Quantitative Analysis of Marker Compounds in <i>Angelica gigas</i>, <i>Angelica sinensis</i>, and <i>Angelica acutiloba</i> by HPLC/DAD

Jeong, Su Yang,Kim, Hye Mi,Lee, Kyu Ha,Kim, Kyu Yeob,Huang, Dae Sun,Kim, Jong Hwan,Seong, Rack Seon Pharmaceutical Society of Japan 2015 Chemical & pharmaceutical bulletin Vol. No.

<P>Although Danggui is the root of Angelica gigas NAKAI in the Korean Pharmacopoeia, it is determined that Danggui is also the root of Angelica sinensis (OLIV.) DIELS in China and Hong Kong, as well as the root of Angelica acutiloba KITAGAWA in Japan. Accordingly, we tried to develop an identification method using the main compounds in A. gigas, A. sinensis, and A. acutiloba through HPLC/diode-array detector (DAD). This method was fully validated for linearity, accuracy, precision, recovery, and robustness. Multivariate analysis was also implemented after pattern analysis and monitoring. As a result, each compound pattern of A. gigas, A. sinensis, and A. acutiloba was identified, making it possible to distinguish them from each other.</P>

<i>In Vitro</i> and <i>in Vivo</i> Antiallergic Effect of the Fructus of <i>Evodia rutaecarpa</i> and Its Constituents

Shin, Yong-Wook,Bae, Eun-Ah,Cai, Xing Fu,Lee, Jung Joon,Kim, Dong-Hyun Pharmaceutical Society of Japan 2007 BIOLOGICAL & PHARMACEUTICAL BULLETIN Vol.30 No.1

<P>The unripe fruit of <I>Evodia rutaecarpa</I> (J<SMALL>USS</SMALL>) B<SMALL>ENTH</SMALL> (ER, Family Rutaceae) has been used frequently as a traditional medicine against inflammatory diseases in Korea, China and Japan. To evaluate antiallergic effect of ER, we isolated its main constituents, evodiamine and rutaecarpine, and evaluated <I>in vivo</I> their inhibitory effects against passive cutaenous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors by compound 48/80. ER and its constituents, evodiamine and rutaecarpine, potently inhibited PCA reaction and scratching behaviors in mice, although ER weakly inhibited scratching behaviors. Evodiamine and rutaecarpine inhibited TNF-α and IL-4 protein expression in RBL-2H3 cells induced by IgE–antigen complex, although these did not inhibit degranulation of RBL-2H3 cells induced by IgE–antigen complex and rat peritoneal mast cells induced by compound 48/80. These findings suggest that ER and its constituents, evodiamine and rutaecarpine, may be effective for IgE-induced allergic diseases such as atopic dermatitis and rhinitis.</P>

Preventive Effect of 7,8-Dihydroxyflavone against Oxidative Stress Induced Genotoxicity

Zhang, Rui,Kang, Kyoung Ah,Piao, Mei Jing,Ko, Dong Ok,Wang, Zhi Hong,Chang, Weon Young,You, Ho Jin,Lee, In Kyung,Kim, Bum Joon,Kang, Sam Sik,Hyun, Jin Won Pharmaceutical Society of Japan 2009 Biological & pharmaceutical bulletin Vol.32 No.2

<P>We elucidated the protective effect of 7,8-dihydroxyflavone against hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>)-induced DNA damage. We found that 7,8-dihydroxyflavone scavenges 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species (ROS). 7,8-Dihydroxyflavone with antioxidant effect prevented the H<SUB>2</SUB>O<SUB>2</SUB>-induced cellular DNA damage, as evidenced by comet tail, 8-hydroxy-2′-deoxyguanosine (8-OHdG) content, and phospho-histone H2A.X protein expression. Hence, 7,8-dihydroxyflavone was shown to protect cell <I>via</I> the inhibition of apoptosis induced by H<SUB>2</SUB>O<SUB>2</SUB>. This was substantiated by decreased apoptotic nuclear fragmentation, decreased sub-G<SUB>1</SUB> cell population, and decreased DNA fragmentation. Furthermore, 7,8-dihydroxyflavone activated the protein kinase B (PKB, Akt) signal pathway, which is a major survival signal pathway. In addition, LY294002, which is phosphatidylinositol 3 kinase (PI3K, upstream of Akt) inhibitor, attenuated the protective effect of 7,8-dihydroxyflavone against H<SUB>2</SUB>O<SUB>2</SUB>-induced cell damage. In conclusion, 7,8-dihydroxyflavone was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing Akt activity.</P>

Proteomic Analysis in Cyclosporin A-Induced Overgrowth of Human Gingival Fibroblasts

