Rhei Rhizoma Research Articles

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

SIMULTANEOUS DETERMINATION AND PHARMACOKINETIC COMPARISONS OF ALOE-EMODIN, RHEIN, EMODIN, AND CHRYSOPHANOL AFTER ORAL ADMINISTRATION OF THESE MONOMERS, RHEI RHIZOMA AND CHAIQIN-CHENGQI-TANG, TO RATS

A study was conducted to compare the pharmacokinetics of aloe-emodin, rhein, emodin, and chrysophanol after oral administration of these monomers, Rhei Rhizoma and Chaiqin-Chengqi-Tang, to rats. Separation of the monomers in rat plasma was accomplished on a YMC C18 stationary phase (4.6 × 150 mm, 5 µm) and a mobile phase of methanol-water containing 0.2% acetic acid (70:30, v/v), with a UV detector setting at 254 nm. The calibration curves for the four compounds were linear in an appropriate concentration ranges. The intra- and inter-day assay coefficients in plasma were less than 10.96% and the recoveries of the method were higher than 90.0%. The validated method was successfully applied in the aforementioned pharmacokinetic study. The results show that the pharmacokinetics of aloe-emodin, rhein, emodin, and chrysophanol in the rat is different when given orally alone, as a Rhei Rhizoma mixture, or in a Chaiqin-Chengqi-Tang prescription. This may be due to unidentified herb-herb or herbal constituent interaction mechanisms.

Steady-state pharmacokinetics and tissue distribution of anthraquinones of Rhei Rhizoma in rats

Aim of the studyRhei Rhizoma, the rhizome of Rheum palmatum L. (RP), is a popular herb in clinical Chinese medicine. RP is abundant in polyphenolic anthraquinones, which have been reported to show various beneficial bioactivities. This study investigated the pharmacokinetics and tissue distribution of anthraquinones following seven-dose administration of RP decoction to rats. Materials and methodsSix Sprague-Dawley rats were given 2.0g/kg of RP twice daily for seven doses and blood samples were collected at designated time after the 7th dose. Another six rats were sacrificed at 30min after the 7th dose and organs including liver, kidney, lung and brain were collected. Serum and tissue specimens were assayed by HPLC before and after hydrolysis with β-glucuronidase and sulfatase, respectively. ResultsPharmacokinetic analysis indicated that the anthraquinones in serum mainly presented as glucuronides/sulfates and contained higher ratio of sulfates when compared with single-dose administration of RP. Contrary to the finding in serum, tissue analysis discovered mainly free form of anthraquinone in most organs assayed, such as aloe-emodin and rhein in kidney, liver, lung; emodin in liver, lung; trace of chrysophanol in kidney and liver. In all brains, neither free forms nor their glucuronides/sulfates have been detected. ConclusionsThe glucuronides/sulfates of anthraquinones were the major forms in bloodstream, whereas the free forms of most anthraquinones were predominant in kidney and liver.

5/6腎摘出ラットの糸球体増殖性変化に対する温脾湯(TJ-8117)と(―)Epicatechin-3-0-gallateの作用.

Onpi-to (TJ-8117) is a herbal medicine composed of five crude drugs (Rhei Rhizoma, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma and Aconiti Tuber). Our previous experiments have demonstrated that TJ-8117 suppressed the development of glomerulosclerosis and retarded the deterioration of renal function in 5/6 nephrectomised rats. In the present study, the effects of TJ-8117 and (-)Epicatechin-3-O-gallate (ECG), which is a component of Rhei Rhizoma, on glomerular cell proliferation, extracellular matrix accumulation and glomerular hypertrophy were investigated in 5/6 nephrectomized rats. Male Wistar rats (170-180 g) were subjected to 5/6 nephrectomy, and TJ-8117 (0.32%, 0.64%) or angiotensin-converting enzyme inhibitor, captopril (CAP 0.08%) was administered daily by mixing in normal chow and ECG (2 mg, 8 mg/100 ml) by drinking water from the day after 5/6 nephrectomy. Following 5/6 nephrectomy, glomerular cell poliferation was increased and reached a maximum at 1 week in the untreated control rats, but was suppressed significantly at 1 and 2 weeks after treatment with TJ-8117 and at only 1 week after treatment with CAP. Extracellular matrix accumulation was detected after 1 week and increased gradually until 4 weeks in the control rats, whereas it was significantly inhibited in both the TJ-8117- and CAP-treated rats. In addition, immunohistochemistry revealed that TJ-8117 significantly inhibited the increase of fibronectin, and tended to reduce type I and type IV collagen at 4 weeks. Furthermore, TJ-8117 suppressed glomerular hypertrophy at 4 weeks. Systolic blood pressure (SBP) and urinary protein excretion (UP) were higher in the control rats than sham-operated rats. TJ-8117 prevented this increase of SBP and UP at 1 week. ECG also suppressed glomerular cell proliferation and the increase of SBP and UP at 1 week after 5/6 nephrectomy. These findings suggest that ECG was one of active components of TJ-8117. These results suggest that TJ-8117 suppressed proliferating changes in glomeruli at an early stage in 5/6 nephrectomized rats, and inhibited the development of glomerulosclerosis.

