Effect of air-drying temperature on antioxidant capacity and stability of polyphenolic compounds in mulberry (Morus alba L.) leaves

The mulberry (Morus alba L.) leaf is a promising dietary source of antioxidants such as quercetin due to its relatively high content of that compound. We investigated effects of an air-drying process on the antioxidant capacity and stability of antioxidant polyphenolic compounds in mulberry leaves. Main compounds playing a central role in antioxidant activities in mulberry leaves are quercetin glycosides and chlorogenic acid. Raw mulberry leaves were air-dried at various temperatures, and antioxidant activity using DPPH radical scavenging assay and levels of antioxidant compounds were measured. DPPH radical scavenging activity and levels of polyphenolic compounds in mulberry leaves air-dried at 60 °C or below were not significantly different from those of freeze-dried mulberry leaves, whereas both values in mulberry leaves air-dried at 70 °C and over decreased significantly. These results indicate that strict temperature control is important in the production of mulberry leaf products to maintain antioxidant activity and levels of polyphenolic compounds.     

Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.)

The aim of the present study was to evaluate the profile of the phenolic constituents of Morus nigra fruits and their antioxidant activity (DPPH) and to compare their contents before and after fermentation. Antioxidant phenolics of black mulberry (M. nigra L.) samples grown in Galicia (NW Spain) were extracted with methanol/formic acid/water (MFW) and determined by high performance liquid chromatography (HPLC). Two major anthocyanins (cyanidin 3-glucoside and cyanidin 3-rutinoside) and two flavonols (quercetin 3-glucoside and rutin) were isolated, together with caffeic acid and other hydroxycinnamic and ellagic acid derivatives. Their chemical structures were identified by spectral analyses with diode array detection (DAD), but also with alkaline saponification and acid hydrolysis of the mulberry phenolics. Good correlations (r² = 0.6229) were observed among total flavonols contents and the IC₅₀ radical scavenging capacities of mulberry fruits. Anthocyanins are the major flavonoids present in mulberry. It would be expected that anthocyanins contribute significantly to their antioxidant activity; nevertheless, alcohol generated during fermentation may also contribute to antioxidant activity. Our results provide useful antioxidant nutritional information of fresh and fermented mulberry fruits.     

Antioxidant flavonoid glycosides from the leaves of Ficus pumila L.

The Okinawan folks in Japan use Ficus pumila L. as a beverage or herbal medicine to treat diabetes and high blood pressure. Four flavonoid glycosides were isolated and identified as rutin (1 and 3), apigenin 6-neohesperidose (2), kaempferol 3-robinobioside (4) and kaempferol 3-rutinoside (5). Among these compounds, rutin exhibited the strongest antioxidant activity in DPPH radical scavenging assay and superoxide radical inhibition assay. The preparation of Ooitabi leaves in water provide sufficient amount of flavonoid glycosides to the Okinawan although 50% of aqueous ethanol extracted these flavonoid glycosides more effectively. These results show the potential of Ooitabi leaves as a natural source of antioxidant for health management.     

Flavonol content in the water extract of the mulberry (Morus alba L.) leaf and their antioxidant capacities

Abstract: The biological activities of the mulberry (Morus alba L.) leaf have been attributed to its flavonoid content. The water extract of the mulberry leaf (WEML) was prepared by autoclaving at 121 °C for 15 min, and the flavonol content of the WEML was determined by HPLC The WEML contained 4 flavonols in the following order: quercetin‐3‐β‐D‐glucose (QT‐G) > quercetin‐3‐O‐glucose‐6″‐acetate (QT‐GA) > rutin (RT) > quercetin (QT). In the oxygen radical absorbance capacity (ORAC) assay, QT had the highest peroxyl radical‐scavenging capacity and a similar hydroxyl radical‐scavenging capacity as its glycosides (QT‐G, QT‐GA, and RT). QT exhibited a stronger cellular antioxidant capacity (CAC) against 2,2′‐Azobis(2‐amidinopropane) dihydrochloride (AAPH)‐ and Cu²⁺‐induced oxidative stress in HepG2 cells compared to its glycosides, indicating that the intracellular antioxidant capacity of QT and its glycosides may depend upon both the permeability across the cell membrane and the peroxyl or hydroxyl radical‐scavenging capacity. Practical Application: The information presented might be used for developing mulberry leaf‐based functional foods.     