Jung, Ji Yeon,Kang, Gi Chang,Jeong, Yeon Jin,Kim, Sun Hun,Kwak, Yong Geun,Kim, Won Jae Pharmaceutical Society of Japan 2009 Biological & pharmaceutical bulletin Vol.32 No.8

<P>Cyclosporin A (CsA) has been used as an immunosuppressive drug to prevent organ transplant rejection and to treat autoimmune diseases. CsA has a proliferative effect on human gingival fibroblasts (HGF) <I>in vitro</I>. However, the molecular mechanisms underlying CsA-induced proliferation in HGF remain to be elucidated. This study was aimed to investigate the CsA responsive proteins in HGF using systematic proteomic approach. Cell viability was determined by MTT assay and reactive oxygen species (ROS) was measured by fluorescent spectrometer. Proteins profiled by two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadrupole time-of-flight mass spectrometry (EIQ-TOF MS). To confirm the expression changes of proteins by proteomics analysis, Western blot was performed using specific antibody. CsA increased the cell viability of HGF in a dose- and time-dependent manner. Significantly, seventeen proteins were overexpressed in the CsA-treated HGF, whereas three proteins were found to be expressed less than the untreated cells. The identified proteins were mainly related with cell proliferation, metabolism, and oxidation. The overexpression of peroxiredoxin 1 (Prx 1) confirmed by Western blotting and reduction of cytosolic reactive oxygen species (ROS) levels in the CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA. Upregulation of Galectin 3 in CsA-treated HGF indicated that it is related to CsA-induced proliferation. These proteomic analysis data will provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA.</P>

A Role for the Val291 Residue within the Transmembrane Domain 2 in Diltiazem- and TMB-8 [3,4,5-Trimethoxybenzoic Acid 8-(Diethylamino)octyl Ester]-Mediated 5-Hydroxytryptamine Type 3A Receptor Regulations

Lee, Byung-Hwan,Choi, Sun-Hye,Pyo, Mi Kyung,Shin, Tae-Joon,Hwang, Sung-Hee,Kim, Bo-Ra,Lee, Jun-Ho,Rhim, Hyewhon,Kim, Hyoung-Choon,Nah, Seung-Yeol Pharmaceutical Society of Japan 2009 Biological & pharmaceutical bulletin Vol.32 No.5

<P>Previous reports have shown that diltiazem and TMB, calcium channel antagonists, inhibit 5-hydroxytryptamine type 3A (5-HT<SUB>3A</SUB>) receptor-mediated currents (<I>I</I><SUB>5-HT</SUB>) in cell lines and in heterologously expressed <I>Xenopus</I> oocytes. In the present study, we sought to elucidate the molecular mechanisms underlying diltiazem- and TMB-induced 5-HT<SUB>3A</SUB> receptor regulations. We used the two-microelectrode voltage clamp technique to investigate the effect of diltiazem and TMB on 5-HT-mediated ion currents in <I>Xenopus</I> oocytes expressing wild-type or 5-HT<SUB>3A</SUB> receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT<SUB>3A</SUB> receptors, diltiazem and TMB dose-dependently inhibited peak <I>I</I><SUB>5-HT</SUB> with an IC<SUB>50</SUB> of 71.4±4.9 and 4.5±0.3 μ<SMALL>M</SMALL>, respectively. Among various mutants of TM2, mutation V291A greatly attenuated and abolished the TMB- and diltiazem-induced inhibition of peak <I>I</I><SUB>5-HT</SUB>, respectively. Mutation V291A also induced constitutively active ion currents in the absence of 5-HT. Diltiazem and TMB inhibited constitutively active ion currents in a dose-dependent manner. The IC<SUB>50</SUB> values of constitutively active ion currents in V291A receptors were 165.3±11.1 and 6.6±0.5 μ<SMALL>M</SMALL> for diltiazem and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), respectively. Results of site-directed mutagenesis experiments suggest that the Val291 residue could be a candidate for common interaction site for diltiazem- and TMB-8-mediated 5-HT<SUB>3A</SUB> receptor regulations.</P>

Mutations Leu427, Asn428, and Leu431 Residues within Transmembrane Domain-I-Segment 6 Attenuate Ginsenoside-Mediated L-Type Ca²⁺ Channel Current Inhibitions

Choi, Sun-Hye,Lee, Jun-Ho,Pyo, Mi Kyung,Lee, Byung-Hwan,Shin, Tae-Joon,Hwang, Sung-Hee,Kim, Bo-Ra,Lee, Sang-Mok,Oh, Jae-Wook,Kim, Hyoung-Chun,Bae, Chun Sik,Rhim, Hyewhon,Nah, Seung-Yeol Pharmaceutical Society of Japan 2009 Biological & pharmaceutical bulletin Vol.32 No.7