Effects of rhubarb tannins on renal function in rats with renal failure.

The effects of tannins purified from Rhei Rhizoma on various parameters of renal function were investigated in rats with adenine-induced renal failure. The glomerular filtration rate, renal plasma flow and renal blood flow were significantly increased in rats given (-)-epicatechin 3-O-gallate at a dose of 5 or 10 mg/kg body weight/day for 24 days. Administration of 5 mg of procyanidin B-2 3,3'-di-O-gallate also led to a significant increase in renal functional parameters. However, unlike the former two components, procyanidin C-1 3,3',3''-tri-O-gallate caused aggravation of renal function.

San‐Huang‐Xie‐Xin‐Tang extract suppresses hepatitis C virus replication and virus‐induced cyclooxygenase‐2 expression

Summary. Chronic hepatitis C virus (HCV) infection is associated with chronic inflammation of liver, which leads to the development of cirrhosis and hepatocellular carcinoma (HCC). Because of severe side effects and only a 50–70% cure rate in genotype 1 HCV‐infected patients upon current standard treatment with pegylated interferon‐α plus ribavirin, new therapeutic regimens are still needed. San‐Huang‐Xie‐Xin‐Tang (SHXT) is a transitional Chinese herbal formula, composed of Rhei rhizoma, Scutellaria radix and Coptidis rhizome, and possesses anti‐inflammatory effect. Here, we describe a (+)‐catechin‐containing fraction extracted from SHXT, referred as SHXT‐frC, exhibited effective inhibition of HCV replication, with selectivity index value (SI; CC50/EC50) of 84, and displayed synergistic anti‐HCV effects when combined with interferon‐α, HCV protease inhibitor telaprevir or polymerase inhibitor 2′‐C‐methylcytidine. The activation of factor‐κB (NF‐κB) and cyclooxygenase‐2 (COX‐2) signalling pathway has particular relevance to HCV‐associated HCC. SHXT‐frC treatment also caused a concentration‐dependent decrease in the induction of COX‐2 and NF‐κB expression caused by either HCV replication or HCV NS5A protein. Collectively, SHXT‐frC could be an adjuvant treatment for patients with HCV‐induced liver diseases.

San‐Huang‐Xie‐Xin‐Tang Protects against Activated Microglia‐ and 6‐OHDA‐Induced Toxicity in Neuronal SH‐SY5Y Cells

San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix and Rhei rhizoma, is a traditional Chinese herbal medicine used to treat gastritis, gastric bleeding and peptic ulcers. This study investigated the neuroprotective effects of SHXT on microglia-mediated neurotoxicity using co-cultured lipopolysaccharide (LPS)-activated microglia-like BV-2 cells with neuroblastoma SH-SY5Y cells. Effects of SHXT on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity were also examined in SH-SY5Y cells. Results indicated SHXT inhibited LPS-induced inflammation of BV-2 cells by downregulation of iNOS, NO, COX-2, PGE2, gp91phox, iROS, TNF-α, IL-1β, inhibition of IκBα degradation and upregulation of HO-1. In addition, SHXT increased cell viability and down regulated nNOS, COX-2 and gp91phox of SH-SY5Y cells co-cultured with LPS activated BV-2 cells. SHXT treatment increased cell viability and mitochondria membrane potential (MMP), decreased expression of nNOS, COX-2, gp91phox and iROS, and inhibited IκBα degradation in 6-OHDA-treated SH-SY5Y cells. SHXT also attenuated LPS activated BV-2 cells- and 6-OHDA-induced cell death in differentiated SH-SY5Y cells with db-cAMP. Furthermore, SHXT-inhibited nuclear translocation of p65 subunit of NF-κB in LPS treated BV-2 cells and 6-OHDA treated SH-SY5Y cells. In conclusion, SHXT showed protection from activated microglia- and 6-OHDA-induced neurotoxicity by attenuating inflammation and oxidative stress.