Isolation and identification of compounds from the ethanolic extract of flowers of the tea (Camellia sinensis) plant and their contribution to the antioxidant capacity

While beneficial health properties of tea leaves have been extensively studied, less attention has been given to that of flowers of the tea (Camellia sinensis) plant. In this work, the ethanolic extract and its ethyl acetate-soluble fraction (EEA) from the tea flowers were found to possess the potent antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay. The compounds present in EEA had comparatively strong DPPH scavenging activity and strongly contributed to the antioxidant activity of the tea flowers. From EEA, besides eight catechins, five flavonol glycosides were isolated and their structures were elucidated on the basis of mass spectrometry and nuclear magnetic resonance spectroscopy as myricetin 3-O-β-d-galactopyranoside, quercetin 3-O-β-d-galactopyranoside, kaempferol 3-O-β-d-galactopyranoside, kaempferol 3-O-β-d-glucopyranoside, and kaempferol 3-O-[α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranoside]. In addition, epigallocatechin gallate and epicatechin gallate were found as the major active components responsible for the antioxidant activity of tea flowers through the use of a combination of preparative liquid chromatography separation and DPPH assay.     

LC-MS–MS characterisation of curry leaf flavonols and antioxidant activity

Curry leaf (Murraya koenegii) is a common flavouring agent in Indian foods. This study characterised the flavonol profile of curry leaf extracted with different solvents and the relative antioxidant capacity of these extracts by quantifying phenolic constituents. Flavonols were extracted using ethanol, methanol, or acetone prior to identification and quantification using liquid chromatography coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry in tandem mode (LC-MS–MS) with negative ion detection. Major curry leaf flavonols included myricetin-3-galactoside, quercetin-O-pentohexoside, quercetin-3-diglucoside, quercetin-3-O-rutinoside, quercetin-3-glucoside, quercetin-3-acetylhexoside, quercetin-O-xylo-pentoside, kaempferol-O-glucoside, and kaempferol-aglucoside. Lag-time and TBARS tests demonstrated that curry leaf phenolics prevent cupric-ion induced oxidation of LDL. The best extraction yield was obtained with 80% ethanol. Acetone extracts provided better antioxidant activity expressed as increased lag-time formation, than did ethanol or methanol extracts. Curry leaf is a rich source of flavonols that have biological activity in vitro and further studies are warranted in regards to the potential health benefits and identification of the novel flavonols whose identities remain unknown.     

Inhibition ofIn VitroHuman LDL Oxidation by Phenolic Antioxidants from Grapes and Wines

Current research suggests that wine contains substances that may reduce the mortality rate from coronary diseases. The oxidation of low-density lipoprotein (LDL) is thought to be a key step in the development of atherosclerosis. Phenolic fractions of a Petite Syrah wine were evaluated for their antioxidant activity in inhibiting LDL oxidation in vitro . The more active fractions contained components of the catechin family. The catechin oligomers and the procyanidin dimers (B₂, B₃, B₄, B₆, B₈) and trimers (C₁, C₂) were extracted, isolated and purified from grapes seeds. These compounds were tested for their inhibition of LDL oxidation, along with other monomeric wine phenolics. The procyanidin dimers B₂ and B₈, and trimer C₁, and the monomers catechin, epicatechin and myricetin had the highest antioxidant activity. The procyanidin dimers B₃, B₄ and C₂ and the monomers gallic acid, quercetin, caffeic acid, and rutin, and a group of compounds that included the dimer B₆, ellagic acid, sinapic acid, cyanidin had lower antioxidant activity and α-tocopherol had the least activity. Thus, the numerous phenolic compounds found in wine are potent antioxidants in inhibiting LDL oxidation in vitro .     

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.     

Antioxidative properties and flavonoid composition of Chenopodium quinoa seeds cultivated in Japan

To evaluate the nutritional advantages of quinoa seeds (Chenopodium quinoa Willd.) cultivated in Japan, antioxidative properties and flavonoid composition were determined and compared to corresponding data for conventionally-used cereals and pseudo-cereals, including quinoa seeds from South America. The antioxidant activities of these grains against DPPH radicals were strongly associated with the total phenolic content of the tested samples. The crude extracts of quinoa seeds cultivated in Japan exhibited higher antioxidative effects than those from South America and other cereals, excluding buckwheat. Four flavonol glycosides were isolated and identified from the Japanese quinoa seeds, and the chemical composition of the flavonoids – quercetin and kaempferol 3-O-(2″,6″-di-O-α-rhamnopyranosyl)-β-galactopyranosides (1 and 4), quercetin 3-O-(2″,6″-di-O-α-rhamnopyranosyl)-β-glucopyranoside (2), and quercetin 3-O-(2″-O-β-apiofuranosyl-6″-O-α-rhamnopyranosyl)-β-galactopyranoside (3) – was evaluated through quantitative determination. Trioside 2 was isolated for the first time from quinoa seeds. These glycosides were not detected in extracts from any of the tested grains except quinoa. The aglycone quercetin content of the Japanese quinoa seeds is higher than in the seeds from South America and buckwheat. The amounts of quercetin and kaempferol formed via acidic hydrolysis in quinoa are much higher than those of conventionally-used edible plants. The quinoa seeds cultivated in Japan are the most effective functional foodstuff – in terms of being a source of antioxidative and bioactive flavonoids – among cereals and pseudo-cereals.     

