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Use of polyphenolic fingerprints established by comprehensive two-dimensional liquid chromatography for the classification of honeys according to their floral origin
Abstract
In this work, the polyphenolic composition of honeys from three different floral origins (chestnut, heather, and thyme), coming from different geographical areas of Spain was investigated. First, samples were characterized in terms of total phenolic content (TPC) and antioxidant capacity, which was established by three different assays. The results revealed that the studied honeys presented similar TPCs and antioxidant capacities, with a wide variability within each floral origin. Next, a comprehensive two-dimensional liquid chromatography method was developed for the first time to establish polyphenol fingerprints of the three types of honeys, after optimizing the separation in terms of column combination and mobile phase gradient programs. After that, the detected common peaks were used for the construction of a linear discriminant analysis (LDA) model able to discriminate honeys according to their floral origin. The LDA model obtained was adequate for the classification of the floral origin of the honeys based on polyphenolic fingerprint data.
... This section will focus on the methodologies developed for honey geographical or botanical origin through liquid chromatography. Table 4 summarizes some selected LC methodologies used for the classification and authentication of honey considering either their geographical or botanical origin [26,65,[68][69][70][71][72]. As can be seen, most of the examples aim to classify honey belonging to only one country using a limited number of samples of each type. ...
... An interesting approach for the characterization and authentication of honey samples was proposed by Vergara-Barberán et al. [72], based on the use of two-dimensional liquid chromatography (LC × LC) for the classification of Spanish honey samples according to the botanical origin. In this case, phenolic compounds were used as the targeted markers for the sample classification. ...
Honey is a traditional food sweetener with a very complex composition, produced naturally by bees (Apis mellifera), highly consumed and appreciated by society not only because of its nutritional value and taste but also due to its beneficial properties on human health. Composed mainly of sugars, water, and proteins, it also contains other bioactive substances, such as vitamins, enzymes, organic acids, and polyphenols, that contribute to a greater or lesser extent to its organoleptic and physicochemical properties, being an important source of antioxidants, exhibiting also an immunomodulator effect in wound healing, as well as
antitumoral, anti-inflammatory, antifungal, and antibacterial properties. These properties are influenced by the botanical origin, geographical production area, the environment, and floral and entomological sources, among other factors. Thus, the characterization of honey based on the different factors that influence its organoleptic and health characteristics, such as geographical origin, is of paramount importance to protect and enhance the beekeeping sector.
The special and unique characteristics of honey have placed it as one of the products most vulnerable to manipulation for illicit purposes, with adulteration and mislabeling being the most common fraudulent practices. This issue is aggravated by the fact that the honey supply is frequently lower than the demand, a situation that has recently been further intensified by the drought environmental conditions in many honey-producing areas. Thus, honey authenticity today awakens the interest of all the actors involved, aiming to avoid economic losses in the beekeeping sector. Hence, among the most relevant honey authenticity issues, we can find the assessment of the botanical species and the geographical production region, especially for those products labelled with a protected designation of origin (PDO) or other equivalent quality attributes, due to the higher prizes they can reach in the market.
Spectroscopic, chromatographic, and mass spectrometry methodologies are widely employed to characterize honey and to solve different honey authentication issues, including fraudulent practices based on adulteration and mislabeling. In this chapter, the role of chromatographic separation techniques, such as liquid chromatography (LC) and gas chromatography (GC), spectroscopy –including simple spectrophotometric assays, such as Folin Ciocalteu (FC) or ferric reducing antioxidant ability (FRAP) assays–, and mass spectrometry to characterize and assess honey botanical and geographical production origin will be addressed by means of relevant examples. An exhaustive review of the topic with coverage of all kinds of publications is beyond the scope of the present contribution, so we will focus on the most relevant applications published in the last five years.
The analysis of anthraquinones was developed using LC × LC-HRMS with continuous shifted gradient in ² D. The method was reproducible both in terms of retention times and peak areas, allowing the detection of 226 peaks in industrial mixtures.