Evaluation of Oxygen Radical Absorbance Capacity in Kampo Medicine

Antioxidant capacity of food has come to be shown in terms of oxygen radical absorbance capacity (ORAC) mainly on vegetables or fruit. However, the evaluation of Kampo in terms of ORAC has not yet been accomplished. It is important that such an investigation is also conducted for Kampo medicine. We measured the ORAC value of almost all the available Kampo formulas used in the Japanese National health insurance system and examined the ORAC value both for the daily prescription, and also the crude herb ingredients. The ORAC value of Kampo medicine ranged 4.65–5913 units/day. The ORAC value was high in Kampo formulas including Rhei Rhizoma, and was relatively high in Kampo formulas including anti-inflammatory herbs other than Rhei Rhizoma. The ORAC value was also high in Kampo formulas including crude herbs that have relaxation effects. The ORAC value of a crude herb would seem to not be fixed but be dependent on combination with other crude herbs from the comparison of different herbs added to the basic Kampo medicine. These results suggest variability and complexity of the antioxidant capacity of Kampo medicine within the similar range of food. On the other hand, investigation of the compound changes of various crude herbs with ORAC may lead to the elucidation of the action mechanism of Kampo medicine.

San‐Huang‐Xie‐Xin‐Tang Prevents Rat Hearts from Ischemia/Reperfusion‐Induced Apoptosis through eNOS and MAPK Pathways

San-Huang-Xie-Xin-Tang (SHXT) is a traditional Chinese medication consisting of three herbs, namely Coptidis rhizome, Scutellariae radix and Rhei rhizome. This study aimed to examine the cardioprotective effects of SHXT in a rat model of acute myocardial apoptosis induced by ischemia/reperfusion (I/R). Vehicle (intravenous saline) or SHXT (intravenous or oral) was administered prior to I/R (occlusion of left coronary artery for 45 min followed by reperfusion for 2 h). In the vehicle group, myocardial I/R caused myocardial infarction with increased plasma cardiac enzymes, severe arrhythmia and mortality. Myocardial apoptosis was induced by I/R as evidenced by DNA ladder and Bcl-2/Bax ratio. In the SHXT group, we found that SHXT significantly reduced plasma levels of cardiac enzymes, arrhythmia scores (from 5 ± 1 to 2 ± 1, P < .01) and mortality rate (from 53 to 0%, P < .01). In addition, pretreatment with intravenous SHXT reduced the infarct size dose-dependently when compared with the vehicle group (10 mg kg−1: 14.0 ± 0.2 versus 44.5 ± 5.0%, and 30 mg kg−1: 6.2 ± 1.2% versus 44.5 ± 5.0%, both P < .01). Similarly, oral administration of SHXT reduced the infarct size dose-dependently. Furthermore, SHXT markedly decreased the apoptosis induced by I/R with increased Bcl-2/Bax ratio. Finally, we found that SHXT counteracted the I/R-induced downstream signaling, resulting in increased myocardial eNOS expression and plasma nitrite, and decreased activation of ERK1/2, p38 and JNK. These data suggest that SHXT has cardioprotective effects against I/R-induced apoptosis, and that these effects are mediated, at least in part, by eNOS and MAPK pathways.

Nanoscale hepatoprotective herbal decoction attenuates hepatic stellate cell activity and chloroform-induced liver damage in mice

BackgroundSan-Huang-Xie-Xin-Tang (SHXXT) decoction, a traditional Chinese medicine containing Rhei rhizome, Coptidis rhizome, and Scutellariae radix, is widely used in hepatoprotective therapy. However, preparation of the decoction requires addition of boiling water that causes loss of numerous effective components.MethodsTo improve the bioavailability of the decoction, nanoscale SHXXT was developed. Chloroform-induced liver injury and hepatic stellate cell activity in mice were used to demonstrate the hepatoprotective characteristics of nanoscale SHXXT decoction.ResultsLiver/body weight ratio and serum aspartate and alanine aminotranferase levels were recovered by the nanoscale SHXXT. TIMP-1 gene expression was inhibited and MMP-2 gene expression was accelerated in activated hepatic stellate cells.ConclusionNanoscale SHXXT decoction prepared in room temperature water could have preserved hepatoprotective ability. The results of this study indicate that nanoscale SHXXT could be extracted easily. The simple preparation of this herbal decoction is more convenient and energy-efficient.