<P>Many lines of evidences have shown that <I>Panax ginseng</I> exhibits beneficial effects on cardiovascular systems. We previously demonstrated that ginsenoside Rg<SUB>3</SUB> (Rg<SUB>3</SUB>), one of active ingredients of <I>Panax ginseng</I>, inhibits Ca<SUP>2+</SUP> channel currents in a stereospecific manner and affects the steady-state activation but not inactivation. This points a possibility that Rg<SUB>3</SUB> regulates Ca<SUP>2+</SUP> channels through specific interaction site(s) for Ca<SUP>2+</SUP> influx inhibition through Ca<SUP>2+</SUP> channels. However, it was not known how Rg<SUB>3</SUB> interacts with Ca<SUP>2+</SUP> channel proteins. In the current study, we sought to identify these site(s) in <I>Xenopus</I> oocytes expressing cardiac wild-type and mutant L(α<SUB>1C</SUB>)-type Ca<SUP>2+</SUP> channels using the two-microelectrode voltage-clamp technique. To this end, we assessed how various point mutations of the L-type Ca<SUP>2+</SUP> channel affected the Rg<SUB>3</SUB> action. Mutations of L427R, N428R and L431K in transmembrane domain-I-segment 6 (IS6) of the channel significantly attenuated the Rg<SUB>3</SUB> action and caused rightward shifts in dose–response curves. Rg<SUB>3</SUB> treatment produced a negative shift in the inactivation voltage but did not alter the steady-state activation voltage, and none of the mutant channels affected the Rg<SUB>3</SUB>-induced negative shift of inactivation voltage. Rg<SUB>3</SUB> had no effects on inactivation time constant in wild-type and mutant channels. These results indicate that Rg<SUB>3</SUB> inhibition of L-type Ca<SUP>2+</SUP> channel currents is attenuated by mutations of Leu427, Asn428 and Leu431 in transmembrane IS6 residues. Leu427, Asn428 and Leu431 residues of the L-type Ca<SUP>2+</SUP> channel play important roles in the Rg<SUB>3</SUB> effect on channel properties.</P>

Fatty Acid Synthase Inhibition by Amentoflavone Induces Apoptosis and Antiproliferation in Human Breast Cancer Cells

Lee, Jin Sun,Lee, Myung Sun,Oh, Won Keun,Sul, Ji Young Pharmaceutical Society of Japan 2009 Biological & pharmaceutical bulletin Vol.32 No.8

<P>Fatty acid synthase (FASN) is highly expressed in breast carcinomas to support their continuous growth and proliferation, but has low expression level in normal tissues. Considerable interest has been developed in searching for novel FASN inhibitors as a therapeutic target for breast cancer. In present study, amentoflavone was isolated from <I>Selaginella tamariscina</I>, a traditional oriental medicine that has been used to treat cancer for many years, and was found to significantly inhibit the <I>in vitro</I> enzymatic activity of FASN at concentrations above 50 μ<SMALL>M</SMALL>. Amentoflavone was also found to decrease fatty acid synthesis by the reduction of [<SUP>3</SUP>H]acetyl-CoA incorporation into lipids in FASN-overexpressed SK-BR-3 human breast cancer cells. Furthermore, this study showed that amentoflavone, at a concentration greater than 75 μ<SMALL>M</SMALL>, increased the cleavage-activity of caspase-3 and poly (ADP-ribose) polymerase (PARP), and administration of pan-caspase inhibitor Z-VAD-FMK completely rescued the SK-BR-3 cells from PARP cleavages. The sequential internucleosomal DNA fragmentation in SK-BR-3 cells was observed at a concentration of 100 μ<SMALL>M</SMALL>. A decrease in breast cancer cell growth was observed in SK-BR-3 cells at 12 and 24 h post treatment with 100 μ<SMALL>M</SMALL> of amentoflavone, followed by a dramatic suppression after 48 h. The inhibition of cancer-growth by amentoflavone was dose-dependent, showing a slight reduction at 50 μ<SMALL>M</SMALL> and significant reduction at concentrations of 75 and 100 μ<SMALL>M</SMALL>. FASN-nonexpressed NIH-3T3 normal cell growth was not decreased by amentoflavone-treatment, both in time- and dose-dependent manners. These data provide evidence that amentoflavone isolated from <I>S. tamariscina</I> induced breast cancer apoptosis through blockade of fatty acid synthesis.</P>

Stereoisomer-specific anticancer activities of ginsenoside Rg3 and Rh2 in HepG2 cells: disparity in cytotoxicity and autophagy-inducing effects due to 20(S)-epimers.

