High-performance liquid chromatographic analysis of bioactive triterpenes in Perilla frutescens

Perilla frutescens (L.) Britt. (Lamiaceae), a famous traditional Chinese medicine, has been used for the treatment of various diseases. To evaluate the quality of P. frutescens, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed for the assessment of three bioactive triterpene acids: tormentic acid (TA), oleanolic acid (OA) and ursolic acid (UA). The HPLC system used an Spherisob octadecylsilyl silica (ODS) column with acetonitrile and aqueous H₃PO₄ as the mobile phase and detection at 206 nm. The method was precise with relative standard deviations for these three constituents that ranged between 0.6–1.5% (intraday) and 0.7–2.6% (interday). The content of these three phytochemicals in the leaves of P. frutescens growing at eight different locations of China was determined to establish the effectiveness of the method.     

LC and UV determination of flavonoids from Passiflora alata medicinal extracts and leaves

The leaves of Passiflora alata Curtis (an official species in the Brazilian Pharmacopoeia) are used to treat anxiety, as antispasmodics and sedatives. The development of new analytical methods for analysing flavonoids in Passiflora species is needed in order to improve the quality assurance of the plant and derived extracts and phytomedicines. A survey on the profile of flavonoids and content of isovitexin in the leaves and fluidextract of P. alata through LC was carried out, comparing its chromatographic profiles with a commercial P. incarnata fluidextract. Also, the concentration of the total flavonoids of extracts and leaves according to phamarcopoeial photometrical methods was determined and discussed. The fluidextract of P. alata was produced in accordance with the Pharmacopoeia Helvetica method; the presence of isovitexin in both species (which have distinct chromatographic profiles) was evidenced, this being the major flavonoid compound in the P. incarnata (1.198 g%), but not in P. alata (0.018 g%) fluidextract. Only traces of vitexin could be observed in the P. alata, as well as the absence of the other tested flavonoids: orientin and swertisin (P. incarnata markers), hyperoside, rutin, hesperidin and clorogenic acid. The LC developed method was shown to be appropriated for the qualitative and quantitative analyses and for differentiate species, suitable to be applied to commercial sample analysis. The spectrophotometrical results of three different methods described for P. incarnata were not comparable, the best performance being the British Pharmacopoeia method.     

Metabolic profile of the bioactive compounds of burdock (Arctium lappa) seeds, roots and leaves

In this work the bioactive metabolic profile, the antioxidant activity and total phenolic content of burdock (Arctium lappa) seeds, leaves and roots were obtained. TEAC values and total phenolic content for hydro-alcoholic extracts of burdock ranged from 67.39 to 1.63 μmol Trolox equivalent/100 g dry weight (DW), and from 2.87 to 45 g of gallic acid equivalent/100 g DW, respectively. Phytochemical compounds were analyzed by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS/MS) in negative mode. The main compounds of burdock extracts were caffeoylquinic acid derivatives, lignans (mainly arctiin) and various flavonoids.The occurrence of some phenolic acids (caffeic acid, chlorogenic acid and cynarin) in burdock seeds; arctiin, luteolin and quercetin rhamnoside in burdock roots; phenolic acids, quercetin, quercitrin and luteolin in burdock leaves was reported for the first time.     

Simultaneous estimation of phenolic acids in sea buckthorn (Hippophaë rhamnoides) using RP-HPLC with DAD

A RP-HPLC-DAD method was developed and validated for the simultaneous analysis of nine phenolic acids including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, p-coumaric acid, cinnamic acid, caffiec acid and ferulic acid in sea buckthorn (SB) (Hippophaë rhamnoides) berries and leaves. The method was validated in terms of linearity, LOD, precision, accuracy and recovery and found to be satisfactory. Phenolic acid derivatives in anatomical parts of SB berries and leaves were separated into free phenolic acids, phenolic acids bound as esters and phenolic acids bound as glycosides and profiled in HPLC. Berry pulp contained a total of 1068 mg/kg phenolic acids, of which 58.8% was derived from phenolic glycosides. Free phenolic acids and phenolic acid esters constituted 20.0% and 21.2%, respectively, of total phenolic acids in SB berry pulp. The total phenolic acid content in seed kernel (5741 mg/kg) was higher than that in berry pulp and seed coat (Table 2). Phenolic acids liberated from soluble esters constituted the major fraction of phenolic acids (57.3% of total phenolic acids) in seed kernel. 8.4% and 34.3% of total phenolic acids in seed kernel were, respectively contributed by free and phenolic acids liberated from glycosidic bonds. The total soluble phenolic acids content in seed coat (448 mg/kg) was lower than that in seed kernel and pulp (Table 2). Proportion of free phenolic acids in total phenolic acids in seed coat was higher than that in seed kernel and pulp. Phenolic acids bound as esters and glycosides, respectively contributed 49.1% and 20.3% of total phenolic acids in seed coat. The major fraction (approximately 70%) of phenolic acids in SB berries was found to be concentrated in the seeds. Gallic acid was the predominant phenolic acid both in free and bound forms in SB berry parts and leaves.     

