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Simultaneous quantification and inhibitory effect on LDL oxidation of the traditional Korean medicine, Leejung-tang
© Seo et al.; licensee BioMed Central Ltd. 2014
Received: 7 May 2013
Accepted: 5 December 2013
Published: 3 January 2014
Leejung-tang (LJT) is a traditional Korean herbal medicine for the treatment of gastrointestinal disorders. In this study, we performed quantification analysis of five marker components, liquiritin (1), ginsenoside Rg1 (2), ginsenoside Rb1 (3), glycyrrhizin (4), and 6-gingerol (5) in LJT using a high performance liquid chromatography-photodiode array (HPLC–PDA). In addition, we investigated the inhibitory effect on low-density lipoprotein (LDL) oxidation by the LJT sample.
Compounds 1–5 were separated within 35 min using a Gemini C18 column. The mobile phase used gradient elution with 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 203 nm, 254 nm, and 280 nm. The inhibitory effect on LDL oxidation conduct an experiment on thiobarbituric acid reactive substance (TBARS) assay, relative electrophoretic mobility (REM) assay, and electrophoresis of ApoB fragmentation of LJT.
Calibration curves of compounds 1–5 showed good linearity (r 2 ≥0.9995) in different concentration ranges. The recoveries of compounds 1–5 were in the range of 98.90–103.39%, with relative standard deviations (RSD) below 3.0%. The RSDs (%) of intra-day and inter-day precision were 0.10–1.08% and 0.29–1.87%, respectively. The inhibitory effect of LJT on Cu2+-induced LDL oxidation was defined by TBARS assay (IC50: 165.7 μg/mL) and REM of oxLDL (decrease of 50% at 127.7 μg/mL). Furthermore LJT reduced the fragmentation of ApoB of oxLDL in a dose-dependent manner.
The established HPLC-PDA method will be helpful to improve quality control of LJT. In addition, LJT is a potential LDL oxidation inhibitor.
Chemicals and materials
Ginsenoside Rg1, ginsenoside Rb1, glycyrrhizin, and 6-gingerol were purchased from Wako (Osaka, Japan). Liquiritin was obtained from NPC BioTechnology Inc. (Daejeon, Korea). The purities of all reference compounds were ≥98.0% according to HPLC analysis. HPLC-grade methanol, acetonitrile, and water were obtained from J.T. Baker (Phillipsburg, NJ, USA). Glacial acetic acid was of analytical reagent grade and procured from Junsei (Tokyo, Japan). The crude herbal medicines from Ginseng Radix Alba, Zingiberis Rhizoma, Glycyrrhizae Radix et Rhizoma, and Atractylodis Rhizoma Alba 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-KE19-1 through KE19-4) have been deposited at the Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine.
Apparatus and conditions
The HPLC analysis was performed using a Shimadzu LC-20A (Shimadzu Co., Kyoto, Japan), which consisted of a pump (LC-20AT), on-line degasser (DGU-20A3), column oven (CTO-20A), autosampler (SIL-20 AC), and PDA detector (SPD-M20A). The data were processed with LCsolution software (Version 1.24, Shimadzu, Kyoto, Japan). The analytes were separated on a Phenomenex Gemini C18 column (250×4.6 mm, 5 μm, Torrance, CA, USA) maintained at 40°C. The gradient elution of mobile phases A (1.0% v/v aqueous acetic acid) and B (acetonitrile with 1.0% v/v acetic acid) was conducted as follows: 15-20% B for 0–10 min, 20-70% B for 10–30 min, 70-100% B for 30–40 min, 100% B for 40–45 min, and 100-15% B for 45–50 min. The flow rate was 1.0 mL/min and injection volume was 10 μL. The PDA detector was monitored at 203 nm, 254 nm, and 280 nm.
The mass spectrometer was operated using a Waters triple quadruple mass spectrometer equipped with electrospray ionization (ESI) source. The MS conditions were as follows: capillary voltage, 3.3 kV; extractor voltage, 3 V; RF lens voltage, 0.3 V; source temperature, 120°C; desolvation temperature, 300°C; desolvation gas, 600 L/h; cone gas, 50 L/h; collision gas, 0.14 mL/min. Data acquisition was processed by Waters MassLynx software (version 4.1, Milford, MA, USA).
Preparation of standard solutions
The reference compounds 1–5 were accurately weighed and dissolved in methanol at a concentration of 1,000 μg/mL. Stock solutions were stored below 4°C and underwent serial dilution with methanol before analysis.
Preparation of sample solutions
Crude components of LJT
Ginseng Radix Alba
Zingiberis Rhizoma Crudus
Calibration curve, limits of detection (LOD), and quantification (LOQ)
Each calibration curve was obtained by assessment of peak areas from standard solutions in the following concentration ranges: compounds 1 and 4, 1.00–500.00 μg/mL and compounds 2, 3, and 5, 5.00–500.00 μg/mL. Stock solutions of reference compounds 1–5 were diluted with methanol to assess LOD and LOQ values. The LOD and LOQ data were determined at signal-to-noise (S/N) ratios of 3 and 10, respectively.
