Identification and quantification of flavonol aglycons in cactus pear (Opuntia ficus indica) fruit using a commercial pectinase and cellulase preparation

The applicability of pectinases and cellulases as soft hydrolysing agents on flavonol glycosides was investigated for identification and quantification of flavonol aglycons in cactus pear fruit. Freeze-dried samples of cactus pear fruit’s peel (cactus pear peels) and onions were treated with commercial pectinase and cellulase preparations at 50 °C for different time periods (up to 16 h). Additionally isorhamnetin-3-O-rutinoside and quercetin-3,4′-O-diglucoside were used as model compounds. In parallel, samples of cactus pear peels and onions were treated by the traditional acidic hydrolysis using HCl. Following hydrolysis, flavonols were characterised using HPLC–DAD. Enzymatically, all model compounds and plant material were highly hydrolysable. Flavonol glycosides were completely hydrolysed after 16 h (cactus pear) and 4 h (onion), respectively. While the acidic hydrolysis caused degradation of the flavonols and produced protocatechuic acid as a degradation product, the enzymatic hydrolysis gave gentle effects and did not produce any protocatechuic acid at all.     

HPLC-DAD-ESIMS analysis of phenolic compounds in bayberries (Myrica rubra Sieb. et Zucc.)

Qualitative analysis of the ethyl acetate extracts from three bayberry cultivars, Xiangshan, Biqi and Dongkui, was performed by means of a hyphenated technique of HPLC coupled to photodiode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Three phenolic compounds were identified (gallic acid, protocatechuic acid, and quercetin 3-glucoside) and seven others (two myricetin hexoside and two myricetin deoxyhexoside derivatives; quercetin hexoside and quercetin deoxyhexoside derivatives; kaempferol hexoside derivative) partially identified. Quantification of phenolic compounds was performed by HPLC-DAD, which revealed that gallic acid (2.6–7.0 mg/kg FW) was the major phenolic acid in all analysed cultivars. Myricetin glycosides (71.1 mg/kg FW) were the major flavonol glycosides in cultivar Xiangshan and quercetin glycosides (117.8 mg/kg FW) were the major ones in cultivar Biqi. Cultivar Dongkui had medium contents of quercetin glycosides (48.0 mg/kg FW) and myricetin glycosides (53.2 mg/kg FW). Kaempferol glyosides (3.1–4.6 mg/kg FW) were the lowest contents of flavonol glycosides in the assayed bayberries. These results are relevant not only from a nutritional point of view, but also in the control of color stability and haze formation during bayberry juice processing and storage.     

The effect of pre-treatment on the anthocyanin and flavonol content of black currant juice (Ribes nigrum L.) in concentration by reverse osmosis

Reverse osmosis process for the concentration of black currant juice was carried using AFC-99 tubular membrane at 30 °C and 45 bar. The contents of selected flavonols and anthocyanins were analyzed after centrifugation; enzyme treatment by Panzym Super E and by Rohapect berry followed by centrifugation; and ultrafiltration black currant juices and juice concentrates. The total soluble solid (TSS) content of the juices increased from the initial 17.6–17.9 °Brix to 24–24.8 °Brix in the case of the centrifuged juice in the concentration process. Similarly, it increased from 14.5–15.5 °Brix to 23.1–23.4 °Brix for the Panzym Super E treated juice, and from 16.1–16.9 °Brix to 22.5–23.1 °Brix for the Rohapect berry treated black currant juices. The ultrafiltered juice had the lowest initial TSS content between 14.1 and 14.9 °Brix and it increased to 22.1–23.1 °Brix. The average permeate fluxes during the concentration process were 7.3 L m⁻² h⁻¹ for the centrifuged juice, 11.9 L m⁻² h⁻¹ for the Panzym Super E treated juice, 9.2 and 13.1 L m⁻² h⁻¹ for the Rohapect berry treated and ultrafiltered juice, respectively. Analysis indicated that the enzymatic treatment resulted in the increase of anthocyanin and flavonol content of the juices. The centrifugation process decreased the amount of anthocyanins and flavonols to some extent. The juice clarified by ultrafiltration had significantly lower concentrations of anthocyanins and flavonols, while the juices treated by Panzym Super E had the highest levels of these flavonoids. This study recommends enzymatic pre-treatment by Panzym Super E, since it improves the permeate flux in reverse osmosis during the concentration process, and results in a juice concentrates highest in anthocyanins and flavonols.     

Identification,quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis)

A novel acylated flavonol glycoside: isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (1), together with two known acylated flavonol glycosides: quercetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (2) and kaempferol (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (3) were isolated from the n-butanol fraction of sea buckthorn (Hippophae rhamnoides ssp. sinensis) berries for the first time by chromatographic methods, and their structures were elucidated using UV, MS, ¹H and ¹³C NMR, and 2D NMR. Compounds 1–3 showed good scavenging activities, with respective IC₅₀ values of 8.91, 4.26 and 30.90 μM toward the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical; respective Trolox equivalent antioxidant capacities of 2.89, 4.04 and 2.44 μM μM⁻¹ toward 2,2′-azino-bis-3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical. The quantitative analysis of the isolated acylated flavonol glycosides was performed by HPLC–DAD method. The contents of compounds 1–3 were in the range of 12.2–31.4, 4.0–25.3, 7.5–59.7 mg/100 g dried berries and 9.1–34.5, 75.1–182.1, 29.2–113.4 mg/100 g dried leaves, respectively.     

Composition and content of flavonol glycosides in broccoli florets (Brassica olearacea) and their fate during cooking

The two main flavonol glycosides present in broccoli florets were identified as quercetin 3-O-sophoroside and kaempferol 3-O-sophoroside. Three minor glucosides of quercetin and kaempferol were also detected, namely isoquercitrin, kaempferol 3-O-glucoside and a kaempferol diglucoside. The sophorosides of quercetin and kaempferol were present in raw florets at a level of 65 mg kg⁻¹ and 166 mg kg⁻¹ fresh weight, respectively. The total content of quercetin and kaempferol glycosides expressed as aglycone was 43 and 94 μg g⁻¹ fresh weight, respectively, and these agree with other recently published data. During the cooking process only 14–28% of the individual glucosides were retained in the cooked tissue, the remainder being largely leached into the cooking water with only a small loss attributed to the formation of the respective aglycones. © 1998 SCI.     

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).     

Optimisation of the aqueous extraction conditions of phenols from meadowsweet (Filipendula ulmaria L.) for incorporation into beverages

Meadowsweet was extracted in water at a range of temperatures (60–100 °C), and the total phenols, tannins, quercetin, salicylic acid content and colour were analysed. The extraction of total phenols followed pseudo first-order kinetics, the rate constant (k) increased from 0.09 ± 0.02 min⁻¹ to 0.44 ± 0.09 min⁻¹, as the temperature increased from 60 to 100 °C. An increase in temperature from 60 to 100 °C increased the concentration of total phenols extracted from 39 ± 2 to 63 ± 3 mg g⁻¹ gallic acid equivalents, although it did not significantly affect the proportion of tannin and non-tannin fractions. The extraction of quercetin and salicyclic acid from meadowsweet also followed pseudo first-order kinetics, the rate constant of both compounds increasing with an increase in temperature up until 90 °C. Therefore, the aqueous extraction of meadowsweet at temperatures at or above 90 °C for 15 min yields extracts high in phenols, which may be added to beverages.     

Identification of flavonol glycosides in American cranberry fruit

Two flavonol glycosides, quercetin galactoside and quercetin arabinoside, have been identified in American cranberry fruit, as a complementary investigation of our previous study. The analysis processes included separation, hydrolysis and structure elucidation of flavonol glycosides. The separation of flavonol glycosides was carried out by solvent extraction, thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). After hydrolysis of the obtained flavonol glycosides, flavonol aglycones and sugars were identified by HPLC and gas chromatography–mass spectrometry (GC–MS), respectively.     

The effect of solar radiation on the flavonol content in broccoli inflorescence

The effect of solar radiation on the quercetin and kaempferol contents in the inflorescence of three broccoli cultivars (‘Lord’, ‘Marathon’ and ‘Fiesta’) was investigated from 1999 to 2001. Great differences in the contents of both flavonols, dependent on growing time and cultivar, were found. Quercetin and kaempferol contents varied from 14.3 to 81.0 mg kg⁻¹ f.w. and from 35.9 to 213 mg kg⁻¹ f.w., respectively. Inflorescences of the cultivar ‘Lord’ were characterised by the highest mean content of quercetin and those of cultivar ‘Fiesta’ of kaempferol. The contents of both flavonols were highly positively correlated with total solar radiation in the period from planting to the harvest of broccoli inflorescences.     

Subcritical water extraction of flavonol quercetin from onion skin

Subcritical water could be an excellent alternative to organic solvent as a medium for extracting flavonol quercetin, due to its temperature-dependent selectivity, safety, efficiency of recovery, and lower cost. This study investigated the application of subcritical water extraction (SWE) of quercetin from onion skin and evaluated the effect of key operation conditions by varying the temperature (100-190 °C), extraction time (5-30 min), and mixture ratio of onion skin and diatomaceous earth (DE) (0.5:3.5-2:2) under high pressure (90-131 bar). The maximum yield of quercetin (16.29 ± 0.75 mg/g onion skin) was obtained at extraction temperature of 165 °C, extraction time of 15 min, mixture ratio of 1.5:2.5 for onion skin and DE. The SWE was compared with three conventional extraction methods in terms of the efficiency. The quercetin yield by SWE was over eight-, six-, and fourfold greater than those obtained using the ethanol, methanol, and water-at-boiling-point extraction methods, respectively.     

Flavonol glycosides from whole cottonseed by-product

Cottonseeds are fed to high-producing dairy cows as a source of fat and highly-digestible fibre. Seven flavonol glycosides have been identified from whole cottonseed by-product. Their structures were established as quercetin 3-O-{β-d-apiofuranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside} (1), kaempferol 3-O-{β-d-apiofuranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside} (2), quercetin 3-O-[β-d-apiofuranosyl-(1 → 2)-β-d-glucopyranoside] (3), quercetin 3-O-β-d-glucopyranoside (4), kaempferol 3-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside] (5), quercetin 3-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside] (6), and kaempferol 3-O-α-l-rhamnopyranoside (7). Gallic acid (8) and 3,4-dihydroxybenzoic acid (9) were also found. All structures were elucidated by ESI-MS and NMR spectroscopic methods. Total polyphenols were assayed by the Folin–Ciocalteu method.     

Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.)

Lipoxygenase (LOX) from pea seeds (Pisum sativum var. Telephone L.) was extracted and studied of biochemical properties. The molecular mass of purified lipoxygenase was 93 kDa. The effects of substrate specificity, pH, and sensibility to various inhibitors: caffeic acid, ferulic acid, benzoic acid, catechin, quercetin and kaempferol of LOX were investigated. Lipoxygenase showed the highest activity toward linoleic acid and the lowest toward oleic acid as substrates. Kinetic studies indicated that V_max of the LOX activity was 151.5 U/min and corresponding K_m value of 0.44 × 10⁻³ M. Optimum pH of lipoxygenase was reported at 5.5. Caffeic acid was the most effective inhibitor and kaempferol was the least effective.     

Extraction and on-line concentration of flavonoids in Brassica oleracea by capillary electrophoresis using large volume sample stacking

Flavonoids are bioactive compounds found in plants. Studies indicate consumption of food containing these compounds may reduce the incidences of cancer and cardiovascular diseases. In broccoli, the flavonoids are present at variable concentrations and so far have mainly been determined using high performance liquid chromatography (HPLC). This paper describes a rapid capillary electrophoresis method, involving large volume sample stacking (LVSS), suitable for the analysis of flavonoids in broccoli. Following acid hydrolysis, the two key flavonoids (kaempferol and quercetin) in a broccoli extract were concentrated on-line by LVSS prior to separation by capillary zone electrophoresis (CZE). Using an optimised method, the extract was injected for 50 s into a 50 μm (internal diameter) × 85 cm (total length) capillary followed by stacking/matrix removal at −5 kV for 83 s. The two analytes were then separated in less than 8 min by CZE using a 10 mM sodium borate buffer (pH 8.40) and a separation voltage of +30 kV at 30 °C. A linear relationship in the range 1–20 ppm was observed for the method (r² = 0.9991–0.9995) with detection limits of 0.9 and 0.6 mg/kg of broccoli for kaempferol and quercetin, respectively. This method demonstrated good repeatability for the standard and extract with relative standard deviations of less than 5% for both peak area and migration time measured over five different days (n = 5). The method was successfully applied to quantitatively determine kaempferol and quercetin contents in a commercial broccoli sample as 11.8 and 14.6 mg/kg fresh weight, respectively. This result was validated by HPLC analysis and is within the ranges reported in the literature.     

