Orthogonal array design for optimizing extraction efficiency of active constituents from Jakyak-Gamcho Decoction, the complex formula of herbal medicines, Paeoniae Radix and Glycyrrhizae Radix.

A complex formula composed of Paeonia lactiflora PALL. and Glycyrrhiza uralensis Fisch., which is called as Jakyak-Gamcho Decoction (JGD), has been used for a pain-relieving function and muscle spasms due to blood deficiency in the traditional medicine. In this study, the anti-inflammatory activity of JGD was evaluated based on the quantitative determinations and the relative proportions of six major constituents in the decoction mixture extracted by orthogonal array methods. Our results suggest that the three parameters are all crucial factors. The optimized conditions for extraction were therefore established [solvent (water); pH value (4); extraction number (4)]. We also optimized the extraction conditions related to anti-inflammatory activity [solvent (70% EtOH); pH value (6); extraction number (4)]. So, we found that the bioactivity was responsible for mixed components but not individual one. It was proportionally associated with the amounts of some components in the extracts of herbal medicines. When the proportion of the active components was similar to each other, they had the similar functions. Furthermore, the results could establish a model system for the quality assurance of herbal preparations, and provided a new paradigm of active components-pharmacodynamics, which is used for illustrating the connections between the bioactivities and the proportion of active constituents in the extracts of herbal medicines.

Effects of constituents of Amomum xanthioides on gastritis in rats and on growth of gastric cancer cells.

In this study we investigated the effects of constituents of Amomum xanthioides (AX) on gastritis in rats and on the growth of human gastric cancer cells. The ethanol extract of Amomum xanthioides significantly inhibited HCl ethanol-induced gastric lesions and the growth of Helicobacter pylori (H. pylon). The ethanol extract of AX was further fractionated with hexane, chloroform, butanol and H20. Among these fractions, oral treatment with the butanol fraction at a dose of 350 mg/kg was the most effective at preventing HCl* ethanol-induced gastric lesions. In pylorus ligated rats, the butanol fraction also decreased the volume of gastric secretion and gastric acid output. We isolated six subfractions of the butanol fraction using open column chromatography. Subfraction 4 (150 mg/kg) significantly inhibited HCl* ethanol-induced gastric lesions and gastric secretion in pylorus ligated rats. Using GC-MS we identified the constituents of subfraction 4 to be five aliphatic compounds, 1-hexadecene, 1-nonadecene, cycloeicosane, 1-octadecene and cyclotetracosane. In addition, subfraction 4 reduced cell viability in a dose-dependent manner in human gastric cancer cells (AGS, KATOIII and SNU638). It also increased intracellular Ca2+ concentration in SNU638 cells, an effect that was significantly inhibited by dantrolene, a Ca2+ release blocker. Moreover, dantrolene significantly inhibited subfraction 4-induced cytotoxicity. Taken together, these results suggest that subfraction 4 of the butanol extract of AX has an anti-gastritic effect in rats and is cytotoxic to human gastric cancer cells. The mechanism of its anti-gastritic action may be associated with the inhibition of secretion of gastric acid and anti-H. pylori action. Its cytotoxicity against human gastric cancer cells may be, at least in part, mediated by intracellular Ca2+ dyshomeostasis. From these results, we suggest that AX may be useful for the treatment of gastritis and gastric cancer.

Anti-inflammatory mechanisms of apigenin: inhibition of cyclooxygenase-2 expression, adhesion of monocytes to human umbilical vein endothelial cells, and expression of cellular adhesion molecules.

The aim of this study was to clarify the anti-inflammatory mechanism of apigenin. Apigenin inhibited the collagenase activity involved in rheumatoid arthritis (RA) and suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a dose dependent manner in RAW 264.7 macrophage cells. Pretreatment with apigenin also attenuated LPS-induced cyclooxygenase-2 (COX-2) expression. In addition, apigenin profoundly reduced the tumor necrosis factor-alpha (TNF-alpha)-induced adhesion of monocytes to HUVEC monolayer. Apigenin significantly suppressed the TNF-alpha-stimulated upregulation of vascular cellular adhesion molecule-1 (VCAM-1)-, intracellular adhesion molecule-1 (ICAM-1)-, and E-selectin-mRNA to the basal levels. Taken together, these results suggest that apigenin has significant anti-inflammatory activity that involves blocking NO-mediated COX-2 expression and monocyte adherence. These results further suggest that apigenin may be useful for therapeutic management of inflammatory diseases.

