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Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1(G146V/I) variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1(G146V/S) and the reported AmGT8 and AmGT8(A394F). The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1(G146V/I) variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1(G146V/S) and the reported AmGT8 and AmGT8(A394F). The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1(G146V/I) variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1(G146V/S) and the reported AmGT8 and AmGT8(A394F). The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1(G146V/I) variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1(G146V/S) and the reported AmGT8 and AmGT8(A394F). The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1(G146V/I) variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1(G146V/S) and the reported AmGT8 and AmGT8(A394F). The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression- like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. (C) 2014 S. Karger AG, Basel

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression- like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. (C) 2014 S. Karger AG, Basel

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression- like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. (C) 2014 S. Karger AG, Basel

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression- like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. (C) 2014 S. Karger AG, Basel

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression- like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. (C) 2014 S. Karger AG, Basel

Danggui Buxue Tang (DBT; 當歸補血湯) is a Chinese medicinal formula, which has been used for hundreds of years in China. Interestingly, the herbal composition of DBT is distinct in different ancient medical books, and three of them are still used today: (i) DBT1155, named after the recorded year composed of four herbs: Angelica Sinensis Radix (ASR), Astragali Radix (AR), Jujuba Fructus and Zingiberis Rhizoma Recens in a weight ratio of 12: 10 : 5 : 4; (ii) DBT1247 composed only two herbs, AR and ASR in a 5 : 1 ratio; (iii) DBT1687 composes of ASR, AR and Rehmanniae Radix Praeparata in a 2 : 1 : 1 ratio. Although all of them are recorded to have the function of nourishing “Qi” and enriching the “Blood”, DBT1247 is the one being used commonly. However, the systemic comparison among these three formulae has not been done. Here, we chose these three DBT as an example to explain the traditional Chinese medicine (TCM) theory by using chemical and biological approaches. The herbal extracts of three different DBT were standardized using rapid resolution liquid chromatography coupled with an electrospray ionization triple quadrupole tandem mass spectrometry. Eight marker chemicals, including AR-derived astragaloside III, astragaloside IV, calycosin, formononetin, ononin, calycosin-7-O-β-D-glucoside and ASR-derived ferulic acid and ligustilide were used to standardize DBT extracts. The highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin, formononetin and ASR-derived ferulic acid were found in DBT1247. Upon the standardization of DBT1247 extract, the dynamic chemical change during the boiling process was studied. According to the traditional record, DBT1247 should be boiled with moderate heating. Under the moderate heating, the AR-derived ononin and calycosin-7-O-β-D-glucoside could be induced to hydrolyze in forming formononetin and calycosin. Both calycosin and formononetin had b

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

目的:研究黄芪中的7个主要活性成分芒柄花素、芒柄花苷、毛蕊异黄酮葡萄糖苷、毛蕊异黄酮、乙酰黄芪皂苷Ⅰ、黄芪皂苷Ⅱ、黄芪甲苷对缺氧诱导因子 (HIF-1) 表达的影响。方法:将包含有高度保留的HIF-1结合位点的缺氧应答原件 (HRE) 插入到含有荧光素酶报告基因的pBI-GL质粒中, 磷酸钙沉降法将构建的质粒转染到人胚肾 (HEK) 293T纤维原细胞。用不同浓度的药物处理48h后测定荧光素酶的活性, 通过荧光素酶报告基因系统评估HIF-1的表达。结果:10μmol·L-1芒柄花素可促进HIF-1的表达。芒柄花苷与HIF-1的表达呈浓度-效应依赖关系, 低浓度促进其表达, 高浓度抑制其表达。其它5种单体与对照比较无明显作用趋势。结论:本试验可为黄芪的抗肿瘤作用及其他疾病的治疗提供一定依据。OBJECTIVE: To explore the effect of formononetin, ononin, calycosin-O-β-D-glucoside, calycosin, acetylastragalosideⅠ, astragalosideⅡ, astragaloside Ⅳ from Astragalus membranaceus on the expression of hypoxia-induced factor-1 (HIF-1) . METHODS: The HRE, derived from EPO containing a highly conserved HIF-1 binding site, were cloned into pBI-GL vectors that contained luciferase reporter genes. The HEK-293T cells were transfected cDNA constructs by calcium phosphate precipitation methods. The activity of luciferase was determined after 48 h treatment. The expression of HIF-1 was evaluated using luciferase report gene system. RESULTS:10 μmol·L-1 formononetin promote expression of HIF-1.Ononin had an effect on the expression of HIF-1 in a concentration-dependent manner. Low concentration could elevate the expression of HIF-1 while high concentration down-regulated the expression. Other 5 monomers had no obvious effect on the expression of HIF-1. CONCLUSION:The study provide reference for the anti-tumor effect of A. membranaceus and therapeutic effect of it for other disease.

