Rapid determination of flavonoids in licorice and comparison of three licorice species

A simple, sensitive and fast method for the simultaneous quantitation of 15 flavonoids in licorice based on an ultra high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry had been established and validated in this study. The analysis was performed on an ACQUITY HSS T3 column with gradient elution using a mobile phase consisted of A (0.1% formic acid in water)/B (acetonitrile) at a flow rate of 0.4 mL/min. Satisfactory separation of these compounds was obtained in less than 9 min. All calibration curves showed good linearity (r² = 0.9940) during the test ranges. The precision, repeatability, accuracy, limit of detection and limit of quantification were also fully investigated. The validated method was successfully applied for the simultaneous quantitation of 15 flavonoids in 106 licorice samples which contained 83 batches of G. uralensis, 14 batches of G. glabra and 9 batches of G. inflata. Furthermore, multivariate statistical analysis (using principal components analysis) was performed to classify the samples based on the contents of the 15 analyzed compounds. The results showed that all of these licorice samples were rich in flavonoids, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of licorice products.     

Rapid identification of chemical compositions of three species of Schisandra chinensis by ultra-high-performance liquid chromatography quadrupole-orbitrap-mass spectrometry

Schisandra chinensis is a traditional Chinese medicine, which has played an important role in the field of medicine and food. In this study, ultra-high-performance liquid chromatography quadrupole-orbitrap-mass spectrometry was used to rapidly classify and identify the chemical compositions. Note that 32, 28, and 30 kinds of compounds were successfully identified from northern Schisandra chinensis, vinegar-processed Schisandra chinensis, and wine-processed Schisandra chinensis, respectively. The cleavage patterns of various components including lignans, organic acids, flavonoids, and terpenoids were summarized, and the effects of different processing methods on Schisandra chinensis were analyzed through chemical composition. This method realized the rapid classification and identification of raw Schisandra chinensis and two different processed products, and provided references for improving the traditional processing methods, strengthening quality control, and ensuring safe clinical application.     

A practical method for rapid discrimination of constituents in Psoraleae Fructus by UPLC-Q-Orbitrap MS

Psoraleae Fructus (PF) is one of the most frequently used traditional Chinese medicine, which has good efficacy in warming kidney to activate yang, promoting inspiration to relieve asthma and warming spleen to stop diarrhea. However, the chemical composition of PF is complex, which makes it difficult to determine its active and toxic components. In order to rapidly classify and identify the chemical components of the extracts from PF, this research was processed with CNKI, PubMed, and PubChem databases and data post-processing technique basing on ultrahigh performance liquid chromatography quadrupole orbitrap mass spectrometry (UPLC-Q-Orbitrap MS) technique. Finally, 73 chemical components were discriminated, including 44 flavonoids, 18 coumarins, and 11 terpenoids, with the cleavage rules of each chemical component summarized. This study established a UPLC-Q-Orbitrap MS method for the separation and discrimination of the chemical constituents of PF, which can lay a foundation for the further study of its medicinal substances and quality control.     

Dynamic Microwave-assisted Extraction of Flavonoids from Radix Scutellariae

IntroductionRadix Scutellariaeis the root ofScutellaria bai-calensis Georgi(Labiatae), which is widely cultivatedand used as traditional Chinese medicine. It has beendemonstrated to be anti-inflammatory, anti-metamor-phic, antiviral, anti-hot, and live pr…     

Quantitative analysis and chromatographic fingerprinting of the semen zizyphi spinosae by ultra-high-performance liquid chromatography coupled with diode-array detector

A simple and sensitive method was developed and validated for fingerprint analysis of semen zizyphi spinosae (SZS) and simultaneous determination of six flavonoids in SZS by ultra‐high‐performance liquid chromatography coupled to diode‐array detector (DAD). The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 RRHD column. The column was maintained at 40°C and the eluents were monitored with DAD at 270 nm. A gradient elution of acetonitrile and water containing 20 mM sodium dihydrogen phosphate was used. The solvent flow rate was 0.4 mL/min. The method was validated. Standard calibration curves showed good linear behaviors (r=1.000) in the range of 0.33–201.00 μg/mL. Acceptable intra‐day precision (RSD<1.9%), inter‐day precision (RSD<4.0%), repeatability (RSD<4.1%) and recovery in the range of 97.4–104% were obtained. The validated method was successfully applied to obtain the chromatographic fingerprints and the contents of six flavonoids in 23 samples of SZS. The principal component analysis (PCA) had been applied for the chromatographic fingerprint analysis and quantitative analysis of six flavonoids to classify and discriminate the 23 samples of SZS. These results demonstrated that the method was very suitable in the analysis and quality control of SZS.     

