Response Surface Methodology in Optimising the Extraction of Polyphenolic Antioxidants from Flower Buds of Magnolia × soulangeana Soul.-Bod. var. 'Lennei' and Their Detailed Qualitative and Quantitative Profiling.

A response surface methodology (RSM) with a central composite design (CCD) was developed to predict and apply the best ultrasound-assisted extraction (UAE) conditions, including the extraction time, the composition of aqueous-ethanolic extractants, and the solvent-to-plant-material ratio, for obtaining the highest yields of different types of polyphenolic components from the dried flower buds of Magnolia × soulangeana Soul.-Bod. var. 'Lennei' (MSL). The novel approach in the RSM procedure resulted from the simultaneous optimisation of UAE conditions to obtain extracts with the highest antioxidant and antiradical potential (examined as dependent variables), using appropriate spectrophotometric assays, with Folin-Ciocâlteu and 2,2-diphenyl-1-picrylhydrazyl reagents, respectively. The use of 66.8% (V/V) ethanol as the extraction solvent during the 55.2 min extraction protocol and the ratio of extractant volume to herbal substance of 46.8 mL/g gave the highest total yield of bioactive antioxidant phenolics in the extract obtained. For this herbal preparation, a qualitative and quantitative analysis was performed using combined chromatographic (LC), spectroscopic (PDA), and tandem mass spectrometric (ESI-QToF-MS/MS) techniques. A detailed phytochemical profiling, conducted for the first time, documented substantial amounts of various polyphenolic antioxidants, especially phenylethanoids and flavonoids, in the MSL flower buds. Their average total content exceeded 30.3 and 36.5 mg/g dry weight, respectively.

Isolation of Mirificin and Other Bioactive Isoflavone Glycosides from the Kudzu Root Lyophilisate Using Centrifugal Partition and Flash Chromatographic Techniques.

Pueraria lobata (Willd.) Ohwi is a legume taxon native to Southeast Asia and widely used in traditional medicine systems of that region. The therapeutic applications of the underground parts of this species (known as kudzu root) are related to its high content of isoflavones, mainly the characteristic C-glycoside derivatives. Within this group, the most explored compound both phytochemically and pharmacologically is puerarin. However, current scientific findings document important anti-biodegenerative effects for some of the minor isoflavones from kudzu roots. Therefore, the main objective of the study was to develop an original preparative method that allowed the efficient isolation of closely related hydrophilic daidzein C-glycosides, including mirificin, from vacuum-dried aqueous-ethanolic extracts of kudzu roots. For this purpose, the combined centrifugal partition (CPC) and flash chromatographic (FC) techniques were elaborated and used. The optimized biphasic solvent system in CPC, with ethyl acetate, ethanol, water, and 0.5% (V/V) acetic acid as a mobile phase modifier, enabled the purification and separation of the polar fraction containing bioactive isoflavones and ultimately the isolation of mirificin, 3'-hydroxy- and 3'-methoxypuerarin, puerarin, and daidzin using FC. The identity of isoflavones was confirmed using spectroscopic (UV absorption and nuclear magnetic resonance) and mass spectrometric methods. The determined purity of isolated mirificin was 63%.

In Vitro Evaluation of the Antioxidant Activity and Chemopreventive Potential in Human Breast Cancer Cell Lines of the Standardized Extract Obtained from the Aerial Parts of Zigzag Clover (Trifolium medium L.).

