Antibacterial activity-guided fractionation of the $CHCl_3-MeOH$ (1:1) extract of Paeonia suffruticosa root bark furnished three monoterpene glycosides, 6-O-vanillyoxypaeoniflorin (1), mudanpioside-H (2), and galloyl-oxypaeoniflorin (3). Of the isolated compounds, compound 1 is a new compound. All isolated compounds showed broad, but moderate, antibacterial activity with minimum inhibitory concentration (MIC) values in the range of 100 to $500\;{\mu}g/mL$ against eighteen pathogenic microorganisms of concern for public health or zoonosis.

Two isoflavone C-glucosides, puerarin (1) and PG-3 (2), a but-2-enolide, $({\pm})-puerol$ B (3), two isoflavone O-glucosides, daidzin (4) and genistin (5), and three pterocarpans, (-)-medicarpin (6), (-)-glycinol (7) and (-)-tuberosin (8), were isolated from a MeOH extract of the roots of Pueraria Iobata, using an in vitro bioassay based on the inhibition of the formation of advanced glycation end products (AGEs) to monitor chromatographic fractionation. The structures of 1-8 were determined by spectroscopic data interpretation, particularly by 1D- and 2D-NMR studies, and by comparison of these data with values in the literature. All of the isolates (1-8) were evaluated for their inhibitory activity on AGEs formation in vitro. Of these, puerarin (1), PG-3 (2), and $({\pm})-puerol$ B (3) exhibited more potent inhibitory activity than the positive control aminoguanidine.

Previously, we synthesized and evaluated several benzofuran derivatives containing heterocyclic ring substituents linked to the benzofuran nucleus at C-2 by a two- to four-atom spacer as potential anti-HIV-1, anticancer and antimicrobial agents. Among these derivatives, NSC 725612 and NSC 725716 exhibited interesting anti-HIV-1 activity. To further investigate the structure-activity relationship, we synthesized several new benzofuran derivatives derived from 2-acetylbenzofuran (2, 3a-c) and 2-bromoacetylbenzofuran (6; 7a,b; 8a,b). The compounds were designed to comprise the heterocyclic substituents directly linked to the benzofuran nucleus at C-2. Moreover, various related benzimidazoles derived from 2-acetylbenzimidazole and from 2-cyanomethylbenzimidazole (12a,b; 13a,b; 15; 16a,b) were also prepared as isosteres. The synthesized compounds were preliminarily evaluated for their in vitro anti-HIV-1, anticancer and antimicrobial activity. Compounds 2, 3a, 3b, and 12b showed weak anti-HIV-1 activity. Compound 6 exhibited mild activity against S. aureus, while compound 15 had mild activity towards S. aureus and C. albicans. However, no significant anticancer activity was observed with any of the tested compounds. From these results, we conclude that the presence of the spacer between the heterocyclic substituent and the benzofuran nucleus may be essential for the biological activity.

Torilin was purified from Torilis japonica (Houtt.) DC., and its effects on a rapidly activating delayed rectifier $K^+$ channel (hKv1.5), cloned from human heart and stably expressed in Ltk cells, as well as the corresponding $K^+$ current (the ultrarapid delayed rectifier, $I_{KUR}$) were assessed in human atrial myocytes. Using the whole cell configuration of the patch-clamp technique, torilin was found to inhibit the hKv1.5 current in time and voltage-dependent manners, with an $IC_50$ value of $2.51{\pm}0.34\;{\mu}M$ at +60 mV. Torilin accelerated the inactivation kinetics of the hKv1.5 channel, and slowed the deactivation kinetics of the hKv1.5 current, resulting in a tail crossover phenomenon. Additionally, torilin inhibited the hKv1.5 current in a use dependent manner. These results strongly suggest that torilin is a type of open-channel blocker of the hKv1.5 channel.