Anti-angiogenic effect and mechanism of rhein from Rhizoma Rhei

Rhein is a major bioactive component in rhubarb (Dahuang), a famous traditional Chinese medicine derived from the rhizome of Rheum palmatum and related species. It was reported to have antitumor and anti-inflammatory properties. Our previous studies found rhein displaying potent anti-angiogenic activities in a zebrafish embryo model. Its action mechanisms need further elucidation. The inhibition effect of vessel formation was checked by microscopic imaging on Tg(fli1a:EGFP)y1 zebrafish embryos. Then the action mechanism of rhein was investigated by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) on wild type zebrafish embryos and further tested on human umbilical vein endothelial cells. At 20μM, rhein could almost completely block intersegmental blood vessels formation at both 48 and 72hpf, and completely inhibit subintestinal vessel plexus formation at 72hpf. Rhein affected multiple molecular targets related to angiogenesis, particularly angpt2 and tie2, and also inhibited endothelial cell migration. Rhein could inhibit angiogenesis, which may play a role in antitumor and anti-inflammatory actions.

Identification of Plant Extracts that Inhibit Cellular Senescence in Human Fibroblasts, Endothelial Cells, and Vascular Smooth Muscle Cells

To identify plant extracts capable of inhibiting cellular senescence, the effects of plant extracts on adriamycin-induced cellular senescence in human fibroblasts, human umbilical vein endothelial cells (HUVECs), and vascular smooth muscle cells were investigated. After adriamcyin treatment, the effects of plant extracts on cellular senescence were evaluated by measuring senescenceassociated β-galactosidase activity of the cells. Among 799 plant extracts, those from Rhei Rhizoma, Cirsii Radix, and Plantagnis Semen repressed adriamycin-induced cellular senescence in fibroblasts. Extracts of Cinnamomi Cortex and Cinnamomi Cortex Spissus were effective in endothelial cells. Extracts of Euonymi Lignum Suberalatum, Salicis Radicis Cortex, Polygoni aviculari Herba, and Chaenomelis langenariae Radix reduced senescence in vascular smooth muscle cells. These results suggested the effectiveness of plant extracts in reducing cellular senescence of human cells. These plant extracts could be used to develop dietary supplements or cosmetics for modulating tissue aging or aging-associated diseases.

In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment

Aim of the study A novel topical paste used for fracture healing (FH), consisting of the extracts of six herbs, Radix Dipsaci, Ramulus Sambucus Williamsii, Rhizoma Notoginseng, Flos Carthami, Rhizoma Rhei and Fructus Gardeniae, was developed according to the classical theory of traditional Chinese medicine. This study aimed to determine the effectiveness of this formula, and some of its important chemical components in the promotion of fracture healing. The transdermal transport of FH was also examined. Materials and methods The osteogenic, angiogenic and nitric oxide suppressing effects of FH and its important chemical marker components were assessed by using osteoblastosacroma UMR-106 cells, human umbilical vein endothelial cells (HUVEC) and murine macrophage RAW264.7 cells, respectively. The bone healing effects of the FH paste and its transdermal absorption were determined using a rabbit fracture model. The callus sizes, bone specific alkaline phosphatase levels and biomechanical properties of the healed bone were assessed. Results FH significantly increased the cell proliferation in UMR-106 and HUVEC cells and inhibited the nitric oxide production in murine macrophage in dose-dependent manner. Its important chemical components asperosaponin VI, ginsenoside Rg1 and emodin were shown to be acting positively in the respective in vitro studies. FH paste significantly improved the bone healing in the rabbit fracture model, as was indicated by the increases in callus size at weeks 2–5, and the elevations in bone specific alkaline phosphatase activities at weeks 5–6. The analysis using LC/MS/MS also showed the presence of important chemical marker components of the FH formula in the plasma after 8 weeks of topical treatment. Conclusion This study presents the first scientific evidence of the efficacy of a herbal paste in the promotion of fracture healing. There were evidences of transdermal transport of the chemical components, control the inflammation through nitric oxide inhibition, promotion of angiogenesis, and bone healing in the in vitro tests, as well as in the experimental animal.

