In vitro biological properties of flavonoid conjugates found in vivo

For some flavonoids such as quercetin, isoflavones and catechins, the pathways of absorption and metabolism are now reasonably well characterised and understood. By definition, for biological activity of flavonoids to be manifest, the target tissue, which includes the blood and vascular system, must respond to the form(s) of flavonoid that it encounters. Bioavailability studies have shown that the circulating form of most flavonoids is as conjugates, with a few notable exceptions. There have been several recent papers on the in vitro biological properties of conjugates that have been found in vivo. This paper reviews the properties of these conjugates. Most of the information currently available is on quercetin glucuronides, but also on isoflavone and catechin conjugates. In addition to the biological properties of the conjugates, the partition coefficients and methods of synthesis are also presented.     

Simultaneous collection of the portal and superior vena cava blood in conscious rats defined that intestinal epithelium is the major site of glucuronidation,but not sulfation and methylation,of quercetin

ABSTRACT

Quercetin is a flavonoid with many physiological effects. Absorbed quercetin is rapidly conjugated in the intestinal epithelium and liver. Different positional isomers of quercetin conjugates have different physiological properties. However, the mechanisms of quercetin conjugation in the intestine are not fully clarified. We examined the regioselective quercetin conjugate formation in the intestine after oral administration of quercetin glycosides, by simultaneous sampling of blood from the portal vein and superior vena cava, and quantifying various positional isomers of quercetin glucuronides and sulfates in conscious rats. Concentrations of quercetin glucuronides were higher in blood from the portal vein than the superior vena cava, showing that glucuronidation mainly occurred in the intestine. Such differences were not observed for quercetin sulfates. Regioselectivity of the intestinal glucuronidation in quercetin hydroxyl groups were 7- >3′- >3- >4′-OH. Quercetin was mainly sulfated on 3′-OH at 30 min, but on 4′-OH at 240 min.     

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.     

In vivo binding and uptake of low-density lipoprotein-gold- and albumin-gold conjugates by parenchymal and sinusoidal cells of the fetal rat liver

Summary To elucidate the participation of fetal rat liver cells in the receptor-mediated internalization of low-density lipoproteins (LDL), rat fetuses were injected with either LDL-gold or albumin-gold conjugates. The degree of binding and uptake of LDL-gold and albumin-gold by parenchymal and sinusoidal cells of the fetal rat liver differs markedly. Endothelial cells exhibit low LDL-gold uptake. In contrast, parenchymal cells internalize LDL-gold more actively (45 ± 8 LDL conjugates/100 m² cytoplasm within 60 min). Kupffer cells exceed this value by a factor of 20. The uptake of albumin-gold by endothelial and Kupffer cells is high, whereas it is extremely low in parenchymal cells. Estradiol pretreatment causes a significant doubling (p<0.05) of the LDL-gold particle density/100 m² cytoplasm both in parenchymal and Kupffer cells, whereas estradiol has no effect on the albumin uptake. The results strongly indicate that LDL uptake by parenchymal and Kupffer cells in the fetal rat liver is mediated by estrogen-inducible receptors, which may correspond to B, E receptors in the adult liver.     

New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis,in vitro biological evaluation,and molecular docking studies

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed K_i of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn²⁺ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

电磁场曝露对生物组织电磁特性的影响

电磁辐射严重影响着人体的健康.电磁场生物效应的发生机制与电磁场本身的特性相关,同时也与生物组织在电磁场作用下电磁特性的改变密切相关.生物体内的信号分子、自由基以及磁颗粒等处于外加电磁场中时其电磁特性会发生变化,尤其是不同频率电磁场曝露作用下生物组织的导电、介电以及磁学等特性会有非常显著的区别.明确不同频率电磁场作用下生物组织电磁特性的变化规律是研究电磁场生物效应发生机制以及预防问题的关键.综述了近年来电磁场对于生物组织电磁特性影响的研究成果,并对未来的研究方向做了展望.     

