Flavonoid Aglycones Research Articles

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism.

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids invitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones invitro prompted us to investigate the potential physiological roles of the enzyme invivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids invivo, we conclude that the major and likely only substrate of Ca10OMTinvivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides.

Characterisation of phenolic compounds by UPLC-QTOF-MS/MS of geopropolis from the stingless bee Melipona subnitida (jandaíra).

Melipona subnitida Ducke (jandaíra) is a stingless bee native to north-eastern Brazil, which produces geopropolis, a mixture of beeswax, plant resins, pollens and earth that is used for sealing beehives. To extend the knowledge on phenolic compounds in fractions obtained by C18-solid phase extraction (SPE) of nine geopropolis samples from Melipona subnitida collected at different times. Chromatographic profiles of nine samples of geopropolis from jandaíra were analysed by ultra-performance liquid chromatography coupled with a diode array detector and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS/MS) and combined with the use of data-independent acquisition (MSE) for the profiling and structural characterisation of the phenolic compounds. The isolated compound was identified by nuclear magnetic resonance of hydrogen and carbon (1 H- and 13 C-NMR). The present study with geopropolis of jandaíra resulted in the characterisation of 51 phenolics by UPLC-DAD-QTOF-MS/MS: four galloyl glucosides, one ellagic acid, 11 acyl-hexosides, 23 acyl-galloyl-hexosides and 12 flavonoids. The structures of two compounds (1,6-di-O-(E)-coumaroyl-2-O-galloyl-β-d-glucopyranoside and 1-O-cinnamoyl-6-O-(E)-coumaroyl-2-O-galloyl-β-d-glucopyranoside) were established by 1 H and the attached proton test (APT) experiments as well as high-resolution electrospray ionisation mass spectroscopy (HR-ESI-MS) analysis. The geopropolis of jandaíra showed phenolic compounds galloyl hexosides, ellagic acid, acyl-(cinnamoyl/coumaroyl)-hexosides, acyl-(cinnamoyl/coumaroyl)-galloyl-hexosides and flavonoids (aglycones and acylated-O-glycosides).

Three Regioselectively Acylated Flavonoid Aglycone Derivatives in Equimolar Yield at One Blow

AbstractA simple and time‐effective chemoenzymatic process to obtain three regioselective acylated quercetin analogues in a 1:1:1 ratio in one pot is described. This process overcomes the inherent scaffold intricacy and synthetic complexity of quercetin. Cell proliferation experiments in three breast cancers cell lines pinpoint the high potency of the generated compounds.

Fermentation and complex enzyme hydrolysis for improving the total soluble phenolic contents, flavonoid aglycones contents and bio-activities of guava leaves tea

There are both soluble and insoluble-bound forms of phenolics in tea-leaf products. In order to increase total soluble phenolics contents, guava leaves tea (GLT) was first fermented with Monascus anka and Saccharomyces cerevisiae, and then hydrolyzed with complex enzymes. The changes in phenolics profiles, antioxidant activities and inhibitory effect on α-glucosidase in processed GLT were investigated. Compared with the un-fermented GLT, fermentation and complex enzymatic processing (FE) significantly increased the total phenolics, total flavonoids, quercetin and kaempferol contents by 2.1, 2.0, 13.0 and 6.8 times, respectively. After the FE, a major proportion of phenolics existed in the soluble form. Quercetin was released in the highest amount among different phenolics. In addition, soluble phenolic extracts from GLT following FE exhibited a highest antioxidant activity and inhibitory effect on α-glucosidase. The paper suggested an improved method for processing GLT into high-value products rich in phenolics and flavonoids aglycones with enhanced health benefits.