Cheong, Jong Hye,Kim, Hyeryung,Hong, Min Jee,Yang, Min Hye,Kim, Jung Wha,Yoo, Hunseung,Yang, Heejung,Park, Jeong Hill,Sung, Sang Hyun,Kim, Hong Pyo,Kim, Jinwoong Pharmaceutical Society of Japan 2015 BIOLOGICAL & PHARMACEUTICAL BULLETIN Vol.38 No.1

<P>Autophagy has been an emerging field in the treatment of hepatic carcinoma since anticancer therapies were shown to ignite autophagy in vitro and in vivo. Here we report that ginsenoside Rg3 and Rh2, major components of red ginseng, induce apoptotic cell death in a stereoisomer-specific fashion. The 20(S)-forms of Rg3 and Rh2, but not their respective 20(R)-forms, promoted cell death in a dose-dependent manner accompanied by downregulation of Bcl2 and upregulation of Fas, resulting in apoptosis of HepG2 cells with poly ADP ribose polymerase cleavage. The LD50 value [45 mu m for Rg3(S), less than 10 mu m for Rh2(S)] and gross morphological electron microscopic observation revealed more severe cellular damage in cells treated with Rh2(S) than in those treated with Rg3(S). Both Rg3(S) and Rh2(S) also induced autophagy when undergoing induced apoptosis. Inhibition of autophagy with lysosomotrophic agents significantly potentiated the cellular damage, implying a favorable switch of the cell fate to tumor cell death. Blocking intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) restored the cell death induced by both Rg3(S) and Rh2(S). Our results suggest that the 20(S)-forms of Rg3 and Rh2 in red ginseng possess more potent antitumor activity with autophagy than their 20(R)-forms via calcium-dependent apoptosis.</P>

Inhibitory Effect of the Compounds Isolated from <i>Rhus verniciflua</i> on Aldose Reductase and Advanced Glycation Endproducts

Lee, Eun Ha,Song, Dae-Geun,Lee, Joo Young,Pan, Cheol-Ho,Um, Byung Hun,Jung, Sang Hoon Pharmaceutical Society of Japan 2008 Biological & pharmaceutical bulletin Vol.31 No.8

<P>The aim of this paper was to evaluate active principles for diabetic complications from <I>Rhus verniciflua</I>. Nine compounds were isolated <I>via</I> bioactivity guided fractionation and isolation and tested for their effects on recombinant human aldose reductase and advanced glycation endproducts. Butein and sulfuretin isolated from ethyl acetate fraction were found to possess strongly both forms of aldose reductase and advanced glycation endproducts inhibition. The inhibitory activity of butein against a recombinant human aldose reductase (IC<SUB>50</SUB> value: 0.5 μ<SMALL>M</SMALL>) was 2.6 times more potent that of epalrestat as a positive control (IC<SUB>50</SUB> value: 1.3 μ<SMALL>M</SMALL>). The inhibitory potency of sulfuretin (IC<SUB>50</SUB> value: 124.7 μ<SMALL>M</SMALL>) on advanced glycation end-products was about 10 times more potent that of aminoguanidine as a positive control (IC<SUB>50</SUB> value: 1231.0 μ<SMALL>M</SMALL>). These compounds all displayed antioxidative activity which was measured by Photochem<SUP>®</SUP> apparatus. It was concluded, therefore, butein and sulfuretin have antioxidative as well as aldose reductase and advanced glycation endproducts inhibitory effects. As a result, these compounds could be proposed as a leading compound for further study as a new natural products drug that could be used for diabetic complications.</P>

High Dosage Sildenafil Induces Hearing Impairment in Mice

Hong, Bin Na,Yi, Tae Hoo,Kim, Sun Yeou,Kang, Tong Ho Pharmaceutical Society of Japan 2008 Biological & pharmaceutical bulletin Vol.31 No.10

<P>Sildenafil is widely administered for the treatment of erectile dysfunction. Recently, sensorineural hearing loss following the ingestion of sildenafil was reported in one male patient. We examined hearing in mice that were administered high doses of sildenafil for up to 105 d. To assess hearing impairment, we evaluated auditory brainstem responses, auditory middle latency responses, and otoacoustic emissions. At high doses, sildenafil increased the hearing threshold shift of auditory brainstem responses. High-dose sildenafil treatment also resulted in delayed latency of both auditory brainstem responses and auditory middle responses. Otoacoustic emissions differed between control and high-dose sildenafil groups with long-term treatment. Collectively, these data demonstrate that high-dose and long-term sildenafil administration can induce hearing impairment in mice.</P>