杭菊叶生长过程中绿原酸类和木犀草苷动态变化分析

目的：建立高效液相色谱（HPLC）法同时测定杭菊叶中5种绿原酸类成分及木犀草苷的方法,并分析不同生长过程杭菊叶中绿原酸类和黄酮类成分的动态变化,比较同期采收的杭菊叶与杭菊花中成分的差异,为杭菊叶的质量控制、合理利用提供参考。方法：采摘不同生长时期的杭菊叶样品及盛花期的杭菊花样品,采用HPLC法对杭菊叶及杭菊花中绿原酸类及黄酮类成分进行检测。结果：不同生长时期杭菊叶中绿原酸类及木犀草苷成分呈不同的规律。新绿原酸、隐绿原酸、异绿原酸B随着生长时间而逐渐累积,到9月含量达到峰值后逐步下降;绿原酸、异绿原酸A则随着成分累积,在9月达到峰值,下降后又在11月出现次峰值。木犀草苷则随着生长过程逐步增加,到11月达到含量峰值。同期采收的杭菊叶中绿原酸类及黄酮类成分高于杭菊花。结论：该研究结果为确定杭菊叶的采收时间及质量测定标准提供了参考和依据,为揭示杭菊叶生长过程中资源性化学成分的变化以及药材品质的控制奠定了一定基础。     

Solubility studies of oleanolic acid and betulinic acid in aqueous solutions and plant extracts of Viscum album L

Mistletoe (Viscum album L.) contains the triterpene acids oleanolic acid (OA) and betulinic acid (BA), which were found to have anti-tumour properties. In this study, the solubilities of OA and BA were studied in water (up to 0.02 microg/mL in each case) and in alkaline solutions of 10 mM trisodium phosphate (pH 11.5; OA: 77.2 microg/mL; BA: 40.1 microg/mL). Furthermore, triterpene acids were quantified in aqueous mistletoe extracts (pH 7.3; drug to extract ratio 1:25). OA (1.1 microg/mL) and BA (0.9 microg/mL) were extracted with a yield of less than 5%. Preparing plant extracts with basic pH values resulted in a triterpene acid content of 9.3 microg/mL OA and 5.2 microg/mL BA (pH 12.1), reaching neither the solubility limits nor a complete extraction of the plant material. The triterpene acid content of neutral plant extracts above the solubility limit could be due to interactions with biocolloids. Interaction studies were performed by gel permeation chromatography. Different mechanisms of the dissolution at pH 7.3 and pH 10.2 are discussed.     

灵芝三萜酸组分高效液相色谱指纹图谱初探

目的：建立灵芝三萜酸组分（GLA）的指纹图谱,为其质量控制提供依据。方法：使用植物组织破碎法提取GLA,利用高效液相色谱法建立GLA的指纹图谱。结果：GLA高效液相色谱指纹图谱中有8个峰为其主要特征,其α值分别为0.68,0.73,0.90,0.95,1.00,1.12,1.36,1.46。结论：所建立的RP-HPLC指纹图谱分析方法可为GLA的质量控制提供理论依据。     

Simultaneous analysis of theanine, chlorogenic acid, purine alkaloids and catechins in tea samples with the help of multi-dimension information of on-line high performance liquid chromatography/electrospray-mass spectrometry

A reverse phase high performance liquid chromatography (RP-HPLC) separation coupled with photo diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS) detection was established for the analyzing of multiple bioactive compounds in tea and tea extracts. Theanine, chlorogenic acid, purine alkaloids and catechins were identified with authentic standard compounds and with MS-spectra. The content of theanine and catechins was measured by employing DAD and caffeine, chlorogenic acid, theobromine and theopylline by protonated molecular ion on selective ion recording (SIR) mode. The unity of LC/ESI-MS provides more qualitative and quantitative information comparing with general HPLC in the analysis of multi-components in tea, and complex extraction or sample pretreatment is unnecessary. The chromatogram acquired by using this method can be used as a bioactive components fingerprint for the quality control of tea and its extracts. With the help of multi-dimension information of HPLC-DAD-ESIMS, the compounds owning different chemical structure such as amino acid, catechins, etc. in tea and its extracts could be identified and determined in one run successfully.     