退化高寒草原土壤生物学性质的变化

对藏北退化高寒草原的土壤生物学性质研究表明:轻度退化草地2~10cm土层微生物(细菌、真菌、放线菌)数量与生物量(碳、氮)、土壤酶(纤维素酶、脲酶、碱性磷酸酶)活性和有机质总体上高于正常草地,中度、严重退化草地则均呈显著降低趋势.微生物生物量碳氮比(BC/BN)与土壤全碳、全氮比(TC/TN)呈极显著正相关(r=0.9088,P≤0.01;n=4);与正常草地相比,轻度、中度退化草地BC/TC、BN/TN值均呈上升趋势,而严重退化草地则呈明显下降趋势.土壤微生物生物量与土壤酶活性呈极显著或显著正相关,但二者均与土壤放线菌数量呈不同程度的负相关;2~10cm土层有机质与土壤微生物生物量、土壤酶活性均呈极显著或显著正相关;随草地退化的加剧,2~10cm和11~20cm土层腐殖质碳占土壤有机碳比重,以及胡敏酸碳占土壤腐殖质碳比重均较正常草地明显上升.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

In vivo and in vitro antioxidant properties of furosemide

The aim of this study was to investigate in vivo and in vitro antioxidant properties of furosemide. In vitro, human red blood cells were submitted to oxidative stress (AAPH), in absence or in presence of different concentrations of furosemide. Potassium efflux was measured in order to quantify the oxidative stress after the action of AAPH on red blood cells. Allophycocyanin assay was also used to investigate antioxidant capacities of furosemide. For the in vivo experiment, male Wistar rats were used. A control group (n = 5) was treated by a daily intraperitoneal injection of saline solution (0.2 ml); 2 other groups (J0 and J+) were treated for 7 days by one daily intraperitoneal injection of furosemide (0.10 mg/kg/day). In the J+group, the injection of furosemide was done one hour before the experiment, while in the J0 group the last injection of furosemide was done on the 6th day and an injection of saline was performed one hour before the experiment. On the day of experiment, a laparotomy was performed under general anesthesia and blood was collected from abdominal aorta. Oxidative stress and antioxidant capacities were evaluated on Wistar rat red blood cells and plasma. In vitro results (oxidative challenge with AAPH) showed that oxidative stress was decreased in presence of furosemide. This was due to a potent free radical scavenging effect of furosemide. In vivo studies confirmed that furosemide had antioxidant properties. These data may be of great relevance in clinical practice, considering the use of large doses of furosemide in patients presenting pathology involving the production of free radicals.     

Design and synthesis of structurally defined heparan sulfate (HS)-FK506 conjugates as an exogenous approach to investigate biological functions of nucleus HS

Although heparan sulfate (HS) is widely implicated in numerous physiological and pathological processes, the biological function of nucleus HS remains underexplored, largely due to its complex structure and high hydrophilic property. To supplement these efforts, ideal vehicles are drawing attention as they combine attractive features including lipid solubility for penetrating cell membrane, high affinity binding to its target receptor, metabolic stability, and no cellular actions resulting in toxicity. Herein, we develop a convenient and promising strategy to prepare HS-FK506 conjugates for membrane transport and entry into nucleus, where click chemistry takes easily place between the exocyclic allyl group of a clinic drug FK506 and thiol as a handle incorporated into HS analogues. HS derivatives for constructing the conjugates were synthesized using a cutting-edge chemoenzymatic method. Meantime, [³⁵S] labeled 3′-phosphoadenosine 5′-phosphosulfate (PAP³⁵S) and [¹⁴C] glucuronic acid (Glc A) were adopted to label HS-FK506 conjugates, respectively, to evaluate their efficiency of nucleus entry, as a result, ¹⁴C Glc A was sensitive, effective and reliable whereas PAP³⁵S gave rise to a mixture of labeled compounds, hampering the understanding of structure-function relationship of nucleus HS. Compared with the corresponding HS, the amount of HS-FK506 conjugates to translocate into nucleus from radioactive assay experiments sharply increased, e.g. tridecasaccharide-FK506 1d increased by approximate 10 folds, offering a simple and robust platform for enabling hydrophilic compounds including carbohydrates to translocate into nucleus and shedding light on their biological functions.     