Quantitative analysis of chondroitin sulfate in raw materials, ophthalmic solutions, soft capsules and liquid preparations.

We performed the quantitative analysis of chondroitin sulfate (CS) obtained from raw materials and various pharmaceutical preparations. To quantify CS content in raw materials and in an ophthalmic solution, each test sample and the authentic CS were first digested by chondroitinase ABC. The CS disaccharides produced were analyzed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC) and CS content was quantified by calculating the total peak areas of the disaccharides derived from a CS calibration curve. In the case of soft capsules, CS was first extracted with hexane followed by phenol-chloroform to remove oil and protein ingredients. The extracted CS samples were depolymerized by chondroitinase ABC and CS content was determined. Quantitative analysis of the disaccharides derived from raw materials and an ophthalmic solution showed the CS contents (%) were 39.5+/-0.1 to 105.6+/-0.1 and 103.3+/-1.2, respectively. In case of CS analysis in soft capsules and liquid preparations, the overall recovery (%) of the spiked CS was 96.79+/-0.53-103.54+/-1.78 and 97.10+/-1.82 to 103.17+/-2.34, respectively. In conclusion, the quantitative analysis of the disaccharides produced by enzymatic digestion can be used in the direct quantitation of CS containing pharmaceutical formulations.

Regulation and quality control of herbal drugs in Korea.

Korea has a great diversity in resources of medicinal plants. The traditional herbal medicines and their preparations have been widely used in Korea as well as in China and Japan for thousands of years. One of the characteristics of Korean herbal medicine preparations is that all the herbal medicines are incorporated into an extractor at the same time and extracted with boiling water during the decoction process. In this process, a variety of interactions between the active components of several herbal medicines may occur. This is the main reason why quality control of oriental herbal drug is more difficult than that of western herbal drug. In this paper, we would like to present an overview of the characteristics of regulation and quality control of herbal medicines in Korea.

Enhancement of paracellular transport of heparin disaccharide across Caco-2 cell monolayers.

The enhancement of paracellular transport of heparin disaccharide using several absorption enhancers across Caco-2 cell monolayers was tested. The cytotoxicity of these enhancers was also examined. The enhancing effects by Quillaja saponin, dipotassium glycyrrhizinate, 18beta-glycyrrhetinic acid, sodium caprate and taurine were determined by changes in transepithelial electrical resistance (TEER) and the amount of heparin disaccharide transported across Caco-2 cell monolayers. Among the absorption enhancers, 18beta-glycyrrhetinic acid and taurine decreased TEER and increased the permeability of heparin disaccharide in a dose-dependent and time-dependent manner with little or negligible cytotoxicity. Our results indicate that these absorption enhancers can widen the tight junction, which is a dominant paracellular absorption route of hydrophilic compounds. It is highly possible that these absorption enhancers can be applied as pharmaceutical excipients to improve the transport of macromolecules and hydrophilic drugs having difficulty in permeability across the intestinal epithelium.

Enhancement of heparin and heparin disaccharide absorption by the Phytolacca americana saponins.

We studied the effects of phytolaccosides, saponins from Phytolacca americana, on the intestinal absorption of heparin in vitro and in vivo. The absorption enhancing activity of these compounds (phytolaccosides B, D2, E, F, G and I) was determined by changes in transepithelial electrical resistance (TEER) and the transport amount of heparin disaccharide, the major repeating unit of heparin, across Caco-2 cell monolayers. With the exception of phytolaccoside G, all of them decreased TEER values and increased the permeability in a dose-dependent and time-dependent manner. In vitro, phytolaccosides B, D2, and E showed significant absorption enhancing activities, while effects by phytolaccoside F and I were mild. In vivo, phytolaccoside E increased the activated partial thromboplastin time (APTT) and thrombin time, indicating that phytolaccoside E modulated the transport of heparin in intestinal route. Our results suggest that a series of phytolaccosides from Phytolacca americana can be applied as pharmaceutical excipients to improve the permeability of macromolecules and hydrophilic drugs having difficulty in absorption across the intestinal epithelium.

