Studies on the flavones using liquid chromatography-electrospray ionization tandem mass spectrometry

Fragmentation pathways of nine flavone compounds have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn). Analyzing the product ion spectra of flavonoids and aglycones, we observed some diagnostic neutral losses, such as *CH3, H2O, residue of glucose and gluconic acid, which are very useful for the identification of the functional groups in the structures. Furthermore, specific retro Diels-Alder (RDA) fragments for flavones with different hydroxyl substitution have also been discussed. The information is helpful for the rapid identification of the location site of hydroxyl substitution on flavones. Fragmentation pathways of C-glycosidic flavonoid have also been discussed using ESI-MSn, demonstrating ions [M-H-60](-), [M-H-90](-), [M-H-120](-) are characteristic ions of C-glycosidic flavonoid. According to the fragmentation mechanism of mass spectrometry and HPLC-MS data, the structures of seven flavones in Scutellaria baicalensis Georgi have been identified on-line without time-consuming isolation. The HPLC-ESI-MSn method for analyzing constituents in the Scutellaria baicalensis Georgi has been established.

Identification of the flavonoid constituents from leaves of Acanthopanax Senticosus Harms

Four flavonoids from leaves of Acanthopanax Senticosus Harms were observed in negative ion mode in the electrospray mass spectra. Two of them were further isolated and identified as quercitrin (quercetin-3-O-alpha-L-rhamnoside) and hyperin (quercetin-3-O-beta-D-galactoside) on the basis of MS' and NMR data. The other two compounds in the mixtures were tentatively established as quercetin and rutin (quercetin-3-O-rutinoside) in terms of their electrospray tandem mass spectrometry (ESI-MSn) data. Three of the four flavonoids (excluding hyperin) haven't been reported in this plant before.

Analysis of flavonoid constituents from leaves of Acanthopanax senticosus Harms by electrospray tandem mass spectrometry

Three known flavonoids, quercetin, quercitrin (quercetin-3-0-rhamnoside) and rutin (quercetin-3-0-rutinoside), have been identified for the first time in the leaves of Acanthopanax senticosus Harms by using electrospray tandem mass spectrometry techniques (ESI-MSn). The flavonoid hyperin (quercetin-3-0-beta-galactoside), already known to be present, was also investigated. The diagnostic fragment ions of the aglycone quercetin were obtained in the ESI-MSn experiments, and a fragmentation mechanism proposed.

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S-franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 mu m i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25 degrees C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples.

The glycosides from Lomatogonium rotatum

A new phenyl glycoside, 2-(3'-O-beta-D-glucopyranosyl) benzoyloxygentisic acid (1), along with seven known glycosides 2-8 was isolated from Tibetan herbal medicine Lomatogonium rotatum. The structures of the compounds were elucidated by spectroscopic methods including 1D and 2D NMR techniques and MS data.

Two new benzochromone glycosides from the stem of Berchemia racemosa

Two new benzochromone glycosides, rubrofusarin 6-O-alpha-L-rhamnosyl- (1 -> 6)-O-beta-D-glucopyranoside (1) and demethylflavasperone 10-O-beta-D-glucopyranoside (2), have been isolated from the stem of Berchemia racemosa Sieb. et Zucc. (Rhamnaceae). Their structures were elucidated on the basis of spectroscopic evidence.

Two new xanthone glycosides from Swertia tetraptera

Two new xanthone glycosides, tetraswerosides A and B, were isolated from the whole plant of Swertia tertraptera. Their structures were determined as 3-O-beta -D-glucopyranosyl-1-hydroxy-4,7-dimethoxyxanthone and 3-O-[beta -D-xylopyranosyl-(1-->6)-beta -D-glucopyranosyl]-1- hydroxy-4,7-dimethoxyxanthone by spectroscopic methods.

Two new iridoid glycosides from the Tibetan folk medicine Swertia franchetiana

Two new iridoid glycosides designated as senburiside III (2) and senburiside IV (3), together with one known iridoid glycoside senburiside I (1) and three known secoiridoid glucosides swertiamarin (4), gentiopicroside (5) and sweroside (6), were isolated from the whole plant of Swertia franchetiana. The structures of the two new compounds were elucidated by spectroscopic methods.