Development of LC-MS method for determination of ursolic acid: application to the analysis of ursolic acid in Staphylea holocarpa Hemsl

Ursolic acid is a hydroxy pentacyclic triterpene, which has a chemoprotective activity in human. A reliable and reproducible liquid chromatography-mass spectrometric assay (LC-MS) was developed for the determination of ursolic acid in laboratory-made mixtures and in leaves and twigs extracts of Staphylea holocarpa Hemsl. The methanolic solution of the extracted ursolic acid was chromatographically analyzed using Shim Pack CLC-CN, C18 (150 x 6 mm, 5 mu) column and a mobile consisting of methanol-1% acetic acid solution (4:1) at a flow rate of 1.0 ml min(-1). The mass spectrometer (LCQ-Finnigan) was programmed in the positive single ion monitoring (SIM) to permit detection and quantitation of ursolic acid in MS-SIM mode at m/z 439.2, 411.2 and 390.9. Linear correlation (r > 0.99) of the peak area and the concentration of ursolic acid over the concentration range 0.25-10 microg ml(-1) was obtained. The relative standard deviation (%R.S.D.) and percentage deviation from the nominal concentrations (%DEV) were found to be 3.03-3.59% and -4.5 to +6.2%, respectively. Analysis of laboratory-made mixtures containing known concentrations of ursolic acid, as quality control samples, gave a mean recovery percentage of 97.8%. Application of the proposed method for the analysis of leaves and twigs extracts of S. holocarpa Hemsl. gave mean percentage contents of ursolic acid of 0.95 and 0.25%, respectively.     

HPLC法测定山楂叶中总黄酮的含量

目的:建立以高效液相色谱法测定山楂叶中总黄酮含量的方法。方法:色谱柱为Shim-packVP-ODSC18(250mm×4.6mm,5μm),流动相为甲醇-2%醋酸溶液(30∶70),流速为1.2mL·min-1,检测波长为355nm。通过测定山楂叶主要化学成分之一金丝桃苷的含量推算出山楂叶中总黄酮的含量。结果:8份山楂叶样品中金丝桃苷与总黄酮的含量成显著正相关(r=0.98,P<0.01),且总黄酮与金丝桃苷的含量比值(换算因子)为5.955±0.300。结论:可以将金丝桃苷的含量乘以换算因子求算出不同产地、不同采收期山楂叶中总黄酮的含量。     

Determination of Flavonoids,Tannins and Ellagic acid in leaves from<Emphasis Type="Italic">Rubus</Emphasis> L. species

This paper describes the quantitative determination of flavonoids, tannins and ellagic acid in the leaves from wild and cultivated variations of Rubus L. species (Rosaceae): raspberry (2 wild and 13 cultivars) and blackberry (3 wild and 3 cultivars). The content of flavonoids was analyzed using spectrophotometric (the Christ-Mullers method) and HPLC analysis after acid hydrolysis. The content of tannins was determined by the weight method, with hide powder, described by German Pharmacopoeia 10 (DAB 10). Ellagic acid content was examined using the HPLC method after acid hydrolysis. Flavonoid content, determined using the Christ-Muller's method was higher for the blackberry leaves than for the raspberry leaves and varied between 0.46% and 1.05%. Quercetin and kaempferol were predominant in all samples analyzed using the HPLC method. The highest flavonoid content was found in the leaves of R. nessensis (1.06%); with results in all of the examined samples varying between 0.27% and 1.06%. The concentration of ellagic acid in all species was determined after acid hydrolysis and ranged from 2.06% to 6.89%. The leaves of raspberries are characterized by greater amounts of tannins (varying between 2.62% and 6.87%) than the leaves of other species. The results from this study indicate that the analyzed species are a rich source of flavonoids, ellagic acid and tannins, which may be used for the quality assessment of Rubus L. species leaves.     

Studies on the constituents of Cimicifuga species

The chemical constituents of Cimicifuga simplex and its related species were reexamined using HPLC and high resolution spectral analysis. From C. simplex, C. acerina and C. japonica, a new alkaloid, 59 new cycloartane triterpene glycosides and 11 new aromatic constituents were isolated with the previously reported compounds. The latter aromatic constituents were identified in HPLC analysis of the extracts from crude drugs due to C. dahurica, C. heracleifolia and C. foetida. Several interesting topics in this study are reviewed about separation of unstable and closely related compounds by HPLC, rearrangement reactions, chemical conversion for confirmation of absolute configuration, isomerism in NMR solutions, X-ray crystal analysis, spectral analysis of complicated structures, isolation of glycoside malonates, high yield of enzymatic hydrolysis, specific CD curve due to a cycloart-7-ene system, and so on. Biological activities of a major glycoside, cimicifugoside, a modified triterpene, acerinol, and benzyltartaric acid derivatives such as fukinolic acid and cimicifugic acids were also mentioned.     