Flavonoid Aglycones and Glycosides from the Leaves of some Japanese Artemisia Species

Sixteen Japanese Artemisia taxa were surveyed for flavonoid aglycones and glycosides. The leaves were rinsed with acetone and then extracted with MeOH. Isolated flavonoids were identified by UV, LC-MS, acid hydrolysis, 1H and 13C NMR, and/or HPLC comparisons with authentic samples. Thus, nine flavone aglycones, apigenin (10), luteolin (5), chrysoeriol (11), hispidulin (6), jaseosidin (7), nepetin (15), sudachitin (8), luteolin 7-methyl ether (13) and eupatilin (14), three flavonol aglycones, axillarin (9), quercetin 3-methyl ether (16) and 5,7,4′-trihydroxy-3,6-dimethoxyflavone (12), one flavanone aglycone, pinocembrin (4), three dihydroflavonol aglycones, taxifolin (1), taxifolin 3-acetate (2) and padmatin (3) were found in their Artemisia taxa with the various combination. On the other hand, four flavonol glycosides, quercetin 3- O-rutinoside (17), quercetin 3- O-glucoside (18), patuletin 3- O-glucoside (19) and patuletin 3- O-rhamnosylglucoside (20), were isolated from a few Artemisia taxa.

Genotype- and provenance-related variation in the leaf surface secondary metabolites of silver birch

The cuticular wax layer on silver birch (Betula pendula Roth) leaves is rich in cyclic secondary metabolites that provide defense against various environmental factors. Micropropagated trees from the southern (60°N), central (62°N), and northern (66°N) latitudes of Finland were grown in a common garden setup and quantified for variation in leaf surface secondary metabolites and other leaf traits, and their association with genotype and provenance was studied. The 12 genotypes studied differed greatly in the quantity of surface secondary metabolites, both for individual flavonoid and triterpenoid aglycones and for the overall metabolite profile. Qualitative differences were observed for one triterpenoid that was present in a single genotype (R3). The variance explained by the provenance was low (between 1% and 36%) for most metabolites, but the profile showed clear separation by provenance. The contents of two alkyl coumarates, reported for the first time in silver birch leaf waxes, displayed differences a...

Investigation of antibacterial and cytotoxic potential of phenolics derived from Cistus incanus L. by means of thin-layer chromatography-direct bioautography and cytotoxicity assay

ABSTRACTCistus incanus L. (hairy rockrose) is a medicinal plant which belongs to the Cistaceae family and the Cistus genus, with a well established position in traditional medicine of the Mediterranean basin and the Middle East. It was the aim of this study to compare antibacterial activity of the phenolics derived from fourteen C. incanus samples of different origin (Turkey, Albania, Greece, and an unknown geographical location) obtained as herbal teas from a local market of diet supplements. This activity was assessed with the use of thin-layer chromatography–direct bioautography (TLC-DB) applied to crude extracts against the Gram negative naturally luminescent marine bacterium Aliivibrio fischeri and the Gram positive soil bacterium Bacillus subtilis as the test microorganisms. It was established that in spite of different origin of the investigated herbal samples, in qualitative terms their antibacterial activity was closely comparable and more strongly pronounced against the Gram positive than the Gram negative bacterium. Crude extract originating from one herbal specimen labelled A3 (sample no. 3 from Albania) underwent selective multi-step extraction of the phenolics, dividing them into six fractions (I to VI) that expectedly contain flavonoid aglycons, free phenolic acids, non-polar flavonoid glycosides, polar flavonoid glycosides, and phenolic acids obtained through the acidic and basic hydrolysis from the respective glycosides. Antibacterial activity of each A3 fraction was then assessed with the use of the same TLC-DB approach and it was established that the strongest effect was exerted by fractions I and II (flavonoid aglycons and free phenolic acids). Moreover, cytotoxic assay was performed for the crude C. incanus extracts against the human colon adenocarcinoma cells and a moderate yet well measurable cytotoxic effect was observed with all investigated C. incanus samples. In analogy to antibacterial activity, also in this case cytotoxic potential of all investigated crude C. incanus extracts was similar.

