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PURPOSE: C-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor that is overexpressed in many cancers. CtBP1 transcriptionally represses a broad array of tumor suppressors, which promotes cancer cell proliferation, migration, invasion, and resistance to apoptosis. Recent studies have demonstrated that CtBP1 is a potential target for cancer therapy. This study was designed to screen for compounds that potentially target CtBP1.METHODS: Using a structure-based virtual screening for CtBP1 inhibitors, we found protocatechuic aldehyde (PA), a natural compound found in the root of a traditional Chinese herb, Salvia miltiorrhiza, that directly binds to CtBP1. Microscale thermophoresis assay was performed to determine whether PA and CtBP1 directly bind to each other. Further, clustered regularly interspaced short palindromic repeats associated Cas9 nuclease-mediated CtBP1 knockout in breast cancer cells was used to validate the CtBP1 targeting specificity of PA.RESULTS: Functional studies showed that PA repressed the proliferation and migration of breast cancer cells. Furthermore, PA elevated the expression of the downstream targets of CtBP1, p21 and E-cadherin, and decreased CtBP1 binding affinity for the promoter regions of p21 and E-cadherin in breast cancer cells. However, PA did not affect the expression of p21 and E-cadherin in the CtBP1 knockout breast cancer cells. In addition, the CtBP1 knockout breast cancer cells showed resistance to PA-induced repression of proliferation and migration.CONCLUSION: Our findings demonstrated that PA directly bound to CtBP1 and inhibited the growth and migration of breast cancer cells through CtBP1 inhibition. Structural modifications of PA are further required to enhance its binding affinity and selectivity for CtBP1.
Subject(s)
Humans , Apoptosis , Asian People , Breast Neoplasms , Breast , Cadherins , Carrier Proteins , Cell Proliferation , Clustered Regularly Interspaced Short Palindromic Repeats , Mass Screening , Promoter Regions, Genetic , Repression, Psychology , Salvia miltiorrhiza , Sensitivity and SpecificityABSTRACT
Objective: To study the secondary metabolites from the fermentation broth of marine-derived fungus Aspergillus sp. SCS-KFD66. Methods: The constituents were isolated and purified by silica gel, Sephadex LH-20 column chromatography and HPLC methods. The structures of the compounds were identified by spectral data analysis. The DPPH radical scavenging, acetylcholinesterase and α-glucosidase inhibitory activities of compounds were evaluated by DPPH method, Ellman colorimetric method and PNPG method, respectively. The inhibitory effect and the minimal inhibitory concentration (MIC) of compounds on Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes were tested using 96-well microtiter plates method. Results: A total of 13 compounds were isolated from the fermentation broth of marine-derived fungus Aspergillus sp. SCS-KFD66 and identified as 3-epi-trans-4-hydroxylinalool 3,6-oxide (1), trans-4-hydroxylinalool 3,6-oxide (2), aloe emodin (3), emodin (4), citreorosein (5), protocatechualdehyde (6), 2,5-dihydroxybenzaldehyde (7), methyl 4-hydroxyphenylacetate (8), 4-hydroxybenzoic acid (9), 4-hydroxyphenylacetic acid (10), 2-(4-hydroxyphenyl) ethanol (11), (E)-p-hydroxycinnamic acid (12) and (E)-ferulic acid (13), respectively. Compounds 1, 6, 7 and 9-13 showed DPPH radical scavenging activities; Compounds 4 and 6 showed acetylcholinesterase inhibitory activity; Compounds 6 and 7 exhibited inhibitory activity against α-glucosidase. Compound 4 has inhibitory activity against S. aureus and B. subtilis with the MIC values of 16 μg/mL and 64 μg/mL, respectively. Compound 6 showed inhibitory activity against E. coli, B. subtilis and L. monocytogenes, with MIC values of 64, 128 and 128 μg/mL, respectively. Conclusion: Twelve known compounds and one new compound were isolated. Compound 1 is a new compound named as aspergfuranol.