Effect of HP-β-cyclodextrins complexation on the antioxidant activity of flavonols

The beneficial effects from phenolic compounds have been attributed to their antioxidant activity. Differences in the chemical structure of flavonols and their degree of substitution will influence phenoxyl radical stability and, thereby, their antioxidant properties. Cyclodextrins (CDs) can be used as a flavonol complexation agent, since they act as a substrate reservoir in a dose-controlled manner. In the present paper, the effect of complexing flavonols, kaempferol, quercetin and myricetin with HP-β-CDs on their antioxidant capacity is studied by means of the oxygen radical absorbance capacity-fluorescein (ORAC-FL) assay. This complexation phenomenon increased the antioxidant activity of the three flavonols, which reached a maximum level when each flavonol had been complexed in the hydrophobic cavity of CDs. The antioxidant activity increased because of the flavonol was protected against rapid oxidation by free radicals.     

Isolation and evaluation of the radical-scavenging activity of the antioxidants in the leaves of an edible plant, Mallotus japonicus

The antioxidative properties of a hot water extract of the leaves of Mallotus japonicus were evaluated. The extract had a high phenolic content and strong antioxidative activity, compared with green tea, rooibos tea, and red wine. Six phenolic compounds were isolated as antioxidative components by HPLC. They were identified as mallotinic acid, mallotusinic acid, corilagin, geraniin, rutin, and ellagic acid. These antioxidative compounds were subjected to DPPH radical-scavenging, superoxide radical-scavenging, and hydroxyl radical-scavenging assays, and compared with other antioxidative compounds. Four of the compounds, mallotinic acid, mallotusinic acid, corilagin and geraniin, exhibited much stronger antioxidative activity than gallic acid, rutin, ellagic acid, quercetin, and chlorogenic acid, and were as active as epigallocatechin gallate (EGCG), a strong antioxidant in green tea. Mallotus japonicus leaves are an excellent source of strong natural antioxidative materials.     

Evaluation of flavonoid and polyphenol constituents in mulberry leaves using HPLC fingerprint analysis

The mulberry leaf is a promising dietary source of antioxidants due to its high levels of beneficial compounds. To further examine its antioxidant properties, twelve batches of authenticated mulberry leaf, with total flavonoid contents (TFC) ranging from 24.34 mg g⁻¹ DW to 58.42 mg g⁻¹ DW and total polyphenol content (TPC) ranging from 11.49 mg g⁻¹ DW to 30.03 mg g⁻¹ DW, were investigated. According to Spearman’s coefficient, antioxidant activities, including the DPPH radical scavenging assay and the ferric reducing antioxidant power (FRAP), were positively correlated with TFC and TPC. The HPLC-DAD analysis results identified the characteristic fingerprint peaks in mulberry leaves as chlorogenic acid, rutin, quercetin 3-O-glucoside and kaempferol 3-O-glucoside, all of which directly contribute to the antioxidant capacity of mulberry leaves. Notably, young mulberry leaves showed higher antioxidant capacity than those of mature leaves. These promising results help create a compelling case for future development of mulberry leaf products.     

Quercetin content in some food and herbal samples

Quercetin is a typical flavonoid ubiquitously present in vegetables and fruits, and its antioxidant effect is implied to be helpful for human health. The efficiency of extraction process and acidic hydrolysis parameters for HPLC analysis of quercetin present in glycosides and aglycone forms was investigated. Hydrolysis for 5 min in the presence of 2.8 M HCl as well as for 10 min with 1.1 M HCl efficiently released quercetin from rutin. The method developed in this study was applied for quantitative determination of quercetin in some food (onion, apple) and herbal (Hypericum perforatum and Sambucus nigra) products.     

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.     

Identification and quantification of phenolic acids and flavonol glycosides in Tunisian Morus species by HPLC-DAD and HPLC–MS

The total polyphenol and flavonoids in leaves of Morus alba var. alba, Morus alba var. rosa and Morus rubra were determined and identification of their components was carried out. The total content of phenolics varied between 345.20 and 631.53 mg gallic acid equivalents (GAE)/100 g dry weight (DW) basis. The total amount of flavonoids ranged between 193.87 and 398.33 mg rutin equivalents (RE)/100 g DW. Thirteen compounds were isolated by chromatography, and their structures determined to be mainly flavonol glycosides and phenolic acids. Three novel components were identified as kaempferol-7-O-glucoside, quercetin-3-O-β-glucoside-7-O-α-rhamnoside and quercetin-3-O-rhamnoside-7-O-glucoside, for the first time from mulberry leaves. Others known compounds were also identified.     