The aboveground parts of Trifolium medium L. (zigzag clover), a little-known representative of the family Fabaceae, collected during flowering in a wild stand (Slawin-Szerokie district, Lublin, Poland), were used in this study. Our previous investigations confirmed the higher content of phytoestrogenic isoflavones (especially biochanin A and formononetin derivatives) in T. medium compared to the closely related medicinal plant T. pratense (red clover) and the involvement of these compounds in anti-osteoporotic effects in ovariectomized female rats. The current study focused on evaluating other antibiodegenerative (antioxidant, chemopreventive, and cytostatic) effects for the lyophilisate (TML) obtained from wild zigzag clover. For this purpose, efficient ultrasound-assisted extraction (UAE) was employed, followed by vacuum drying and phytochemical standardization using a newly developed reversed-phase high-performance liquid chromatography (RP-LC) coupled with a PDA detection. Malonylglycosides of biochanin A and formononetin were the predominant compounds and were found to contribute more than 54% to the total isoflavone content determined in the standardized extract of zigzag clover. The antioxidant potential of TML was examined in vitro using the Folin-Ciocalteu and cupric ion-reducing (CUPRAC) methods in addition to the free radical (DPPH• and ABTS•+) scavenging assays. The cytotoxic effects of TML, formononetin, and ononin were evaluated on MCF-7 (estrogen-dependent) and MDA-MB-231 (estrogen-independent) human breast cancer cell lines using the MTT assay. The important role of malonyl isoflavone derivatives has been indicated both in chemoprevention and potential cytotoxic effects of TML against certain types of breast cancer.

Comparison of Phenolic Metabolites in Purified Extracts of Three Wild-Growing Herniaria L. Species and Their Antioxidant and Anti-Inflammatory Activities In Vitro.

The work is aimed at phytochemical characterization and In Vitro evaluation of antioxidant actions, anti-inflammatory effects, and cytotoxicity of purified extracts from three rupturewort (Herniaria L.) species, i.e., Herniaria glabra (HG), H. polygama (HP), and H. incana herb (HIh). The total phenolic content established in the purified extracts (PEs) of HIh, HP, and HG was 29.6, 24.0, and 13.0%, respectively. Thirty-eight non-saponin metabolites were identified using LC-HR-QTOF-ESI-MS; however, only 9 were common for the studied Herniaria species. The most abundant phenolic compound in HG-PE was narcissin (7.4%), HP-PE shared 3 major constituents, namely cis-2-hydroxy-4-methoxycinnamic acid 2-O-ß-glucoside (cis-GMCA, 5.8%), narcissin (5.4%), and rutin (5.3%). Almost half of HIh phenolic content (14.7%) belonged to oxytroflavoside A (7-O-methylkaempferol-3-O-[3-hydroxy-3-methylglutaryl-(1→6)]-[α-rhamnopyranosyl-(1→2)]-ß-galactopyranoside). Antioxidant properties of the Herniaria PEs were evaluated employing an experimental model of human blood plasma, exposed to the peroxynitrite-induced oxidative stress. The assays demonstrated significant reduction of oxidative damage to protein and lipid plasma components (estimated by measurements of 3-nitrotyrosine, protein thiol groups, thiobarbituric acid-reactive substances), and moderate protection of its non-enzymatic antioxidant capacity. Anti-inflammatory properties of the Herniaria PEs were evaluated In Vitro as inhibitory effects against cyclooxygenases (COX-1 and -2) and concanavalin A-induced inflammatory response of the peripheral blood mononuclear cells (PBMCs). None of the studied plants showed inhibitory effects on COXs but all purified extracts partly reduced the release of interleukin 2 (IL-2) and tumor necrosis factor-alpha (TNF-α) from PBMCs, which suggested their prospective ability to up-regulate inflammatory response of the cells. The purified extract from H. glabra turned out to be the most efficient suppressor of PBMCs' inflammatory response. Additionally, cytotoxicity of purified Herniaria extracts on PBMCs was ruled out based on In Vitro studies.

In-depth phytochemical and biological studies on potential AChE inhibitors in red and zigzag clover dry extracts using reversed-phase liquid chromatography (RP-LC) coupled with photodiode array (PDA) and electron spray ionization-quadrupole/time of flight-mass spectrometric (ESI-QToF/MS-MS) detection and thin-layer chromatography-bioautography.