In order to investigate the biochemical mechanism of Dang-Gui Buxue Tang (DBT) involved in its cardioprotective action, the effects of DBT and related preparations on the cellular level of reduced glutathione (GSH) and on susceptibility to menadione-induced toxicity were examined in H9c2 cardiomyocytes. Treatment with herbal extract prepared from the fresh root of Astragalus membranaceus (RAM) or Angelica sinensis (RAS) alone and their combinations (D-1:1-D-10:1) in varying ratios of RAM to RAS (1 : 1 to 10:1, respectively) increased cellular GSH in a concentration-dependent manner, with the effect produced by the D-5:1 extract, an authentic formula of DBT, being the most potent. The enhancement of cellular GSH was found to correlate positively with the degree of cytoprotection against menadione toxicity. Both GSH-enhancing and cytoprotective effects of DBT were largely abolished by GSH depletion as a result of buthionine sulfoximine (BSO)/phorone treatment. The DBT-induced increase in the cellular GSH level and the associated cytoprotection were also suppressed by the treatment with BSO, an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of GSH regeneration. The results indicate that DBT treatment protects against oxidant injury in H9c2 cells, and that the cytoprotective action is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.

In order to investigate the biochemical mechanism of Dang-Gui Buxue Tang (DBT) involved in its cardioprotective action, the effects of DBT and related preparations on the cellular level of reduced glutathione (GSH) and on susceptibility to menadione-induced toxicity were examined in H9c2 cardiomyocytes. Treatment with herbal extract prepared from the fresh root of Astragalus membranaceus (RAM) or Angelica sinensis (RAS) alone and their combinations (D-1:1-D-10:1) in varying ratios of RAM to RAS (1 : 1 to 10:1, respectively) increased cellular GSH in a concentration-dependent manner, with the effect produced by the D-5:1 extract, an authentic formula of DBT, being the most potent. The enhancement of cellular GSH was found to correlate positively with the degree of cytoprotection against menadione toxicity. Both GSH-enhancing and cytoprotective effects of DBT were largely abolished by GSH depletion as a result of buthionine sulfoximine (BSO)/phorone treatment. The DBT-induced increase in the cellular GSH level and the associated cytoprotection were also suppressed by the treatment with BSO, an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of GSH regeneration. The results indicate that DBT treatment protects against oxidant injury in H9c2 cells, and that the cytoprotective action is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.

In order to investigate the biochemical mechanism of Dang-Gui Buxue Tang (DBT) involved in its cardioprotective action, the effects of DBT and related preparations on the cellular level of reduced glutathione (GSH) and on susceptibility to menadione-induced toxicity were examined in H9c2 cardiomyocytes. Treatment with herbal extract prepared from the fresh root of Astragalus membranaceus (RAM) or Angelica sinensis (RAS) alone and their combinations (D-1:1-D-10:1) in varying ratios of RAM to RAS (1 : 1 to 10:1, respectively) increased cellular GSH in a concentration-dependent manner, with the effect produced by the D-5:1 extract, an authentic formula of DBT, being the most potent. The enhancement of cellular GSH was found to correlate positively with the degree of cytoprotection against menadione toxicity. Both GSH-enhancing and cytoprotective effects of DBT were largely abolished by GSH depletion as a result of buthionine sulfoximine (BSO)/phorone treatment. The DBT-induced increase in the cellular GSH level and the associated cytoprotection were also suppressed by the treatment with BSO, an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of GSH regeneration. The results indicate that DBT treatment protects against oxidant injury in H9c2 cells, and that the cytoprotective action is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.