Honey is a rich source of nutrients, which might be affected by bees foraging on different plants. Therefore, the current study aimed to discriminate honey samples based on their contents of antioxidants, chemical components, and pesticide residues. The antioxidant scavenging activity and total phenolic contents were measured spectrophotometrically. Bioactive chemical components and pesticide residues were estimated using gas chromatography-mass spectrometry. The data were statistically analyzed using analysis of variance and clustering and discriminant models to differentiate the different kinds of honey. The results revealed significant differences between the examined kinds of honey in terms of total phenolic contents, with the greatest values for sider and acacia (138.14 mg gallic acid equivalent/100 g fresh weight). Moringa and fennel unifloral honey exhibited the highest scavenging activities of the free radical α, α-diphenyl-β-picrylhydrazyl. Discriminate analysis revealed differences among honey samples of diverse botanical origins based on their content of sugar, glycoside, flavonoid, fatty acid, and terpenoids.
Honey consumption and imports have increased in recent years, and it is considered by consumers to be a healthy alternative to more commonly used sweeteners. Honey contains a mixture of polyphenols and antioxidant compounds, and the botanical origin and geographical area of collection play an important role on its chemical composition. The present study investigated the physicochemical properties, total phenolic content and antioxidant capacity of Spanish thyme honey and chestnut honey, and their mixtures with royal jelly (2% and 10%) and propolis (2% and 10%). The analysis of the physicochemical parameters of both honey samples showed values within the established limits. Propolis showed the highest value of total phenolic content (17.21–266.83 mg GAE/100 g) and antioxidant capacity (DPPH, ORAC and ABTS assays; 0.63–24.10 µg eq. Tx/g, 1.61–40.82 µg eq. Tx/g and 1.89–68.54 µg eq. Tx/g, respectively), and significantly reduced ROS production in human hepatoma cells. In addition, mixtures of honey with 10% of propolis improved the results obtained with natural honey, increasing the value of total phenolic content and antioxidant capacity. A significant positive correlation was observed between total phenolic compounds and antioxidant capacity. Therefore, the antioxidant capacity could be attributed to the phenolic compounds present in the samples, at least partially. In conclusion, our results indicated that thyme and chestnut honey supplemented with propolis can be an excellent natural source of antioxidants and could be incorporated as a potential food ingredient with biological properties of technological interest, added as a preservative. Moreover, these mixtures could be used as natural sweeteners enriched in antioxidants and other bioactive compounds.
The study of electrical impedance applied to food has become a method with great potential for use in the food industry, which allows the monitoring and control of quality processes in a safe and non-invasive way. Recent research has shown that this technique can be an alternative method to determine the floral origin of the honey bee (Apis mellifera L.) and acquire information on chemical and physical properties such as conductivity, ash content and acidity. In this work, the electrical impedance of six monofloral honey samples from diverse origins and one commercial multi-floral honey were measured using a low-cost impedance meter, obtaining 101 samples (reactance (X) versus resistance (R)), with a frequency sweep between 1 Hz and 25 MHz in all the honeys analyzed. This shows that it is possible, by using a multilayer neural network trained from these data, to classify with 100% accuracy between these honeys and, thereby, quickly and easily determine the floral origin of the honey. This is without the need to use the chemical data or equivalent electrical models.
The polyphenolic composition of Algerian date palm (Phoenix dactylifera L) leaves (in Hamray, Safray, Ghars, Horra, Deglet Nour (DN) cultivars), and pollen (in DN cultivars), were investigated for the first time in these cultivars. The polyphenolic fractions were extracted using three solvents (acetone, methanol and ethanol), and characterized in terms of antioxidant activity and contents of total polyphenols (TPC), flavonoids (TFC), and tannins (TCTC). All the extracts exhibited high antioxidant activity and TPC, TFC and TCTC values, DN being the cultivar that provided the best results without significant differences among the three solvents. When the extracts were analyzed by HPLC-MS, 21 and 23 phenols were identified in leaf and pollen extracts, respectively. Furthermore, polyphenolic fingerprints of leaf extracts were obtained by comprehensive two-dimensional liquid chromatography (LC×LC), which made it possible to discriminate samples according to their cultivar by linear discriminant analysis.