查尔酮异构酶基因过表达对新疆雪莲类黄酮生物合成的调控

新疆雪莲（Saussurea involucrata Kar. et Kir.）是我国名贵中药材，其主要药用活性成分为黄酮类化合物。目前人们对新疆雪莲及它的黄酮类化合物的需求日益增多，但雪莲的人工栽培技术尚未成熟，在野生状态下，新疆雪莲只能生长在海拔4,000到5,000米的雪山上，现在由于过度采挖已濒临灭绝。为解决雪莲资源匮乏，提高雪莲中黄酮类成分的含量，本研究通过基因工程手段利用发根农杆菌将黄酮代谢途径中的关键酶－查尔酮异构酶（CHI）基因导入新疆雪莲，产生转基因新疆雪莲毛状根及再生苗，以期提高新疆雪莲的黄酮类物质含量，进行新疆雪莲黄酮类物质的生产。主要结果如下： 　　1．对克隆到的水母雪莲查尔酮异构酶基因（Smchi）进行功能分析。转Smchi正义、反义烟草的CHI酶活性实验结果表明，转Smchi正义的烟草CHI酶活性比对照提高3-6倍，而转反义Smchi基因的烟草CHI酶活性比对照则显著降低。分析不同株系的转基因烟草和对照烟草的黄酮含量和花色素含量表明，转Smchi正义的烟草积累比对照显著增高水平的总黄酮，其中株系CS-5黄酮含量是对照的6倍，转Smchi反义的烟草则积累较低水平的总黄酮，而且转基因烟草的总黄酮含量与Smchi基因的表达水平和CHI酶活性成正相关。但不论转Smchi基因正义或反义方向的烟草，其花色素含量和对照相比均没有发生显著变化。进一步对转基因烟草的黄酮成分进行分析，发现烟草中的主要黄酮成分芦丁在转Smchi正义烟草中有很高的积累。 　　2．发根农杆菌介导法将Smchi基因导入新疆雪莲，得到转Smchi基因的新疆雪莲毛状根。实验发现，35S-chi转基因对毛状根的生长没有显著影响，但35S-chi转基因毛状根能够合成显著提高水平的芹菜素和总黄酮，其中根系C46经过35 d培养，能产生32.1 mg/L的芹菜素和647.8 mg/L的总黄酮，分别是对照根系的12倍和4倍;不同根系的Smchi基因表达水平、CHI酶活性和芹菜素含量成正相关。本研究为通过基因工程手段提高新疆雪莲毛状根芹菜素和总黄酮含量提供了一个有效方法。 　　3．在1/2MS附加GA1.5 mg/L的培养基上，新疆雪莲毛状根的不定芽再生频率高且不定芽生长健壮。再生苗在MS+BA1.0 mg/L+NAA0.1 mg/L的培养基上继代培养生长量较大，经过20 d的培养，35S-chi转基因新疆雪莲再生苗株系（C17、C27、C46）、对照再生苗（Control-1）和正常试管苗（Control-2）之间生长量差异不显著，增殖倍数都在7倍左右;实验还发现，毛状根再生苗比各自来源的毛状根的芹菜素和总黄酮含量下降了20-30%，但转基因再生苗的芹菜素和总黄酮含量比Control-1和Control-2都有显著提高，其中C46芹菜素和总黄酮含量分别为1.86 mg/g 干重和37.3 mg/g干重，分别是Control-1的12倍和 2.4 倍，Control-2的4 倍和1.6倍。这些结果表明由毛状根诱导出的再生苗可作为增强目标次生代谢产物生产的另外一个有效来源。 　　