In the study, Trifolium medium L. and T. pratense L. were used to obtain lyophilisates which were named as TML and TPL, respectively. The former clover taxon represents a little explored species, while the latter one is often found in dietary supplements and functional foods due to the content of isoflavones that alleviate various menopausal symptoms. Detailed phytochemical profiles of both lyophilisates were examined and compared using coupled chromatographic (RP-LC) and spectroscopic PDA/ESI-QToF/MS-MS methods. A total of 54 and 55 compounds were identified in TML and TPL, respectively. Close chemotaxonomic similarities were confirmed for both clover taxa examined, especially in terms of the most abundant isoflavones and hydroxycinnamates. For the first time, neurotropic polyphenols (AChE inhibitors) were identified in clover extracts using TLC-bioautography. In this group, caffeoylmalic acid exhibited significant AChE inhibitory effect confirming the potential of TPL and TML to support physiological functions of the endocrine and nervous systems.

Apigenin and hesperidin augment the toxic effect of doxorubicin against HepG2 cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies, with an increasing incidence. Despite the fact that systematic chemotherapy with a doxorubicin provides only marginal improvements in survival of the HCC patients, the doxorubicin is being used in transarterial therapies or combined with the target drug - sorafenib. The aim of the study was to evaluate the effect of natural flavonoids on the cytotoxicity of the doxorubicin against human hepatocellular carcinoma cell line HepG2. METHODS: The effect of apigenin and its glycosides - cosmosiin, rhoifolin; baicalein and its glycosides - baicalin as well as hesperetin and its glycosides - hesperidin on glycolytic genes expression of HepG2 cell line, morphology and cells' viability at the presence of doxorubicin have been tested. In an attempt to elucidate the mechanism of observed results, the fluorogenic probe for reactive oxygen species (ROS), the DNA oxidative damage, the lipid peroxidation and the double strand breaks were evaluated. To assess impact on the glycolysis pathway, the mRNA expression for a hexokinase 2 (HK2) and a lactate dehydrogenase A (LDHA) enzymes were measured. The results were analysed statistically with the one-way analysis of variance (ANOVA) and post hoc multiple comparisons. RESULTS: The apigenin and the hesperidin revealed the strongest effect on the toxicity of doxorubicin. Both flavonoids simultaneously changed the expression of the glycolytic pathway genes - HK2 and LDHA, which play a key role in the Warburg effect. Although separate treatment with doxorubicin, apigenin and hesperidin led to a significant oxidative DNA damage and double strand breaks, simultaneous administration of doxorubicin and apigenin or hesperidin abolished these damage with the simultaneous increase in the doxorubicin toxicity. CONCLUSION: The obtained results indicate the existence of a very effective cytotoxic mechanism in the HepG2 cells of the combined effect of doxorubicin and apigenin (or hesperidin), not related to the oxidative stress. To explain this synergy mechanism, further research is needed, The observed intensification of the cytotoxic effect of doxorubicin by this flavonoids may be a promising direction of the research on the therapy of hepatocellular carcinoma, especially in a chemoembolization.

High Antimicrobial Efficacy, Antioxidant Activity, and a Novel Approach to Phytochemical Analysis of Bioactive Polyphenols in Extracts from Leaves of Pyrus communis and Pyrus pyrifolia Collected During One Vegetative Season.

Dried leaf samples of Pyrus communis L. var. 'Conference' and Pyrus pyrifolia Burm. f. (Nakai) var. 'Shinseiki' were subjected to the successful extraction procedures using various solvents, followed by filtering and/or drying liquid plant preparations under reduced pressure. As a result of this, for each Pyrus leaf sample examined, four dried residues were obtained, including methanolic (EA), ethyl acetate (EC), water (EB), and the residue obtained from aqueous solution (ED). Antiradical activity of these preparations was measured using the ABTS+• assay, and antimicrobial activity was examined using various strains of bacteria and yeasts. The highest antiradical activity was observed for EC from leaves of P. communis var. 'Conference' collected in May, but the highest average antibacterial activity was noted for EC residues from P. pyrifolia var. 'Shinseiki' collected in May. Antibacterial activity positively correlated with concentration of hydroquinone in extracts. No antifungal activity was observed for any extract. In addition, qualitative and quantitative analyses of active polyphenolic components in extracts from Pyrus were performed. Hydroquinone and hydroxycinnamic acid derivatives were analyzed using a new optimized method comprising reversed-phase high-performance liquid chromatography (RP-LC) coupled with simultaneous photodiode-array and fluorescence detection.