For the exploration of pharmacophoric moiety of malloapelta B (1) possessing the inhibitory activity of $NF-{\kappa}B$ activation, structural variation of ${\alpha},{\beta}-unsaturated$ carbonyl motif was attempted. 1 was reduced by catalytic hydrogenation, sodium borohydride, and lithium aluminumhydride. Catalytic hydrogenation with 30 psi or 15 psi of $H_2$ gas of 1 generated 8-butyl-5,7-dimethoxy-2,2-dimethylchroman (2) and 1-(5,7-dimethoxy-2,2-dimethylchroman-8-yl)butan-1-one (3), respectively. Reduction with sodium borohydride occurred at the double bond of ${\alpha},{\beta}-unsaturated$ ketone of 1 to give 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)butan-1-one (4). Reduction of 1 with lithium aluminumhydride and then quenched with methanol and water produced unexpected products, 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)-3-methoxy-1-butene (5) and 1-(5,7-dimethoxy-2,2-dimethyl-2H-chromen-8-yl)-3-hydroxy-1-butene (6). These are formed from the isomerization of initial product 9 through the continuous conjugate carbocation intermediate 11. Addition of ethylmagnesium bromide and dimethyl malonate anion to 1 gave the conjugate adducts 7 and 8. Ethylmagesium bromide and sodium borohydride reduction unusually gave the conjugate addition due to steric congestion around carbonyl group of 1. Compound 2 exhibits the reduced inhibitory activity against $NF-{\kappa}B$ activation and the others do not show the activity. Therefore ${\alpha},{\beta}-unsaturated$ carbonyl group of 1 should be important for its inhibitory activity.

Three new sesquiterpene hydroperoxides, 1-[3-(2-hydroperoxy-3-methylbut-3-en)-4-hydroxyphenyl]ethanone (2), $7{\beta}-hydroperoxy-eudesma-11-en-4-ol$ (3), and $7{\alpha}-hydroperoxymanool$ (4), together with three known compounds, germacrone (1), ent-germacra-4(15),5,10(14)-trien-$1{\alpha}$-ol (5) and teucdiol A (6) were isolated from the aerial parts of Aster spathulifolius (Compositae). Their structures were characterized using chemical and spectroscopic methods. The isolated compounds were tested for their cytotoxicity against five human tumor cell lines in vitro using a SRB method. The two new compounds, 3 and 4, showed moderate cytotoxicity against human cancer cells with $ED_{50}$ values ranging from 0.24 to $13.27\;{\mu}g/mL$.

Previous screening of the pharmacological action of Gastrodia elata (GE) root (Orchidaceae) showed that methanol (MeOH) extracts have significant anti-inflammatory properties. The antiinflammatory agents of GE, however, remain unclear. In this experiment, MeOH extracts of GE were fractionated with organic solvents for the anti-inflammatory activity-guided separation of GE. Eight phenolic compounds from the ether (EtOEt) and ethyl acetate (EtOAc) fractions were isolated by column chromatography: 4-hydroxybenzaldehyde (I), 4-hydroxybenzyl alcohol (II), benzyl alcohol (III), bis-(4-hydroxyphenyl) methane (IV), 4(4'-hydroxybenzyloxy)benzyl-methylether (V), 4-hydroxy-3-methoxybenzyl alcohol (VI), 4-hydroxy-3-methoxybenzaldehyde (VII), and 4-hydroxy-3-methoxybenzoic acid (VIII). To investigate the anti-inflammatory and anti-oxidant activity of these compounds, their effects on carrageenan-induced paw edema, arachidonic acid (AA)-induced ear edema and analgesic activity in acetic acid (HAc)-induced writhing response were carried out in vivo; cyclooxygenase (COX) activity, reactive oxygen species (ROS) generation in rat basophilic leukemia (RBL 2H3) cells and 1,1-diphenyl-2-picryl-hydroazyl (DPPH) scavenging activity were determined in vitro. These phenolic compounds not only had anti-inflammatory and analgesic properties in vivo, but also inhibited COX activity and silica-induced ROS generation in a dose-dependent manner. Among these phenolic compounds, compound VII was the most potent anti-inflammatory and analgesic. Compound VII significantly inhibited silica-induced ROS generation and compound VI significantly increased DPPH radical scavenging activity. Compounds I, II and III significantly inhibited the activity of COX-I and II. These results indicate that phenolic compounds of GE are anti-inflammatory, which may be related to inhibition of COX activity and to anti-oxidant activity. Consideration of the structure-activity relationship of the phenolic derivatives from GE on the anti-inflammatory action revealed that both C-4 hydroxy and C-3 methoxy radicals of benzyl aldehyde play an important role in anti-inflammatory activities.

