Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography

An HPLC method is reported for the separation and quantification of five major polyphenolic groups found in fruits and related products: single ring phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid derivatives), flavan-3-ols, flavonols, anthocyanins, and dihydrochalcones. A binary mobile phase consisting of 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55) (solvent A) and acetonitrile (solvent B) was used. The use of sodium acetate was new and key to the near baseline separation of 25 phenolics commonly found in fruits. A photodiode array detector was used and data were collected at four wavelengths (280, 320, 360, and 520 nm). This method was sensitive and gave good separation of polyphenolics in apple, cherry, strawberry, blackberry, grape, apple juice, and a processing by-product. The improved separation has led to better understanding of the polyphenolic profiles of these fruits. Individual as well as total phenolic content was obtained, and the latter was close to and correlated well with that obtained by the Folin-Ciocalteu method (FC). The HPLC data can be used as a total phenolic index (TPI) for quantification of fruit phenolics, which is advantageous over the FC because it has more information on individual compounds.     

New possibilities for the valorization of olive oil by-products

In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200 °C (EC(50) 18.6 μg/mL) and liquid ethanol at 150 °C (EC(50) 27.4 μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542 mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819 mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves.     

Evaluation of high-performance liquid chromatography for determination of phenolic compounds in pear horticultural cultivars

Summary An analytical evaluation of an HPLC method with diode array detection to separate and quantify polyphenolic compounds from pears has been made. The method was applied to the quantitative analysis of phenolics from five pear horticultural cultivars (“Agua”, “Blanquilla”, “Conference”, “Pasagrana” and “Decana”) in both peel and pulp matrices and evaluated for precision and accuracy. Precision was taken as the reproducibility in peak area of the polyphenols of interest as well as in the slope of calibration graphs. Values ranged 2–5%. Accuracy was evaluated by recovery of all polyphenolic compounds from both peel and pulp in all pears investigated. Accuracy values ranged 92–102%, and were independent of the polyphenolic structure, horticultural cultivar and matrix. Identification was by comparing retention times and UV spectra with those of standards when commercially available. When not available commercially, provisional identification was according to spectral characteristics as well as from isolation and hydrolysis data. Application of the method revealed differences between peel and pulp in all cases studied; the higher levels of phenolics were found in the peels. “Decana” and “Pasagrana” cultivars showed the highest phenolic content compounds whereas “Conference” showed the lowest.     

Fast separation of (poly)phenolic compounds from apples and pears by high-performance liquid chromatography with diode-array detection

Polyphenolic compounds in apples and pears were analysed by HPLC on C₁₈-modified silica. Gradient elution with phosphoric acid–methanol mixtures and phosphoric acid–acetonitrile mixtures gave complete separation of all polyphenolics of interest. The use of methanol as modifier was preferred because it provides a more rapid separation (20 min). Diode-array detection was used for the provisional identification of polyphenolic compounds not available as standards.     

Rapid analysis of stilbenes and derivatives from downy mildew-infected grapevine leaves by liquid chromatography-atmospheric pressure photoionisation mass spectrometry

Resveratrol, trans-epsilon-viniferin and trans-delta-viniferin are the major stilbenes induced in downy mildew infected grapevine leaves. In addition, nine minor polyphenolic compounds, described as stilbenes derivatives, have been separated and detected among known stilbenes after a methanolic microextraction of small pieces (1-2 mg) from infected grapevine leaves with a rapid, qualitative and optimized HPLC method coupled to mass spectrometry using atmospheric pressure photoionisation (APPI-MS(n)). The characterization of unknown stilbenic derivatives as six resveratrol dimers, two dimethylated resveratrol dimers and a resveratrol trimer are reported. Therefore, structures have been proposed for the dimethylated resveratrol dimers. Use of an easy sample treatment and the LC-APPI-MS(n) method results in spectral data of these minor naturally occurring viniferin analogues.     