The effects of pretreatment with Dang-Gui Buxue Tang (DBT, a decoction of Astragali and Angelica roots) and its component herb extracts on myocardial ischaemia-reperfusion (IR) injury were examined in rats er vivo. DBT and its component herb extracts could protect against myocardial IR injury in a dose-dependent manner. The more potent cardioprotection afforded by DBT pretreatment than that of a mixture of Astragali and Angelica root extracts was associated with a much higher extraction yield of active ingredients from Angelica root in the herbal decoction. The high level of active ingredients might increase their bioavailability after oral administration. DBT pretreatment could enhance myocardial mitochondrial as well as red blood cell (RBC) glutathione status, thereby increasing their resistance to oxidative stress-induced injury in rats. The measurement of RBC glutathione status may serve as a useful index for the antioxidant effect produced by DBT treatment in human subjects. Copyright (c) 2006 John Wiley & Sons, Ltd.

The effects of pretreatment with Dang-Gui Buxue Tang (DBT, a decoction of Astragali and Angelica roots) and its component herb extracts on myocardial ischaemia-reperfusion (IR) injury were examined in rats er vivo. DBT and its component herb extracts could protect against myocardial IR injury in a dose-dependent manner. The more potent cardioprotection afforded by DBT pretreatment than that of a mixture of Astragali and Angelica root extracts was associated with a much higher extraction yield of active ingredients from Angelica root in the herbal decoction. The high level of active ingredients might increase their bioavailability after oral administration. DBT pretreatment could enhance myocardial mitochondrial as well as red blood cell (RBC) glutathione status, thereby increasing their resistance to oxidative stress-induced injury in rats. The measurement of RBC glutathione status may serve as a useful index for the antioxidant effect produced by DBT treatment in human subjects. Copyright (c) 2006 John Wiley & Sons, Ltd.

The effects of pretreatment with Dang-Gui Buxue Tang (DBT, a decoction of Astragali and Angelica roots) and its component herb extracts on myocardial ischaemia-reperfusion (IR) injury were examined in rats er vivo. DBT and its component herb extracts could protect against myocardial IR injury in a dose-dependent manner. The more potent cardioprotection afforded by DBT pretreatment than that of a mixture of Astragali and Angelica root extracts was associated with a much higher extraction yield of active ingredients from Angelica root in the herbal decoction. The high level of active ingredients might increase their bioavailability after oral administration. DBT pretreatment could enhance myocardial mitochondrial as well as red blood cell (RBC) glutathione status, thereby increasing their resistance to oxidative stress-induced injury in rats. The measurement of RBC glutathione status may serve as a useful index for the antioxidant effect produced by DBT treatment in human subjects. Copyright (c) 2006 John Wiley & Sons, Ltd.