诱导子对水母雪莲次生代谢的影响及雪莲查耳酮合成酶基因克隆

水母雪莲(Saussurea medusa Maxim)为菊科凤毛菊属植物,是名贵中药材。为解决雪莲资源匮乏，我们实验室通过植物组织培养技术，成功的建立起水母雪莲细胞和毛状根体系。通过对它的药理实验及化学成分分析，主要成分为黄酮类物质和紫丁香甙单体。为了进一步提高这些物质在水母雪莲培养物中的含量，本文开展通过添加外源诱导子手段来调控水母雪莲次生代谢合成途径。 利用水杨酸（SA）和酵母提取物（YE）作为外源诱导子，添加到水母雪莲细胞系和毛状根系培养基中，研究诱导子不同添加浓度和不同添加时间对水母莲细胞系和毛状根系的生长及次生物质合成的诱导效应。实验结果发现：对于细胞系来说，SA比YE的诱导效果要好，低浓度SA处理时，不仅能促进细胞的生长，还能提高水母雪莲细胞中黄酮化合物和紫丁香甙的含量。其中，在细胞生长周期的第6天添加终浓度为20 μM的SA，诱导效果表现最佳。在此条件下，细胞内总黄酮产量达到532 mg/l，紫丁香甙为630 mg/l，分别比对照提高了130%，和150%。对于毛状根体系来说，SA和YE生长早期添加会抑制毛状根生长。总体上，YE的诱导效果比SA明显。在第10天添加终浓度为40 μg/ml的YE，总黄酮达到741 mg/l，紫丁香甙达到303 mg/l，分别是对照的2.8和2.5倍。 同时研究了20 μM和100 μM SA诱导下，黄酮合成途径中相关酶的变化。发现，低浓度的SA能在短时间内诱导CHS和CHI表达，24h后PAL酶活性升高到对照的7.5倍，而48 h总黄酮的含量检测到最高值。因此可以初步断定，SA诱导苯基苯丙烷类物质的积累与CHS和CHI表达，PAL酶活性提高有关。 另外，从水母雪莲cDNA中克隆到雪莲黄酮合成途径的第一个关键酶—查耳酮合成酶基因（SmCHS）全长cDNA。此cDNA序列全长为1313bp，其编码的蛋白为389个氨基酸，推测的氨基酸序列与许多物种都高度同源，同源性高达88%。生物信息学分析，SmCHS具有CHS－like保守结构域，其二级结构与苜蓿的CHS十分相似，且苜蓿中的CHS酶活性中心的关键氨基酸位点在SmCHS也一致对应相同，没有突变。因此可以初步推测这个SmCHS应该具有查耳酮合成酶功能。并进一步构建SmCHS植物表达载体，转化拟南芥chs突变体，通过功能互补分析研究此基因的功能。由于时间关系这部分研究尚在进行中。