With an approach to study the anti-tumor effects and mechanism of selenium compound, we investigated the anti-tumor activity and mechanism of $Na_5SeV_5O_{18}{\cdot}3H_2O$ (NaSeVO) in K562 cells. The results showed that $0.625{\sim}20\;mg/L$ NaSeVO could significantly inhibit the proliferation of K562 cells in vitro in a time- and concentration-dependent manner as determined by microculture tetrazolium (MTT) assay, the IC50 values were 14.41 (4.45-46.60) and 3.45 (2.29-5.22) mg/L after 48 hand 72 h treatment with NaSeVO respectively. In vivo experiments demonstrated that i.p. administration of 5, 10 mg/kg NaSeVO exhibited an significant inhibitory effect on the growth of transplantation tumor sarcoma 180 (S180) and hepatoma 22 (H22) in mice, with inhibition rate 26.8% and 58.4% on S180 and 31.3% and 47.4% on H22, respectively. Cell cycle studies indicated that the proportion of G0/G1 phase was increased at 2.5 mg/L while decreased at 10 mg/L after treatment for 24, 48 h. Whereas S phase was decreased at 2.5-5 mg/L and markedly increased at 10 mg/L after treatment for 48 h. After treatment for 24 h, 10 mg/L NaSeVO also markedly increased S and G2/M phases. Take together, the result clearly showed that NaSeVO markedly increased S and G2/M phases at 10 mg/L. The study of immunocytochemistry showed that the expression bcl-2 is significantly inhibited by 10 mg/L NaSeVO, and bax increased. Morphology observation also revealed typical apoptotic features. NaSeVO also significantly caused the accumulation of $Ca^{2+}$ and $Mg^{2+}$, reactive oxygen species (ROS) and the reduction of pH value and mitochondrial membrane potential in K562 cells as compared with control by confocal laser scanning microscope. These results suggest that NaSeVO has anti-tumor effects and its mechanism is attributed partially to apoptosis induced by the elevation of intracellular $Ca^{2+}$, $Mg^{2+}$ and ROS concentration, and a reduction of pH value and mitochondria membrane potential (MMP).

When patients with cancers are treated with chemotherapeutic agents a long time, some of the cancer cells develop the multidrug resistance (MDR) phenotype. MDR cancer cells are characterized by the overexpression of multidrug resistance1 (MDR1) gene which encodes P-glycoprotein (Pgp), a surface protein of tumor cells that functions to produce an excessive efflux and thereby an insufficient intracellular concentration of chemotherapeutic agents. A variety of studies have sought potent MDR modulators to decrease MDR1 gene expression in cancer cells. Our previous study has shown that curcumin exhibits characteristics of a MDR modulator in KB-V1 multidrug-resistant cells. The aim of this study was to further investigate the effect of curcumin on MDR1 gene expression in patient leukemic cells. The leukemic cells were collected from 78 childhood leukemia patients admitted at Maharaj Nakorn Chiang Mai Hospital, Chiang Mai, Thailand, in the period from July 2003 to February 2005. There were 61 cases of acute lymphoblastic leukemia (ALL), 14 cases of acute myeloblastic leukemia (AML), and 3 cases of chronic myelocytic leukemia (CML). There were 47 males and 31 females ranging from 1 to 15 years old. Bone marrows were collected. The leukemic cells were separated and cultured in the presence or absence of $10{\mu}M$ curcumin for 48 hours. MDR1 mRNA levels were determined by RT-PCR. It was found that curcumin reduced MDR1 gene expression in the cells from 33 patients (42%). Curcumin affected the MDR1 gene expression in 5 of 11 relapsed cases (45%), 10 of 26 cases of drug maintenance (38%), 7 of 18 cases of completed treatment (39%), and 11 of 23 cases of new patients (48%). The expression levels of MDR1 gene in leukemic patient cells as compared to that of KB-V1 cells were classified as low level (1-20%) in 5 of 20 cases (25%), medium level (21-60%) in 14 of 32 cases (44%), and high level (61-100%) in 14 of 20 cases (70%). In summary, curcumin decreased MDR1 mRNA level in patient leukemic cells, especially in high level of MDR1 gene groups. Thus, curcumin treatment may provide a lead for clinical treatment of leukemia patients in the future.