Yip Pui Ying., Thesis submitted in: November 2004., Thesis (M.Phil.)--Chinese University of Hong Kong, 2005., Includes bibliographical references (leaves 147-184)., s in English and Chinese., p.i, 摘要 --- p.iii, Acknowledgment --- p.v, Abbreviations --- p.vii, Table of contents --- p.viii, List of Figures --- p.xii, List of Tables --- p.xvii, Chapter Chapter 1 --- Introduction --- p.1, Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1, Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5, Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8, Chapter 1.3.1. --- DNA sequencing --- p.9, Chapter 1.3.2. --- DNA fingerprinting --- p.14, Chapter 1.3.3. --- Nucleic acid hybridization --- p.19, Chapter 1.4. --- Objectives --- p.20, Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22, Chapter 2.1. --- Introduction --- p.22, Chapter 2.2. --- Materials and Methods --- p.22, Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22, Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22, Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33, Chapter 2.2.2. --- Extraction of total DNA --- p.35, Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35, Chapter 2.2.4. --- Purification of PCR products --- p.38, Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38, Chapter 2.2.5.1. --- Polishing --- p.38, Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38, Chapter 2.2.5.3. --- Transformation --- p.40, Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40, Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41, Chapter 2.2.6. --- Sequencing of ITS regions --- p.41, Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41, Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41, Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42, Chapter 2.2.7. --- Sequence analysis and alignment --- p.42, Chapter 2.3. --- Results --- p.42, Chapter 2.3.1. --- Extraction of total DNA --- p.42, Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44, Chapter 2.3.2.1. --- Interspecific ITS study --- p.44, Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46, Chapter 2.3.3. --- Sequence analysis and alignment --- p.47, Chapter 2.3.3.1. --- Interspecific ITS study --- p.47, Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56, Chapter 2.4. --- Discussions --- p.60, Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60, Chapter 2.4.2. --- The results agreed with previously published works --- p.60, Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60, Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60, Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61, Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting, Chapter 3.1 --- Introduction --- p.62, Chapter 3.2 --- Materials and Methods --- p.62, Chapter 3.2.1 --- Extraction of total DNA --- p.62, Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63, Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63, Chapter 3.3 --- Results --- p.63, Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63, Chapter 3.3.2 --- Fingerprint analysis --- p.69, Chapter 3.4 --- Discussion --- p.85, Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85, Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85, Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86, Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88, Chapter 3.4.5 --- Explanation of results --- p.88, Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89, Chapter 3.4.7 --- Further studies --- p.90, Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91, Chapter 4.1 --- Introduction --- p.91, Chapter 4.2 --- Materials and Methods --- p.92, Chapter 4.2.1 --- Samples for microarray study --- p.92, Chapter 4.2.2 --- Extraction of total DNA --- p.95, Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95, Chapter 4.2.4 --- Preparation of labeled probe --- p.95, Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97, Chapter 4.2.6 --- Preparation of slides --- p.103, Chapter 4.2.7 --- Hybridization and washing --- p.104, Chapter 4.2.8 --- Scanning and data analysis --- p.105, Chapter 4.3 --- Results --- p.105, Chapter 4.3.1 --- DNA extraction --- p.105, Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107, Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112, Chapter 4.3.4 --- Preparation of slides --- p.112, Chapter 4.3.5 --- Scanning and data analysis --- p.116, Chapter 4.4 --- Discussion --- p.134, Chapter 4.4.1 --- Implications --- p.134, Chapter 4.4.2 --- Applying the findings --- p.134, Chapter 4.4.3 --- Ways to maximize specificity --- p.137, Chapter 4.4.4 --- Optimisation --- p.138, Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138, Chapter Chapter Five --- General Discussion and Summary --- p.140, Chapter 5.1. --- Objectives of this study --- p.140, Chapter 5.2. --- rDNA ITS sequencing --- p.140, Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140, Chapter 5.2.2. --- Implications from the results --- p.140, Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141, Chapter 5.3. --- AP-PCR fingerprinting --- p.141, Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141, Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142, Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143, Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143, Chapter 5.4.2. --- Implications from the results --- p.143, Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144, Chapter 5.5. --- Overall summary --- p.144, Chapter 5.6. --- Future studies --- p.146, References --- p.147, Appendix --- p.185, http://library.cuhk.edu.hk/record=b5896535, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Law Sui-Lin., Thesis (M.Phil.)