Preparation of Synthetic and Natural Derivatives of Flavonoids Using Suzuki–Miyaura Cross-Coupling Reaction

Herein, we report the use of the Suzuki–Miyaura cross-coupling reaction for the preparation of a library of synthetic derivatives of flavonoids for biological activity assays. We have investigated the reactivity of halogenated flavonoids with aryl boronates and with boronyl flavonoids. This reaction was used to prepare new synthetic derivatives of flavonoids substituted at C-8 with aryl, heteroaryl, alkyl, and boronate substituents. The formation of flavonoid boronate enabled a cross-coupling reaction with halogenated flavones yielding biflavonoids connected at C-8. This method was used for the preparation of natural compounds including C-8 prenylated compounds, such as sinoflavonoid NB. Flavonoid boronates were used for the preparation of rare C-8 hydroxyflavonoids (natural flavonoids gossypetin and hypolaetin). A series of previously unknown derivatives of quercetin and luteolin were prepared and fully characterized.     

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods

A novel method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed‐phase C₁₈ column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.     

Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection

A high-performance liquid chromatography (HPLC) method with photo-diode array (DAD) detection was developed to separate and quantify flavonoids in sugarcane leaves and bagasse (= the crushed sugarcane refuse from juice extraction), and in sugarcane juice. Sugarcane flavonoids consist of a complex mixture of aglycones and glycosides (including flavonolignan glycosides), and the HPLC-UV method herein proposed is suitable for their quantification as total flavonoids. This method was applied to analyze samples of cultivated sugarcane, commercial juice and transgenic sugarcane leaves. Sugarcane leaves proved a promising source of flavonoids: an average of 1.10 mg of total flavonoids/g plant material was found in fresh leaves. Moreover, the flavonoid content of sugarcane juice (0.6 mg/mL) is comparable to other food sources of flavonoids previously reported. Transgenic sugarcane leaves ("Bowman-Birk" and "Kunitz") were compared with non-modified ("control") plant samples using the proposed HPLC-UV method, which indicated that the content of total flavonoids in transgenic plants is different from that in non-modified sugarcane.     

4种黄酮小分子对DPPH自由基的清除作用及构效关系研究

通过紫外可见光谱测定了4种黄酮小分子芦丁、牡荆素、山奈素、金丝桃苷对DPPH自由基的清除率、稳定性及半抑制浓度(IC50),并以常用的天然抗氧化剂抗坏血酸作为对照,考察了其抗氧化效果,探讨了黄酮类化合物的抗氧化性与结构的关系。结果表明：不同的抗氧化剂清除DPPH自由基达到平衡的时间不同,芦丁所需时间最长。4种黄酮小分子及抗坏血酸均对DPPH自由基有清除效果,并存在一定的量效关系。对DPPH自由基的清除能力从大到小依次为金丝桃苷、抗坏血酸、芦丁、山奈素、牡荆素。结构分析表明,B环邻二酚羟基是黄酮类化合物抗氧化所必需的基团,其羟甲基化及A环羟基糖苷化不利于黄酮类化合物的抗氧化活性。而C环3-OH的糖苷化对抗氧化活性有利,且单糖苷优于双糖苷。     

Response surface optimized ultrasonic-assisted extraction of flavonoids from Sparganii rhizoma and evaluation of their in vitro antioxidant activities

An efficient ultrasound-assisted extraction technique was employed to extract total flavonoids from Sparganii rhizoma. The optimum extraction conditions for the highest yield of total flavonoids were ethanol concentration 53.62%, ultrasonication time 29.41 min and ultrasound power 300 W, which were determined using response surface methodology. The extraction yields of the optimal ultrasound-assisted extraction were higher than using conventional extraction. The crude extract was then purified on a polyamide resin, whereby the flavonoids content in the purified extract increased to 94.62%. The antioxidant activities of the purified flavonoids including DPPH radical scavenging activity, ABTS+ radical scavenging activity, reducing power, hydroxyl radical scavenging activity and superoxide anion scavenging activity, were evaluated in vitro, which suggested that the flavonoids showed significant antioxidant activities. Rutin, kaempferol and formononetin were identified in the extract by comparing relative retention times and UV-Vis spectra with those of reference standards.     