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Objective:To establish an HPLC method for the simultaneous determination of six active constituents ( ferulic acid, chlorogenic acid,ephedrine hydrochloride,protocatechuic acid,protocatechualdehyde and naringin) in Jizhi syrups. Methods:A Zorbax XDB-C18(4.6 mm×250 mm,5 μm)column was used. The mobile phase consisted of acetonitrile (A)-0.9% acetic acid (B) with gradient elution at the flow rate of 1. 0 ml·min-1 . The detection wavelength was changed as follows:257 nm for 0-22. 0 min, 326 nm for 22. 0-30. 0 min, 320 nm for 30. 0-52. 0 min, 210 nm for 52. 0-58. 0 min and 283 nm for 58. 0-60. 0 min. The column temperature was 30 ℃ and the injection volume was 10 μl. Results:The separation of the six active constituents was good. The linear range ( r>0. 9990) was 2. 817-112. 670, 2. 342-93. 670, 0. 710-28. 415, 0. 776-31. 035, 0. 694-27. 755 and 1. 279-51. 175 ng for ferulic acid, chlorogenic acid, ephedrine hydrochloride, protocatechuic acid, protocatechualdehyde and naringin, respectively. The average recover-ies varied from 99.3% to 99.8%(RSD varied from 0.18% to 0.28%). Conclusion: The method is rapid with high sensitivity, promising accuracy and good specificity, which can provide scientific basis for the quality control of Jizhi syrups.
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Objective: To study the chemical compositions from the root tuber of Tetrastigma hemsleyanum and their anti-oxidative activities. Methods: The compounds were isolated by various column chromatography. Their structures were identified by spectroscopic methods including 1H-NMR, 13C-NMR, MS, HR-MS, etc. The anti-oxidative activities were evaluated by DPPH radical scavenging assay. Results: Fourteen compounds were isolated from the root tuber of T. hemsleyanum, they were identified as kaempferol (1), quercetin (2), salicylic acid (3), benzoic acid (4), oxyresveratrol (5), catechin (6), L-epicatechin (7), epigallocatechin (8), procyanidin B2 (9), procyanidin B1 (10), 3-O-caffeoylquinic acid (11), protocatechualdehyde (12), p-hydroxybenzoic acid (13), and protocatechuic acid (14). The anti-oxidative IC50 values of compounds 2, 8, 9, 10, and 12 were 14.15, 14.19, 15.99, 12.4, and 15.98 μmol/L, respectively, better than the positive control Vit C (IC50 value was 23.0 μmol/L). Conclusion: Compounds 4-12 are isolated from T. hemsleyanum for the first time. It has been demonstrated that the root tuber contain flavonoids and also is rich in organic acids. Moreover, compounds 2, 8, 9, 10, and 12 have the certain prospects in the development of natural anti-oxidative agents.
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Objective:To establish an HPLC method to simultaneously determine the content of protocatechualdehyde, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza Bunge. Methods: The chromatographic column was a Linksil-ODS ( 250 mm × 4. 6 mm,5 μm) column,the mobile phase was 1% acetic acid water solution-acetonitrile (7∶3) with the flow rate of 0. 8 ml·min-1 . The detection wavelength was 280nm , the column temperature was 30℃ and the injection volume was 20 μl. Results: The linear range of protocatechualdehyde, rosmarinicacid and salvianolic acid A was 3-300 μg·ml-1(r>0. 999 0), and the recovery was within the range of 95. 22%-99. 68% with RSD below 2% (n=5). Conclusion:The method is accurate, rapid and reproducible, and can be used to control the quality of Salvia miltiorrhiza Bunge.