--Chinese University of Hong Kong, 2003., Includes bibliographical references (leaves 99-109)., s in English and Chinese., CONTENTS --- p.i, p.v, 撮要 --- p.vii, ACKNOWLEDGEMENTS --- p.ix, LIST OF FIGURES & TABLES --- p.xi, ABBREVIATIONS --- p.xv, Chapter Chapter 1 --- INTRODUCTION --- p.1, Chapter 1.1 --- The Transition of Hyperplastic to Hypertrophic Growth During Heart Development --- p.1, Chapter 1.2 --- The Controversial Capability of Heart Regeneration --- p.3, Chapter 1.3 --- Challenges in Treating Heart Diseases --- p.5, Chapter 1.4 --- A New Insight Behind Traditional Chinese Medicine (TCM) for Treating Heart Diseases --- p.7, Chapter 1.5 --- The Potential Mitogenic TCMs on Cardiomyocytes --- p.10, Chapter 1.5.1 --- Radix Astragali --- p.11, Chapter 1.5.2 --- Radix Ophiopogonis --- p.12, Chapter Chapter 2 --- MATERIALS & METHODS --- p.14, Chapter 2.1 --- Materials --- p.14, Chapter 2.2 --- Cell Culture --- p.16, Chapter 2.2.1 --- Primary neonatal rat cardiomyocytes cell culture --- p.16, Chapter 2.2.1.1 --- Mayer's hemalum-eosin staining --- p.17, Chapter 2.2.2 --- Primary rat fibroblasts cell culture --- p.18, Chapter 2.2.3 --- H9C2 cardiac cell culture --- p.18, Chapter 2.3 --- TCMs Preparation and Treatment --- p.19, Chapter 2.3.1 --- Preparation of TCMs powder from aqueous extracts --- p.19, Chapter 2.3.2 --- Preparation of culture medium with TCMs powder --- p.19, Chapter 2.3.3 --- Pre-treatment of undifferentiated and differentiated H9C2 cardiac cells with TCMs --- p.20, Chapter 2.3.4 --- Post-treatment of differentiated H9C2 cardiac cells with TCMs --- p.20, Chapter 2.4 --- Assessment of DNA Synthesis and Proliferation --- p.21, Chapter 2.4.1 --- Tritiated thymidine incorporation assay --- p.21, Chapter 2.4.2 --- 5-Bromo-2'-deoxy-uridine (BrdU) assay --- p.22, Chapter 2.4.3 --- Cell counting --- p.23, Chapter 2.4.4 --- Statistical analysis --- p.23, Chapter 2.5 --- Screening of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by cDNA Microarray --- p.25, Chapter 2.5.1 --- Total RNA extraction --- p.25, Chapter 2.5.2 --- RNA labeling --- p.26, Chapter 2.5.2.1 --- Synthesis of fluorescence labeled probe --- p.26, Chapter 2.5.2.2 --- Purification of fluorescence labeled probe --- p.27, Chapter 2.5.3 --- Microarray hybridization --- p.28, Chapter 2.5.3.1 --- Concentration of fluorescence labeled probe --- p.28, Chapter 2.5.3.2 --- Hybridization --- p.28, Chapter 2.5.3.3 --- Post-hybridization treatment --- p.29, Chapter 2.5.4 --- Data collection --- p.29, Chapter 2.5.4.1 --- Scanning of slide --- p.29, Chapter 2.5.4.2 --- Image processing: spots finding and quantification --- p.30, Chapter 2.5.5 --- Data normalization and analysis --- p.30, Chapter 2.6 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after TCM Treatment by RT-PCR --- p.32, Chapter 2.6.1 --- DNase I digestion of total RNA sample --- p.32, Chapter 2.6.2 --- First-strand cDNA synthesis --- p.32, Chapter 2.6.3 --- RT-PCR of the candidate genes --- p.33, Chapter Chapter 3 --- RESULTS --- p.36, Chapter 3.1 --- Neonatal Primary Rat Cardiomyocytes --- p.36, Chapter 3.1.1 --- Preparation of high-purity neonatal primary rat cardiomyocytes --- p.36, Chapter 3.1.2 --- Neonatal primary rat cardiomyocytes ceased to undergo DNA replication after 6-day in vitro culturing --- p.38, Chapter 3.1.3 --- Both MD and HQ promoted the growth of day 1 primary rat cardiomyocytes in dose- and time-dependent manners --- p.40, Chapter 3.1.4 --- HQ is more potent than MD in promoting the growth of day 7 primary rat cardiomyocytes --- p.43, Chapter 3.2 --- H9C2 Cardiac cells --- p.45, Chapter 3.2.1 --- Proliferative effect of MD and HQ on undifferentiated H9C2 cardiac cells --- p.45, Chapter 3.2.2 --- Pre-treatment of HQ on H9C2 cardiac cells during differentiation --- p.50, Chapter 3.2.3 --- Pre-treatment of MD and HQ on differentiated H9C2 cardiac cells --- p.52, Chapter 3.2.4 --- Post-treatment of MD on differentiated H9C2 cardiac cells…… --- p.55, Chapter 3.3 --- Primary Rat Fibroblasts --- p.57, Chapter 3.3.1 --- Proliferative effect of MD and HQ on primary rat fibroblasts --- p.58, Chapter 3.4 --- Screening of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by cDNA Microarray --- p.60, Chapter 3.4.1 --- Differentially expressed genes in undifferentiated H9C2 cardiac cells after HQ treatment --- p.60, Chapter 3.4.2 --- Differentially expressed genes in differentiated H9C2 cardiac cells after HQ treatment --- p.66, Chapter 3.4.3 --- Comparison of differentially expressed genes in both undifferentiated and differentiated H9C2 cardiac cells after HQ treatment --- p.72, Chapter 3.5 --- Confirmation of Differentially Expressed Genes in H9C2 Cells after HQ Treatment by RT-PCR --- p.73, Chapter 3.5.1 --- "Preferential up-regulation of N-G, N-G-dimethylarginine dimethylaminohydrolase mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment " --- p.74, Chapter 3.5.2 --- Preferential up-regulation of heme oxygenase-3 mRNA expression level in undifferentiated H9C2 cardiac cells after HQ treatment --- p.75, Chapter 3.5.3 --- Preferential up-regulation of cyclin B mRNA expression level in differentiated H9C2 cardiac cells after HQ treatment --- p.76, Chapter Chapter 4 --- DISCUSSION --- p.77, Chapter 4.1 --- HQ Being a More Effective Mitogenic TCM than MD on Cardiomyocytes Exerted its Effect in Dose- and Time Dependent --- p.79, Chapter 4.2 --- Mitogenic Effect of Both MD and HQ might Possibly Due to the Regulation of Intrinsic Factors --- p.82, Chapter 4.3 --- HQ Rather Than MD Showed a Higher Specificity in Promoting DNA Synthesis in Cardiomyocytes --- p.83, Chapter 4.4 --- The Differentially Expressed Genes were Supported by The Clinical Functions of HQ --- p.85, Chapter 4.5 --- Relating the Differentially Expressed Genes with Cardiac Growth and Development --- p.87, Chapter 4.6 --- The Hypothetic Mechanisms of Action that HQ Exerted on Cardiac Growth and Development --- p.92, Chapter 4.7 --- Future Prospect --- p.94, Chapter 4.7.1 --- In vivo study of HQ on the proliferation of rat cardiomyocytes from neonatal to postnatal development --- p.94, Chapter 4.7.2 --- The study of transgenic mice carrying the target gene regulated by HQ on cardiac growth and development --- p.96, Chapter 4.7.3 --- The determination of active component of HQ on cardiac growth and development --- p.97, REFERENCES --- p.99, APPENDIX --- p.110, http://library.cuhk.edu.hk/record=b5891630, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