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.     

Analysis of flavonoids: tandem mass spectrometry, computational methods, and NMR

Due to the increasing understanding of the health benefits and chemopreventive properties of flavonoids, there continues to be significant effort dedicated to improved analytical methods for characterizing the structures of flavonoids and monitoring their levels in fruits and vegetables, as well as developing new approaches for mapping the interactions of flavonoids with biological molecules. Tandem mass spectrometry (MS/MS), particularly in conjunction with liquid chromatography (LC), is the dominant technique that has been pursued for elucidation of flavonoids. Metal complexation strategies have proven to be especially promising for enhancing the ionization of flavonoids and yielding key diagnostic product ions for differentiation of isomers. Of particular value is the addition of a chromophoric ligand to allow the application of infrared (IR) multiphoton dissociation as an alternative to collision-induced dissociation (CID) for the differentiation of isomers. CID, including energy-resolved methods, and nuclear magnetic resonance (NMR) have also been utilized widely for structural characterization of numerous classes of flavonoids and development of structure/activity relationships.The gas-phase ion chemistry of flavonoids is an active area of research particularly when combined with accurate mass measurement for distinguishing between isobaric ions. Applications of a variety of ab initio and chemical computation methods to the study of flavonoids have been reported, and the results of computations of ion and molecular structures have been shown together with computations of atomic charges and ion fragmentation. Unambiguous ion structures are obtained rarely using MS alone. Thus, it is necessary to combine MS with spectroscopic techniques such as ultraviolet (UV) and NMR to achieve this objective. The application of NMR data to the mass spectrometric examination of flavonoids is discussed.     

Analysis of flavonoids by capillary zone electrophoresis with electrokinetic supercharging

On-line concentration via Electrokinetic Supercharging (EKS) was used to enhance the sensitivity of the capillary electrophoretic separation of the four flavonoids naringenin, hesperetin, naringin and hesperidin. Separation conditions, including the background electrolyte pH and concentration, the length and choice of terminator and the electrokinetic injection time were optimized. The optimum conditions were: a background electrolyte of 30 mM sodium tetraborate (pH 9.5) containing 5% (v/v) of methanol, electrokinetic injection of the sample (130 s, -10 kV) followed by hydrodynamic injecting of 100 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES) (17 s, 0.5 psi) as terminator, and separation with -20 kV. Under these conditions the four flavonoids could be separated with a sample-to-sample time of 15 min and detection limits from 2.0 to 6.8 ng mL(-1). When compared to a conventional hydrodynamic injection the sensitivity was enhanced between 824 and 1515 times which is 7.6-16 times higher than other CE methods for the on-line concentration of flavonoids. The applicability of the developed method was demonstrated by the detection of the four flavonoids in an aqueous extract of Clematis hexapetala pall.     

天然产物黄酮类化合物的高效液相色谱分析

引用了自1997年以来的45篇文献,综述了黄酮类化合物的高效液相色谱（HPLC）分析方法,着重介绍了黄酮、黄酮醇和双氢黄酮等的定性和定量分析,同时简要介绍了指纹图谱及药代动力学研究,为黄酮类化合物今后在分离分析,质量控制和药理药效研究等方面的深入研究提供理论依据。     

Enrichment of flavonoid-rich extract from Bidens bipinnata L. by macroporous resin using response surface methodology,UHPLC–Q-TOF MS/MS-assisted characterization and comprehensive evaluation of its bioactivities by analytical hierarchy process