柱前衍生RP-HPLC法测定桑叶中16种游离氨基酸的含量

目的:建立柱前衍生法测定桑叶中游离氨基酸的含量。方法:以水超声提取桑叶中的氨基酸,以异硫氰酸苯酯柱前衍生,采用反相高效液相色谱法测定氨基酸的含量。结果:桑叶中含有苏氨酸、缬氨酸、脯氨酸、蛋氨酸、谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸、天门冬氨酸、γ-氨基丁酸、丙氨酸、组氨酸、胱氨酸、色氨酸、异亮氨酸等16种游离氨基酸;其中人体必需氨基酸有7种,人体半必需氨基酸有1种,含量占氨基酸总量的20%左右。结论:本法不需要专门的氨基酸分析仪,操作简便、灵敏度高,结果准确、可靠。桑叶中氨基酸的种类齐全、含量丰富,具有很高的开发利用价值。     

不同产地蒺藜药材中氨基酸成分比较分析

目的建立检测蒺藜氨基酸成分的高效液相色谱测定法,并比较不同产地的蒺藜药材中氨基酸的含量,为评价蒺藜药材的质量提供参考依据。方法柱前衍生化HPLC,应用色谱柱ELite-AAK;以乙腈-水(1∶1):4%醋酸钠缓冲盐为流动相,流速为1.2 mL/min,梯度洗脱,检测波长为360 nm,柱温为27℃。结果不同产地药材中,均含所检测的18种氨基酸,其中包括8种人体必需氨基酸,以河南产的必需氨基酸和总氨基酸含量为最高。结论以蒺藜药材中氨基酸的含量对其药材质量进行评价,具有一定的意义。     

银杏酸的高效液相色谱法测定

目的建立银杏酸的简便预净化处理和HPLC含量测定方法。方法用LC/DAD/ESI/MS对银杏酸进行定性鉴定。银杏叶经正己烷提取、硅胶柱色谱净化处理,HPLC测定。色谱分析条件:色谱柱为Inertsil ODS-2,流动相为甲醇-3% HAc溶液(92∶8),流速1.0 mL·min^-1,柱温40℃,紫外检测波长310 nm。结果银杏叶中存在5种银杏酸C13∶0,C15∶0,C15∶1,C17∶1和C17∶2,其中C15∶1和C17∶1约占银杏酸的85%,C17∶2银杏酸未见国内文献报道。银杏叶提取物经硅胶柱色谱处理后,其HPLC谱中除银杏酸峰外,几无其他杂质峰。平均回收率97.0%,RSD为1.7%(N=6)。结论该方法准确、简便、可靠,可用于银杏酸的定量分析。     

蒲公英质量标准提升及不同产地药用蒲公英质量评价

目的:提升蒲公英药材质量标准,并评价不同产地药用蒲公英药材的质量。方法:在2020年版《中国药典》(一部)"蒲公英"项下规定的基础上,增加水溶性浸出物(热浸法)、醇溶性浸出物(热浸法)、总黄酮、绿原酸、咖啡酸、菊苣酸和异绿原酸A的含量测定方法,同时以8个产地42批药用蒲公英药材为对象建立高效液相色谱(HPLC)指纹图谱,并基于上述结果进行主成分分析。结果:42批药用蒲公英药材的醇溶性浸出物含量为15.30%~30.40%,水溶性浸出物含量为27.59%~38.96%。总黄酮(紫外-可见分光光度法)和绿原酸、咖啡酸、菊苣酸、异绿原酸A(HPLC法)的质量浓度分别在0.016~0.096、0.003~0.196、0.004~0.117、0.025~1.578、0.002~0.152 mg/mL范围内线性关系良好(R~2均大于0.9990);精密度、稳定性、重复性试验的RSD均小于2.00%(n=6);平均加样回收率为98.97%~103.53%,RSD为1.19%~1.58%。42批药用蒲公英药材中总黄酮、绿原酸、咖啡酸、菊苣酸、异绿原酸A的含量分别为0.734%~3.700%、0.004%~0.123%、0.006%~0.087%、0.073%~1.499%、0.005%~0.109%。42批药用蒲公英样品的HPLC指纹图谱共有峰相对保留时间的RSD为0~0.94%,相对峰面积的RSD为0~125.57%,其中39批样品相似度大于0.900。主成分分析结果显示,陕西产药用蒲公英药材的综合质量较优,河北产药材次之。结论:拟定蒲公英药材中醇溶性浸出物、水溶性浸出物、总黄酮、绿原酸、咖啡酸、菊苣酸、异绿原酸A的含量分别不得低于17.0%、27.0%、1.383%、0.024%、0.021%、0.450%、0.021%。陕西产药用蒲公英药材除咖啡酸含量相对较低外,其余各项指标均较优;河北产药材的咖啡酸含量高于陕西产药材,其他各项指标略低于陕西产药材;其余产地的药用蒲公英药材综合质量相对较差,且同一产地不同批次间的药材质量不稳定。     