HPTLC–densitometric and HPTLC–MS methods for analysis of flavonoids

ABSTRACTHPTLC silica gel plates without and with fluorescence indicator F254 in combination with n-hexane–ethyl acetate–formic acid (20:19:1, v/v/v) as a developing solvent were explored for the HPTLC–densitometric and HPTLC–MS/(MSn) analyses of flavonoids. Pre-development of the plates with chloroform–methanol (1:1, v/v) was needed for reliable HPTLC–densitometric analyses of flavonoid aglycones in the whole RF range, while 2-step pre-development (1st methanol–formic acid (10:1, v/v), 2nd methanol), that decreased background signals of formic acid adducts, was required for HPTLC–MS analyses. Optimization with conditioning of the adsorbent layer with water before development and saturation of the twin trough chamber resulted in required decrease of the RF values of studied flavonoids (flavone, apigenin, luteolin, chrysin, quercetin dihydrate, myricetin, kaempferide, kaempferol, naringenin, pinocembrin).Detection was performed based on fluorescence quenching (on the plates with F254), natural fluorescence and after post-chromatographic derivatization with natural product reagent without or with further enhancement and stabilization of fluorescent zones with polyethylene glycol (PEG 400 or PEG 4000) or paraffin–n-hexane reagents. For all three reagents, drying temperature and time passed after drying influenced the intensity, which was increasing the first 20 min, and the stability (less than 2 h for PEGs and at least 24 h for paraffin–n-hexane) of the standards’ zones.Optimal wavelengths for densitometric evaluation were selected based on in-situ absorption spectra scanned before and after derivatization and after stabilization. The developed method was tested via analyses of propolis, roasted coffee, rose hip, hibiscus, rosemary and sage crude extracts. To further increase the reliability of the obtained densitometric results HPTLC–MS/(MSn) analyses of all crude extracts were performed. Several phenolic and non-phenolic compounds were tentatively identified.Some possible interferences with phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) that are often present in the extracts together with flavonoids were also examined.

Cytochromes P450 Involved in Metabolism of Flavonoid Aglycones

Flavonoids are secondary plant metabolites to which we are exposed through everyday diet (vegetables, fruits, dietary supplements, herbal drugs). They represent a wide class of compounds that have extensively been studied due to beneficial biological effects. As we have evolutionary been exposed to these compounds, human body has developed mechanisms how to excrete these compounds (metabolism, transporters). Objective of this study was to determine cytochromes P450 involved in metabolism of flavonoid aglycones and characterize generated metabolites. Metabolism of 30 flavonoids was assessed using human liver microsomes and recombinant liver cytochrome P450 enzymes. HPLC method coupled with UV detector and MS TOF was used for detection and quantification. Flavonoids could be separated based on retention time even though there are subtle differences in flavonoid structures. Structures of metabolites were elucidated using accurate mass data, retention time and confirmed using flavonoid standards. Flavonoid aglycones are susceptible to hydroxylation and demethylation reactions. The most significant enzyme involved in metabolism of analyzed flavonoids was cytochrome P450 1A2 which is in accordance with previously reported data. Other enzymes involved in metabolism of flavonoid aglycones were cytochromes P450 2D6, 2C19, and 3A4. As data on pharmacokinetics of flavonoids is limited to few well studied flavonoids, understanding metabolism of flavonoids will improve efforts to obtain flavonoid candidates less susceptible to metabolic elimination.Support or Funding InformationThis work was supported by the Croatian Science Foundation under the project UIP‐2014‐09‐5704 (M.B.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insight into the composition and surface corona reliant biological behaviour of quercetin engineered nanoparticles

Physicochemical properties of nanoparticles have noteworthy influence on their interaction with biological systems. In the present study, we have developed gold, silver and their bimetallic alloy nanoparticles of different compositions using plant-derived aglycone flavonoid quercetin as a reducing and stabilizing agent. It is interesting that a simple biomolecule can provide a handle to control the composition in a facile manner besides suitable biomolecular corona. Moreover, the presence of quercetin corona in conjugation with specific metal composition exhibited in-vitro radical scavenging capabilities, inherent in-vitro peroxidase like behaviour and antibacterial activities in selective manner. In addition, the cytotoxic effects of these nanoparticles on skin fibroblast cells (M5S) were assessed by membrane leakage of lactate dehydrogenase (LDH), reactive oxygen species (ROS) generation and cellular morphology analysis. All the nanoparticles were found to have composition and surface corona reliant effects on M5S cells. Furthermore, quercetin capping increased cell number and induced the collagen type-1A mRNA expression, confirming collagen synthesis in cells upon treatment. This study reveals that the investigated biological actions of engineered nanoparticles are driven by metal composition of nanoparticles in addition to their surface corona; and fine-tuning of these properties can open new opportunities for a wide range of applications.

