液相色谱法同时测定维药蜀葵花中芦丁、槲皮素和山柰酚

维药是祖国医药学不可分割的组成部分。维药现代化,即利用现代技术研究维药的有效成分,是维药科学化、标准化、规范化、商品化和产业化的必经之路。本文建立了维药蜀葵花中有效成分芦丁、槲皮素和山柰酚的选择性提取方法,优化了高效液相色谱法(HPLC)同时测定这3种有效成分的分析条件。采用HC-C₁₈色谱柱(250 mm×4.6 mm, 5 μm)和甲醇-0.4%磷酸(50:50, v/v)流动相,在柱温30 ℃和流速1.00 mL/min的条件下实现了3种物质之间以及和干扰物之间的基线分离。维药蜀葵花中芦丁、槲皮素及山柰酚的线性范围分别为12.5~150 μg/mL (r=0.9998), 12.5~125 μg/mL (r=0.9999)及12.5~125 μg/mL (r=0.9988),加标回收率(n=5)分别为100.3%(RSD=1.1%)、97.60%(RSD=0.47%)、97.75%(RSD=0.71%)。该方法实现了同时测定维药蜀葵花中芦丁、槲皮素及山柰酚,为其他黄酮类物质的开发应用提供了科学依据,同时也可为其他维药分析提供借鉴。     

反相高效液相色谱法对秦岭21种蕨类植物中槲皮素与山柰酚含量的测定

运用反相高效液相色谱(RP/HPLC)法对21种蕨类植物中槲皮素、山柰酚的含量进行测定。使用Shimadzu C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇-水溶液为流动相进行等度洗脱,流速1.0 mL/min,检测波长360 nm,进样量20μL,柱温28℃。各对照品的质量浓度与色谱峰面积线性关系良好,具有较好的精确度和重复性,槲皮素、山柰酚的加标回收率分别为93%和95%。采用该方法分别对采自秦岭的21种蕨类植物的地上和地下部分进行测定,地上部分有19种含槲皮素、15种含山柰酚,其中毡毛石韦中槲皮素含量最高(2.11 mg/g),蜈蚣草中山柰酚含量最高(19.80 mg/g);而地下部分除有边瓦韦、大瓦韦含槲皮素(含量分别为0.11、0.12 mg/g)外,其余根状茎中几乎没有这两种黄酮类化合物;表明槲皮素与山柰酚在蕨类植物的地上部分广泛存在。     

高效液相色谱法同时测定不同产地及不同药用部位景天三七中4种黄酮类成分的含量北大核心CSCD

建立了同时测定不同产地及不同药用部位景天三七中槲皮素、木犀草素、山奈酚和异鼠李素含量的高效液相色谱(HPLC)法。采用TOP ODS-AQ色谱柱(250×4.6mm,5μm),以甲醇-0.1%磷酸溶液为流动相梯度洗脱,检测波长为365nm。结果表明,槲皮素、木犀草素、山奈酚和异鼠李素的浓度分别在1.90~189.90μg·mL-1(r=0.99996),1.12~112.00μg·mL-1(r=0.99998),3.71~370.56μg·mL-1(r=0.99995)和0.98~97.60μg·mL-1(r=0.99996)范围内与其色谱峰面积呈良好线性关系;平均加标回收率分别为99.79%、100.06%、100.19%和100.00%,且不同产地及不同药用部位的4个黄酮类成分在数量上或质量上有明显差异。该方法快速、准确,重现性好,可用于同时测定景天三七中槲皮素、木犀草素、山奈酚和异鼠李素含量。     

鱼腥草中黄酮类成分的高效液相色谱指纹图谱分析

采用均匀实验设计和信息理论评价方法,建立了鱼腥草中黄酮类成分的高效液相色谱(HPLC)指纹图谱的分析方法。采用建立的方法和本研究室提出的指纹图谱评价软件,对同样种植条件下10个批次的鱼腥草指纹图谱进行了相似性评价,相似度均大于0.90;同时测定了芦丁、槲皮甙和槲皮素3个成分在10批鱼腥草药材中的含量分别为0.25%～0.34%、0.27%～0.37%、0.012%～0.016%。另外对不同采收季节和不同部位的鱼腥草药材中的黄酮类成分进行了指纹图谱的测定、主成分分析以及成分含量测定,结果表明,不同季节、不同部位的鱼腥草中黄酮类化合物的指纹图谱及成分含量存在较大的差异,且药用部位的差异大于采收季节的差异。该方法为规范鱼腥草中黄酮类成分在制药和用药的实际应用提供了一些可靠的基础信息。     