Metabolomic strategy for the classification and quality control of phytomedicine: a case study of chamomile flower (Matricaria recutita L.)

In order to improve the accuracy and consistency of control phytomedicine preparations worldwide, regulatory authorities are requesting research into new analytical methods for the stricter standardisation of phytomedicines. Such methods have to be both objective and robust, and should address the reproducibility of the content of the chemical profiles. NMR-based metabolomics, which combines high-resolution (1)H-NMR spectroscopy with chemometric analysis, has been employed as an innovative way to meet those demands. In this paper, chamomile flowers from three different geographical regions, namely, Egypt, Hungary and Slovakia were characterised using 1H-NMR spectroscopy followed by principal component analysis. It was found that the origin, purity and preparation methods contributed to the differences observed in prepared chamomile extracts. In addition, this method also enabled the elucidation of the molecular information embedded in the spectra responsible for the observed variability. The metabolomic strategy employed in the current study should provide an efficient tool for the quality control and authentication of phytomedicines.     

红树林耐酸真菌OUCMDZ-4736次生代谢产物研究

目的 探究红树林耐酸真菌OUCMDZ-4736的次生代谢产物及其抗甲型流感病毒H1N1活性。方法 菌株在pH 2.5条件下规模发酵,通过现代色谱学方法（硅胶柱色谱、凝胶柱色谱、半制备以及制备型HPLC）对发酵产物进行分离,运用现代波谱学技术（紫外、质谱、核磁共振等）鉴定分离获得的化合物结构,采用MTT法评价化合物的抗流感病毒H1N1的活性。 结果 从红树林根部泥土样品来源的真菌OUCMDZ-4736中分离鉴定了10个化合物：asterric acid（1）、questinol（2）、parietinic acid（3）、endocrocin（4）、isorhodoptilometrin（5）、sulochrin（6）、monochlorsulochrin（7）、dihydrogeodin（8）、yicathin C（9）、2, 5-dimethyl-6, 8-dihydroxy-chromone（10）。活性评价结果首次报道化合物1、3、4和7显示有弱的抗甲型流感病毒H1N1活性。结论 红树林耐酸真菌OUCMDZ-4736在酸性调节下能代谢产生具有抗甲型流感H1N1病毒活性的化合物。     

α-Tocopherol,Flavonoid, and Phenol Contents and Antioxidant Activity of Ficus carica. Leaves

Abstract

The aim of the current study was to evaluate the α.-tocopherol content and to investigate the antioxidant capacities of the extracts prepared from the leaves of Ficus carica. L. (Moraceae). The antioxidant capacities of the extracts were evaluated by the phosphomolibdenum spectrophotometric method. α.-Tocopherol content was determined by using a high-performance liquid chromatography (HPLC)-UV method. Total flavonoid content was determined by using the aluminium chloride method. Total phenol content was estimated by a modified colorimetric method using Folin-Ciocalteau reagent. The results clearly demonstrate that these extracts have antioxidant capacity. Antioxidant capacity results are consistent with total flavonoid and phenol contents. The α.-tocopherol content of the n.-hexane extract was found to be 3.2788%, whereas it was calculated as 0.0570% on the dry-weight basis of the leaves.     

HPLC determination of phenolic acids and antioxidant activity in concentrated peat extract--a natural immunomodulator

The aim of this study was to devise a method for identification and quantification of phenolic acids in concentrated peat extract samples. The simple reversed-phase HPLC method for simultaneous determination of several phenolic acids was developed. The method was validated and it was suitable for the analysis of phenolic acids in peat extracts. This method allowed identifying eight phenolic acids in peat extracts. Phenolic profiles of two samples of peat extract obtained from different medicinal peats were similar, although variations in amounts of individual phenolic acids were observed. Also, slight variations in total phenolic content were detected. The antioxidant activity of peat extracts was evaluated with spectrophotometric ABTS assay. Differences in antioxidant activity were observed for two samples of peat extract produced from different peat varieties. This differences probably reflected phenolic composition of peat extracts.