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Objective To develop an RP-HPLC method for determination of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid, tanshinone II A and astragaloside in blood-invigorating and stasis-removing prescription (BSP). Methods The analysiswas performed with a column of Waters Symmetry Shield™ RP C18(150 mm × 4. 6 mm, 3. 5 μm), and the mobile phase consisted of methanol-0. 25% acetic acid. The flow rate was 0. 8 mL/min and the column temperature was 30°C. UV was employed to determine the contents of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid and tanshinone II A, and the detection wavelength was set at 280 nm. Evaporative light scattering detection (ELSD) was employed to determine the contents of astragaloside. The temperature of drift tube was 90°C and the gas flow was 2. 8 L/min (compressed air). Results The linearity was obtained over 0. 01-0. 80 μg (r = 0. 999 8) for tanshinol, 0. 005-0. 4 μg (r=0. 999 7) for protocatechualdehyde, 0. 05-4 μg (r = 0. 999 8) for paeoniflorin, 0. 005-0. 4 μg (r = 0. 999 7) for puerarin, 0.006-0.5 μg (r=1) for ferulic acid, 0. 005-0. 4 μg (r=1) for tanshinone II A, and 0. 031-2. 46 μg (r= 0. 999 3) for astragaloside. The recoverieswere all between 97. 0%-101. 0%, and RSDs were all less than 2%. The contents (mean) of tanshinol, protocatechualdehyde, paeoniflorin, puerarin, ferulic acid, tanshinone H A and astragaloside in three batches of samples were 1. 15, 0. 13, 4. 48, 0. 80, 0. 72, 0. 31 and 3. 12 mg/g, respectively. Conclusion The method in our study is convenient, accurate and sensitive, and it provides a reference for the determination of active ingredients in BSP.
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OBJECTIVE: To establish a LC-MS method for simultaneous determination of 5 compounds (astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1) in Qishenyiqi Dropping Pills. METHODS: The chromatographic separation was carried out at 30°C on a Wonda Sil C18 column (4.6 mm × 150 mm, 5 μm) eluted in a gradient program. The mobile phase consisted of water containing 10 mmol · L-1 ammonium formate and 0.2% formic acid-acrtonitrile containing 0.2% formic acid. The mass spectra were obtained by Agilent 6410 triple quad mass spectrometer with electrospray ionization source in negative ion mode. RESULTS: The linear ranges for astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1 were 10.72-536.0, 89.10-4 455, 1.337-66.87, 22.60-1 130 and 23.55-1 177 ng · mL-1, respectively. The average recoveries ranged between 98%-102%. CONCLUSION: The established method is convenient, sensitive and accurate. It can be used for the determination of the contents of astragaloside IV, danshensu, protocatechualdehyde, ginsenosides Rg1 and Rb1 in Qishenyiqi Dropping Pills for quality control.
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OBJECTIVE: To establish an high performance capillary electrophoresis (HPCE) method for simultaneous determination of rosmarinic acid, protocatechualdehyde, danshensu, salvanic acid B, and salvianolic acid A in Salviae Miltiorrhizae Radix et Rhizoma. And to determine the optimum drying temperature for the herb. METHODS: 10 mmol · L-1 boric acid -12 mmol · L-1 monosodium phosphate-10 mmol · L-1 SDS was used as buffer solution, an uncoated fused silica capillary column (75 μm × 64.5 cm, 56 cm of effective length) was used, the separation voltage was 18 kV, detection wavelength was 206 nm, column temperature was maintained at 20°C, and sample was injected at 5 kPa × 6 s. RESULTS: Five components showed good linearity (r>0.9990) in the ranges of the tested concentrations. The optimum drying temperature was 100°C. CONCLUSION: The method is simple, accurate and reproducible, and can be used for the quality control of Salviae Miltiorrhizae Radix et Rhizoma.
