Design, synthesis, and primary activity assays of baicalein derivatives as cyclin-dependent kinase 1 inhibitors.

Malignant tumor is a disease with high mortality. Traditional treatment methods have many disadvantages, such as side-effects, drug resistance. Because cyclin-dependent kinase 1 (CDK1) plays an indispensable role in cell cycle regulation, it became an attractive target in rational anti-cancer drug discovery. Herein, we reported a series of baicalein derivatives, which remarkably repressed the proliferation of MCF-7 tumor cells and the activity of CDK1/cyclin B kinase. Among them, compound 4a displayed better inhibition rate than flavopiridol against MCF-7 proliferation at the concentration of 50 µg/ml, comparable to compound CGP74514A, while compound 3o possessed the best activity against CDK1/cyclin B kinase (IC50  = 1.26 µM). The inhibitory activities toward the kinase well correlated with anti-proliferative activities. Molecular docking results suggested that compound 3o can interact with the key amino acid residues, E81, L83, and D146, of CDK1 through hydrogen bond just like flavopiridol does. And it can also form an extra hydrogen bond with D146 by its introduced 7-acrylate group, which flavopiridol does not have. These findings proved that baicalein derivatives can be used as CDK1 inhibitors fighting against cancer.

Aurones: A Golden Resource for Active Compounds.

Deemed as poorly represented in nature, aurones have been often overlooked by researchers compared to other members of the flavonoid superfamily. However, over the past two decades, they have been reassessed by the scientific community, who are increasingly appreciating their ability to modulate several biological pathways. This review summarizes the recent literature on this class of compounds, which has been analyzed from both a chemical and a functional point of view. Original articles, reviews and editorials featured in Pubmed and Scifinder over the last twenty years have been taken into account to provide the readers with a view of the chemical strategies to obtain them, their functional properties, and their potential of technological use. The resulting comprehensive picture aims at raising the awareness of these natural derivatives as effective drug candidates, fostering the development of novel synthetic analogues.

Microwave irradiation followed by zinc oxide based dispersive solid-phase extraction coupled with HPLC for simultaneous extraction and determination of flavonoids in Veronicastrum latifolium (Hemsl.) Yamazaki.

In this study, a sample preparation method involving microwave irradiation followed by dispersive solid-phase extraction was used to extract and separate flavonoids from Veronicastrum latifolium (Hemsl.) Yamazaki. Ethyl lactate was selected as the extraction solvent during microwave irradiation. Zinc oxide and ethanol were used as the adsorbents and the eluent, respectively, in dispersive solid-phase extraction. Several experimental parameters affected the extraction performance, such as the extraction solvent and method, the microwave irradiation time, temperature, and solid-to-liquid ratio, the type of adsorbent, the amount of sample solution, the adsorption time and method, and the type and volume of the eluent, and were investigated by single-factor and Box-Behnken design experiments in the pretreatment process. The recommended method was applied to extract flavonoids in Veronicastrum latifolium (Hemsl.) Yamazaki. The enriched flavonoids were analyzed for the first time by high-performance liquid chromatography, and seven flavonoids-vitexin, luteolin, wogonoside, apigenin, chrysoeriol, acacetin, and pectolinarigenin-were identified. Additionally, the method exhibited good linearity (r2 ≥ 0.9992) for flavonoids under the optimum conditions. Moreover, the relative standard deviation of the intraday and interday precision was less than 3.97%. The mean recoveries for flavonoids were more than 98.10%. This work is the first to report a method to extract, separate, and analyze flavonoids from Veronicastrum latifolium (Hemsl.) Yamazaki. Graphical Abstract Microwave irradiation followed by ZnO-based dispersive solid-phase extraction for the pretreatment of Veronicastrum latifolium (Hemsl.) Yamazaki samples, and high-performance liquid chromatography (HPLC) for analysis of enriched flavonoids.

TLC-MALDI-TOF-MS-based identification of flavonoid compounds using an inorganic matrix.

INTRODUCTION: Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC-mass spectrometry (MS) coupling allows a more detailed characterisation of samples. OBJECTIVES: To demonstrate the potential for the flavonoid dereplication using an inorganic matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) method with and without TLC separation. MATERIAL AND METHODS: Samples derived from wine, apple or rose were deposited on an aluminium-backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu-Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. RESULTS: Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in-source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. CONCLUSION: TLC-MALDI-TOF-MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples.