HPLC Screening of Bioactives Compounds and Antioxidant Capacity of Different Hypericum Species

Our study compares the content in polyphenolic compounds and hypericin, in four species of Hypericum - H. perforatum L., H. maculatum Cr., H. hirsutum L., H. tetrapterum Fr. (syn. Hypericumacutum Mnch.) harvested from spontaneous flora in the north-western area of Transylvania, Romania. These species represent an important source of such compounds with different biological actions. After making the extracts, they were subjected to HPLC-SM analysis. The presence of rutoside in the largest amount (462.82 mg %) in the H. perforatum extract was observed, this containing most of the flavonoid heterosides. For the species H. maculatum, the presence in a much higher amount of the hyperoside (976.36 mg %) is characteristic compared to the other species. Quercetol is the best represented of the flavonoid aglycons, its concentration being the highest in H. hirsutum (659.66 mg %). The hypericin content ranges from 0.2171 g % in the H. tetrapterum extract, to 0.0314 g % in the methanol extract of H. maculatum.The highest antioxidant properties measured by FRAP method were recorded in the case of H. perforatum and H. maculatum.

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis).

Fruits of sweet orange (Citrus sinensis), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O-glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O-rutinosides or O-neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7-O-rhamnoside, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside, implying that the enzyme has flavonoid 7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities in vivo.

Pharmacokinetic Study of Bioactive Flavonoids in the Traditional Japanese Medicine Keigairengyoto Exerting Antibacterial Effects against Staphylococcus aureus.

Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by β-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT flavonoids and to identify active flavonoids contributing to the adjuvant effects of KRT. KRT was evaluated at pharmacokinetic analysis to quantify absorbed flavonoids, and cutaneous infection assay induced in mice by inoculation of Staphylococcus aureus. Preventive or therapeutic KRT administration reduced the number of bacteria in the infection site as well as macroscopic and microscopic lesion scores with efficacies similar to antibiotics. Pharmacokinetic study revealed low plasma levels of flavonoid aglycones after KRT administration; however, plasma concentrations were enhanced markedly by β-glucuronidase treatment, with baicalein the most abundant (Cmax, 1.32 µg/mL). In random screening assays, flavonoids such as bacalein, genistein, and apigenin enhanced bacteria phagocytosis by macrophages. Glucuronide bacalin was converted to aglycone baicalein by incubation with living macrophages, macrophage lysate, or skin homogenate. Taken together, the adjuvant effect of KRT may be due to some blood-absorbed flavonoids which enhance macrophage functions in host defense. Flavonoid-rich KRT may be a beneficial treatment for infectious skin inflammation.

Major chemical constituents and antioxidant activities of different extracts from the peduncles of Hovenia acerba Lindl

ABSTRACTHovenia acerba Lindl. is a perennial tree of Rhamnaceae family. In the present study, four solvents (80% methanol, ethyl acetate, n-hexane, and water) were used to extract the compounds of Hovenia acerba Lindl. peduncles (HAP). The total contents of phenolics and tannins (TPC and TTC), as well as their antioxidant activities (DPPH, ABTS, and FRAP) of these four different extracts including 80% methanol extract (ME), ethyl acetate extract (EE), n-hexane extract (HE) and water extract (WE) were compared. The antioxidant activities of HAP showed positive correlations with TPC and TTC, indicating that phenolics maybe a major contributor to the antioxidant activities of HAP. The significant difference between antioxidant activities and TPC/TTC implied the respective contribution extent of phenolics for the antioxidant activities of four solvent extracts. Besides, a total of 117 compounds, including 7 oligosaccharides, 11 organic acids, 41 phenolics, 1 steroids, 25 terpenes, 11 fatty acids, 9 amino acids, and 12 other compounds were identified or tentatively characterized in HAP for the first time. Among them, 23 bioactive constituents, including 4 organic acids, 5 phenolic acids, and 14 flavonoids, were quantified. The comparison of characterization and quantitation of 4 extracts demonstrated that 80% methanol showed the widest range of selectivity for both HAP flavonoid aglycones and glycosides, while ethyl acetate preferred to extract the flavonoids aglycone from HAP. Secondly, water and 80% methanol were excellent solvents for the extraction of phenolic acids. And both ethyl acetate and 80% methanol perform well at the extraction of terpenes and steroids.