Honey is a highly consumed natural product produced by bees which is susceptible to fraudulent practices, some of them regarding its botanical origin. Two HPLC-UV non-targeted fingerprinting approaches were evaluated in this work to address honey characterization, classification, and authentication based on honey botanical variety. The first method used no sample treatment and a universal reversed-phase chromatographic separation. On the contrary, the second method was based on an off-line SPE preconcentration method, optimized for the isolation and extraction of polyphenolic compounds, and a reversed-phase chromatographic separation optimized for polyphenols as well. For the off-line SPE method, the use of HLB (3 mL, 60 mg) cartridges, and 6 mL of methanol as eluent, allowed to achieve acceptable recoveries for the selected polyphenols. The obtained HPLC-UV fingerprints were subjected to an exploratory principal component analysis (PCA) and a classificatory partial least squares-discriminant analysis (PLS-DA) to evaluate their viability as sample chemical descriptors for authentication purposes. Both HPLC-UV fingerprints resulted to be appropriate to discriminate between blossom honeys and honeydew honeys. However, a superior performance was accomplished with off-line SPE HPLC-UV polyphenolic fingerprints, being able to differentiate among the different blossom honey samples under the study (orange/lemon blossom, rosemary, thyme, eucalyptus, and heather). In general, this work demonstrated the feasibility of HPLC-UV fingerprints, especially those obtained after off-line SPE polyphenolic isolation and extraction, to be employed as honey chemical descriptors to address the characterization and classification of honey samples according to their botanical origin.
Italian honeys from different floral sources (acacia, orange, honeydew, chestnut, strawberry tree, sulla, eucalyptus, dandelion, linden, polyfloral) were analysed in terms of colour, total phenolic content, in vitro antioxidant capacity and content of 15 phenolic compounds. Physicochemical parameters were also examined to assess the overall quality of honey. Dark honeys demonstrated to have the highest content in bioactive compounds and in antioxidant activity with the highest values in strawberry tree and honeydew honeys. Data were processed using principal component analysis (PCA), hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). The overall classification capacity for the 9 unifloral honey varieties obtained by LDA was 100.00%, with a very low level of prediction misclassification in cross validation (less than 5%). This study demonstrates the strong relation between honey floral origin and bioactive compounds profile and amount, together with the importance of colour attributes as a simple approach for a preliminary evaluation of the antioxidant properties and floral origin discrimination.
Inflammation is a defense process triggered when the body faces assaults from pathogens, toxic substances, microbial infections, or when tissue is damaged. Immune and inflammatory disorders are common pathogenic pathways that lead to the progress of various chronic diseases, such as cancer and diabetes. The overproduction of cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, is an essential parameter in the clinical diagnosis of auto-inflammatory diseases. In this review, the effects of bee products have on inflammatory and autoimmune diseases are discussed with respect to the current literature. The databases of Google Scholar, PubMed, Science Direct, Sci-Finder and clinical trials were screened using different combinations of the following terms: “immunomodulatory”, “anti-inflammatory”, “bee products”, “honey”, “propolis”, “royal jelly”, “bee venom”, “bee pollen”, “bee bread”, “preclinical trials”, “clinical trials”, and “safety”. Honey bee products, including propolis, royal jelly, honey, bee venom, and bee pollen, or their bioactive chemical constituents like polyphenols, demonstrate interesting therapeutic potential in the regulation of inflammatory mediator production as per the increase of TNF-α, IL-1β, IL-6, Il-2, and Il-7, and the decrease of reactive oxygen species (ROS) production. Additionally, improvement in the immune response via activation of B and T lymphocyte cells, both in in vitro, in vivo and in clinical studies was reported. Thus, the biological properties of bee products as anti-inflammatory, immune protective, antioxidant, anti-apoptotic, and antimicrobial agents have prompted further clinical investigation.
Honey is a natural food product very famous for its health benefits for being an important source of antioxidant and phenolic compounds. Euphorbia honeys obtained from different regions of Morocco were evaluated for their ability to inhibit acetylcholinesterase, lipoxygenase, tyrosinase and xanthine oxidase activities. Their antioxidant properties were evaluated using the: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity, nitric oxide scavenging activity (NO) and scavenging ability of superoxide anion radical. Then, the phenolic extracts of the same entire honey samples were evaluated by liquid chromatography coupled to diode array detection and mass spectrometry (LC-DAD-MS) and tested for the biological activities previously evaluated on the entire honeys, in order to conduct a comparative study between both (honey and phenolic extracts). The chromatographic profiles for the studied Euphorbia honey extracts were different. Phenolic compounds gallic acid, 4-hydroxybenzoic acid and p-coumaric acid were detected in all samples, whereas kampferol was only present in two samples. Physicochemical parameters and total phenolic content were also determined. Entire honey that recorded the highest rate of phenols was sample M6 (E. resinifera) = 69.25 mg GAE/100 g. On the other hand, the phenolic extracts had better antioxidant and enzyme inhibitory activities than the entire honeys, regardless the monofloral honey type. In conclusion, the studied Euphorbia honeys may have a great potential as antioxidant, anti-inflammatory and anti-tyrosinase sources for pharmaceutical and cosmetic applications.
Bee products have been known for centuries for their versatile healing properties. In recent decades they have become the subject of documented scientific research. This review aims to present and compare the impact of bee products and their components as antimicrobial agents. Honey, propolis, royal jelly and bee venom are bee products that have antibacterial properties. Sensitivity of bacteria to these products varies considerably between products and varieties of the same product depending on their origin. According to the type of bee product, different degrees of activity were observed against Gram-positive and Gram-negative bacteria, yeasts, molds and dermatophytes, as well as biofilm-forming microorganisms. Pseudomonas aeruginosa turned out to be the most resistant to bee products. An analysis of average minimum inhibitory concentration values for bee products showed that bee venom has the strongest bacterial effectiveness, while royal jelly showed the weakest antibacterial activity. The most challenging problems associated with using bee products for medical purposes are dosage and safety. The complexity and variability in composition of these products raise the need for their standardization before safe and predictable clinical uses can be achieved.
Honey has exerted a high impact in the field of alternative medicine over many centuries. In addition to its wound healing, anti-microbial and antioxidant properties, several lines of evidence have highlighted the efficiency of honey and associated bioactive constituents as anti-tumor agents against a range of cancer types. Mechanistically, honey was shown to inhibit cancer cell growth through its pro-apoptotic, anti-proliferative and anti-metastatic effects. However, the potential of honey to regulate anti-tumor immune responses is relatively unexplored. A small number of in vitro and in vivo studies have demonstrated the ability of honey to modulate the immune system by inducing immunostimulatory as well as anti-inflammatory effects. In the present review, we summarize the findings from different studies that aimed to investigate the immunomodulatory properties of honey and its flavonoid components in relation to cancer. While these studies provide promising data, additional research is needed to further elucidate the immunomodulatory properties of honey, and to enable its utilization as an adjuvant therapy in cancer.
Adulteration of honey is a major problem in the food industry. The purpose of the present study was to classify different types of monofloral honey based on physicochemical characterization and analysis of phenolic compounds coupled with chemometrics methods. The methods for classification were trialed on a wide range of honey samples from different floral origins. For thyme, jujube, coriander, barberry, acacia and alfalfa honey samples, principal component analysis combined with discriminant analysis (PCA-DA) and partial least squares combined with discriminant analysis (PLS-DA) were trialed. The results indicate that the botanical origin of the honey affects the profile of flavonoids and phenolic compounds. For example, jujube honey samples had the highest amounts of hesperetin and chrysin, while thyme honey had the maximum amount of caffeic acid; the highest levels of quercetin and p-coumaric acid were found in coriander honey. To reduce the numbers of independent variables for modeling, the principal component analysis (PCA) algorithm was used. The three scores extracted from PCA had 83.17% variance. The classification results show that PLS-DA was successfully used to predict the class membership of honey samples (100%), but PCA-DA gave the lowest correct classification rate (97%).
Honey color and other physicochemical characteristics depend mainly on the botanical and geographical origin. The study of these properties could make easier a correct classification of unifloral honey. This work determined the palynological characteristics and some physicochemical properties such as pH, electrical conductivity, and color (Pfund scale and the CIELa*b* coordinates), as well as the total content of the bioactive compounds phenols and flavonoids of ninety-three honey samples. Samples were classified as chestnut, blackberry, heather, eucalyptus, and honeydew honey. The study showed a close relationship between the physicochemical variables and the botanical origin. The five types of honey presented different physicochemical properties among them. A principal component analysis showed that Hue, lightness, b*, and Chroma variables were important for the honey types classification, followed by Erica pollen, pH, Cytisus, and Castanea variables. A forward stepwise regression analysis was performed introducing as dependent variables the color (mm Pfund) and the Chroma and the Hue variables. The regression models obtained explained 86%, 74%, and 86% of the variance of the data, respectively. The combination of the chromatic and physicochemical and pollen variables through the use of multivariable methods was optimal to characterize and group the honey samples studied.