Neuroprotective Effects of a Standardized Flavonoid Extract from Safflower against a Rotenone-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is a major age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra par compacta (SNpc). Rotenone is a neurotoxin that is routinely used to model PD to aid in understanding the mechanisms of neuronal death. Safflower (Carthamus tinctorius. L.) has long been used to treat cerebrovascular diseases in China. This plant contains flavonoids, which have been reported to be effective in models of neurodegenerative disease. We previously reported that kaempferol derivatives from safflower could bind DJ-1, a protein associated with PD, and that a flavonoid extract from safflower exhibited neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD. In this study, a standardized safflower flavonoid extract (SAFE) was isolated from safflower and found to primarily contain flavonoids. The aim of the current study was to confirm the neuroprotective effects of SAFE in rotenone-induced Parkinson rats. The results showed that SAFE treatment increased body weight and improved rearing behavior and grip strength. SAFE (35 or 70 mg/kg/day) treatment reversed the decreased protein expression of tyrosine hydroxylase, dopamine transporter and DJ-1 and increased the levels of dopamine and its metabolite. In contrast, acetylcholine levels were decreased. SAFE treatment also led to partial inhibition of PD-associated changes in extracellular space diffusion parameters. These changes were detected using a magnetic resonance imaging (MRI) tracer-based method, which provides novel information regarding neuronal loss and astrocyte activation. Thus, our results indicate that SAFE represents a potential therapeutic herbal treatment for PD.

[Separation and authentication of tilianin and quality standards of semen of Dracocephalum moldavia].

Tilianin was separated and authenticated from the seeds of Dracocephalum moldavia, a Uygur medicine, by chromatographic technique and spectroscopic method. The purity of tilianin is more than 98% determined by HPLC area normalization method. Thin layer chromatography (TLC) method was used to separate tilianin from D. moldavia by mixture of chloroform-methanol (5: 1) as a developing solvent on high performance silicagel precoated plate (SGF254) and using aluminium trichloride as a chromogenic agent for qualitative identification of D. moldavia. To establish a HPLC method for quantitative analysis of D. moldavia, tilianin was used as a Quantitative marker and separated on a C18 (4.6 mm x 250 mm, 5 µm) column with acetonitrile-01% formic acid (25: 75) as the mobile phase and detected at 330 nm. The calibration curve of tilianin displayed ideal linearity over the range of 0.617 2-123.44 µg x mL(-1) with a regression equation of Y = 33.773X - 0.824 8 (r = 1). The average recovery of tilianin was 101.0% with RSD of 3.7%. The RSD values of intra-day and inter-day precision were less than 2%. The content of tilianin in 4 batches of the authenticated semen of D. Moldavia was between 0.016 and 0.187 mg x g(-1). The qualitative and quantitative method established is suitable for the quality evaluation and assessment of semen of D. Moldavia.

Advancement in the chemical analysis and quality control of flavonoid in Ginkgo biloba.

Flavonoids are the main active constituents in Ginkgo biloba L., which have been suggested to have broad-spectrum free-radical scavenging activities. This review summarizes the recent advances in the chemical analysis of the flavonoids in G. biloba and its finished products (from 2009 to 2014), including chemical composition, sample preparation, separation, detection and different quality criteria. More than 70 kinds of flavonoids have been identified in this plant. In this review, various analytical approaches as well as their chromatographic conditions have been described, and their advantages/disadvantages are also compared. Quantitative analyses of Ginkgo flavonoids applied by most pharmacopeias start with an acidic hydrolysis followed by determination of the resulting aglycones using HPLC. But increasing direct assay of individual flavonol glycosides found that many adulterated products were still qualified by the present tests. To obtain an authentic and applicable analytical approach for quality evaluation of Ginkgo and its finished products, related suggestions and opinions in the recent publications are mainly discussed in this review. This discussion on chemical analyses of Ginkgo flavonoids will also be found as a significant guide for widely varied natural flavonoids.

Different extraction pretreatments significantly change the flavonoid contents of Scutellaria baicalensis.