HPLC separation of flavonols, flavones and oxidized flavonols with UV-, DAD-, electrochemical and ESI-ion trap MS detection

The cation-induced or electrochemical oxidation of flavonols has been reported to yield 2-(hydroxybenzoyl)-2-hydroxy-3(2H)-benzofuranones. Two new gradient reversed phase HPLC methods are presented which allow the determination of those oxidized flavonols simultaneously with flavonols and flavones. UV and electrochemical detection are used because of their high sensitivity. Qualitative detection together with quantification of all compounds is achieved with photodiode-array detection. An electrospray ionization ion trap mass spectrometric method is presented for unique identification of the benzofuranones after HPLC separation.     

HPLC separation of flavonols, flavones and oxidized flavonols with UV-, DAD-, electrochemical and ESI-ion trap MS detection

The cation-induced or electrochemical oxidation of flavonols has been reported to yield 2-(hydroxybenzoyl)-2-hydroxy-3(2H)-benzofuranone. Two new gradient reversed phase HPLC methods are presented which allow the determination of those oxidized flavonols simultaneously with flavonols and flavones. UV and electrochemical detection are used because of their high sensitivity. Qualitative detection together with quantification of all compounds is achieved with photodiode-array detection. An electrospray ionization ion trap mass spectrometric method is presented for unique identification of the benzofuranones after HPLC separation.     

Bioaccumulation and biotransformation of flavonols by erythrocytes

A model for the complete system of bioaccumulation, transport, and biotransformation of polyphenolic compounds (flavonols) that includes hemoglobin-containing red blood cells and serum albumin was proposed. The distribution of flavonols between the erythrocyte fraction and albumin was studied. Hemoglobin was shown to play a role in the biotransformation of flavonols. The formation of several intermediate and final products of pseudoperoxidase oxidation of flavonols catalyzed by methemoglobin was established by UV spectrophotometry and RP-HPLC. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 153-157, March-April, 2009.     

Complete NMR signal assignments of flavonol derivatives

Common substitution positions of flavonols are at C-5 and C-7; 6-substituted flavonol derivatives are rarely found in natural sources. Here, we report complete assignments of 1H and 13C chemical shifts of eight flavonol derivatives including four 6-substituted flavonols.     

Identification and mass spectrometric characterization of glycosylated flavonoids in Triticum durum plants by high-performance liquid chromatography with tandem mass spectrometry

A mass spectrometric method for extensive detection and semi-quantitative determination of flavonoid glycosides in stem and leaves of young Triticum durum plants is presented. About 100 g of sample were lyophilized and ground, and the compounds of interest were then extracted, cleaned-up, and fractionated using high-performance liquid chromatography (HPLC). Tandem mass spectrometry analyses were performed using a quadrupole-linear ion trap instrument with an information-dependent data acquisition (IDA) protocol that looped two experiments, enhanced MS scan and enhanced product ion scan. Various glycoconjugates, which are all derivatives of only four flavones, apigenin, luteolin, chrysoeriol and tricin, were identified and belong to the following categories: 7 monoglycosides, 31 diglycosides, 15 triglycosides and 1 tetraglycoside. Among these some acylated glycosides were found. Tricin derivatives are present exclusively as O-glycosides, while apigenin and luteolin are present always as C-glycosides. Semi-quantitative estimation was performed by using the monoglycoside and diglycoside of quercetin as internal standards.     

A validated chromatographic method for the determination of flavonoids in Copaifera langsdorffii by HPLC

Hydroethanolic extracts of C. langsdorffii leaves have therapeutic potential. This work reports a validated chromatographic method for the quantification of polar compounds in the hydroethanolic extract of C. langsdorffii leaves. A reliable HPLC method was developed using two monolithic columns linked in series (100 x 4.6 mm - C18), with nonlinear gradient elution, and UV detection set at 257 nm. A procedure for the extraction of flavonols was also developed, which involved the use of 70% aqueous ethanol and the addition of benzophenone as the internal standard. The developed method led to a good detection response as the values for linearity were between 10.3 and 1000 microg/mL, and those for recovery between 84.2 and 111.1%. The detection limit ranged from 0.02 to 1.70 microg/mL and the quantitation limit from 0.07 to 5.1 microg/mL, with a maximum RSD of 5.24%. Five compounds, rutin, quercetin-3-O-alpha-L-rhamnopyranoside, kaempferol-3-O-alpha-L-rhamnopyranoside, quercetin and kaempferol, were quantified. This method could, therefore, be used for the quality control of hydroethanolic extracts of Copaifera leaves and their cosmetic and pharmaceutical products.     