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.     

Antioxidant flavonol glycosides in mulberry (Morus alba L.) leaves isolated based on LDL antioxidant activity

Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. We investigated LDL antioxidant activity and extracted compounds of mulberry (Morus alba L.) leaves. The LDL antioxidant activity of 60% ethanol extracted of mulberry leaves, which inhibits human LDL oxidation induced by copper ion, was determined on the basis of oxidation lag time and calculated as epigallocatechin 3-gallate equivalents (58.3 μmol of EGCG equivalent/g of dry weight). Three flavonol glycosides [quercetin 3-(6-malonylglucoside), rutin (quercetin 3-rutinoside) and isoquercitrin (quercetin 3-glucoside)] were identified as the major LDL antioxidant compounds by LC-MS and NMR. The amounts of these flavonol glycosides in mulberry leaves and mulberry-leaf tea were determined by HPLC. Our results showed that quercetin 3-(6-malonylglucoside) and rutin were the predominant flavonol glycosides in the mulberry leaves.     

Antithrombotic activity of fractions and components obtained from raspberry leaves (Rubus chingii)

The 70% ethanol fraction from an aqueous extract of raspberry leaves was shown to be the most antithrombotic fraction in in vitro and in vivo tests. The total flavonoids and phenolics in this fraction were 0.286 g/g and 0.518 g/g by colorimetry. Six compounds, including salicylic acid, kaempferol, quercetin, tiliroside, quercetin 3-O-β-d-glucopyranoside and kaempferol 3-O-β-d-glucopyranoside, were isolated from the active fraction. Among them, kaempferol, quercetin and tiliroside obviously delayed plasma recalcification time (PRT) in blood.     

Flavonol glycosides in wild and cultivated berries of three major subspecies of Hippophaërhamnoides and changes during harvesting period

Flavonol glycosides are an important group of bioactive components of sea buckthorn (Hippophaë rhamnoides). The content and profile of flavonol glycosides of some major subspecies and most cultivars as well as the variation amongst the harvesting years and dates are largely unknown. This study investigated flavonol glycosides in wild berries of two major subspecies H. rhamnoides ssp. rhamnoides and ssp. sinensis and berries of eight cultivars of ssp. rhamnoides and mongolica by reverse phase high performance liquid chromatography combined with diode array detection. The major flavonol glycosides were isorhamnetin-3-O-glucoside-7-O-rhamnoside, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-sophoroside-7-O-rhamnoside, quercetin-3-O-rutinoside, quercetin-3-O-glucoside and quecertin-3-O-sophoroside-7-O-rhamnoside. The total content of flavonol glycosides fell in the range of 27–130 mg per 100 g fresh berries with considerable variation amongst the origins and the harvesting years. Compared with the berries of ssp. sinensis and ssp. mongolica, the berries of ssp. rhamnoides contained high levels of isorhamnetin-3-O-glucoside-7-O-rhamnoside and isorhamnetin-3-O-glucoside and lower levels of quercetin-3-O-rutinoside and quercetin-3-O-glucoside. In the wild berries of ssp. sinensis, the contents of flavonol glycosides reached maxima around late September to early October and decreased thereafter, whereas a general decreasing trend was seen in the cultivated berries of ssp. rhamnoides from the end of August to the end of October.     

Thermal properties and resistant starch content of green banana flour (Musa cavendishii) produced at different drying conditions

The objective of this research was to verify the effect of drying conditions on thermal properties and resistant starch content of green banana flour (Musa cavendishii). The green banana flour is a complex-carbohydrates source, mainly of resistant starch, and quantifying its gelatinization is important to understand how it affects food processing and the functional properties of the flour. The green banana flour was obtained by drying unripe peeled bananas (first stage of ripening) in a dryer tunnel at 52 °C, 55 °C and 58 °C and air velocity at 0.6 m s⁻¹, 1.0 m s⁻¹ and 1.4 m s⁻¹. The results obtained from differential scanning calorimetry (DSC) curves show a single endothermic transition and a flow of maximum heating at peak temperatures from (67.95 ± 0.31) °C to (68.63 ± 0.28) °C. ANOVA shows that only drying temperature influenced significantly (P < 0.05) the gelatinization peak temperature (Tp). Gelatinization enthalpy (ΔH) varied from 9.04 J g⁻¹ to 11.63 J g⁻¹ and no significant difference was observed for either temperature or air velocity. The resistant starch content of the flour produced varied from (40.9 ± 0.4) g/100 g to (58.5 ± 5.4) g/100 g, on dry basis (d. b.), and was influenced by the combination of drying conditions: flour produced at 55 °C/1.4 m s⁻¹ and 55 °C/1.0 m s⁻¹ presented higher content of resistant starch.     

Immunomodulatory and anticancer activities of phenolics from emblica fruit (Phyllanthus emblica L.)

There is a paucity of studies on the immunomodulatory properties of fruit extracts of emblica with the emphasis on lymphocytes. Therefore, the aim of the study was to evaluate the immunomodulatory properties and anticancer potential of six phenolic compounds from emblica fruit by in vitro proliferation assay. Effects of these compounds on splenocyte proliferation and the cytotoxicity to both human breast cancer cell (MCF-7) and human embryonic lung fibroblast cell (HELF) were determined by the MTT method. Significantly stimulatory effects (P < 0.05) were found for geraniin and isocorilagin. The concentration of geraniin, quercetin 3-β-d-glucopyranoside, kaempferol 3-β-d-glucopyranoside, isocorilagin, quercetin, kaempferol and rutin to obtain 50% of stimulatory effect was 56, 123, 242, 42, 73, 93 and 92 μg/ml, respectively. The assay of anticancer activities suggested that geraniin and isocorilagin exhibited higher cytotoxicities than other compounds against MCF-7 with IC₅₀ of 13.2 and 80.9 μg/ml, respectively. Isocorilagin exhibited a strong cytotoxicity to HELF cell with IC₅₀ of 51.4 μg/ml. Geraniin, quercetin, kaempferol and their glycosides had weak cytotoxicity against HELF cells. Paclitaxel showed a strong cytotoxicity to MCF-7 and HELF with IC₅₀ of 6.8 and 14.5 μg/ml, respectively. These findings are in line with the reported potent antioxidant activity. These results suggested that the antitumour activity of these compounds might be achieved by immunomodulatory properties which could partially be attributed to their antioxidant activity.     

Preliminary evaluation of nutraceutical and therapeutic potential of raw Spondias pinnata K., an exotic fruit of India

The underutilized, edible green raw (whole) fruits of amra, Spondias pinnata K. (Anacardiaceae family) from the eastern region of India were investigated for their nutraceutical and therapeutic potential. A thorough nutritional characterization of this fruit demonstrated it as a source of energy (348 kcal/100 g DM), phenolic compounds, natural antioxidants and minerals. It is also a moderate source of ascorbic acid, malic acid, calcium, phosphorus and other nutrients. The phytochemical screening revealed 5.12 ± 0.32 mg 100 g^− 1 DM of alkaloids followed by saponins and tannins. All assays were carried out in three different solvent extracts of the fruit. Total phenolic, flavonoid and flavonol contents were obtained as 210 ± 1.89 mg GAE, 28.0 ± 0.91 mg CE and 9.97 ± 0.72 mg RE respectively in 100 mg mix solvent extract (MSE). Antioxidant activity of different extracts (as DPPH scavenging) ranged from 0.73 to 0.59 mg ml^− 1 as IC₅₀ value, ABTS with a trolox equivalent antioxidant concentration (TEAC) value as 0.68 to 0.83 and FRAP 5.97 to 7.93 mg TE 100 mg^− 1 extract. LC- MS/MS analysis revealed the presence of gallic acid, salicylic acid, chlorogenic acid, ellagic acid, p-coumaric acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, quercetin, catechin, myrecetin and rutin. The MSE showed the highest antimicrobial activity (against Listeria monocytogenes (MIC 1.8 mg ml^− 1)) and ??-amylase inhibition capacity (as IC₅₀: 29.3 mg ml^− 1 extract). GC/MS screening showed the presence of vitamin E, furfural, phytosterol, campesterol and fatty acids. Analysis of volatile flavor showed isopropyl myristinate as a major compound followed by the other monoterpenes and sesquiterpenes. The current study explains the nutritional as well as medicinal utility of the fruit which is a rich source of minerals and antioxidants such as phenols and flavonoids.