Co-contamination of Aflatoxins with Ochratoxin A and Zearalenone in Thuja orientalis Semen.

Korea is representative of a country that consumes herbal medicines; most of the herbal medicines circulating in South Korea have been imported from developing countries in Southeast Asia, such as China and Indonesia. Recently, domestic hygiene and safety are issues that have come to the forefront, because herbal medicines currently in circulation could possibly contain contaminants or residues. Furthermore, the appearance or discovery of harmful new species due to environmental and industrial developments is becoming a social problem. Therefore, it may be necessary to consider and investigate these matters on a continual basis. Recently, mycotoxin contaminations in such foods as cereals, nuts, and powdered red pepper have been reported. They have become a problematic issue; the possibility of contamination in herbal medicines has also been considered. Nevertheless, recognition of and research into mycotoxin contamination in herbal medicines has been scarce because herbal medicine is used in only a few nations. In this research, we identified contamination by aflatoxin which is known to be the most potent mutagenic, carcinogenic, and teratoge-nic mycotoxin in Thujae Semen, a herbal medicine. We also found co-contaminations involving other mycotoxins, including ochratoxin A and zeraleanone.

Effects of saponins from the root bark of Aralia elata on the transport of chondroitin sulfate in Caco-2 cell monolayers and rats.

We investigated the intestinal absorption enhancing effect of the saponins from the root bark of Aralia elata (SRBAE) in Caco-2 cell monolayers and rats. SRBAE at concentrations of 0.04% and 0.08% (w/v) decreased the transepithelial electrical resistance (TEER) values and increased the paracellular uptake of chondroitin sulfates (CSs) having different molecular weights (MW 500, 4500, and 18000) in a dose-dependent manner. We also evaluated the cytotoxicity of SRBAE to determine its proper concentration as an absorption enhancer. MTT assay and trypan blue exclusion test indicated that the cytotoxicity of SRBAE at concentrations of 0.04% and 0.08% was negligible. CS (MW 18000) was orally administered with or without SRBAE to rats. The oral administration of SRBAE (250 mg/kg) in 1 h increased the intestinal absorption of CS, by 4.9-fold versus the control (CS alone). Histological examination of the gastrointestinal tissues showed that SRBAE did not cause any damage to tissues. In conclusion, our results suggest that SRBAE acts as an efficient absorption enhancer and makes it easier for hydrophilic molecules to penetrate the intestinal epithelium.

Effects of low molecular weight chondroitin sulfate on type II collagen-induced arthritis in DBA/1J mice.

In order to evaluate the improvement in the treatment of chronic arthritis, we investigated chondroitin sulfate depolymerization product (low molecular weight chondroitin sulfate, LMWCS) and intact chondroitin sulfate (CS) in vitro and in vivo. LMWCS was prepared by a chemical depolymerization process induced by hydrogen peroxide in the presence of copper salts. LMWCS (300 mg/kg) and CS (1200 mg/kg) were orally administered to DBA/1J mice once daily for 14 d prior to initial immunization with type II collagen. Their elastase activities and the production of cytokines in sera were examined on type II collagen-induced arthritis in DBA/1J mice. We also compared the paracellular transport of LMWCS and CS across Caco-2 cell monolayers and examined the inhibitory effects on elastase activities. LMWCS inhibited elastase activity slightly, but CS did not show inhibition. Hind paw edema was significantly decreased by LMWCS treatment. Levels of anti-type II collagen antibody and tumor necrosis factor-alpha (TNF-alpha) in sera were also reduced by LMWCS treatment but not in case of CS, although no significant difference was observed between LMWCS and CS on interleukin-6 (IL-6) induction. The LMWCS preparation showed preventive effects on the type II collagen-induced arthritis in DBA/1J mice and better permeability through Caco-2 cells.