Analysis of ellagitannins and conjugates of ellagic acid and quercetin in raspberry fruits by LC-MSn

The use of gradient reversed phase HPLC with diode array and MS(n) detection for the analysis of ellagitannins, ellagic acid conjugates and quercetin conjugates in raspberries (Rubus idaeus L.) is described. MS(n) is a particularly powerful tool for the analysis of trace levels of natural products in impure extracts as interpretation of fragmentation patterns, coupled in some instances with knowledge of HPLC retention properties, can facilitate the partial identification of components when reference compounds are unavailable.     

Validated methods for direct determination of hydroquinone glucuronide and sulfate in human urine after oral intake of bearberry leaf extract by capillary zone electrophoresis

Bearberry leaf extracts are used in herbal medicinal products for the treatment of lower urinary tract infections. Two metabolites of the major phenolic constituent in the extract, arbutin (hydroquinone-1-O-β- -glucoside), must be assumed to be precursors of the active disinfectant principle hydroquinone. In order to assay the renal elimination of these two metabolites, i.e., hydroquinone conjugates with glucuronic and sulfuric acid, two separate capillary electrophoresis methods have been developed. Both methods were validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998. As there is little sample preparation necessary, both methods are very suitable for urine analysis with large sample numbers as frequently coming up in the course of pharmaceutical bioavailability, bioequivalence and pharmacokinetic studies.     

In vivo immunomodulatory effects of plant flavonoids in lipopolysaccharide-challenged broilers

Plant flavonoids are generally regarded as natural replacers of synthetic growth promoters in poultry production. This study investigated the immunomodulatory effects of plant flavonoids, such as genistein and hesperidin, in lipopolysaccharide (LPS)-challenged broilers. A total of 700 21-day-old commercial Arbor Acres broiler chicks were randomly assigned into six treatment groups, each having six pens of 20 chicks/pen. Chicks were fed a basal diet without any additive (control, CON), 5 mg genistein/kg feed (G5), 20 mg hesperidin/kg (H20), or a basal diet with a combination of genistein and hesperidin (1 : 4) with doses of 5 mg/kg feed (GH5), 10 mg/kg (GH10) and 20 mg/kg (GH20) for 6 weeks. Half of the birds from each treatment were separated, and either challenged with 0·9% sodium chloride solution or Escherichia coli LPS (250 μg/kg BW) on days 16, 18 and 20. The results showed that both genistein and hesperidin improved (P<0.01) the plasma antioxidant status of growing broilers, by increasing total antioxidant capacity (TAOC), superoxide dismutase (SOD) activity and decreasing malondialdehyde production. LPS challenge further increased (P<0.05) TAOC and SOD levels. Regardless of LPS challenge, both genistein and hesperidin improved the humoral and mucosal immunity by increasing the intestinal intraepithelial lymphocyte numbers (P<0.01), as well as anti-Newcastle disease and anti-avian influenza antibody titers (P<0.05). Supplementation of both the plant flavonoids generally increased (P<0.05) the immune organs indices (spleen, thymus and bursa). Thus, supplementation of basal diet of broiler chicks, either with genistein or hesperidin, improved immune and antioxidant status of growing broilers. In addition, combined supplementation of both the flavonoids showed further improvement than individual compounds.     

In vitro properties of various growth factors and in vivo effects

This article summarizes some of the data that have been accumulated on several growth factors. Biochemical and biological properties of the Epidermal, Fibroblast, Astrocytes and Tumor growth factors (EGF, FGF, AGF, TGF) and those of growth factors derived from Platelets (PDGF), Brain (BDGF, ECGF), Eye (EDGF) and Cartilage (CDGF) are reviewed, as well as the in vitro mechanism of action of EGF and PDGF. The in vivo effects of these growth factors, particularly the experiments achieved to understand the physiological or physiopathological significance are described. The potential interest of these molecules in pharmacology and their use as wound healing agents is discussed.     

