mL), and then the solution was passed through a 0.2 μm syringe filter (Woongki Science, Seoul, Korea) before analysis by HPLC.
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
The ABTS radical scavenging activity of the samples was determined by using the method described Re et al. [18] with slight modifications. Briefly, the ABTS radical cation was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate, then the solution was stored in the dark at room temperature for 16 h. Prior to the assay, the solution was diluted with phosphate buffer saline (PBS, pH 7.4) to an absorbance of 0.7 at 734 nm. The ABTS •+ solution was then added to a 96well plate containing the test sample. After 5 min incubation, the absorbance was immediately measured at 734 nm by using a microplate reader (Benchmark Plus, Bio-Rad. Hercules, CA, USA). The extent of decolorization was calculated as the percentage reduction of absorbance. The scavenging capability of test compounds was calculated by using the equation: where A control is the absorbance of the negative control and A sample is the absorbance of the sample. RC 50 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. [19] 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 CuSO 4
We examined the oxidation of LDL by CuSO 4 by using a previously described method [20]. 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 CuSO 4 (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 [21]. 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 [22].

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% CO 2 , 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 analysis
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).

Results and discussion
Optimization of chromatographic conditions HPLC conditions such as column type, column temperature, and mobile phases were optimized to achieve the simultaneous separation of five analytes including one iridoid, geniposide (1), one flavonoid, baicalin (2), and three alkaloids, coptisine (3), palmatine (4), and berberine (5). For the separation of compounds 1-5, columns types (Phenomenex Gemini C 18 , Waters SunFire C 18 , and OptimaPak C 18 column), column temperatures (30, 35, and 40°C), and various mobile phases (acids including acetic acid and phosphoric acid and buffers such as SDS and ammonium acetate, and organic solvent with methanol and acetonitrile) were examined. By comparing the peak shape, resolution, and baselines of the target compounds under different conditions, the most satisfactory conditions were selected as Phenomenex Gemini C 18 column (250 × 4.6 mm, 5 μm) with gradient elution of 10% v/v, acetonitrile in 0.2% SDS with phosphoric acid 200 μL/L-acetonitrile at 35°C for the separation. Quantitation was achieved by using PDA  detection at 240 nm for compounds 1 and 3-5 and 277 nm for compound 2 based on retention time and UV spectra compared with those of the standards. By using the optimized HPLC conditions, the five analytes eluted within 40 min and afforded good specificity without interference from other components. Representative HPLC chromatograms of standards and the HHT extract are shown in Figure 2.

Regression equation, linearity, LOD, and LOQ
The regression equations were calculated by plotting the peak area (y) versus concentration (x, μg/mL) of each compound by using serial dilutions of the stock solution.
The correlation coefficients (r 2 ) of compounds 1-5 were ≥ 0.9997, which showed good linearity. The LODs and LOQs of the investigated compounds 1-5 were in the range 0.34-0.87 and 1.12-2.89 μg/mL, respectively ( Table 2). The results showed that the developed HPLC method was acceptable for the quantitative determination of compounds 1-5.

Accuracy and precision
The recovery and precision of the developed method are shown in Table 3. The recoveries of compounds 1-5 were in the range of 98.90-103.39% and the RSD values were less than 2.53%. The repeatability of the developed assay was evaluated based on peak responses and retention time by using the standard solution. The RSDs of peak responses and retention time for repeatability were < 0.44% and < 0.09% (data not shown), respectively, indicating that the HPLC assay showed good repeatability under the optimized conditions. The precisions of intra and interday variation of compounds 1-5 in HHT were less than 1.08% and 1.87%, respectively ( Table 4). The results described above indicate that the established HPLC method was accurate and precise for the quantitative determination of HHT extract.

HHT sample analysis
The five compounds in HHT were well separated by using the developed HPLC method. The retention times   Table 5.

Antioxidant activity of HHT and its components
To evaluate the antioxidant activity of HHT and of compounds 1-5, their respective scavenging activities on ABTS and DPPH radicals were tested. HHT showed clear antioxidant properties, which were concentration dependent ( Figure 3A and C). The estimated concentration required for 50% reduction (RC 50 ) against ABTS and DPPH was 25.76 ± 0.16 μg/mL and 92.97 ± 2.05 μg/mL, respectively.

Effect of HHT and its components on Cu 2+ -mediated oxidation of LDL
The effect of the compounds on Cu 2+ -mediated oxidation of LDL was determined by two methods. First, the degree of LDL oxidation was evaluated by the TBARS assay. Lipid peroxidation was quantified by measuring Figure 5 Effects of HHT and its five components on PDGF-induced VSMC proliferation. (A) Cytotoxicity of HHT and its five components in VSMCs. Cells were incubated with the indicated concentrations for 24 h. Cell viability was determined by using the CCK-8 assay. (B) Antiproliferative effects of HHT and its five components in PDGF-treated VSMCs. Quiescent VSMCs were stimulated with PDGF-BB (10 ng/mL) in the presence of the indicated concentrations of samples for 24 h and the proliferation was examined by using the CCK-8 assay. Geniposide (1), baicalin (2), coptisine (3), palmatine (4), and berberine (5). The data are mean values of three experiments ± SEM (n = 3). ** P < 0.01 compared with the control group, # P < 0.05, ## P < 0.01 compared with the PDGF group. the amount of degradation by product MDA [23]. As shown in Figure 4A and B, when LDL was incubated with CuSO 4 for 6 h, a significant increase in TBARS was detected. In contrast, HHT significantly reduced, in a concentration-dependent manner, the amount of TBARS fromed ( Figure 4A). Alteration of agarose gel electrophoretic mobility reflects the increase in negative charge of LDL particles that occurred during oxidation [22]. When the oxidation was carried out in the presence of HHT, the increased electrophoretic mobility of oxidized LDL was significantly reduced ( Figure 4C). As shown in Figure 4B and D, compound 2 only reduced the Cu 2+ -induced LDL oxidation.

Effect of HHT and its components on PDGF-induced VSMC proliferation
To determine whether HHT and its five components had any effect on cell viability, CCK-8 assays were performed on cultured rat VSMCs treated with various concentrations of samples for 24 h. As shown in Figure 5A, HHT and compounds 1 and 2 had no significant effect on the viability of cells under the experimental conditions, whereas compounds 3-5 induced cell proliferation. VSMCs were pretreated with different concentrations of HHT (125-500 μg/mL) and compounds 1-5 (50-200 μM) followed by stimulation with PDGF-BB (10 ng/mL) for 24 h. HHT and compound 2 inhibited PDGF-BB-induced proliferation of VSMCs in a concentration-dependent manner ( Figure 5B). The proliferative effects of compounds 3-5 on PDGF-treated VSMCs were accomplished by themselves. These observations suggest that the inhibitory effect of HHT on PDGF-induced VSMC proliferation was partly attributed to compound 2.

Conclusions
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.