雪莲细胞培养物中黄酮类物质代谢调控及其生物活性成份分析

水母雪莲（Saussurea medusa Maxim.）和新疆雪莲（Saussurea involucrata Karel. et Kir.）是我国珍稀的药用植物资源，具有清热解毒、止痉镇痛、敛伤、消肿及治疗热病、风湿等多种功效。雪莲的主要药用成份为紫丁香甙（Syringin）、芦丁(Rutin)、高车前素（Hispidulin）和Jaceosidin等苯基丙酸类（phenylpropanoid）和黄酮类（flavonoids）物质。最新的药理研究表明，上述物质还具有抗菌消炎、保肝降压、延缓衰老和抑制癌细胞增殖等重要的研发价值。 雪莲生境恶劣，生长缓慢，人工引种困难，加上长期掠夺性采挖，已使雪莲处于灭绝的边缘。为了保存国家珍稀植物品种，保护生态环境，满足临床上对雪莲药物的需求，本研究在雪莲组织培养的基础上，应用诱导子添加技术和毛状根培养技术对雪莲中具有重要药用价值的次生代谢物质进行调控，并对雪莲MYB类转录因子的功能进行了初步探索，为保护珍稀植物资源、维护生态环境、开发野生雪莲替代产品、缩短雪莲药用成份的生产周期奠定了基础。另外，分析了野生雪莲和雪莲培养物中主要生物活性成份的种类及含量，为今后雪莲药理药效研究及品质评价奠定了基础。 为了提高雪莲黄酮的产量，满足工业化生产的需要，在细胞培养水平上，通过添加茉莉酸甲酯（MJ），对雪莲黄酮类物质的代谢进行调控。研究了诱导子的添加时间、添加浓度对水母雪莲红色系悬浮细胞的生物量和总黄酮产量的影响。发现在细胞培养的指数期（第9天）添加5.0 µmol/L的MJ，可以使总黄酮产量提高2.4倍（1134.5 ± 63.86 mg/L），而雪莲细胞干重（dw）仅比对照提高23.8 %（20.4 ±0.27 g/L）。另外，细胞中苯丙氨酸裂解酶（PAL）的活性分析表明，MJ添加后PAL活性的增加与雪莲总黄酮含量增长之间存在相关性。 在器官培养水平上，对雪莲毛状根的诱导频率及其培养条件进行了研究。结果表明，选择发根农杆菌R1601侵染预培养2天的新疆雪莲根段外植体，毛状根的诱导效率可达到83 %。毛状根的冠瘿碱检测、PCR和Southern分析表明，Ri质粒中的T-DNA已整合到植物基因组中并稳定表达。以新疆雪莲毛状根为外植体，能够容易地获得再生芽。在含有1.0 mg/L 6-BA的MS固体培养基上，其再生频率高达91 ± 5.9 %，是其正常根的2.4倍。而水母雪莲在该培养条件下，仅有少量的畸形芽出现。进而对毛状根的培养条件进行初步研究，结果表明在无激素附加的MS液体培养基中，新疆雪莲的HR1601根系在一个培养周期内（32 天），其生物量能够达到接种量的16倍，而紫丁香甙含量（43.5 ± 1.13 mg/g dw）能够达到野生雪莲的83倍。从而显示了雪莲毛状根培养体系的优良特性。 在基因水平上，对雪莲黄酮类物质代谢调控的研究已经展开。玉米P基因编码的Myb类转录因子能够调节黄酮类物质代谢途径关键酶基因的表达。根据P基因的保守序列设计引物，从雪莲细胞培养物中获得了SmP基因。核酸序列分析表明，SmP基因与烟草中涉及苯丙素类物质代谢途径的LBM 1、LBM 3和MybAS 1基因具有较高的一致性，分别为66 %、60 %和61 %。因此为了研究雪莲SmP基因的功能，构建了正义表达载体，并与先前构建好的反义表达载体分别导入烟草，分析了转基因植株的形态特征及黄酮类物质的含量变化。其中，约有30 %转反义SmP基因的株系表现叶片皱缩、叶脉紊乱、主侧脉角度缩小、叶片、花瓣失去对称性以及花粉败育等性状。 另外，通过正交试验设计优化了雪莲提取工艺的条件，并对雪莲细胞提取物进行了分离纯化。正交试验设计结果表明，温度对雪莲黄酮提取效率的影响极为显著，而分批多次提取比一次性浸提，能够收到较好的提取效果。考虑到工业生产中的实际问题，推荐在60 ℃水浴条件下，采用50 %乙醇对雪莲样品连续浸提2次的方案。对雪莲提取物的纯化研究表明，雪莲成份复杂，仅依靠单一的分离手段，往往难以奏效。另外，野生雪莲及雪莲培养物中生物活性成份的比色法、HPLC（High Performance Liquid Chromatography）、LC-ESI-MS（Liquid Chromotagraphy Electrospray Ionization Mass Spectrometry）分析表明，传统的NaNO2-AlCl3 法测定雪莲总黄酮的含量，结果偏高，不利于雪莲黄酮的实验室研究分析与今后工业化生产的质量监控。而AlCl3 法的显色反应较为特异，今后有望取代NaNO2-AlCl3 法，作为雪莲类药材品质评价的标准。而HPLC-DAD结合LC-ESI-MS可以对雪莲中的主要生物活性成份进行较为准确的定性分析，从而解决了由于缺乏相应的雪莲化合物标准品而难以对雪莲中的成份进行定性定量分析及比较的难题。最后综合利用上述分析方法，对雪莲细胞培养物中的花素类物质进行了分析。结果表明，雪莲细胞中至少含有7种花色素类物质，分别为矢车菊素-3-O-葡萄糖甙及其衍生物、天竺葵素糖甙衍生物和芍药色素糖甙衍生物。