`Dang-Gui Decoction for Enriching the Blood' (BE), a traditional Chinese formulation comprising Angelica sinensis and Astragalus membranaceus, is used for stimulating red blood cell production as well as enhancing cardiovascular function. In the present study, we have demonstrated the myocardial protection afforded by BE pretreatment against ischaemia-reperfusion (IR) injury in isolated-perfused rat hearts, A more complete and potent myocardial protection against IR injury was also shown by a Polygonum multiflorum extract supplemented BE preparation (BEA), The results suggest that the more potent cardioprotective action of BEA may be related to its ability to sustain the myocardial glutathione antioxidant status under conditions of IR-induced oxidative stress, which may possibly in turn result from the synergistic interaction between the BE and Polygonum extract. Copyright (C) 2000 John Wiley & Sons, Ltd.

`Dang-Gui Decoction for Enriching the Blood' (BE), a traditional Chinese formulation comprising Angelica sinensis and Astragalus membranaceus, is used for stimulating red blood cell production as well as enhancing cardiovascular function. In the present study, we have demonstrated the myocardial protection afforded by BE pretreatment against ischaemia-reperfusion (IR) injury in isolated-perfused rat hearts, A more complete and potent myocardial protection against IR injury was also shown by a Polygonum multiflorum extract supplemented BE preparation (BEA), The results suggest that the more potent cardioprotective action of BEA may be related to its ability to sustain the myocardial glutathione antioxidant status under conditions of IR-induced oxidative stress, which may possibly in turn result from the synergistic interaction between the BE and Polygonum extract. Copyright (C) 2000 John Wiley & Sons, Ltd.

`Dang-Gui Decoction for Enriching the Blood' (BE), a traditional Chinese formulation comprising Angelica sinensis and Astragalus membranaceus, is used for stimulating red blood cell production as well as enhancing cardiovascular function. In the present study, we have demonstrated the myocardial protection afforded by BE pretreatment against ischaemia-reperfusion (IR) injury in isolated-perfused rat hearts, A more complete and potent myocardial protection against IR injury was also shown by a Polygonum multiflorum extract supplemented BE preparation (BEA), The results suggest that the more potent cardioprotective action of BEA may be related to its ability to sustain the myocardial glutathione antioxidant status under conditions of IR-induced oxidative stress, which may possibly in turn result from the synergistic interaction between the BE and Polygonum extract. Copyright (C) 2000 John Wiley & Sons, Ltd.