Evaluation of the phytochemical composition and protective activities of methanolic extracts of Centaurea borysthenica and Centaurea daghestanica (Lipsky) Wagenitz on cardiomyocytes treated with doxorubicin.

Centaurea L. is a genus of the family Asteraceae that comprises over 600 taxa. Representatives of the Centaurea genus were used as natural medications for many diseases. Methanolic-aqueous extracts from aerial parts of two Centaurea species: C. borysthenica Gruner and C. daghestanica (Lipsky) Wagenitz were studied for their polyphenolic composition and potential protective effect on cardiomyocytes treated with doxorubicin. Effectiveness of doxorubicin in cancer therapy is limited by a dose-dependent cardiotoxicity. Oxidative stress is a widely recognized mechanism of this phenomenon. One of the most important strategies has been an application of drug together with antioxidant agents. A cardioprotective effect of selected extracts of Centaurea species was suspected in this study. Cell viability, oxidative stress, and mitochondrial membrane potential analyses showed protective activity of the methanolic extract of C. borysthenica and C. daghestanica on rat cardiomyocytes treated with doxorubicin. Although C. borysthenica is more effective as a cardiomyocyte protective agent, in higher concentrations it weakened the drug activity. C. daghestanica extract did not change the doxorubicin efficacy in the evaluated experiment. Interestingly, both tested extracts were cytotoxic for myeloma cells. The detected antioxidant activity of the studied extracts can be used in the prevention of doxorubicin-induced cardiotoxicity.

LC-QTOF-MS Analysis and Activity Profiles of Popular Antioxidant Dietary Supplements in Terms of Quality Control.

The dietary supplements with claimed antioxidant activity constitute a substantial part of the dietary supplement market. In this study, we performed the LC-QTOF-MS analysis and investigated the activity profiles of popular antioxidant dietary supplements from different chemical groups in terms of quality control. The commonly used antioxidant tests and statistical analysis revealed that substantial part of the results was comparable if 1 g sample was considered, but while comparing single and daily doses, significant differences in antioxidant values were noticed in all assays. The best antioxidant activity was obtained in ORAC assay (from 142 to 13814 µM of Trolox equivalents per 1 g of sample), and the strongest correlation occurred between TPC and ORAC. The LC-QTOF-MS analysis revealed that catechins were present in samples having the best antioxidant activity and that dietary supplements showing the weakest activity contained very small amount of any chemical constituents.

Analysis of Polyphenolic Compounds in Extracts from Leaves of Some Malus domestica Cultivars: Antiradical and Antimicrobial Analysis of These Extracts.

In this study, methanol, ethyl acetate, water extracts, and precipitate were obtained from leaves of Malus domestica cultivars: Golden delicious, Jonagold, Elstar, Ligol, and Mutsu. Antiradical activity of these extracts was measured using the ABTS+∙ radical, and antimicrobial activity was measured with the disk-diffusion method. Phenolic compounds were measured with the colorimetric method and identified with high performance liquid chromatography (HPLC). The highest antiradical activity was observed for the Jonagold variety, and in particular strong activity was noted for ethyl acetate extracts. Antimicrobial activity was observed against strains of Staphylococcus aureus, Enterococcus faecalis, and the fungus Candida glabrata. Particularly susceptible to the extracts activity appeared to be Staphylococcus aureus, but the growth of Candida glabrata was inhibited in the presence of ethyl acetate extracts. With the HPLC method we identified a high amount of phloridzin (above 500 mg per g of ethyl acetate extracts), lower amounts of hyperoside, isoquercitrin, and quercitrin, and traces of p-hydroxybenzoic and chlorogenic acids. The contribution of phloridzin to antiradical activity of methanol and ethyl acetate extracts was very high (above 90%). In water extract the contribution of phloridzin was between 38.9 and 55.2%, chlorogenic acid 22.7 and 36.1%, and hyperoside 12.2 and 13.3%.