Simultaneous analysis of nine components in patch preparations of Ru-Yi-Jin-Huang-San by high-performance liquid chromatography

A facile high-performance liquid chromatography (HPLC) method for the resolution and quantitative measurement of nine marker substances, the active ingredients in patch preparations of Ru-Yi-Jin-Huang-San, was established using gradient elution in the reversed-phase mode. These marker substances included berberine (Phellodendri Cortex), curcumin (Curcumae Rhizoma), imperatorin (Angelicae Dahuricae Radix), magnolol (Magnoliae Cortex), hesperidin (Citri Leiocarpae Exocarpium), glycyrrhizin (Glycyrrhizae Radix), and emodin, sennoside A, sennoside B (Rhei Rhizoma). The ingredients in the water-based and oil-based patches of the formula from different manufactures were also analyzed for quality evaluation. Extracted samples were analyzed by HPLC using a reversed-phase column (Inertsil 5 ODS-2, 4.6-mm I.D. x 250 mm) at 30 degrees C and eluted with a mixture of 20 and 70% acetonitrile aqueous solution in gradient manner at a flow rate of 1.0 ml/min. The detection wavelength varied with time as follows: 275 nm, 0-72 min; 250 nm, 72-105 min; 220 nm, 105-145 min. Relative coefficients of variations of intra- and interday analysis were less than 5%. All the recoveries were 93.30-113.63%. This method could be applied for the simultaneous determination of nine marker substances in Ru-Yi-Jin-Huang-San.

Active ingredients of traditional Japanese (kampo) medicine, inchinkoto, in murine concanavalin A-induced hepatitis

Aim of the study The traditional Japanese (kampo) medicine inchinkoto (ICKT) is used in Eastern Asia as a choleretic and hepatoprotective agent. Previously, we reported that ICKT ameliorates murine concanavalin A (con A)-induced hepatitis via suppression of interferon (IFN)-γ and interleukin (IL)-12 production. In the present study, we investigated the active ingredients of ICKT. Materials and methods ICKT and extracts of its component herbs were fractionated, and their effects on liver injury and cytokine production in vivo (biochemical markers of liver injury and cytokine levels in serum) and in vitro (cytokine and nitrite production in the cultures of splenocytes and peritoneal macrophages). Results Decoctions of component herbs, Artemisiae Capillari Spica (Artemisia capillaris Thunberg: ‘Inchinko’ in Japanese), Gardeniae Fructus (Gardenia jasminoides Ellis: ‘Sanshishi’) and Rhei Rhizoma (Rheum palmatum Linné: ‘Daio’) were administered orally. Inchinko and Sanshishi decreased serum transaminases and IFN-γ concentrations. Examination of fractions of component herbs suggested that capillarisin, a component of Inchinko, has potent hepatoprotective activity in vivo. In in vitro studies, capillarisin and genipin, an intestinal metabolite of geniposide that is contained in Sanshishi, were examined. IFN-γ production was significantly suppressed by capillarisin and genipin in con A-stimulated splenocyte culture. Genipin also suppressed IL-1β, IL-6, and IL-12p70 synthesis. Capillarisin and genipin decreased nitrite release from IFN-γ-stimulated macrophages. Conclusions These results suggested that both Inchinko and Sanshishi may contribute to the protective effects of ICKT against con A hepatitis. Capillarisin was found to be potently hepatoprotective, and genipin may also contribute, especially via modulation of cytokine production.

Determination of the Major Chemical Components in Different Combinations of Dahuang Huanglian Xiexin Decoction by HPLC-DAD

Abstract Objective The aim of this study is to determine the major chemical components in different herbs and combinations of the Chinese medicine formula Dahuang Huanglian Xiexin Decoction (DHXD), which is composed of Rhizoma rhei, Rhizoma coptidis , and Radix scutellariae , with High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) and Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) methods. Methods A HPLC-DAD method was established with Luna octadecyl silane (ODS) column (250 mm × 4.6 mm, 5 μm). The mobile phase was acetonitrile-0.5% triethylamine (adjusted by phosphoric acid, pH 3.5) gradient elution with the flow rate of 1.0 mL/min. The sample size was 20 μL. The chromatographic peaks of the individual herbs and the DHXD formula were identified by comparing the relative retention time and ultraviolet absorption spectrum with the reference compounds, together with LC/MS/MS. Results The HPLC-DAD method was accurate and reproducible. A total of 20 chromatographic peaks were observed. Eleven peaks, including berberine from Coptis , were identified using reference compounds, and four peaks, including coptisine, were determined by LC/MS/MS. Conclusion The HPLC-DAD method is suitable for qualitative analysis of major chemical components in DHXD. The identification of the 15 chemical components and determination of their source herbs in the formula provides new analytical evidence on the chemical basis and combinational principles and quality control of DHXD.