Precision and accuracy
Intra-day and inter-day precisions were determined using a standard addition method to prepare spiked samples, employing both standards and controls. To confirm the repeatability, six replicates using the mixed standard solutions were measured and evaluated. The relative standard deviation (RSD) of peak areas and retention times of each compound were used to evaluate the method repeatability. Accuracy tests, which were evaluated by a recovery test, were performed by adding three different concentration levels (low, middle, and high) of reference compounds 1–5 to 200 mg of LJT sample. This test was evaluated using the calibration curve.
Determination of LDL oxidation
Oxidation of LDL by CuSO 4
We performed oxidation of LDL using CuSO4-mediated 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 sample. After 5 min, the oxidation was initiated by the addition of CuSO4 (25 μM). After 6 h oxidation, lipid peroxidation, electrophoretic mobility, and Apo B fragmentation of the LDL were measured as described below.
Determination of thiobarbituric acid reactive substance (TBARS)
Lipid peroxidation of LDL was estimated by the determination of the level of malondialdehyde (MDA) using a TBARS assay kit (BioAssay Systems, 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. After completing the reaction, the absorbance at 535 nm was measured using a microplate reader.
Relative electrophoretic mobility (REM) assay
The electrophoretic mobility of LDLs was measured 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 .
Electrophoresis of ApoB fragmentation
After oxidation, 20 μg of LDLs were denatured with 3% sodium dodecylsulfate (SDS), 10% glycerol, and 5% 2-mercaptoethanol at 100°C for 5 min. SDS-polyacrylamide gel electrophoresis (6% SDS-PAGE) was performed to detect the ApoB fragmentation. The electrophoresis proceeded at 100 V for 6 h. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 to visualize ApoB of LDLs .
Statistical evaluation of the results was performed using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test using GraphPad InStat 3.05 software (Graphpad Software Inc, CA, USA).
Results and discussion
Optimization of extraction methods
The extraction conditions were optimized to obtain a satisfactory extraction efficiency examining extraction method (ultra-sonication and shaking), extraction solvent (0%, 50%, 70%, and 90% methanol, v/v), and extraction time (10, 20, 30, 60, 90, and 120 min). By comparing the peak area of the target compounds for different conditions, the most satisfactory conditions were selected as ultra-sonication with 50% methanol for 90 min.
Optimization of chromatographic conditions
Calibration curves, LODs, LOQs, and the detected ions of the five marker compounds
Linear range (μg/mL)
Regression equation a
Correlation coefficient (r 2)
LOD b (μg/mL)
LOQ c (μg/mL)
y?=?15003.57x – 8421.59
y?=?3944.65x – 9262.82
y?=?7952.91x – 4715.75
y?=?5737.29x – 3255.18
Linearity, range, LOD, and LOQ
The linearity of the peak area (y) versus concentration (x, μg/mL) curve for each component was used to calculate the amount of each main component in LJT. The calibration curves for compounds 1–5 showed good linearity (r 2?≥?0.9995). The LODs and LOQs were less than 2.44 μg/mL and 8.13 μg/mL, respectively (Table 2).
Accuracy and precision
Recoveries for the assay of three investigated compounds in LJT
Original amount (μg/mL)
Spiked amount (μg/mL)
Found amount (μg/mL)
The precision and accuracy of the analytical results (n?=?5)
Fortified conc. (μg/mL)
Observed Conc. (μg/mL)
Observed Conc. (μg/mL)
The amount of marker compounds 1–5 in the LJT sample (n?=?3)
Effect of LJT on Cu2+-mediated oxidation of LDL
In this study, we evaluated the anti-atherosclerotic potential of LJT by their ability to inhibit Cu2+-mediated LDL oxidation models. LDL is the major carrier in the blood stream. Oxidative modification of LDL has been known to play a key role during early atherosclerosis. Protection of LDL from oxidation should be an effective strategy to prevent the development or progression of atherosclerosis [14–16]. Liquiritin (1) and glycyrrhizin (4) in Glycyrrhizae Radix et Rhizoma and 6-gingerol (5) in Zingiberis Rhizoma were detected from LJT by HPLC analysis (Table 5). The root extracts of Glycyrrhiza glabra (licorice) decreased the susceptibility of LDL to oxidation [17, 18]. The extract of Zingiberis Rhizoma reduced cell-mediated oxidation of LDL  and gingerol isolated from Zingiber was shown to inhibit AAPH- or hemin-mediated LDL oxidation .
A simple and accurate HPLC-PDA method has been developed for simultaneous separation and determination of fiver major components in the traditional Korean herbal medicine, LJT. The developed method showed good linearity, precision, and accuracy to evaluate the quality of LJT. The proposed method will be valuable for quality control of LJT. In addition, we evaluated the inhibitory effect on LDL oxidation of LJT at a concentration of 1,000 μg/mL and the results showed inhibitory activity. Consequently, LJT may have an inhibitory effect on LDL oxidation.