Phytochemical Content and Antioxidant Activity of Malus domestica Borkh Peel Extracts

Apple is an important dietary source of carotenoids and phenolic compounds, and its regular consumption is associated with several health benefits. The aim of this study was to evaluate the phytochemical composition of fresh peels of four red-skinned (“Champion”, “Generos”, “Idared”, “Florina”) and two yellow-skinned (“Golden Delicious”, “Reinette Simirenko”) apple varieties. Antioxidant activity of apple peel extracts was determined by ferric reducing antioxidant power (FRAP) and ABTS radical scavenging capacity assays. Total carotenoid and polyphenolic contents were determined spectrophotometrically, while the profile of individual carotenoids and anthocyanins (in red-skinned varieties) was analyzed using high-performance liquid chromatography coupled to a photodiode array detector (HPLC-PDA). Carotenoid composition was specific for each variety, and total carotenoid content was slightly higher in yellow-skinned apple peels compared to red-skinned varieties. In contrast, total phenolic content was higher in the peels of red-skinned cultivars. Anthocyanin profile was predominated by cyanidin-3-O-galactoside. Antioxidant potential followed the trend of the total polyphenolic content, being highest in “Florina”, as measured by both FRAP and ABTS assays. Our results demonstrated apple peels have high phytochemical content with diverse compositions, and their regular consumption can be an excellent source of antioxidants.     

Evaluation of diphenylamine derivatives in apple peel using gradient reversed-phase liquid chromatography with ultraviolet-visible absorption and atmospheric pressure chemical ionization mass selective detection

A method was developed for extracting, identifying, and quantifying diphenylamine (DPA) derivatives in the peel of DPA-treated apples using gradient reversed-phase liquid chromatography with ultraviolet-visible absorption and atmospheric pressure chemical ionization detection (LC-UV-vis-APCI-MS). Compounds routinely analyzed using this method included hydroxylated, nitrosated, nitrated, and methoxylated diphenylamine derivatives. Analysis of peel treated with 0-8 g L(-1) DPA showed that peel DPA content was a limiting factor in derivative production and that recovery of most compounds over this range was linear.     

Deployment of response surface methodology to optimise recovery of grape (Vitis vinifera) stem polyphenols

A 2³-full factorial design and response surface methodology were deployed to assess some basic factors (time, % ethanol and pH) affecting profoundly the extractability of polyphenolic phytochemicals from grape (Vitis vinifera) stems. In an effort to obtain a thorough insight into the applicability of the models established, stem extracts from three different varieties were tested, by determining several indices of the polyphenolic composition, such as total polyphenol (TP), total flavanol (TFl), total flavone (TFn) and proanthocyanidin (PC) concentration. It was shown that the models generated can adequately predict the recovery levels for each polyphenol group, but the optimal conditions predicted for TP, TFl, TFn and PC recovery varied significantly. Notable differences were also seen among the different varieties. Correlation of the polyphenol indices with the antiradical activity and reducing power of the extracts indicated that the PC fraction might exert strong effects, while the influence of other groups was not apparent. Examination of the optimally obtained extracts using liquid chromatography-mass spectrometry revealed that the most prominent compounds were caftaric acid, flavanols and derivatives thereof, as well as dehydroflavonols and flavonols.     