Potential ex vivo immunomodulatory effects of San-Huang-Xie-Xin-Tang and its component herbs on mice and humans

Aim of the study San-Huang-Xie-Xin-Tang (SHXXT), an important Chinese medicine formula, contains Rhei Rhizoma (RR), Scutellariae Radix (SR) and Coptidis Rhizoma (CR). RR and SR are abundant in anthraquinone and flavonoid polyphenols. Pharmacokinetic study of SHXXT indicated that glucuronides were the predominant forms of polyphenols in rats. Materials and methods As an extension of pharmacokinetic study, the serum metabolites of SHXXT, RR, SR and CR were prepared from rats and quantitated, then the immunomodulation effects were examined by culturing these serum metabolites with murine and human immune cells. Results The results indicated that the inhibitions on nitric oxide (NO) and cytokine production from mitogen-activated peritoneal macrophages by the serum metabolites of SHXXT, RR, SR and CR were through reducing the protein expression of inducible NO synthase (iNOS) and the IC 50 were 0.8%, 1.5%, 3.0% and 0.8% of their blood concentrations, respectively. In addition, the serum metabolites of SHXXT, RR, SR and CR significantly decreased the ratios of interferon-gamma (IFN-γ) to interleukin (IL)-4 in mitogen-stimulated mice spleen cells and human peripheral blood mononuclear cells (PBMCs). Moreover, the serum metabolites of SHXXT and SR significantly arrested the mitogen-stimulated mice spleen cells at G2/M stage. Conclusions In conclusion, the serum metabolites of SHXXT and the component herbs exerted promising modulation activities on the immune functions and the cell cycle distribution of mice and human immune cells. We suggest that SHXXT is a promising remedy for immunomodulation through Th1/Th2 regulation.

Identification of Rhei Rhizoma by DART-TOF-MS: chemotypes discrimination

Identification of Rhei Rhizoma by DART-TOF-MS: chemotypes discrimination

만성신부전 치료제 개발을 위한 WHW 엑스의 기준 및 시험방법 설정

This study is to specify the criteria and testing methods of WHW extract which has a potency as a therapeutic agent for chronic renal failure. The determination of specifications of WHW extract is mostly important because of the quality assurance. Three batches of WHW extract were obtained by the extraction at C for 3 hr using water from mixture of 15 herbal medicines including Codonopsis Pilosulae Radix, Salviae Radix, Pinelliae Rhizoma, Coptidis Rhizoma, Evodiae Fructus, Epimedii Herba, Rhei Rhizoma, Perillae Herba, Glycyrrhizae Radix, Artemixiae capillaris Herba, Alimatis Rhizoma, Hoelen, Atractylodies Rhizoma alba, Polyporus and Cinnamomi Ramulus, subsequently, vaccum-dried for 15 hr. The yield of WHW extracts was 24.53% on the average. The identification of each herbal medicine of WHW extract was performed by modification of Korean Pharmacopeia IX (KP IX). The assay of WHW extract was performed using standard such as berberine, icariin, glycyrrhizin, and cinnamic acid of indicative herbal medicines by modification of KP IX, too. As well as, paticle size classification test was carried out to indicate the boundary of particle size of WHW extract and the particle size of WHW extract more than 50% showed the 140 m. Taken together, WHW extract could be prepared reproducibly and assurable if follows the presented extraction and drying steps and its specifications were satisfied with the indicated criteria.

Chunghyuldan attenuates brain microglial inflammatory response

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target stroke and neurodegenerative diseases. Chunghyuldan, a combinatorial drug consisting of Scutellariae Radix, Coptidis Rhizoma, Phellodendri Cortex, Gardeniae Fructus, and Rhei Rhizoma, has an inhibitory effect on stroke recurrence in patients with small-vessel disease. It has also been reported to confer antihypertensive, antihyperlipidemic, and antiinflammatory effects. The aim of this study was to examine whether Chunghyuldan suppresses microglial activation. Chunghyuldan was effective at inhibiting LPS-induced nitric oxide (NO) release from rat brain microglia. Real-time reverse transcriptase PCR analysis revealed that pretreatment of rat brain microglia with Chunghyuldan attenuated the LPS-induced expression of mRNAs encoding inducible NO synthase, tumor necrosis factor (TNF)-alpha, interleukin-1beta, and cyclooxygenase-2. In rat brain microglia, Chunghyuldan reduced the LPS-stimulated production of TNF-alpha and prostaglandin E2. In addition, Chunghyuldan significantly decreased LPS-induced phosphorylation of the ERK1/2 and p38 signaling proteins. These results suggest that Chunghyuldan provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia.