A critical evaluation on the reliability of two aluminum chloride chelation methods for quantification of flavonoids

Flavonols kaempferol, quercetin, myricetin and gossypetin, and flavones apigenin, acacetin, luteolin, orientin and tricin, are subjected to two AlCl(3) spectrophotometric methods used for determination of total flavonoid content. The method developed by Woisky and Salatino involves addition of AlCl(3) solution to the flavonoid solution and recording of optical density at 420nm. All flavonols except kaempferol have absorption maxima above 440nm and so readings at 420nm are erroneous. Among flavones, all except for luteolin and orientin, have absorption maxima below 400nm. Further, addition of CH(3)COOK and recording the absorbance at 415nm, as modified by Chang et al., works well for flavonols kaempferol, quercetin and myricetin, but not for gossypetin. The flavones luteolin and orientin absorbed above 400nm, whereas all others absorbed below 400nm. Examination of the results of both methods indicates they are inadequate, and should not to be considered as universal and standard methods for total flavonoid determination.     

The flavone,flavonol and flavan-3-ol content of the Greek traditional diet

Flavonoids are an important category of plant antioxidants and evidence is accumulating on their favorable effects against the development of heart disease and certain forms of cancer. We analytically determined the flavonol (quercetin, kaempferol, myricetin, isorhamnetin), flavone (luteolin, apigenin) and flavan-3-ol (catechin, epicatechin, epigallocatechin, epigallocatechin gallate, epicatechin gallate) content of a weekly menu representative of the Greek traditional diet. The overall daily average content was found 79.01 mg of which flavonols contribute 47% (37.17 mg/day), flavan-3-ols 40% (31.67 mg/day) and flavones 13% (10.17 mg/day). The levels of agreement between the analytical results and the respective theoretical flavonoid calculations conducted previously on the same weekly menu ranged widely, indicating that caution should be taken when calculated flavonoid values are to be used in epidemiological studies. Compared to northern European and American diets, the traditional Greek diet has a higher flavonoid content, at least with respect to flavones, flavonols and flavan-3-ols.     

Flavonols and flavones of parsley cell suspension culture change the antioxidative capacity of plasma in rats

The flavonoid aglycones from an illuminated parsley (Petroselinum crispum (Mill.) Nym.) cell suspension culture were identified and quantified as the flavones apigenin, luteolin and chrysoeriol and the flavonols kaempferol, quercetin and isorhamnetin. Flavonoid extracts from these cultures were purified by solid phase extraction from RP C-18 phase and given by gavage to rats. Only extract from illuminated culture increased the antioxidative capacity (AOC) of blood plasma temporarily with maximum values after 1 h. It is concluded that the course of AOC reflects changes in the plasma content of flavonoids.     

Evaluation of antioxidant and inhibitory activities for different subclasses flavonoids on enzymes for rheumatoid arthritis

Antioxidant activities of flavonoids were decreased in the order of flavonols > flavanones > flavones. Inhibitory intensities for hyaluronidase and collagenase reaction differed clearly according to flavonoid subclasses. Kaempferol, quercetin, myricetin, and rutin in flavonols inhibited hyaluronidase reaction specifically, while apigenin, luteolin, baicalin, and baicalein in flavones showed specific inhibition to collagenase reaction. In addition, the flavonoids, except baicalin and catechin, inhibited potently LPS-induced nitrite production in a dose-dependent manner, which might be mainly due to the suppression of inducible nitric oxide (NO) synthase. Quercetin and luteolin showed the strongest inhibitory activities on 15-lipoxygenase (LOX), and quercetin showed relatively potent inhibition on cyclooxygenase-1 (COX-1) reaction. Otherwise, all tested flavonoids possessed the inhibitory activity to COX-2 reaction, and especially luteolin, kaempferol, hesperetin, and naringin showed relatively the potent inhibition on COX-2 reaction. This report elucidated the anti-inflammatory activities, such as the antioxidant property, inhibition of NO production, and inhibition of inflammatory enzymes (hyaluronidase, collagenase, LOX, and COXs) of several subclass flavonoids.     

Changes of flavonol synthase and flavonol contents during grape berry development

Flavonols as an important kind of flavonoids not only have many defense-related functions, but also play an important role as potent antioxidants. Flavonol synthase (FLS) as the key enzyme in flavonol synthesis determines the final contents of flavonols in plant materials. The objective of this work was to study the changes of total flavonol content, free flavonol content, the individual flavonol aglycone contents and the two flavones (luteolin and apigenin) contents during grape berry development. The activity and the amount of the changes of FLS during grape berry development were also estimated. The results showed that quercetin, myricetin, kaempferol, isorhamnetin and galangin were detectable during grape berry development, and quercetin aglycone predominated in the total flavonol content in rapid growth phase, lag growth phase and the latter part of the ripening phase, myricetin aglycone witnessed a great increase at veraison, although kaempferol, isorhamnetin and galangin only accounted for a little part of the total flavonol contents but their individual contents all increased after veraison and kaempferol witnessed its highest content at the ripening phase. When it came to the contents of total phenols, total flavonoids, total flavonols and free flavonols, all of them showed accumulated peaks at both the rapid growth phase and the ripening phase. The result of FLS activity had a highly positive correlation with the total contents of flavonols, and the immunoblotting analysis detected two proteins whose signal intensities were in accordance with the FLS activities.     

Study of flavonoids in aqueous spinach extract using positive electrospray ionisation tandem quadrupole mass spectrometry

The presence of three flavonols (quercetin, kaempferol, myricetin) and two flavones (apigenin, luteolin) were investigated in the extract of fresh spinach leaves. Aqueous spinach extracts were prepared with a leaf/water ratio of 1:2 at 80 °C for 30 min stirring. Ferric ammonium sulphate method was used for measuring total polyphenols in the extracts and expressed as catechins and tannic acid equivalents. The flavonoids glycosides in the extract were hydrolysed to their aglycons with 1.2 M HCl in boiling 50% water methanol solution. The resulting aglycons were identified and quantified by a C18 reverse phase high-performance liquid chromatography (RP-HPLC). Furthermore, the results were confirmed by HPLC coupled to an electrospray ionisation tandem triple-quadrupole mass spectrometer ESI-MS performing low-energy collision induced dissociation (CID-MS/MS) in the collision cell (HPLC-ESI-MS/MS). Analyses were made in the multiple reaction monitory (MRM) mode. Results showed that total polyphenols contents in fresh spinach leaves were 270 mg kg⁻¹ and 390 mg kg⁻¹ as tannic acid and catechin equivalents, respectively, in which, major flavonoids aglycons were apigenin (170 mg kg⁻¹), quercetin (50 mg kg⁻¹) and kaempferol (30 mg kg⁻¹).     

Flavonoids and phenolic acids from Labisia pumila (Kacip Fatimah)

Both total phenolic content (TPC) and total flavonoid content (TFC) of Labisia pumila extracts were determined spectrophotometrically. L. pumila leaves extracted in 60% methanol (MeOH) were fractionated on C18 cartridge and the antioxidant property of each fraction was determined by measuring free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. The 40% MeOH fraction exhibited the highest scavenging activity. Nine flavonols (quercetin, myricetin and kaempferol), two flavanols (catechin and epigallocatechin) and nine phenolic acids were identified from this active fraction by UPLC–ESI-MS/MS, and confirmed by comparison with the mass spectra of standard aglycones, theoretical fragments generated from MS Fragmenter software, and literature values.     

Determination of red wine flavonoids by HPLC and effect of aging

A new method for simultaneous determination of 10 flavonols and 2 flavones by high performance liquid chromatography was developed in this paper. The identified compounds contained quercetin, kaempferol, myricetin, rhamnetin, isorhamnetin, quercetrin, rutin, morin, galangin, fisetin, apigenin and luteolin. The chromatographic separation of these flavonoids was performed in a single run by using the mobile phase gradient elution of acetonitrile–methanol–water mixture (1% tetrahydrofuran, THF) at 20 °C, with the flow rate at 1.0 ml/min and the detection wavelength at 360 nm. With direct injection of wine samples, seven red wine samples, differing in their origin of producing places and time, were analyzed for flavonoids content by this method. The results showed the presence of myricetin, luteolin, quercetin, kaempferol, isorhamnetin and galanin. Additionally, the changes of flavonoids in red wines stored in the three types of oak barrels with aging time were investigated, which indicated that the component of flavonoids in red wine is related to wine aging greatly. These provide a substantial basis for the further research on control of flavonoids during winemaking.     

Metabolic pathways of the colonic metabolism of flavonoids (flavonols,flavones and flavanones) and phenolic acids

Flavonoids are metabolized by the gut microflora giving rise to a wide range of phenolic acids that may exert systemic effects in the body. Nevertheless, the colonic metabolism pathways and the function of the metabolites formed are poorly studied. In the present study, the individual colonic metabolism of three subclasses of flavonoids (flavonols, flavones and flavanones) and phenolic acids was evaluated. For this, seven standards of flavonoids (quercetin, quercetin–rhamnoside, quercetin–rutinoside, myricetin, luteolin, naringenin and kaempferol–rutinoside) and two phenolic acids (protocatechuic acid and gallic acid) were submitted to an in vitro fermentation model using rat colonic microflora. Simultaneously, a nuts–cocoa cream enriched with these standards of flavonoids was incubated and the colonic metabolism of these compounds was evaluated. The results showed that the greatest number of colonic metabolites came from the fermentation of quercetin and quercetin–rhamnoside, and the maximum concentration of fermentation products was observed after 48 h of fermentation. On the other hand, a smaller number of fermentation products were observed after the colonic fermentation of kaempferol–rutinoside, naringenin, luteolin and myricetin. The phenolic acids were slightly metabolized by the colonic microflora.     

Flavonols and flavones of vegetables. V. Flavonols and flavones of root vegetables (author's transl)]

Root vegetables contain flavon(ol) glycosides in tracers up to small amounts, while the level of their leaves are in part considerable (to more than 1 g/kg, calculated as aglycon). Radish, rutabagas, scorzoneras, and beets contain less than 1 mg/kg kaempferol and/or quercetin; carrots less than 1 mg/kg apigenin and luteolin; celery roots ca. 75 mg apigenin/kg and 14 mg luteolin/kg; horseradish about 20 mg kaempferol/kg and small radish 1-10 mg kaempferol/kg, whereby all these flavones and flavonols occur as glycosides in the vegetables. In leaves of small radish, variety "Eiszapfen", we found besides isoquercitrin (quercetin-3-glucoside) a quercetin-3-0-diglycoside and a kaempferol-0-diglycoside, both with the sugars rhamnose and arabinose, by tlc.     

Use of HPLC–DAD–ESI/MS to profile phenolic compounds in edible wild greens from Portugal

In some Mediterranean areas traditional wild greens are responsible for a significant percentage of total dietary antioxidant intakes. Asparagus acutifolius L. (wild asparagus), Bryonia dioica Jacq. (white bryony) and Tamus communis L. (black bryony) are important examples of those edible wild greens widely consumed. This study aimed to determine the phenolic profile and composition of edible vernal early shoots of those species. Wild asparagus and black bryony revealed glycosides of flavonols as the main phenolic compounds, while white bryony contained C-glycosylated flavones. Black bryony was the wild green that possessed the highest content of phenolic compounds (2200 mg/kg). Amongst the 11 flavonols found in this sample, kaempferol glycosides were the main compounds (1760 mg/kg). In the sample of wild asparagus, quercetin 3-O-rutinoside was the main flavonol found (263 mg/kg). Five flavones and one flavonol were found in the white bryony sample, apigenin 6-C-glucoside-7-O-glucoside being the major compound (1550 mg/kg).     

A new flavone from antioxidant extracts of Pistacia terebinthus

Acetone and methanol extracts of the fruits of Pistacia terebinthus L. subsp. terebinthus L. were studied for their antioxidant activity by investigating their total phenolic and flavonoid contents, β-carotene bleaching potential, DPPH radical scavenging effect, scavenging activity on superoxide anion radical, reducing power, and metal chelating effect on ferrous ion. Both extracts showed very similar chemical profile by checking on TLC plates, and exhibited high scavenging activity on superoxide anion radical and DPPH radical. Due to these similarities they were combined and fractionated on a silica gel column for their constituents, and the most active three fractions in DPPH assay were purified to afford a new flavone 6′-hydroxyhypolaetin 3′-methyl ether (1) besides a group of known flavonoids apigenin, luteolin, luteolin 7-O-glucoside, quercetin, quercetagetin 3-methyl ether 7-O-glucoside, isoscutellarein 8-O-glucoside. Their structures were established by UV, UV shift reagents, and ¹H NMR spectroscopic techniques. Antioxidant activity of the new flavone was investigated by β-carotene bleaching and DPPH radical scavenging activity methods, and it showed a high activity in the first system, but not so good in the latter.     

Antioxidant activities of Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr: Identification of free flavonoids and cinnamic acid derivatives

Different solvent extracts of endemic Sideritis (Labiatae) species, Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr, were analyzed for free flavonoids (quercetin, apigenin, myricetin and kaempferol) and cinnamic acid derivatives (rosmarinic acid, ferulic acid, caffeic acid, p-coumaric acid and chlorogenic acid) using HPLC-DAD. All the phenolics were quantified in acid-hydrolyzed extracts, except rosmarinic acid, chlorogenic acid and myricetin which were quantified in raw samples. Antioxidant activities of extracts of these two plants and many of their components in pure form were evaluated based on DPPH^. and ABTS^.+ assays. In general, S. arguta extracts displayed higher antioxidant activity than S. congesta extracts possibly due to their richness in antioxidant components of strong activity. Acetone extract of S. arguta, with its strikingly high TEAC value of 3.2 mM trolox and low IC₅₀ value of 38.3 ??g/mL showed the highest antioxidant potency among all extracts. ??-tocopherol, the positive control, displayed IC₅₀ and TEAC values of 33.8 ??g/mL and 2.9 mM trolox, respectively. No direct correlation was found between antioxidant activities and total phenolic contents of the plant extracts studied.     

Phenolic composition and antioxidant activity of culms and sugarcane (Saccharum officinarum L.) products

The present work reports amounts of flavonoids and phenylpropanoids of culms of three sugarcane varieties and of raw juice, syrup, molasse and VHP sugar. The antioxidant activity of those materials was evaluated by the DPPH and β-carotene/linoleic acid methods. The predominant phenolics in culms were phenylpropanoids (caffeic, chlorogenic and coumaric acids), while flavones (apigenin, tricin and luteolin derivatives) appeared in lower amounts. Differences were noted either among phenolic profiles of sugarcane culms or between culms and sugarcane products. The antioxidant activities of solutions from most samples were similar or higher than a 80 μM Trolox solution.     

北京市售30种蔬菜中类黄酮物质含量分析

目的测定北京地区30种蔬菜中5种类黄酮物质的含量。方法采用高效液相色谱法检测槲皮素、山奈酚、杨梅黄酮、木犀草素、芹菜配基类黄酮物质的含量,蔬菜样品均购自北京当地菜市场。结果30种蔬菜中全部检出了槲皮素,含量在2.1～85.9mg/kg鲜重,9种蔬菜检出杨梅黄酮,8种蔬菜检出芹菜配基,8种蔬菜检出木犀草素,仅在胡萝卜和西红柿中检出山奈酚。紫洋葱、白洋葱、胡萝卜、甜椒和苦瓜等蔬菜类黄酮含量较高。结论不同蔬菜类黄酮物质的组成和含量差别较大,槲皮素为蔬菜中主要的类黄酮物质。     

Phenolic compound content of fresh and dried figs (Ficus carica L.)

Eighteen fig cultivars commonly grown in the south-eastern Spain were collected in two crops (spring and summer) and their polyphenolic profile was determined. Fruit from the first crop generally showed higher phenolic values than those from the second. Higher concentrations of total phenolics were found in skin than in flesh. LC–UV-DAD/ESI-MSⁿ analysis of the figs pointed to a high anthocyanin content, mainly cyanidin-3-rutinoside, flavonols such as quercetin-rutinoside, phenolic acids such as chlorogenic acid and flavones like luteolin 6C-hexose-8C-pentose and apigenin-rutinoside. The c-glycosides have not been previously described in figs. Moreover, to compare intact proanthocyanidins and proanthocyanin cleavage products, an acid-catalysis was made in the presence of phloroglucinol (phloroglucinolysis) and the mean degree of polymerisation was calculated. Finally, three cultivars of dried figs were also studied.     

Flavonoid content and antioxidant activity of vegetables from Indonesia

Extracts from 11 vegetables of Indonesian origin were screened for flavonoid content, total phenolics, and antioxidant activity. The flavonols myricetin, quercetin, and kaempferol and flavones luteolin and apigenin were quantified by HPLC. Flavonoid content in mg/100 g fresh weight (fw) was apparently initially reported for Cosmos caudatus H.B.K. (52.19), Polyscias pinnata (52.19), Pluchea indica Less. (6.39), Nothopanax scutellarius (Burm.f.) Merr (5.43), Talinum triangulare (Jacq.) Willd. (3.93), Pilea melastomoides (Poir.) Bl. (2.27), and Etlingera elatior (Jack) R.M.Sm (1.18). The flavonoid content of the vegetables studied were mainly quercetin and kaempferol and ranged from 0.3 to 143 mg/100 g fw, with the highest level found in Sauropus androgynus (L) Merr. C. caudatus H.B.K. had the greatest total phenols among the vegetables analysed, with 1.52 mg GAE/100 g fw. P. indica Less. and C. caudatus H.B.K. had the highest antioxidant activity as measured by ferric cyanide reducing power, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) scavenging, and inhibition of linoleic acid oxidation. Therefore, S. androgynus (L) Merr, C. caudatus H.B.K., and P. pinnata were identified as potentially rich sources of dietary flavonoids and antioxidants.     

The flavonol content of peas as influenced by variety and light,and a note on the flavonol content of broad beans

The flavonol contents of the leaves and pods of peas are influenced particularly by the amount of light irradiation received, while varietal differences appear to be of lesser importance. The seeds contain only traces of flavonols (< 1 part/10⁶). Broad beans on the other hand contain ca 30–70 parts/10⁶ of flavonols, determined as kaempferol, quercetin and myricetin after hydrolysis of the respective glycosides. The flavonols are localised mainly in the skin. No myricetin could be detected in the pods and leaves.     

Production of flavonoid and lignan aglycones from flaxseed and soy extracts by Bifidobacterium strains

Flaxseed and soybean are an important source of phytochemical compounds, mainly lignans and isoflavones, but also of flavones, flavanones and flavonols. These compounds appear mainly glycosylated in plant food and are converted into aglycones by the intestinal microbiota, increasing their bioavailability. In this work, we analyse the ability of Bifidobacterium strains (n = 25) to produce lignan and flavonoids aglycones from flaxseed and soybean extracts. Most of the Bifidobacterium strains increased the concentrations of secoisolariciresinol, daidzein, genistein, naringenin, eriodyctiol, luteolin and apigenin. Moreover, Bifidobacterium pseudocatenulatum and Bifidobacterium breve strains showed high production of herbacetin, increased the kaempferol concentration and produced quercetin and quercetagetin. B. pseudocatenulatum INIA P815 and B. breve INIA P367 produced 32.37 ± 2.81 and 28.64 ± 3.36 mg respectively of herbacetin g⁻¹ of lignan extract. Bifidobacterium strains transformed the glycosides of a wide range of flavonoids into their aglycones, increasing the antioxidant activity and improving their bioavailability.     

Nutrients,phytochemicals and bioactivity of wild Roman chamomile: A comparison between the herb and its preparations

Roman chamomile, Chamaemelum nobile L. (Asteraceae), has been used for medicinal applications, mainly through oral dosage forms (decoctions and infusions). Herein, the nutritional characterisation of C. nobile was performed, and herbal material and its decoction and infusion were submitted to an analysis of phytochemicals and bioactivity evaluation. The antioxidant activity was determined by free radicals scavenging activity, reducing power and inhibition of lipid peroxidation, the antitumour potential was tested in human tumour cell lines (breast, lung, colon, cervical and hepatocellular carcinomas), and the hepatotoxicity was evaluated using a porcine liver primary cell culture. C. nobile proved to be an equilibrated valuable herb rich in carbohydrates and proteins, and poor in fat, providing tocopherols, carotenoids and essential fatty acids (C18:2n6 and C18:3n3). Moreover, the herb and its infusion are a source of phenolic compounds (flavonoids such as flavonols and flavones, phenolic acids and derivatives) and organic acids (oxalic, quinic, malic, citric and fumaric acids) that showed antioxidant and antitumour activities, without hepatotoxicity. The most abundant compounds in the plant extract and infusion were 5-O-caffeoylquinic acid and an apigenin derivative. These, as well as other bioactive compounds, are affected in C. nobile decoction, leading to a lower antioxidant potential and absence of antitumour potential. The plant bioactivity could be explored in the medicine, food, and cosmetic industries.