高效液相色谱法同时测定枸杞中槲皮素、山柰酚和异鼠李素

建立了高效液相色谱同时检测枸杞中槲皮素、山柰酚和异鼠李素的分析方法。样品经过甲醇超声提取后,用甲醇-25%HCl水解1 h,采用Inertsustain C18色谱柱进行分离,以甲醇-0.4%H3PO4溶液(48∶52,V/V)为流动相,进行等度洗脱,流速为1.0 m L/min,二极管阵列检测器检测,检测波长为360 nm,柱温为40℃。槲皮素,山柰酚,异鼠李素在40 min内实现分离,并分别在0.053~21.2μg/m L,0.053~4.24μg/m L和0.046~3.72μg/m L范围内具有良好的线性关系,相关系数为0.9972~0.9992,测得槲皮素、山柰酚、异鼠李素的加标回收率为99.2%~103.1%,95.6%~101.8%,93.2%~109.1%;相对标准偏差分别为0.95%~2.8%,0.55%~2.3%,0.81%~2.4%。对槲皮素、山柰酚和异鼠李素的检出限分别为0.04,0.05,0.03 mg/kg。方法可用来测定枸杞中3种黄酮苷元的含量。     

大孔树脂-高速逆流色谱分离纯化薇甘菊中的黄酮类化合物

该文建立了大孔树脂-高速逆流色谱分离薇甘菊中黄酮类物质的方法。分离条件为:采用大孔树脂AB-8,洗脱液为50%（v/v）乙醇水溶液,高速逆流色谱溶剂体系为正丁醇-乙酸-水（4：1：5,v/v）。从薇甘菊中分离到4种黄酮类物质:槲皮素-3-O-芸香糖苷（纯度90.2%）、山奈酚-3-O-芸香糖苷（纯度98.55%）、木犀草苷（纯度98.33%）和紫云英苷（纯度99.23%）。建立的大孔树脂-高速逆流色谱方法简单、高效,可扩展应用于从其他植物中分离黄酮类物质。     

固相萃取-液相色谱-串联质谱法同时测定蜂蜜中16种黄酮类化合物和阿魏酸

建立了固相萃取净化-液相色谱-串联质谱法（SPE-LC-MS/MS）同时测定蜂蜜中芦丁、杨梅素、桑黄素、槲皮素、柚皮素、橙皮素、木犀草素、染料木素、山柰酚、异鼠李素、芹菜素、松属素、汉黄芩素、白杨素、高良姜素、芫花素和阿魏酸含量的方法。蜂蜜经pH 2的盐酸溶液稀释,C₁₈固相萃取柱净化,液相色谱-串联质谱法检测,外标法定量。以空白蜂蜜基质溶液配制0~200 μg/kg的系列标准溶液,线性相关系数大于0.997,方法定量限为20 μg/kg。在蜂蜜样品中进行加标水平为20、40、100 μg/kg的添加回收试验,回收率为64.5%~113%,相对标准偏差为1.4%~14.5%。该方法取样量少、操作简便、快捷,可用于蜂蜜中黄酮类化合物的测定。     

高效液相色谱-质谱联用法同时测定脑心清原料药浸膏及其片剂中的8种有效成分

建立了高效液相色谱-质谱联用（HPLC-MS/MS）同时测定脑心清原料药及其片剂中原儿茶酸、金丝桃苷、山柰酚-3-O-β-D-吡喃葡萄糖苷、山柰酚-3-O-β-D-吡喃半乳糖苷、杨梅素、槲皮素、柚皮素和山柰酚8种有效成分的分析方法。以乙腈和0.1%（v/v）甲酸水溶液为流动相进行梯度洗脱,经Dikma Diamonsil C₁₈柱分离,采用电喷雾离子源以负离子多反应监测模式进行质谱检测。结果表明,8种有效成分在线性范围内线性关系良好（r > 0.99）,回收率为99.0%~102.1%,相对标准偏差为0.9%~3.3%（n=6）。该方法高效、快速、准确,为脑心清原料药浸膏及其片剂的质量标准研究提供了一定的参考。     

双波长分光光度-K系数法同时测定荷花玉兰花叶中三种黄酮成分

应用双波长分光光度K系数法测定了荷花玉兰花叶中主要黄酮类成分芦丁、槲皮素和山萘酚.选择297 nm/293 nm,381 nm/397 am,428 nm/451 mn为分析波长对,三种组分的线性范围分别为0.635～12.7,0.576～11.5,0.456～9.12 mg·L-1;样品测定结果的相对标准偏差(n=5)分别为0.1%,0.9%,2.1%;样品加标回收率分别为103.0%,98.4%,97.7%.     