CONTEXT: Scutellaria baicalensis Georgi (Labiatae) is one of the most commonly used medicinal herbs, especially in traditional Chinese medicine. However, compared to many pharmacological studies of this botanical, much less attention has been paid to the quality control of the herb's pretreatment prior to extract preparation, an issue that may affect therapeutic outcomes. OBJECTIVE: The current study was designed to evaluate whether different pretreatment conditions change the contents of the four major flavonoids in the herb, i.e., two glycosides (baicalin and wogonoside) and two aglycones (baicalein and wogonin). MATERIALS AND METHODS: A high-performance liquid chromatography assay was used to quantify the contents of these four flavonoids. The composition changes of four flavonoids by different pretreatment conditions, including solvent, treatment time, temperature, pH value and herb/solvent ratio were evaluated. RESULTS: After selection of the first order time-curve kinetics, our data showed that at 50 °C, 1:5 herb/water (in w/v) ratio and pH 6.67 yielded an optimal conversion rate from flavonoid glycosides to their aglycones. In this optimized condition, the contents of baicalin and wogonoside were decreased to 1/70 and 1/13, while baicalein and wogonin were increased 3.5- and 3.1-fold, respectively, compared to untreated herb. DISCUSSION AND CONCLUSION: The markedly variable conversion rates by different pretreatment conditions complicated the quality control of this herb, mainly due to the high amount of endogenous enzymes of S. baicalensis. Optimal pretreatment conditions observed in this study could be used obtain the highest level of desired constituents to achieve better pharmacological effects.

[Study on in situ intestinal absorption of baicalin contained in Tiangou Jiangya capsules].

OBJECTIVE: To study in situ intestinal absorption kinetics of baicalin contained in Tiangou Jiangya capsules, and the effect of different intestinal segments, pH value, drug concentration and P-gp inhibitor on the absorption. METHOD: The in situ intestinal perfusion test was adopted, and HPLC method was used to determine the content of baicalin in samples at different time points. Ultra-violet (UV) spectrophotometry was used to determine the content of phenol red in samples at different time points. RESULT: When pH value was at 5. 0, 6. 5, 7. 4, the absorption of baicalin was not impacted. P-gp inhibitor verapamil could enhance the absorption of baicalin. When the quality concentration of the test solution ranged between 5-20 g L -1 , the linearity of the absorption amount of baicalin increased. The absorption kinetic equation of baicalin was Y = -0. 073 7X +0. 118 7 (r = 0. 994 8) , K. 0. 073 7 h -1 , t1/2 9. 40 h. CONCLUSION: Baicalin is mainly absorbed in colon. The absorption of baicalin shows the first-order kinetics process, with the absorption mechanism of passive diffusion. Baicalin is a substrate for P-gp.

[Preparation of flavonoid reference standards from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis].

Flavonoid reference standards were targeted-prepared from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. With HPLC-MS analysis of Scutellariae Radix, 19 flavonoid components were identified by analyzing and comparing their retention times, ultraviolet spectra, and mass spectrometry data with literature. The separation and purification protocols of all targeted flavonoid reference standards were optimally designed according to the results of HPLC-MS analysis and related literature. The ethanol extract of Scutellariae Radix was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol successively. The ethyl acetate extract and n-butanol extract were separately subjected to primary separation by low pressure reverse phase preparative chromatography. Then the fractions containing targeted compounds were further purified by low pressure reverse and normal phases preparative chromatography. Finally, baicalin and wogonoside reference standards were obtained from n-butanol extract; baicaelin, wogonin, and oroxylin A reference standards were obtained from ethyl acetate extract. The structures of the 5 reference standards were identified by mass spectrometry (MS) and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The HPLC analytical results showed that the purities of the 5 reference standards were all above 98%. It is demonstrated that the rapid targeted-preparation method under the guidance of the HPLC-MS analysis is applicable for the isolation and preparation of chemical components in traditional Chinese medicines.

Identification of antioxidants in Fructus aurantii and its quality evaluation using a new on-line combination of analytical techniques.

A new on-line method for simultaneous identification and monitoring of antioxidants in Fructus aurantii was established by coupling high performance liquid chromatography-diode array detector-electrospray ionisation-ion trap-time of flight-mass spectrometry with post-column derivatisation and luminol-potassium ferricyanide chemiluminescence (HPLC-DAD-ESI-IT-TOF-MS-PCD-LPFCL). While the HPLC fingerprint, structural identification and radical scavenging profile were rapidly obtained by an on-line assay using ultraviolet (UV) absorption, MS and LPFCL, details of the precise substitution patterns of various structures were achieved through UV absorption using PCD addition of shift reagents. Twenty-five flavonoids were identified by either their PCD and MS data or comparison with reference substances. Data collected both from chromatograms and activity profiles of 12 samples revealed significant differences among samples from different habitats. The results showed that this method was rapid and precise, and therefore would be an effective and sensitive method for biocompounds analysis and quality evaluation for complex food and medicinal samples.