Therapeutic potential of Achillea fragrantissima extracts in amelioration of high-fat diet and low dose streptozotocin diabetic rats

Achillea fragrantissima is a perennial herb used by Arabic Bedouin for the treatment of several pathologies. Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is usually associated with hyperlipidemia into what is nowadays known as the metabolic syndrome epidemic. Aim: is to evaluate the effects of alcoholic and ethyl acetate extracts of A. fragrantissima in metabolically manipulated T2DM rats. Materials and Methods: Induction of T2DM in rats was through high–fat diet (HFD) for twelve consecutive weeks followed by a single injection of streptozotocin (STZ). The activities of both A. fragrantissima extracts were assessed in comparison with glibenclamide and fenofibrate as reference drugs. Results: Elevated blood glucose levels, serum lipid profile, liver functions and kidney functions witnessed a significant reduction by treatment with the extracts. Meanwhile, Oxidative stress markers (MDA, GPx and SOD), pro-inflammatory biomarkers (TNF-α and IL-6 )and adhesion molecules (ICAM-1 and VCAM-1) assessments recorded significant improvement after treatment with A. fragrantissima extracts.The results were also associated with improvement in histopathological examination of pancreas, liver and kidney tissues. The total polyphenolic and flavonoid contents were determined spectrophotometrically. Seven phenolic compounds were isolated and identified in the ethyl acetate extract’s fractions; three flavonoid glycosides, four flavonoid aglycones and a triterpene. Conclusions: The current study suggests that A. fragrantissima extract might be considered as a promising natural supplement for management of T2DM manifestations.

The investigation of bioactive secondary metabolites of the methanol extract of eryngium amethystinum

Eryngium amethystinum L. belonging to the Apiaceae family, is a perennial plant distributed in Southeast Europe. Even though this plant is used in traditional medicine, its phytochemical characterization is still incomplete. In this study composition of bioactive constituents of the methanol extract are reported for the first time. By means of the UPLC-LTQ-Orbitrap-MSn method, altogether sixty-three constituents were characterized: eight hydroxybenzoic acid derivatives (7-13, 32), fifteen cinnamic acid derivatives (14, 17-19, 21, 24-26, 28, 30, 39-42 and 44), four flavonoid aglycones (45, 51, 52, 54), twenty-four flavonoid derivatives (23, 27, 29, 31, 33-38, 43, 46-50, 53, 55-59, 61 and 62), three coumarin derivatives (15, 16 and 22) and nine other compounds (1-6, 20, 60 and 63).

Optimization of Acid Hydrolysis of Myricetin-3-O-rhamnoside Using Response Surface Methodology

This study aimed to optimize the acid hydrolysis of glycosylated flavonols, to apply the optimum conditions for hydrolyzing Inga edulis extract (IEE), rich in glycosylated flavonols, and evaluating its impact on the antioxidant capacity. To evaluate the influence of three independent variables on the aglycone obtained after the reaction, the response surface methodology was applied using myricetin-3-O-rhamnoside (M3R) as a pure compound. The phenolic compound profile and antioxidant capacity were determined by high-performance liquid chromatography (HPLC) and the Trolox-equivalent antioxidant capacity (TEAC) assay, respectively. The maximum content of the aglycone myricetin (81.15%) occurred with 2.5 M HCl at 75 oC for 60 min. Under these conditions, the IEE produced the aglycones myricetin, quercetin and cyanidin. The TEAC values of the M3R samples and IEE before and after acid hydrolysis did not show significant differences (p > 0.05). These results suggest that the hydrolytic process is effective to produce aglycone flavonoids from leaf extracts of I. edulis, and probably for other plant extracts rich in M3R.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography – direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones’ components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.