高效液相色谱法测定野葱中黄酮类化合物

建立了测定野葱中芦丁、黄酮醇类化合物的高效液相色谱方法。80%乙醇超声提取,高效液相色谱分析野葱中芦丁和黄酮醇类化合物的含量。芦丁检测条件:V(甲醇)∶V(0.2%磷酸水)=45∶55,检测波长:360nm;槲皮素、山奈酚、异鼠李素检测条件:V(甲醇)∶V(0.2%磷酸水)=40∶60,检测波长:360 nm。结果表明,野葱中芦丁含量0.22%,槲皮素、山奈酚、异鼠李素含量依次为0.42%、0、0.23%,总黄酮醇类化合物含量为1.63%。     

Precision improvement for the analysis of flavonoids in selected Thai plants by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method for the analyses of kaempferol in Centella asiatica and Rosa hybrids and rutin in Chromolaena odorata was developed. The optimization was performed on analyses of flavonoids (e.g., rutin, kaempferol, quercetin, myricetin, and apigenin) and organic carboxylic acids (e.g., ethacrynic acid and xanthene-9-carboxylic acid) by investigation of the effects of types and amounts of organic modifiers, background electrolyte concentrations, temperature, and voltage. Baseline separation (R(s) = 2.83) of the compounds was achieved within 10 min in 20 mM NaH2PO4 - Na2HPO4 (pH 8.0) containing 10% v/v ACN and 6% v/v MeOH using a voltage of 25 kV, a temperature of 30 degrees C, and a detection wavelength set at 220 nm. The application of the corrected migration time (t(c)), using ethacrynic acid as the single marker, was efficient to improve the precision of flavonoid identification (% relative standard deviation (RSD) = 0.65%). The method linearity was excellent (r2 > 0.999) over 50-150 microg/mL. Precision (%RSD < 1.66%) and recoveries were good (> 96% and %RSDs < 1.70%) with detection and quantitation limits of 2.23 and 7.14 microg/mL, respectively. Kaempferol in C. asiatica and R. hybrids was 0.014 g/100 g (%RSD = 0.59%) and 0.044 g/100 g (%RSD = 1.04%), respectively, and rutin in C. odorata was 0.088 g/100 g (%RSD = 0.06%).     

Statistical optimization of an RP‐HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities

An isocratic RP‐HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin‐7‐O‐glucoside, kaempferol and kaempferol‐3‐O‐glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water–acetonitrile, pH 2.8 (adjusted with formic acid), flow rate 0.5 mL min⁻¹ and column temperature 35°C. The obtained levels of bioflavonoids (mg per 100 mL of juice) were as follows: for quercetin, ca. 0.21–5.12; for kaempferol, ca. 0.05–1.2; for rutin, ca. 0.4–6.5; for luteolin‐7‐O‐glucoside, ca. 5.6–10.2; and for kaempferol‐3‐O‐glucoside, ca. 0.02–0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC₅₀ values varied from 8.56 to 14.05 mg L⁻¹. These values are ~2.5–3 times lower than quercetin, ascorbic acid and Trolox®, but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.     

高效液相色谱法测定北柴胡地下部分黄酮类化合物的含量

建立了高效液相色谱法测定北柴胡地下部分黄酮类化合物含量的方法。采用SepaxGP-C18色谱柱(250×4.6 mm,5μm),流动相为乙腈-0.4%磷酸(体积比35∶65),检测波长360 nm,柱温30℃,流速1.0 mL/min。结果表明,北柴胡地下部分含有槲皮素。芦丁、木犀草素、槲皮素、山奈酚、芹菜素分别在0.0050～0.0248、0.0050～0.0248、0.0051～0.0256、0.0046～0.0232、0.0054～0.0272 mg/mL范围内线性关系良好,相关系数分别为0.9957、0.9995、0.9998、0.9998、0.9998,槲皮素的平均回收率为98.34%,相对标准偏差(RSD)为0.76%。该法简便,快速,准确,重复性好,可作为北柴胡药材质量控制的方法。     

Rapid screening and evaluation of XOD inhibitors and O2•− scavenger from total flavonoids of Ginkgo biloba leaves by LC–MS and multimode microplate reader

Superoxide anion radical scavenger and xanthine oxidase inhibitor play an important role in the treatment of several relevant human diseases. In the present study, ultrafiltration liquid chromatography–mass spectrometry coupled to microplate reader was applied to screen and identify superoxide anion radical scavengers and xanthine oxidase inhibitors from total flavonoids of Ginkgo biloba leaves. As a result, four compounds (quercetin, apigenin, kaempferol and isorhamnetin) were screened as xanthine oxidase inhibitors by ultrafiltration LC–MS, and the 50% scavenging concentration values of the screened flavonoids were lower than those for allopurinol. Lineweaver–Burk plot results indicated that kaempferol was a competitive xanthine oxidase inhibitor; the other flavonoids were all anticompetitive inhibitors. Four flavonoids—rutin, quercetin, kaempferol and isorhamnetin—were screened as superoxide anion radical scavengers by LC–MS. The results demonstrate that the method for screening and evaluation of superoxide anion radical scavenger and xanthine oxidase inhibitor from a complex mixture system is feasible and efficient.     