Orthogonal test design for optimization of the extraction of flavonid from the Fructus Gardeniae.

OBJECTIVE: It is imperative to provide some consistent experimental results for the extraction of flavonid from Fructus Gardeniae. METHODS: The key extraction parameters that influenced the yield of flavonid from Fructus Gardeniae were optimized by employing an orthogonal experiment [L(9)(3)(4)], including the ratio of buffer solution (Na(2)B(4)O(7)·10H(2)O) to raw material, concentration of Fructus Gardeniae in extracting solution, extraction time and pH of buffer solution. An UV/Vis detector was used to perform the qualitative and quantitative analyses of the extracted flavonid with the using of the standard sample. RESULTS: The maximum extraction yield of the crude extract was 5.0533 (mg/g) after 20 min when the mass ratio of Na(2)B(4)O(7)·10H(2)O to raw material was 0.4%, the concentration of Fructus Gardeniae in the extraction solution was 1/12 (g/mL), and pH of buffer solution was 4.5. The positive reactions to the Molish and HCl-Mg tests suggested that the extracted compound was flavonoid, and FTIR measurements also identified the presence of flavonoid in the extracts. CONCLUSION: This work is expected to provide a basis for further research, development, and utilization of Fructus gardenia in flavonid extraction.

Determination of quercetin and resveratrol in whole blood--implications for bioavailability studies.

Resveratrol (trans-3,4',5-trihydroxystilbene) and quercetin (3,3',4',5,7-pentahydroxyflavone) are two naturally occurring polyphenols with the potential to exert beneficial health effects. Since their low bioavailability is a major obstacle to biomedical applications, efforts are being made to improve their absorption and slow down phase II metabolism. An accurate evaluation of the corresponding levels in the bloodstream is important to assess delivery strategies, as well as to verify claims of efficacy based on in vitro results. In the present work we have optimized a simple method ensuring complete stabilization and extraction of resveratrol and quercetin from whole blood. The suitability of different protocols was evaluated by measuring the recovery of polyphenol and internal standard from spiked blood samples via HPLC/UV analysis. The optimized procedure ensured a satisfactory recovery of both internal standards and compounds. Comparing plasma and whole blood, up to 76% of the analyte, being associated with the cellular fraction, was unaccounted for when examining only plasma. This indicates the importance of analysing whole blood rather than plasma to avoid underestimating polyphenol absorption in bioavailability studies.

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi.

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 µg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 µg/ml. The half maximal inhibitory concentration (IC(50)) of naringenin for motility of adult females was 2.5 µg/ml. Flavone immobilized female adult worms at 31.2 µg/ml (MTT>80%) and microfilariae at 62.5 µg/ml. Rutin killed microfilariae at 125 µg/ml and inhibited MTT-reduction in female worms for >65% at 500 µg/ml. Naringin had adulticidal effects at 125 µg/ml while chrysin killed microfilariae at 250 µg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin>flavone=hesperetin>rutin>naringin>chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.

The hidden face of food phenolic composition.

Plant polyphenols are extremely diverse, due to the occurrence of several basic structures, numerous substitutions and, for some groups, of polymers (tannins). Plant polyphenol composition depends on the plant species and organ, with some molecules specific of particular plant families while others are ubiquitous. The polyphenol content is classically assessed by global analysis methods, which lack specificity and accuracy. These methods have been replaced with high performance liquid chromatography (HPLC), that enables accurate determination of individual molecules, provided they can be unambiguously identified and calibration curves can be established. However, HPLC analysis is restricted to simple compounds and difficult to apply in the case of complex extracts. Further difficulties encountered in the case of polymers include irreversible adsorption on the stationary phases. Proanthocyanidin analysis by HPLC after acid-catalysed depolymerisation in the presence of a nucleophile permits to overcome these problems and shows that proanthocyanidins predominate in the polyphenol composition of most plants. Large varietal differences in tannin quantitative and qualitative composition were observed for all plant species studied. Moreover, analysis is usually performed after extraction, which may lead to significant underestimation of the polyphenol content, since a large proportion is not extracted by usual solvents. This may be due to covalent binding to other plant constituents and to non-covalent adsorption on plant solids. Such matrix effect also influences the taste perception of polyphenols and their fate in the digestive tract, from in-mouth interactions with salivary proteins to their metabolism by colon microflora, with potential influence on bioavailability.