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Objective: To study and determine the Water-soluble substances of Salvia chinensis. Methods: The constituents of the n-Butanol-soluble portion of the water extractive were isolated and purified by means of Sephadex LH20 column chromatographic methods. HPLC was used to determine the contents. Results: Two compounds were isolated and identified as danshensu and rosmarinic acid. The linear range of danshensu was 1.48～7.40 μg. The average recovery was 102.8% and RSD was 1.04% ; The linear range of protocatechualdehyde was 0.05～0.26μg. The average recovery was 101.5% and RSD was 0.72%. Conclusion: The method can provide useful references to quality control of Herba Salvia chinensis. Danshensu was firstly isolated from Herba Salvia chinensis.
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OBJECTIVE: To develop an HPLC method for content determination of protocatechualdehyde in Tong-jingkang oral liquid.METHODS:Zorbax Eclipse XDB-C18(250 mm?4.6 mm,5 ?m) was used as chromatographic column with mobile phase consisted of methanol-water-formic acid(20∶79.5∶0.5) at a flow rate of 1.0 mL?min-1.The detection wavelength was set at 280 nm and the temperature of column was kept at room temperature.RESULTS: The linear range of protocatechualdehyde was 0.025～0.5 ?g(r=0.999 9) with an average recovery of 101.1%(RSD=2.29%,n=6).CONCLUSION: The established method is simple,accurate and it is applicable for the quality control of Tongjingkang oral liquid.
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OBJECTIVE: To study the mechanism of intestinal absorption of protocatechualdehyde .METHODS: Using invivo rat intestinal absorption model and HPLC, the apparent small intestine absorption rate of protocatechualdehyde was mea-sured. RESULTS: Under low, medium and high concentrations of drug, the apparent small intestine absorption rates of proto-catechualdehyde were 1.1 341/h, 1.1 637/h and 1.0 847/h respectively .CONCLUSION: The protocatechualdehyde absorptionmechanism is passive diffusion.
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OBEJECTIVE:To study quality differences of compound danshen injections of different batch numbers from different manufactures.METHODS:The fingerprints of compound danshen injections were obtained through gradient elution by HPLC;The contents of danshensu,protocatechualdehyde and savianolic acid B in samples of different batch numbers and different manufactures were determined,and the insoluble fine particle,protein,tannin,resin etc.in samples were studied ac-cording to the specification of China Pharmacopoeia2005.RESULTS:The fingerprints of compound danshen injections were specific;Great differences were found in the contents of danshensu,protocatechualdehyde and salvianolic acid B of the samples from different manufactures,quality discrepancies were found between some of the related substances in some of the prepara-tions and the specifications.CONCLUSION:Quality differences were found in compound danshen injections from different manufactures,therefore,the quality control standards of compound danshen injections should be enhanced and unified,and with regard to the controlling of the insoluble fine particles,the technics and techniques of which should be improved and given a further study.
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Object To establish a new method to determine protocatechualdehyde in Maodongqing Sustained- release Tablet. Methods The RP- HPLC method was applied with CH3OH and 1 % acetic acid as mobile phase, flowrate at 1.0 mL/min, and detection wavelength at 312 nm. Results In the range from 11.7 ng to 140.4 ng, protocatechualdehyde was in a good line with peak area, r=0.99995, the average recovery ratio was 96.41 % , RSD=1.04 % (n=5). Conclusions It was the first time to establish the RP- HPLC method to determine and control the protocatechualdehyde in Maodongqing Sustained- release Tablet with accuracy and a good reproducibility.
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Objective To establish a RP- HPLC method for the determinatio n of protocatechualdehyde in Danqi tablet. Methods LiChrospher RP- 18 column w as used, the mobile phase was MeOH- 1.5 % Hac(1∶ 9) with a flow rate of 1.0 mL/ min, and the detection wavelength was at 280nm. Results There was a goo d linearity within the range of 0.030～ 0.150 ? g of protocatechualdehyde. The average recovery was 103.08 % and RSD was 0.796 % (n=5). Conclusion This method can be used for the quality control of Danqi tablet .