Identification of flavonoids and their glycosides by high-performance liquid chromatography with electrospray ionization mass spectrometry and with diode array ultraviolet detection

Identification of flavonoids and flavonoid glycosides was carried out on Psidium guajava Linn leaves by means of high-performance liquid chromatography ultraviolet (HPLC-UV) analysis and HPLC mass spectrometry. By using HPLC-UV, two known phenolics (gallic acid and quercetin) and five newly reported ones (procatechuic acid, chlorogenic acid, caffeic acid, kaempferol and ferulic acid) were identified in alcohol guava leaf extract. Structural information about the compounds was obtained from the retention times, the UV spectra and mass spectra without the need to isolate the individual compounds. Two flavonoids (quercetin and kaempferol) and four flavonoid glycosides (three known components, quercetin 3-O-alpha-L-arabinoside, quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-galactoside, along with one novel compound, kaempferol-glycoside) and three other unknown compounds have been identified in the fractions.     

Separation and determination of flavonoids in three traditional chinese medicines by capillary electrophoresis with amperometric detection

Flavonoids are important active ingredients in many traditional Chinese medicines. In this paper, capillary electrophoresis with amperometric detection was employed to separate and detect eight flavonoids, rutin, quercetrin, quercetin, kaempferol, kaempferide, catechin, apigenin, and luteolin, in a home‐made capillary electrophoresis device. Under the separation voltage of 2000 V, the eight flavonoids could be completely separated within 33 min in 18 mM borax running buffer at pH 10.2. Good linear relationships were obtained for all analytes and the detection limits for flavonoids ranged from 0.46 to 0.85 μM. Then, the method was applied to separate and determine the flavonoids in three traditional Chinese medicines, hippophae rhamnoides, hypericum perforatum, and cacumen platycladi. Finally, rutin, kaempferol, quercetin, and quercetrin were discovered in these medicines and the concentrations ranged from 0.28 to 9.94 mg/g. The recoveries of flavonoids ranged from 84.7 to 113%, which showed the high reliability of this method.     

Analysis of selected constituents in methanolic extracts of Hypericum perforatum collected in different localities by capillary ITP-CZE

The on-line combination of CZE with capillary ITP (ITP-CZE) was used for the separation and quantification of selected flavonoids and phenolic acids in Hypericum perforatum leaves and flowers collected in six different localities in Slovakia. The leading electrolyte in the ITP preseparation step was 10 mM HCl with Tris as counterion (pH* 7.2). The terminating electrolyte was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The BGE in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 65 mM boric acid, pH* 8.3. The content of methanol in all electrolytes was 20% v/v. The total time of the analysis (including the preseparation step) was approximately 35 min. The rectilinear calibration ranges were between 0.125 and 5.0 microg/mL with kaempferol as internal standard. The correlation coefficients ranged between 0.9912 (for quercitrin and chlorogenic acid) and 0.9988 (for isoquercitrin). The RSD values are between 0.86 and 7.78% (n = 6) when determining rutin and quercetin (4 microg/mL). The optimized method was employed for the assay of flavonoids in medicinal plant extract of different collections of Hypericum perforatum haulm. The variability of the content of the active components depending on the place of collection was confirmed.     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Fingerprint profiles of flavonoid compounds from different Psidium guajava leaves and their antioxidant activities

Flavonoids are the main active components in Psidium guajava leaves and have many multi‐physiological functions. In this study, the flavonoid compositions were identified in the Psidium guajava leaves samples using a high‐performance liquid chromatography with time‐of‐flight electrospray ionization mass spectrometry method. A high‐performance liquid chromatography fingerprint method, combined with chemometrics, was used to perform a quality assessment of the Psidium guajava leaves samples. The eight identified flavonoid compounds including rutin, isoquercitrin, quercetin‐3‐O‐β‐d ‐xylopyranoside, quercetin‐3‐O‐α‐l ‐arabinopyranoside, avicularin, quercitrin, quercetin, and kaempferol were used as the chemical markers. The antioxidant activity of 15 batches of samples was examined using three different methods, and the results revealed the Psidium guajava leaves samples that had higher contents of the flavonoid compounds, glycoside and aglycone, possessed the highest antioxidant capacities. Consequently, a combination of chromatographic fingerprints and chemometric analyses was used for a quality assessment of Psidium guajava leaf tea and its derived products, which can lay the foundation for the development of plant tea resources or other herbs.