芍药花色形成的化学机制及其化学分类研究

本文以9 个芍药野生种（15 份种质）、104 个品种及2 个牡丹芍药组间杂种的花瓣为材料，利用液质联用技术鉴定了花瓣中的色素成分并探讨了芍药花色形成的化学机制和化学分类法。 结果表明，芍药花中主要含有5 种花青素，即芍药花素-3，5-二葡糖苷（ peonidin-3,5-di-O-glucoside ， Pn3G5G ）; 矢车菊素-3 ， 5- 二葡糖苷（ cyanidin-3,5-di-O-glucoside ， Cy3G5G ）; 天竺葵素-3 ， 5- 二葡糖苷（ pelargonidin-3,5-di-O-glucoside ， Pg3G5G ）; 芍药花素-3- 葡糖苷（peonidin-3-O-glucoside，Pn3G）和矢车菊素-3-葡糖苷（cyanidin-3-O-glucoside，Cy3G）。此外，3 种微量的花青素首次在芍药中发现：它们分别为芍药花素-3-葡萄糖-5-阿拉伯糖苷（peonidin-3-O-glucoside-5-O-arabinoside，Pn3G5Ara）、矢车菊素-3- 葡萄糖-5- 半乳糖苷（ cyanidin-3-O-glucoside-5-O-galactoside ，Cy3G5Gal）和天竺葵素-3-葡萄糖-5-半乳糖苷（pelargonidin-3-O-glucoside-5-Ogalactoside，Pg3G5Gal）。特征花青素Cy3G5Gal 和Pg3G5Gal 仅在新疆芍药（Paeonia anomala L.）及其亚种川赤芍（P. anomala subsp. veitchii(Lynch) D. Y.Hong & K. Y. Pan）中被检测出来，表明它们属于同一个种。Pn3G5Ara 仅存在于欧洲的野生芍药花瓣中，表明中国野生芍药和欧洲芍药的花青素代谢途径不同。 芍药花瓣中主要含有11 种花黄素，均为黄酮醇类物质。包括栎精-3，7 二葡糖苷（ quercetin-3,7-di-O-glucoside ）、山奈酚-3 ， 7 二葡糖苷（kaempferol-3,7-di-O-glucoside）、异鼠李素-3，7 二葡糖苷（isorhamnetin-3,7-di-Oglucoside）、栎精-3-O-(6”-没食子酰基)-葡糖苷 [quercetin-3-O-（6”-O-galloyl）-glucoside] 、栎精-3- 葡糖苷（ quercetin-3-O-glucoside ）、山奈酚-7- 葡糖苷（ kaempferol-7-O-glucoside ）、山奈酚-3-O- （ 6”- 没食子酰基） - 葡糖苷[kaempferol-3-O-（6”-O-galloyl)-glucoside]、异鼠李素-3-O-（6”-没食子酰基）-葡糖苷 [isorhamnetin-3-O- （ 6”-O-galloyl ） -glucoside] 、山奈酚-3- 葡糖苷（kaempferol-3-O-glucoside）、异鼠李素-3-葡糖苷（isorhamnetin-3-O-glucoside）和山奈酚-丙二酰葡糖苷（kaempferol-malonyl-glucoside）。此外，查耳酮在黄色的栽培品种‘黄金轮’和牡丹芍药组间杂交种‘伊藤杂种’中首次被检测到。其化学结构为查耳酮-2’-葡糖苷（chalcononaringenin 2’-O-glucoside），它是花瓣表现出黄色的主要色素，它与黄色牡丹野生种‘滇牡丹’（P. delavayi Franchet）花瓣中主要黄色色素成分一致。 通过对所有芍药野生种和栽培品种的色素分析，研究发现花青素是芍药花瓣中主要的色素，其中Pn3G5G 是花瓣中含量最高的花青素苷，其次为Cy3G5G。3G 型糖苷仅在少数品种中检测出来。此外，黄酮醇是芍药花瓣中重要的辅助色素。山奈酚苷是花瓣中含量最高的黄酮醇类，其次是栎精。 多元线性回归分析的结果表明，芍药花色的形成主要与花瓣中Pn3G5G、Cy3G5G 和Pg3G5G 的含量及总花青素量（TA）有关。根据8 种花青素结构与花色组成，将国内的野生种和大部分品种进行了化学分类：所有样本聚成3 大类，聚类后的树状图与其花色、花色素组成数据相一致，直观反映了野生种和栽培品种花色形成的化学背景和表型相似性程度。 芍药成色机理和化学分类的初步研究，对芍药新花色育种具有重要意义：芍药鲜红色花的育种中，育种亲本应具有高的Cy3G 含量、低的辅助色素效应指数。选育深紫色花或紫黑色花的品种，亲本应具有高的Pn3G5G 含量和低的Pg3G5G 含量。