Effects of Extracts from Trifolium medium L. and Trifolium pratense L. on Development of Estrogen Deficiency-Induced Osteoporosis in Rats.

Some plant species belonging to Trifolium L. genus are a source of isoflavones considered to exert phytoestrogenic activities. The aim of the present study was to examine the effects of standardized extract obtained from aerial parts of Trifolium medium L., in comparison with the extract of Trifolium pratense L., on the development of estrogen deficiency-induced osteoporosis in rats. Both Trifolium extracts, at doses corresponding to 10 and 20 mg/kg of isoflavone aglycones daily, or estradiol (0.2 mg/kg daily), were administered orally to ovariectomized (OVX) rats for 4 weeks. Serum bone turnover markers, bone mass, mineralization, and mechanical properties were studied. In OVX control rats, mechanical properties of the tibial metaphysis and femoral neck were strongly worsened in comparison with sham-operated control rats, and those of femoral diaphysis were unaffected. Estradiol counteracted the worsening of the tibial strength and increases in bone turnover markers. Both extracts significantly increased the strength of the femoral diaphysis and calcium and phosphorus content in the bone mineral, but only T. pratense extract increased the strength of the tibial metaphysis. In conclusion, effects of both Trifolium extracts differed from those of estradiol. It is possible that other than isoflavone extract constituents contributed to their skeletal effects.

Studies on phytoestrogenic and nonphytoestrogenic compounds in Trifolium incarnatum L. and other clover species using pressurized liquid extraction and high performance column liquid chromatography with photodiode-array and fluorescence detection.

HPLC coupled with photodiode array (PDA) and fluorescence (FL) detectors has been used for the identification and determination of phytoestrogenic (isoflavones and coumestrol) and nonphytoestrogenic (flavones) compounds in hydrolyzed and nonhydrolyzed extracts obtained from aerial parts of Trifolium incarnatum L. and related clover species. The effective isolation technique of pressurized liquid extraction (PLE) was used. Various types of extraction solvents, i.e., ethanol, n-propanol, n-butanol, and water solutions (75%, v/v) of methanol or ethanol at changeable or constant temperatures were tested, taking into account the chemical character of isolated compounds. Higher PLE efficiency in relation to isoflavone aglycones was found for ethanol. Predominant isoflavone glycosides determined in clover samples were sissotrin and ononin, reaching levels above 1.4% dry weight (wt) in T. medium. The presence of flavone compounds (apigenin and luteolin) in aerial parts of T. incarnatum, occurring in amounts exceeding 0.4% dry wt, documented chemotaxonomic distinction of this species from other clovers examined. Additionally, within the group of phytoestrogenic isoflavones, only biochanin A and formononetin derivatives were identified in above-ground parts of T. incarnatum. The application of simultaneous PDA and FL detection enabled unambiguous confirmation of the lack of a strong phytoestrogen, coumestrol, in all hydrolyzed and nonhydrolyzed clover extracts.

Pressurized liquid extraction versus other extraction techniques in micropreparative isolation of pharmacologically active isoflavones from Trifolium L. species.