Phenolic profile of edible honeysuckle berries (genus lonicera) and their biological effects

The current status of research on polyphenolic compounds in the berries of edible honeysuckle and their biological effects, including recommended utilization, are reviewed. The major classes of phenolic compounds in the blue berried honeysuckle are flavonols (quercetin, rutin, quercitrin) and flavanes (proanthocyanidins, catechins) and anthocyanins. Cyanidin-3-glucoside and cyanidin-3-rutinoside are considered as major anthocyanidins in edible honeysuckle berries. Such a high level of antioxidant activity in the berries of different species of the genus Lonicera is especially due to the high level of polyphenolic compounds, especially anthocyanins. These berries seem to be prospective sources of health-supporting phytochemicals that exhibit beneficial anti-adherence and chemo-protective activities, thus they may provide protection against a number of chronic conditions, e.g., cancer, diabetes mellitus, tumour growth or cardiovascular and neurodegenerative diseases.     

Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan‐3‐ols and Flavonols in Fresh Vegetables

A fast high‐performance liquid chromatography method was used for analysis of prominent flavan‐3‐ols and flavonols in vegetables. Gradient elution with phosphoric acid‐acetonitrile mixtures and phosphoric acid‐methanol mixtures allowed fast and complete separation of the studied phenolic compounds within analysis times less than 10 min. The development of two elution gradients using methanol and acetonitrile as modifiers proved to be an excellent approach for the verification of the real polyphenolic composition in vegetables samples because the two optimized methods allowed the separation of the same number of compounds in the same elution order. Diode‐array detection was employed for the provisional identification of phenolic compounds that were not available as standards. We preferred methanol as a modifier because it was less toxic and cheaper than acetonitrile. Detection limits ranged between 0.12 and 0.59 μg mL^–1. High recoveries of phenolics from fresh vegetables were measured in all studied cases, independent of the phenolic structure, matrix, and vegetable in question. High levels of procyanidins between 150 and 450 mg kg^–1 were found in all studied vegetables. Quantification of quercetin and kaempferol glycosides was only possible in marrow and onion, respectively.     

Evaluation of flavonols and derivatives as human cathepsin B inhibitor

Cathepsin B (catB) is a cysteine protease involved in tumour progression and represents a potential therapeutic target in cancer. Among the 15 evaluated extracts from cerrado biome, Myrcia lingua Berg. (Myrtaceae) extract demonstrated to be a source of compounds with potential to inhibit catB. Using bioactivity-guided fractionation, we have found flavonols as inhibitors and also some other derivatives were obtained. From the evaluated compounds, myricetin (5) and quercetin (6) showed the most promising results with IC₅₀ of 4.9 and 8.2 μM, respectively, and mode of inhibition as uncompetitive on catB. The results demonstrated polyhydroxylated flavonols as promising inhibitors of catB.     

Benefits and Pitfalls of HPLC Coupled to Diode-Array,Charged Aerosol,and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.     

Rapid screening and identification of antioxidants in the leaves of Malus hupehensis using off‐line two‐dimensional HPLC–UV–MS/MS coupled with a 1,1′‐diphenyl‐2‐picrylhydrazyl assay

The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off‐line two‐dimensional high‐performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1′‐diphenyl‐2‐picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two‐dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB‐C₁₈ column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.     

The Biological Effects of Forsythia Leaves Containing the Cyclic AMP Phosphodiesterase 4 Inhibitor Phillyrin

Forsythia fruit (Forsythia suspensa Vahl (Oleaceae)) is a common component of Kampo medicines for treating the common cold, influenza, and allergies. The main polyphenolic compounds in the leaves of F. suspensa are pinoresinol β-d-glucoside, phillyrin and forsythiaside, and their levels are higher in the leaves of the plant than in the fruit. It is known that polyphenolic compounds stimulate lipid catabolism in the liver and suppress dyslipidemia, thereby attenuating diet-induced obesity and polyphenolic anti-oxidants might attenuate obesity in animals consuming high-fat diets. Recently, phillyrin was reported as a novel cyclic AMP phosphodiesterase 4 (PDE4) inhibitor derived from forsythia fruit. It was expected that the leaves of F. suspensa might display anti-obesity effects and serve as a health food material. In this review, we summarized our studies on the biological effects of forsythia leaves containing phillyrin and other polyphenolic compounds, particularly against obesity, atopic dermatitis, and influenza A virus infection, and its potential as a phytoestrogen.