Xanthohumol, a novel anti-HIV-1 agent purified from Hops Humulus lupulus

Xanthohumol, prenylchacone flavonoid, is a natural product with multi-biofunctions purified from Hops Humulus lupulus. Its anti-HIV-1 activity was tested in the present study. Results showed that xanthohumol inhibited HIV-1 induced cytopathic effects, the production of viral p24 antigen and reverse transcriptase in C8166 lymphocytes at non-cytotoxic concentration. The EC50 values were 0.82, 1.28 and 0.50 mug/ml, respectively. The therapeutic index (TI) was about 10.8. Xanthohumol also inhibited HIV-1 replication in PBMC with EC50 value of 20.74 mug/ml. The activity of recombinant HIV-1 reverse transcriptase and the HIV-1 entry were not inhibited by xanthohumol. The results from this study suggested that xanthohumol is effective against HIV-1 and might serve as an interesting lead compound. It may represent a novel chemotherapeutic agent for HIV-1 infection. However, the mechanism of its anti-HIV-1 effect needs to be further clarified. (C) 2004 Elsevier B.V. All rights reserved.

Anti-human Immunodeficiency Virus-1 Constituents of the Bark of Poncirus trifoliata

A total of 36 compounds (1-36) were obtained from the stem bark of Poncirus trifoliata including three new prenylated flavonoids, (-)-5,4'-dihydroxy-7,8-[(3 '',4 ''-cis-dihydroxy-3 '',4 ''-dihydro)-2 '',2 ''-dimethylpyrano]-flavone (1), (-)-5,4'-dihydroxy-7,8-[(3 ''-hydroxy-4 ''-one)-2 '',2 ''-dimethylpyrano]-flavone (2), and (-)-5,4'-dihydroxy-7,8-[(cis-3 ''-hydroxy-4 ''-ethoxy-3 '',4 ''-dihydro)-2 '',2 ''-dimethylpyrano]-flavone (3). The new structures were elucidated by means of spectroscopic methods. Compounds 1-20 were evaluated for their anti-human immunodeficiency virus-1 (HIV-1) activity, in which 2 showed significant anti-HIV-1 activity with high therapeutic index (T1) of 143.65.

Effects of ginkgo biloba extract on cation currents in rat ventricular myocytes

Ginkgo biloba extract (GBE), a valuable natural product for cerebral and cardiovascular diseases, is mainly composed of two classes of constituents: terpene lactones (e.g., ginkgolide A and B, bilobalide) and flavone glycosides (e.g., quercetin and kaempferol). Its electrophysiological action in heart is yet unclear. In the present study, using whole-cell patch clamp technique, we investigated electrophysiological effects of GBE on cation channel currents in ventricular myocytes isolated from rat hearts. We found that GBE 0.01-0.1% inhibited significantly the sodium current (I-Na), L-type calcium current (I-Ca) and transient outward potassium current (IKto) in a concentration-dependent manner. Surprisingly, its main ingredients, ginkgolide A (GB A), ginkgolide B (GB B) and bilobalide (GB BA) at 0.1 mM did not exhibit any significant effect on these cation channel currents. These results suggested that GBE is a potent non-selective cation channel modulator in cardiaomyocytes. Other constituents (rather than GB A, GB B and GB BA) might be responsible for the observed inhibitory effects of GBE on cation channels. (C) 2004 Elsevier Inc. All rights reserved.