Methyl gallate (MG) is a medicinal herbal product that is isolated from Paeonia lactiflora that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin $D_2\;(PGD_2)$ generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with an $IC_{50}$ values of $17.0\;{\mu}M$. This compound also found inhibited the COX-2-dependent conversion of the exogenous arachidonic acid to $PGD_2$ in a dose-dependent manner with an $IC_{50}$ values of $190\;{\mu}M$, using a COX enzyme assay kit. However, at concentrations up to $80\;{\mu}M$, MG did not inhibit COX-2 protein expression in BMMC, indicating that MG inhibits COX-2 activity directly. Furthermore, MG consistently inhibited the production of leukotriene $C_4\;(LTC_4)$ in a dose dependent manner, with an $IC_{50}$ value of $5.3\;{\mu}M$. These results demonstrate that MG has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity, which might provide the basis for novel anti-inflammatory drugs.

To develop a $^{13}C-urea-containing$ capsule for more economic and sensitive diagnosis of Helicobacter pylori infection, the $^{13}C-urea-containing$ capsules were prepared with various additives such as polyethylene glycol, microcrystalline cellulose, sodium lauryl sulfate and citric acid. Their dissolution test and $^{13}C-urea$ Breath Test in human volunteers were then performed. Polyethylene glycol increased the initial dissolution rates of urea and difference ${\sigma}$ $^{13}C$ values from $^{13}C-urea$, while microcrystalline cellulose and sodium lauryl sulfate decreased them. Irrespective of addition of citric acid, the compositions with polyethylene glycol showed higher values from $^{13}C-urea$ compared to a commercial 76 mg $^{13}C-urea-containing$ capsule due to higher initial dissolution rate. The capsules with 38 mg $^{13}C-urea$ and 1.9 mg polyethylene glycol, which showed higher Helicobacter pylori-positive value of about $8{\textperthousand}$ at 10 min, improved the sensitivity of $^{13}C-urea$ in human volunteers. Thus, the $^{13}C-urea-containing$ capsule with polyethylene glycol would be a more economical and sensitive preparation for diagnosis of Helicobacter pylori infection.

Mutation in the presenilin 2 (PS2mt) is known to be one of factors involved in the development of Alzheimer's disease (AD). It was recently revealed that an abnormality of lipid metabolism is a phenomenon occurring in AD. Therefore, the aim of this study was to investigate the potential relationship between the mutation of PS2 and alterations of the lipid profile within the brain. The results showed there increases in the levels of cholesterol, low density lipoprotein and triglyceride, but a decrease in the level of high density lipoprotein in brain tissues expressing mutant PS2. These findings indicated that PS2mt is involved in the abnormalities of the lipid profile, which could cause or result in the development of AD.

In inflammatory responses, induction of cytokines and other immune regulator genes in macrophages by pathogen-associated signal such as lipopolysaccharide (LPS) plays a crucial role. In this study, the gene expression profile changes by LPS treatment in the macrophage/monocyte lineage cell line RAW264.7 was investigated. A 60-mer oligonucleotide microarray of which probes target 32381 mouse genes was used. A reverse transcription-in vitro translation labeling protocol and a chemileuminescence detection system were employed. The mRNA expression levels in RAW264.7 cells treated for 6 h with LPS and the control vehicle were compared. 747 genes were up-regulated and 523 genes were down-regulated by more than 2 folds. 320 genes showing more than 4-fold change by LPS treatment were further classified for the biological process, molecular function, and signaling pathway. The biological process categories that showed high number of increased genes include the immunity and defense, the nucleic acid metabolism, the protein metabolism and modification, and the signal transduction process. The chemokine-cytokine signaling, interleukin signaling, Toll receptor signaling, and apoptosis signaling pathways involved high number of genes differentially expressed in response to LPS. These expression profile data provide more comprehensive information on LPS-target genes in RAW264.7 cells, which will be useful in comparing gene expression changes induced by extracts and compounds from anti-inflammatory medicinal herbs.

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosis in vivo, and platelet aggregation in vitro and ex vivo. Administration of KRG to rats not only prevented carotid artery thrombosis in vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregation ex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its anti platelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with $IC_{50}$ values of $620{\pm}12$, $823{\pm}22$, $722{\pm}21$ and $650{\pm}14\;{\mu}g/mL$, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effect in vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.