Elucidating the Skin Delivery of Aglycone and Glycoside Flavonoids: How the Structures Affect Cutaneous Absorption.

Flavonoids are bioactive phytochemicals that exhibit protective potential against cutaneous inflammation and photoaging. We selected eight flavonoid aglycones or glycosides to elucidate the chemistry behind their skin absorption capability through experimental and computational approaches. The skin delivery was conducted using nude mouse and pig skins mounted on an in vitro Franz cell assembly. The anti-inflammatory activity was examined using the O2•– and elastase inhibition in activated human neutrophils. In the equivalent dose (6 mM) application on nude mouse skin, the skin deposition of naringenin and kaempferol was 0.37 and 0.11 nM/mg, respectively, which was higher than that of the other flavonoids. Both penetrants were beneficial for targeted cutaneous therapy due to their minimal diffusion across the skin. The absorption was generally greater for topically applied aglycones than glycosides. Although naringenin could be classified as a hydrophilic flavonoid, the flexibility of the chiral center in the C ring of this flavanone could lead to better skin transport than the flavonols and flavones with a planar structure. An optimized hydrophilic and lipophilic balance of the flavonoid structure was important for governing the cutaneous delivery. The hydrogen bond acceptor and stratum corneum lipid docking estimated by molecular modeling showed some relationships with the skin deposition. The interaction with cholesteryl sulfate could be a factor for predicting the cutaneous absorption of aglycone flavonoids (correlation coefficient = 0.97). Baicalin (3 µM) showed the highest activity against oxidative burst with an O2•– inhibition percentage of 77%. Although naringenin displayed an inhibition efficiency of only 20%, this compound still demonstrated an impressive therapeutic index because of the high absorption. Our data are advantageous to providing the information on the structure–permeation relationship for topically applied flavonoids.

The Influence of Compatibility of Rhubarb and Radix Scutellariae on the Pharmacokinetics of Anthraquinones and Flavonoids in Rat Plasma.

Rhubarb-Radix scutellariae is a classic herb pair, which is commonly used to clear away heat and toxin in clinic. The aim of this study was to investigate the influence of compatibility of Rhubarb and Radix scutellariae on the pharmacokinetic behaviors of anthraquinones and flavonoids in rat plasma. Eighteen rats were randomly divided into three groups, and were orally administered Rhubarb and/or Radix scutellariae extracts. A sensitive and rapid UPLC-MS/MS method was developed and validated to determine the concentrations of baicalin, baicalein, wogonside, wogonin, rhein, and emodin in rat plasma. The concentrations of phase II conjugates of flavonoid aglycones and anthraquinone aglycones were also determined after hydrolyzing the plasma with sulfatase. Compared with administration of Radix scutellariae alone, co-administration of Rhubarb significantly decreased the first maximum plasma concentration (C max1) of baicalin, wogonside, and the phase II conjugates of baicalein, wogonin to 46.40, 61.27, 41.49, and 20.50%, respectively. The area under the plasma concentration-time curve from time zero to infinity (AUC0-∞) was significantly decreased from 82.60±20.22 to 51.91±7.46μM·h for rhein and 276.83±98.02 to 175.42±86.82μM·h for the phase II conjugates of wogonin after compatibility. The time to reach the first maximum plasma concentration (T max1) of anthraquinones was shortened and the second peak of anthraquinones disappeared after compatibility. Compatibility of Rhubarb and Radix scutellariae can significantly affect the pharmacokinetic behaviors of characteristic constituents of the two herbs. The cause of these pharmacokinetic differences was further discussed combined with the in vivo ADME (absorption, disposition, metabolism, and excretion) processes of anthraquinones and flavonoids.