The extract of Bidens bipinnata L. exhibited wide spectrum of bioactivities owing to the presence of flavonoids. In this study, a purification process was developed to enrich the flavonoid-rich extract from B. bipinnata L. (BBTF). AB-8 resin was selected for the purification of total flavonoids. Response surface methodology coupled with Box–Behnken design was employed to optimize the purification condition; it was optimized as pH 5.1, volume of ethanol 80 ml, flow rate of ethanol 1.8 bed volume per hour (BV/h) and concentration of ethanol 76.0%. The total flavonoid content of BBTF was 56.48% under the optimal conditions. The identification of flavonoids in BBTF was conducted using UHPLC–ESI-Q-TOF MS. A total of 14 compounds, including 12 flavonoids, were identified in BBTF. Finally, the in vitro antioxidant activities, α-glucosidase and α-amylase inhibitory activities of BBTF were comprehensively analyzed by an analytical hierarchy process. The results indicated that it exhibited higher bioactivities than the crude extract. These findings suggested that the optimized process could significantly enhance the purity of flavonoids and their bioactivities. This study showed a comprehensive analysis of a total flavonoid extract of B. bipinnata L. for the first time, which could provide a useful approach for its purification process and quality control as well as bioactivities.     

Optimization for quantitative determination of four flavonoids in Epimedium by capillary zone electrophoresis coupled with diode array detection using central composite design

Herba Epimedii (family Berberidaceae), Ying-Yang-Huo in Chinese, is a famous Chinese herbal medicine. Flavonoids are thought to be the major active components in it. A capillary zone electrophoresis (CZE) separation were developed for simultaneous determination of four flavonoids including icariin, epimedin A, epimedin B and epimedin C in Epimedium. The effects of the experimental variables on CZE had been optimized by using central composite design (CCD). The best separation of four flavonoids could be obtained using 50 mM borate buffer (pH 10.0) containing 22% acetontrile as modifier, while separation voltage was 15 kV and temperature was at 25 degrees C. The method developed is accurate, simple and reproducible, which could be used for quality control of Epimedium and its medical preparations.     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Chemical composition and antioxidant activities of extracts from Apocyni Veneti Folium

An expeditious and effective HPLC-UV method has been developed for the simultaneous determination of seven major flavonoids in Apocyni Veneti Folium (AVF) extract. The chemical profile of seven flavonoids, including quercetin-3-O-β-D-glc(2?→?1)-β-D-glucoside, rutin, isoquercetin, kaempferol-3-O-β-D-glucoside, quercetin-3-O-(6″-O-malonyl)-β-D-glucoside, quercetin and kaempferol was acquired by HPLC-UV. The analysis was performed on a Diamosil C18 analytical column with a gradient solvent system of acetonitrile-0.1% aqueous acetic acid. Full validation of the method was carried out (linearity, reproducibility, repeatability, accuracy and limit of detection). The results indicated that the contents of investigated flavonoids in Apocyni Veneti Folium varied significantly from habitat to habitat, with contents ranging from 0.01 to 5.57 mg g?1. The antioxidant activity results demonstrate that the seven flavonoids showed great efficiency in scavenging DPPH radicals. The high content of flavonoid components of AVF could be responsible for its high antioxidant activity. This study provides powerful evidence for the relationship between the chemical ingredients of and bioactivity in AVF.     

Bio-guided isolation of Centaurea bruguierana subsp. belangerana cytotoxic components

Centaurea bruguierana subsp. belangerana was extracted by 80% ethanol. The total extract was then partitioned into four fractions including chloroform, ethyl acetate and methanol. Cytotoxic effect of fractions was examined by MTT assay in K562 (chronic myelogenous leukemia), AGS (gastric adenocarcinoma), MCF-7 (breast adenocarcinoma) and SW742 (colon adenocarcinoma) cell lines. The Chloroform fraction, with the lowest LC₅₀ against K-562 cell lines, was partitioned into 14 subfractions and subjected to further purification by reversed-phase (C18) silica gel and sephadex LH-20 column chromatography. Three flavonoids including cirsimaritin, cirsilinelol and eupatilin were isolated for the first time from the species and the structures were confirmed by spectroscopic data. The high selectivity index of the purified flavonoids indicates valuable components with potential few side effects for normal cell lines. However, solubility tests for isolated components indicates the need for novel pharmaceutical dosage forms, in the case for using natural flavonoids as chemotherapeutic agents.