The inhibitory effects of paeonol, a major compound of Paeoniae radix, on the development of locomotor sensitization, conditioned place preference (CPP) and dopamine receptor supersensitivity induced by the repeated administration of morphine were investigated through behavioral experiments. A single administration of morphine produces hyperlocomotion. Repeated administration of morphine develops sensitization (reverse tolerance), a progressive enhancement of locomotion, which is used as a model for studying the drug-induced drug-seeking behaviors, and CPP, which is used as a model for studying drug reinforcement. Paeonol inhibited morphine-induced hyperlocomotion, sensitization and CPP. In addition, paeonol inhibited the development of postsynaptic dopamine receptors supersensitivity, which may be an underlying common mechanism that mediates the morphine-induced dopaminergic behaviors such as sensitization and CPP. Apomorphine (a dopamine agonist)-induced climbing behaviors also were inhibited by a single direct administration of paeonol. These results provide evidence that paeonol exerts anti-dopaminergic activity, and it is suggested that paeonol may be useful for the prevention and therapy of these adverse actions of morphine.

Phenolic antioxidant butylated hydroxyanisole (BHA) is a commonly used food preservative with broad biological activities, including protection against chemical-induced carcinogenesis, acute toxicity of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, as well as its undesirable potential tumor-promoting activities. Understanding the molecular basis underlying these diverse biological actions of BHA is thus of great importance. Here we studied the pharmacokinetics, activation of signaling kinases and induction of phase II/III drug metabolizing enzymes/transporter gene expression by BHA in the mice. The peak plasma concentration of BHA achieved in our current study after oral administration of 200 mg/kg BHA was around $10\;{\mu}M$. This in vivo concentration might offer some insights for the many in vitro cell culture studies on signal transduction and induction of phase II genes using similar concentrations. The oral bioavailability (F) of BHA was about 43% in the mice. In the mouse liver, BHA induced the expression of phase II genes including NQO-1, HO-1, ${\gamma}-GCS$, GST-pi and UGT 1A6, as well as some of the phase III transporter genes, such as MRP1 and Slco1b2. In addition, BHA activated distinct mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), as well as p38, suggesting that the MAPK pathways may play an important role in early signaling events leading to the regulation of gene expression including phase II drug metabolizing and some phase III drug transporter genes. This is the first study to demonstrate the in vivo pharmacokinetics of BHA, the in vivo activation of MAPK signaling proteins, as well as the in vivo induction of Phase II/III drug metabolizing enzymes/transporters in the mouse livers.

Prolonged release micro particles of clarithromycin (CL) were prepared using Eudragit RL 100 and RS 100 by spray-drying and casting-drying techniques. For the characterization of those microparticles, preparation yield, particle size distribution, X-ray diffraction, thermal behavior, active agent content and in vitro dissolution from the microparticles were performed. HPLC was used for the assay of clarithromycin and the assay method was validated. All the formulations obtained showed prolonged release when compared to pure clarithromycin. Microparticles prepared by spray-drying method had a slower release compared to those of casting drying method. Spray-drying method seems to be a more suitable method to prepare microparticles for prolongation in release.

Percutaneous delivery of NSAIDs has advantages of avoiding hepatic first pass effect and delivering the drug for extended period of time at a sustained, concentrated level at the inflammation site that mainly acts at the joint and the related regions. To develop the new topical formulations of pranoprofen that have suitable bioadhesion, the gel was formulated using hydroxypropyl methylcellulose (HPMC) and poloxamer 407. The effects of temperature on drug release was performed at $32^{\circ}C$, $37^{\circ}C$ and $42^{\circ}C$ according to drug concentration of 0.04%, 0.08%, 0.12%, 0.16%, and 0.2% (w/w) using synthetic cellulose membrane at $37{\pm}0.5^{\circ}C$. The increase of temperature showed the increased drug release. The activation energy (Ea), which were calculated from the slope of lop P versus 1000/T plots was 11.22 kcal/ mol for 0.04%, 10.79 kcal/mol for 0.08%, 10.41 kcal/mol for 0.12% and 8.88 kcal/mol for 0.16% loading dose from the pranoprofen gel. To increase the drug permeation, some kinds of penetration enhancers such as the ethylene glycols, the propylene glycols, the glycerides, the non-ionic surfactants and the fatty acids were incorporated in the gel formulation. Among the various enhancers used, propylene glycol mono laurate showed the highest enhancing effects with the enhancement factor of 2.74. The results of this study suggest that development of topical gel formulation of pranoprofen containing an enhancer is feasible.