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8-methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4',8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 microM in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.     

Quercetin cumulatively enhances copper induction of metallothionein in intestinal cells

Wilson’s disease, a genetic copper-overload condition, is currently treated with zinc because of the ability of zinc to induce metallothionein. We are interested in nonmetal chemicals that may alter intestinal copper metabolism and thus help to alleviate copper toxicity. Previously, we have shown that quercetin, a dietary flavonoid, can chelate copper. This study further examined the interaction of quercetin and copper in intestinal epithelial cells. We found that quercetin enhanced metallothoinein induction by copper and the effect was dose dependent. Quercetin also exerted a cumulative effect after repeated exposure. Repeated low-dose treatment (3–10 μM) of cells with quercetin can lead to the same effect on metallothoinein as one higher concentration treatment (100 μM). This property of quercetin is distinct from its chemical interaction with copper, but both can contribute to a reduction of copper toxicity. Among other flavonoids tested, two other copper chelators, catechin and rutin, did not increase copper induction of metallothionein, whereas genistein, an isoflavone that does not interact with copper chemically, increased copper induction of metallothionein. The effect of quercetin on copper metabolism is unique. Quercetin decreased zinc-stimulated metallothionein expression and had no effect on the cadmium induction of metallothionein. The clinical application of our observation needs to be explored.     

In vitro cytotoxicity and in vivo distribution after direct delivery of PEG-camptothecin conjugates to the rat brain

Low water solubility and rapid elimination from the brain inhibits local delivery via implants and other delivery systems of most therapeutic drugs to the brain. We have conjugated the chemotherapy drug, camptothecin (CPT), to poly(ethylene glycol) (PEG) of molecular weight 3400 using previously established protocols. These new conjugates are very water-soluble and hydrolyze at a pH-dependent rate to release the active parent drug. We have studied the uptake of these conjugates by cells in vitro and quantified their cytotoxicity toward gliosarcoma cells. These conjugates were loaded into biodegradable polymeric controlled-release implants, and their release characteristics were studied in vitro. We implanted similar polymeric disks into rat brains and used a novel sectioning scheme to determine the concentration profile of CPT in comparison to conjugated CPT in the brain after 1, 7, 14, and 28 days. We have found that PEGylation greatly increases the maximum achievable drug concentration and greatly enhances the distribution properties of CPT, compared to corelease of CPT with PEG. Although only one percent of CPT in the conjugate system was found in the hydrolyzed, active form, drug concentrations were still significantly above cytotoxic levels over a greater distance for the conjugate system. On the basis of these results, we believe that PEGylation shows great promise toward increasing drug distribution after direct, local delivery in the brain for enhanced efficacy in drug treatment.     

Synthesis and biological activity of new glycyrrhizic acid conjugates with amino acids and dipeptides

New glycyrrhizic acid (GA) conjugates were synthesized with the use of tert-butyl esters of amino acids or benzyl esters of dipeptides; they contained two residues of L-amino acids (Met, Phe, Pro, and Ile or dipeptides Gly-Leu and Gly-Phe). Activation of GA carboxy groups was carried out with the help of N-hydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, or N-hydroxybenzotriazole with dicyclohexylcarbodiimide. A proline-containing GA derivative is a low-toxic substance; it raises the level of agglutinins by 3.7 times in the blood of mice and 3 times that of hemolysins compared with the control. Dipeptide GA derivatives possess an expressed anti-HIV-1 activity in cultures of MT-4 cells and are 90-70 times less cytotoxic than azidothymidine. The selectivity index of the compounds exceeds those of GA by 110 and 34 times, respectively.