iridoids and wogonin

To observe the protective effect of active fractions of Huanglian Jiedu Decoction (HJD) on primary cortical neuron injury after oxygen-glucose deprivation (OGD)/reperfusion (R) injury. Methods Using macroporous resin method, HJDFE30, HJDFE50, HJDFE75, and HJDFE95 with 30%, 50%, 75%, and 95% alcohol were respectively prepared. Then the content of active components in different HJD fractions was determined with reverse phase high-performance liquid chromatography (RP-HPLC). The OGD/R injury model was induced by sodium dithionite on primary cortical neurons in neonate rats. MTT assay was used to observe the effect of four fractions (HJDFE30, HJDFE50, HJDFE75, and HJDFE95) and seven index components of HJD on the neuron viability.. RP-HPLC showed active component(s) contained in HJDFE30 was geniposide; baicalin, palmatine, berberine, and wogonside contained in HJDFE50; baicalin, berberine, baicalein, and wogonin contained in HJDFE75. The neuron viability was decreased after OGD for 20 min and reperfusion for 1 h, (P <0. 01), and significantly increased after administered with HJD, HJDFE30, HJDFE50, and HJDFE75 (P <0. 05, P <0. 01). Geniposide, baicalin, baicalein, palmatine, wogonside, and wogonin could increase the cortical neuron viability (P <0. 05, P <0. 01).. HJDFE30, HJDFE50, and HJDFE75, as active fractions of HJD, had protective effect on primary cortical neuron injury after OGD/R. Furthermore, geniposide, baicalin, and baicalein were main active components of HJD.

Topics: Animals; Berberine; Berberine Alkaloids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Flavanones; Flavonoids; Glucose; Iridoids; Models, Animal; Neurons; Oxygen; Rats; Reperfusion Injury

This study was performed to develop methods for the chromatographic determination of biomarkers in Hwangryunhaedok-tang (HHT) and the quantitative evaluation of commercial HHT. To develop an analytical method, an RP-amide column (2.7 μm, 4.6 × 100 mm, Halo: Supelco, Bellefonte, PA) was used with a gradient solvent system of mixed acetonitrile and 0.1 % phosphoric acid/water and an ultra performance liquid chromatography-diode array detector. The method was validated by specificity, linearity, accuracy (recovery) and precision tests (repeatability, intra and inter-day). The correlation coefficients (R (2)) of biomarkers were calculated as 0.9998-1.000 and their ranges were as follows: geniposide (62.5-1,000.0 μg/ml), berberine (31.3-500.0 mg/ml), palmatine (31.3-500.0 μg/ml), baicalin (125.0-1,500.0 μg/ml), baicalein (15.6-250.0 μg/ml) and wogonin (5.2-125.0 μg/ml), respectively. The limit of detection was 0.34-4.01 μg/ml, and the limit of quantification was 1.02-12.16 μg/ml. The intra-day and inter-day precision of six components were revealed as 0.02-2.48 % as a relative standard deviation (RSD). The repeatability value of biomarkers in three different concentrations of HHT was 0.29-2.98 % (RSD value) and recovery was 95.72-104.90 %. Among several extraction methods tested, biomarker content was higher with the 20 times extraction (20TE) and mixture of extract powder (MEP) methods than with any other method, and some differences among diverse pharmaceutical medicines were revealed. The validation data indicated that the method developed is suited to the determination of six marker compounds in HHT. The content of biomarkers by simultaneous analysis was evaluated in 20TE, MEP, USA formula and Taiwan formula.

Topics: Berberine; Drugs, Chinese Herbal; Flavanones; Flavonoids; Iridoids

Scutellaria baicalensis extract (SbE) has been shown to exert chemopreventive effects on several types of cancer. Baicalin, a hydrophilic flavonoid found in SbE, may have opposing effects that decrease the antitumor potential of SbE against colorectal cancer. In this study, after removing baicalin, we prepared an aglycone-rich fraction (ARF) of SbE and evaluated its anti-proliferative activity and mechanisms of action. The flavonoids found in ARF, baicalin fraction (BF) and SbE were determined by high-performance liquid chromatography (HPLC). The effects of ARF, BF, SbE and representative flavonoids on the proliferation of HCT-116 and HT-29 human colorectal cancer cells were determined by an MTS assay. The cell cycle, the expression of cyclins A and B1 and cell apoptosis were assayed using flow cytometry. Apoptosis-related gene expression was visualized by quantitative real-time polymerase chain reaction (PCR), and mitochondrial membrane potential was estimated following staining with JC-1. HPLC analysis showed that ARF contained two hydrophobic flavonoids, baicalein and wogonin, and that BF contained only baicalin. SbE had little anti-proliferative effect on the colorectal cancer cells; cancer cell growth was even observed at certain concentrations. ARF exerted potent anti-proliferative effects on the cancer cells. By contrast, BF increased cancer cell growth. ARF arrested cells in the S and G2/M phases, increased the expression of cyclins A and B1, and significantly induced cell apoptosis. Multiple genes in the mitochondrial pathway are involved in ARF-induced apoptosis, and subsequent cellular functional analysis validated the involvement of this pathway. These results suggest that removing baicalin from SbE produces an ARF that significantly inhibits the growth of colorectal cancer cells, and that the mitochondrial apoptotic pathway plays a role in hydrophobic flavonoid-induced apoptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Chemoprevention; Colorectal Neoplasms; Cyclin A; Cyclin B1; Enzyme Inhibitors; Flavanones; Flavonoids; G2 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Iridoids; Membrane Potential, Mitochondrial; Mitochondria; Phytotherapy; Plant Extracts; S Phase Cell Cycle Checkpoints; Scutellaria baicalensis

High-performance liquid chromatography was employed to determine the contents of several marker substances such as gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in Sann-Joong-Kuey-Jian-Tang. The separation was performed on a Cosmosil 5C18-AR column by gradient elution with 0.03% (v/v) phosphoric acid-acetonitrile (0 min, 90:10; 10 min, 87:13; 17-27 min, 77:23; 40 min, 62:38; 50 min, 55:45) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. n-Propylparaben was used as the internal standard and seven regression equations revealed linear relationships between the peak-area ratios (marker substances/internal standard) and concentrations. The repeatability and reproducibility (relative standard deviation) of the method were in the ranges 0.02-1.78% and 1.44-4.95%, respectively.

Topics: Berberine; Berberine Alkaloids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Flavanones; Flavonoids; Glucosides; Glycyrrhetinic Acid; Glycyrrhizic Acid; Iridoid Glucosides; Iridoids; Pyrans; Reproducibility of Results; Xanthenes; Xanthones