[Manufacturing process and quality control of total flavonoid from Chrysanthemum morifolium and Sophora japonica and its quality control].

OBJECTIVE: To optimize the manufacturing process of the total flavonoid from Chrysanthemum morifolium and Sophora japonica, and establish the quality control methods for total flavonoid and its indicative constituents. METHOD: The solvent for extraction was investigated and L9 (3(4)) orthogonal experiment was used to optimize the extraction process of the total flavonoid. Colorimetric method was used to determine the content of total flavonoid, and HPLC was used to determine the content of the indicative constituents. RESULT: The optimized manufacturing process of the total flavonoid was that the prescribed crude drugs were extracted by using 70% ethanol for 3 times, 1 h each time. The extract was concentrated to the final concentration of 0.5 g x mL(-1), and was subjected to HPD-600 macroporous resin column chromatography at the weight ratio 1 : 0. 5 of crude drugs to macroporous resin. The sample was eluted with water in the volume of 10 times of the resin's, and then was desorpted with 85% ethanol in the volume of 5 times of the resin's. The content of total flavonoid was determined as 64.98%-66.79%, and the content of indicative constituents was determined as 5.87% - 6.93% for luteolin-7-O-glucoside and 14.09%-16.62% for rutin. CONCLUSION: Extracting with aqueous alcohol in combination with purifying with macroporous resin column chromatography is an efficient way to prepare the total flavonoid from Ch. morifolium and S. japonica, and the colorimetric and the HPLC methods are useful for its quality control.

Chromatographic fingerprint analysis of Pycnogenol dietary supplements.

The bark of maritime pine (Pinus pinaster Aiton) has been widely used as a remedy for various degenerative diseases. A standard high-performance liquid chromatographic (HPLC) procedure for Pycnogenol analysis is a method specified in the United States Pharmacopeia (USP) monograph, which requires measurement of peak areas and identification of four components of the extract: caffeic acid, catechin, ferulic acid, and taxifolin. In this study, a fingerprint analysis using an HPLC method based on the USP monograph has been developed to provide additional qualitative information for the analysis of Pycnogenol-containing dietary supplements (PDS). Twelve commercially available PDS samples were purchased and analyzed along with a standard Pycnogenol extract. Their chromatographic fingerprints were analyzed using principal component analysis. The results showed that two of the samples were not consistent with the standard reference Pycnogenol extract. One contained other active ingredients in addition to Pycnogenol, and the other may have resulted from a quality control issue in manufacturing.

Stability of active ingredients of traditional Chinese medicine (TCM).

Studies on stability of active ingredients are fundamental and critical for the rational development of Traditional Chinese Medicine (TCM) in view of its modernization and worldwide use. The stability of both active and marker constituents of plants used in TCM is reviewed for the first time. More than 100 papers, mostly written in Chinese, have been reviewed. Studies concerning plant constituents were analyzed according to their chemical classification of active ingredients. In addition, several crude drugs of animal origin are also reported. Stability of active ingredients is summarized during extraction and/or storage of the herbal drug preparations, and under stress conditions (pH, temperature, solvents, light, and humidity) and in the presence of preservatives, antioxidants, and metals.

[Contribution to the phytochemical study of polyphenols in ten hydroalcoholic extracts of Chamomile floss]. / Contributii la analiza fitochimica a polifenolilor existenti in extracte hidroalcoolice preparate din mostre comerciale de Chamomillae flos.

UNLABELLED: Continuing a series of studies that intend to evaluate the pharmaceutical quality of 10 commercial samples of chamomile, we tried to investigate the chemical composition of the hydroalcoholic extracts obtained in our laboratory, starting from this raw material. MATERIAL AND METHOD: The qualitative and semiquantitative analysis of the extracts was done by HPLC means. RESULTS: All extractive solutions have a high content in ferulic acid, whereas the caffeic acid level is the lowest. Regarding the flavonoids, there are many quantitative differences between the samples: one extract lacking the rutoside and two of them having low apigenin-7-glucoside contents.