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AIM: To establish a method for the quality control of Danqi Tablet(Radix et Rhizoma Salviae Miltiorrhizae,Radix et Rhizoma Notoginseng,etc). METHODS: The content of salvianic A and protocatechualdehyde,notoginsenoside R_1 and ginsenoside Rg_1 in Danqi Tablet were determined simultaneously respectively by HPLC. RESULTS: The methodological study showed that a good linear correlation existed in the range of 0.164-3.28 ?g(r=0.999 99) of sodium salvianic A,0.013-0.52 ?g(r=0.999 999) of protocatechualdehyde,0.2-6 ?g(r=0.999 997) of notoginsenoside R_1 and 0.824-24.72 ?g(r=0.999 96) of ginsenoside Rg_1,respectively.The average recovery of sodium salvianic A was 98.34%(n=9),RSD was 2.03%,100.65% for protocatechualdehyde(n=9),RSD was 2.96%,98.97% for notoginsenoside R_1(n=9),RSD was 2.81%;98.93% for ginsenoside Rg_1(n=9),RSD was 0.57%. CONCLUSION: The determination methods are simple,accurate and reproducible.The method can be used for quality control of Danqi Tablet.
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AIM: To establish an external standard HPLC method for the determination of protocatechuic acid and protocatechualdehyde in Compound Shijiqing Tablets(Folium Ilicis Chinensis, etc.). METHODS:Alltima C 18(5?m, 4.6mm?250mm)was used. The mobile phase was consisted of methanol-1% acetate(18∶82). The flow rate was 1.0mL?min -1. The UV detection wavelength was at 280nm. RESULTS: The two components has good resolution. The liner ranges were in the range of 7.6～127.1?g?mL -1 for protocatechuic acid and in the range of 2.62～43.6?g?mL -1 for protocatechualdehyde with the correlation coefficients 0.9997 and 0.9994, respectively. The average recoveries were 99.9% and 97.5% with RSD 2.1%,2.2%(n=5), respectively CONCLUSION: The method is accurative, reproducible and can be used to control the quality of Compound Sijiqing Tablets.
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Objective: To prepare Danshen Injection (Radix Salviae Miltiorrhizae) by micelle-ultrafiltration. Methods: For preparing Danshen Injection, we packed the impurities in the macrofiltered liquor of Radix Salviae Miltiorrhizae with micelle of Tween-80, and ultrafiltered the compounds with ultrafiltration membrane withholding molecular weight more than 50000. Then we evaluated the process with the comprehensive indexes including loss rate of UV absorbance, loss rate of Protocatechualdehyde and the stability of transparence. Conclusion: The process of Danshen Injection prepared by micelle-ultrafiltration is better than that of ordinary ultrafiltration.
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OBJECTIVE:To make an investigation on the quality of decoctions of traditional Chinese medicine prepared by decocting device and fire heating decocting method.METHODS:To compare the chemical compositions in decoctions of Salvia miltiorrhiza Bunge prepared by above-mentioned two methods and to take the contents of danshensu and protocatechualdehyde as indices,both of which were determined by HPLC.RESULTS:There were no evident difference between the two methods in extracting chemical compositions.In addition,the contents of danshensu and protocatechualdehyde prepared by decocting device were as2.97times and5.6times as those prepared by fire heating decocting method respectively.CONCLUSION:The prepa?ration of decoction of Saliva miltiorrhiza Bunge by decocting device can double the effective constituents.This provides scien?tific basis for making rational use of herbs.
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OBJECTIVE:To find the optimal extraction method for Danshen granules METHODS:According to the orthogonal design L9(34),four factors in extraction were optimized,i e soak duration,decocting times,decocting duration and amount of water added The content of salvianolic acid and the weight of solids were taken as indices RESULTS:The best extracting condition was that 20 mesh size of particles was soaked in eight times amount of water for 1 5h,decocted for 1 5h at first,and then six times amount of water for 1 0h CONCLUSION:This technique is highly repeatable in pilot-experiment