This research was supported by a grant (no. K13030) from the Korea Institute of Oriental Medicine.
- Hur J: Donguibogam. 2007, Seoul: Namsandang, 382-Google Scholar
- Seo HY, Han JK, Kim YH: The effect of Lizhongtang on the suppression of Th2 differentiation by IFN-r response in IGE hyperproduction and atopic dermatitis-like skin lesions induced in NC/Nga mouse. J Kor Orient Pediatr. 2009, 23: 1-22.Google Scholar
- Seo HY, Han JK, Kim YH: Therapeutic effects of Yijungtang on atopic dermatitis-like skin lesions of NC/Nga mouse induced by mite antigen. J Kor Orient Pediatr. 2011, 25: 1-27.Google Scholar
- Ha JY, Lee J: Experiment on the antitumor and immunomodulatory effects of Gagamijungtang in BALB/c mice. Kor J Orient Med Pathol. 1998, 12: 73-81.Google Scholar
- Jung YP, Hwang YH, Lee JH, Yim NH, Cho WK, Ma JY: A study on the acute toxicity of Leejung-tang (Lizhong-tang) and fermented Leejung-tang (Lizhong-tang) extract in ICR mice. Kor J Herbology. 2012, 27: 95-100. 10.6116/kjh.2012.27.3.95.View ArticleGoogle Scholar
- Shin IS, Lee MY, Lim HS, Seo CS, Ha HK, Shin HK: Gastroprotective effects of Leejung-tang, an oriental traditional herbal formula, on ethanol-induced acute gastric injury in rats. Afr J Tradit Complement Altern Med. 2013, 10: 316-323.Google Scholar
- Li J, Xie ZZ, Tang YB, Zhou JG, Guan YY: Ginsenoside-Rd, a purified component from panax notoginseng saponins, prevents atherosclerosis in apoE knockout mice. Eur J Pharmacol. 2011, 10: 104-110.View ArticleGoogle Scholar
- Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M: Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-deficient mice. J Nutr. 2000, 130: 1124-1131.PubMedGoogle Scholar
- Visavadiya NP, Soni B, Dalwadi N: Evaluation of antioxidant and anti-atherogenic properties of Glycyrrhiza glabra root using in vitro models. Int J Food Sci Nutr. 2009, 60 (Suppl 2): 135-149.View ArticlePubMedGoogle Scholar
- Barnhart RL, Busch SJ, Jackson RL: Concentration-dependent antioxidant activity of probucol in low density lipoproteins in vitro: probucol degradation precedes lipoprotein oxidation. J Lipid Res. 1989, 30: 1703-1710.PubMedGoogle Scholar
- Buege JA, Aust SD: Microsomal lipid peroxidation. Methods Enzymol. 1978, 52: 302-310.View ArticlePubMedGoogle Scholar
- Sparks DL, Phillips MC: Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis. J Lipid Res. 1992, 33: 123-130.PubMedGoogle Scholar
- Miura S, Watanabe J, Tomita T, Sano M, Tomita I: The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low density lipoprotein. Biol Pharm Bull. 1994, 17: 1567-1572. 10.1248/bpb.17.1567.View ArticlePubMedGoogle Scholar
- Steinberg D, Witztum JL: Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted to date refute the hypothesis?. Circulation. 2002, 105: 2107-2111. 10.1161/01.CIR.0000014762.06201.06.View ArticlePubMedGoogle Scholar
- Stocker R, Keaney JF: Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004, 84: 1381-1478. 10.1152/physrev.00047.2003.View ArticlePubMedGoogle Scholar
- Jialal I, Grundy SM: Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein. J Lipid Res. 1992, 33: 899-906.PubMedGoogle Scholar
- Vaya J, Belinky PA, Aviram M: Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free Radic Biol Med. 1997, 23: 302-313. 10.1016/S0891-5849(97)00089-0.View ArticlePubMedGoogle Scholar
- Belinky PA, Aviram M, Fuhrman B, Rosenblat M, Vaya J: The antioxidative effects of the isoflavan glabridin on endogenous constituents of LDL during its oxidation. Atherosclerosis. 1998, 137: 49-61. 10.1016/S0021-9150(97)00251-7.View ArticlePubMedGoogle Scholar
- Lam RY, Woo AY, Leung PS, Cheng CH: Antioxidant actions of phenolic compounds found in dietary plants on low-density lipoprotein and erythrocytes in vitro. J Am Coll Nutr. 2007, 26: 233-242. 10.1080/07315724.2007.10719606.View ArticlePubMedGoogle Scholar
- Wallin B, Rosengren B, Shertzer HG, Camejo G: Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants. Anal Biochem. 1993, 208: 10-15. 10.1006/abio.1993.1002.View ArticlePubMedGoogle Scholar
- Esterbauer H, Gebicki J, Puhl H, Jürgens G: The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992, 13: 341-390. 10.1016/0891-5849(92)90181-F.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/3/prepub
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