As a new sample preparation technique, pressurized liquid extraction (PLE), in combination with reversed-phase liquid chromatography (RP-LC) and photodiode-array (PDA) detection were used for the isolation and determination of phytoestrogenic isoflavones in hydrolyzed extracts obtained from aerial parts of five Trifolium L. (clover) species. To optimize the effectiveness of PLE procedure, variable extraction parameters: methanol and acetone (or their 75% aqueous solutions), as extraction solvents, temperatures (75, 100 and 125 degrees C) and the changeable number of static extraction cycles were tested. Additionally, two other micropreparative techniques: ultrasound-assisted extraction (UAE), and conventional solvent extraction (CSE), under optimized conditions, were also used and compared. Optimum extraction efficiency, expressed in the highest yield of biochanin A, formononetin, daidzein and genistein from plant material, with PLE, using methanol-water (75:25, v/v) as an extraction solvent, an oven temperature of 125 degrees C and three 5-min static extraction cycles, was obtained.

Ultrasound-assisted solid-phase extraction coupled with photodiode-array and fluorescence detection for chemotaxonomy of isoflavone phytoestrogens in Trifolium L. (Clover) species.

Detailed chemotaxonomic studies were undertaken to establish the qualitative profile and real amounts of the pharmacologically active isoflavone aglycones genistein, daidzein, formononetin, and biochanin A in aerial parts of thirteen Trifolium L. (clover) species, native to Poland. A newly elaborated micropreparative technique--SPE--on BakerBond octadecyl, cyclohexyl, and phenyl cartridges was used in combination with ultrasound-assisted extraction for isolation of isoflavone aglycones from hydrolyzed samples. The effectiveness of all three SPE sorbents in the purification of plant extracts was compared and very high recoveries (>96%) were documented for four isoflavones. Classical photodiode-array and very sensitive fluorescence detection, coupled with reversed-phase high-performance liquid chromatography (RP-HPLC), were employed to obtain the most reliable qualitative and quantitative results. Chemotaxonomic differences combined with flower color variability were demonstrated within thirteen clover species. Concentration levels of particular isoflavones in ten Trifolium species possessing flowers with white, pink, or purple-red corolla ranged from approximately 3 to approximately 3300 microg/g dry weight, while in three yellow flowering clovers (T. aureum, T. dubium, and T. campestre) isoflavone compounds have not been detected at all. RSD values, determined for intra- and inter-day precision of the quantitative results, were not higher than 6.2% and 7.1%, respectively.

Retention behavior of silica-bonded and novel polymeric reversed-phase sorbents in studies on flavones as chemotaxonomic markers of Scutellaria L. genus.

In this study, the effectiveness of both classical and novel polymeric sorbents used in solid-phase extraction (SPE) for isolation of pharmacologically active flavones (baicalin, luteolin, apigenin, wogonin and chrysin) from aerial parts of 13 species of Scutellaria L. (Skullcap) genus was assessed. The highest recoveries of hydrophilic (baicalin, luteolin) flavones for Oasis HLB were obtained, whereas for medium hydrophobic (apigenin) and hydrophobic (wogonin, chrysin) flavones better quantitative results for BakerBond phenyl cartridges were stated. Eluates were analysed using reversed-phase high-performance liquid chromatography with diode array detection (RP-HPLC-DAD). For the five target compounds the determined concentrations ranged from approximately 4 to approximately 15,500 microg/g dry wt. Very good linearities (r(2)>0.9995) of calibration curves were achieved for each flavone. The accuracy was below 5% for most compounds examined. This is the first method reported that enabled simultaneous qualification and quantitation of five flavones (being chemotaxonomic markers) in 13 species of Scutellaria L. genus.

Phenolic acids from Symphoricarpos albus (L.) Blake (Caprifoliaceae).

The leaves, flowers and fruits of Symphoricarpos albus (L.) Blake (Caprifoliaceae) were analysed for the presence of phenolic acids. Eleven free and liberated by hydrolysis phenolic acids were identified by TLC, HPLC and spectral (UV) methods. Moreover, the HPLC method was applied for the quantitative determination of phenolic acids in the analysed fractions.