Anti-melanogenesis properties of quercetin- and its derivative-rich extract from Allium cepa

In an effort to find a new whitening agent, we have found that the methanol extract of the dried skin of Allium cepa showed inhibition of melanin formation. Bioassay-guided fractionation led to the isolation of quercetin (1) and quercetin 4’-O-β-glucoside (3) from A. cepa as the inhibitors of melanin formation in B16 melanoma cells with IC₅₀ values of 26.5 and 131 μM, respectively. In addition, we evaluated the effect of some quercetin derivatives, such as isoquercitrin (2), quercetin 3,4’-O-diglucoside (4), rutin (5) and hyperin (6) on B16 melanoma cells. These quercetin derivatives did not show any inhibition of melanin formation. Furthermore, the ORAC values of compounds 1–6 were 7.64, 8.65, 4.82, 4.32, 8.17 and 9.34 μmol trolox equivalents/μmol, respectively. Dried skin of red onion showed inhibitory activity against melanin formation in B16 melanoma cells, as well as antioxidant properties.     

Inhibition of oxidation of omega-3 polyunsaturated fatty acids and fish oil by quercetin glycosides

The antioxidant properties of naturally occurring flavonols, quercetin glycosides, were examined and compared with common food antioxidants butylated hydroxytoluene (BHT) and α-tocopherol. Antioxidants were incorporated into selected polyunsaturated fatty acids (PUFA) or fish oil in aqueous emulsions and bulk oil systems. The effectiveness of quercetin was similar to or greater than quercetin glycosides in inhibiting lipid oxidation in the oil-in-water emulsion systems when oxidation was induced by heat, light, peroxyl radical or ferrous ion. In bulk fish oil, C-3 glycosylation enhanced the antioxidant activity of quercetin. The effectiveness of quercetin and its glycosides was greater than that of α-tocopherol in the emulsions. Quercetin and quercetin-3-O-glucoside exhibited a better antioxidant activity than BHT in bulk fish oil; however, the reverse was observed in the emulsions of omega-3 PUFA and fish oil systems in agreement with the polar paradox theory. Quercetin and its glycosides were more effective than α-tocopherol in emulsion systems.     

Activity guided isolation of antioxidants from the leaves of Ricinus communis L.

An activity-guided isolation and purification process was used to identify the DPPH (l,l-diphenyl-2-picrylhydrazyl) free radical scavenging components of the food plant (Ricinus communis L.) of Eri silkworm. Dry leaves of R. communis L. were extracted with different solvents and tested for their antioxidant activity against DPPH^•. The MeOH:water (8:2) extract showed strong DPPH radical-scavenging activity, and was subjected to column chromatography over silica gel. Gallic acid, quercetin, gentisic acid, rutin, epicatechin and ellagic acid were isolated as active components and characterised by different spectroscopic techniques.     

Preparation and properties of rutin-hydrolyzing enzyme from tartary buckwheat seeds

A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70 kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37 °C for 30 min in the presence of 20% ethanol, and its K_m value for rutin is 1.04 × 10⁻³ M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by ¹H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.     

A remarkable influence of microwave extraction: Enhancement of antioxidant activity of extracted onion varieties

Four (red, yellow, white and grelot onion) varieties of Allium cepa, a rich source of quercetin (flavonol) glycosides, were studied for their total content of reducing compounds (TCRC), flavonol content and antioxidant activity evaluation. Extracts obtained by solvent free microwave hydrodiffusion and gravity (MHG) technique and conventional solvent extraction (CSE) were analysed with high performance liquid chromatography (HPLC) for quantification of flavonoids. Three different methods were selected for evaluating the antioxidant capacity of the different onion varieties (after the determination of their phenolic content by the Folin–Ciocalteu method): the reduction of the stable DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, the ORAC (oxygen radical absorbance capacity) method, and the inhibition of the AAPH-induced peroxidation of linoleic acid in SDS micelles. The highest antioxidant capacity was observed for red onion, followed by yellow, white and grelot onion. In spite of the low recovery of extractable flavonoids (quercetin 3,4′-diglucoside, 4′-glucoside and 3-glucoside), MHG remained the preferred extraction method in comparison to the conventional method, as all the samples obtained under microwave-assisted extraction (MAE) exhibited the highest antioxidant activities in all the tests. Also the microscopic observations of extracted tissues showed that at cellular level, microwaves induced disruptions of vacuoles and cell walls thus promoting the effectiveness of this method.