The emergent market for honeydew honeys in Europe prompt to increasing requirements of consumers and honey industry for the characterisation of this type of honey. The aim of this study was to characterise 59 samples of Spanish oak honeydew honeys. Physicochemical properties showed values within the limits established by the legislation and typical for honeydew honeys. Honeys were differentiated into two groups according to the hue (hab) and all were classified as dark honeys (L* < 55). A total of 14 minerals were determined, with K, P, Mg, and Ca being the most abundant. The development and validation of an HPLC method allowed the determination of the contents of two monosaccharides, five disaccharides, and two trisaccharides. Total phenolic and flavonoid contents showed mean values of 130.2 mg/100 g and 11.3 mg/100 g of honey, respectively. Honeydew honeys showed ability to scavenge free radicals and to inhibit lipid peroxidation, which is very interesting, because, as far as we know, there are no previous studies for this type of honey. Results showed that all honeydew honeys are a source of chemical compounds with nutritional and antioxidant properties that could be of interest for consumers and food industry.
Background
Globally, cancer ranks among the most common causes of death. Multiple experimental and clinical studies have investigated anticancer effects of honey with promising results. This study focused on potential background mechanisms of this effect.
Methods
The current literature was reviewed for potential anticancer pathways which are suggested for honey and its ingredients.
Results
Flavonoids (kaempferol, catechin, and quercetin) and phenolic acids (caffeic acid and gallic acid) are the most important ingredients of honey with known anti-cancer activity. The main suggested mechanisms for anti-cancer activity of honey and its ingredients are antioxidant, apoptotic, tumor necrosis factor inhibiting, antiproliferative, immunomodulatory, anti-inflammatory and estrogenic effects.
Conclusion
This review collates the current scientific understanding on the mechanism of anti-cancer activity of honey.
https://www.sciencedirect.com/science/article/pii/S0261561418325871
Honey is a natural substance appreciated for its therapeutic abilities since ancient times. Its content in flavonoids and phenolic acids plays a key role on human health, thanks to the high antioxidant and anti-inflammatory properties that they exert. Honey possesses antimicrobial capacity and anticancer activity against different types of tumors, acting on different molecular pathways that are involved on cellular proliferation. In addition, an antidiabetic activity has also been highlighted, with the reduction of glucose, fructosamine, and glycosylated hemoglobin serum concentration. Honey exerts also a protective effect in the cardiovascular system, where it mainly prevents the oxidation of low-density lipoproteins, in the nervous system, in the respiratory system against asthma and bacterial infections, and in the gastrointestinal system. A beneficial effect of honey can also be demonstrated in athletes. The purpose of this review is to summarize and update the current information regarding the role of honey in health and diseases.
The antioxidant and antimicrobial components of honey vary based on sourced of nectar. Medicinal plants with the therapeutic value have potential to produce honey with greater bioactivity. The aim of the present study was to characterize the physico-chemical and antioxidant capacities of Agastache honey produced from Agastache rugosa and compare them with other popular commercial honeys sold in Australia. The total phenolics, total flavonoids, moisture content, colour, pH, protein content and antioxidant capacity were evaluated for Agastache, Manuka, Jelly bush, Tea tree, Super manuka and Jarrah honeys. The results reveal that the moisture content ranged from 17–21%, pH ranged from 3.8–4.3 and estimated protein content ranged from 900–2200 µg/g. The DPPH•, ABTS•+, ORAC and FRAP methods were used to measure the antioxidant capacity of the honey samples. The DPPH• % inhibition, ABTS•+, ORAC and FRAP values for Agastache honey were 9.85 (±1.98 µmol TE/g), 26.88 (±0.32 µmol TE/g), 19.78 (±1.1 µmol TE/g) and 3.61 (±0.02 µmol TE/g) whereas the highest antioxidant capacity values obtained were 18.69 (±0.9 µmol TE/g), 30.72 (±0.27 µmol TE/g), 26.95 (±0.9 µmol TE/g) and 3.68 (±0.04 µmol TE/g), respectively. There was a positive correlation between colour, total phenolic content and DPPH• scavenging activity for most of the honeys except Tea tree honey. However, there was no clear correlation with ABTS•+, ORAC and FRAP values. The measured antioxidant capacity of samples varied with the assays used. The DPPH• assay clearly indicated that the phenolic compounds contribute to the scavenging activity of the honeys. Nevertheless, all assays confirm that Agastache honey has significant antioxidant capacity. Therefore, Agastache honey can be important to human nutrition and health.
Honey is a functional food widely consumed. Thus, the evaluation of honey samples to determine its phenolic content and antioxidant capacity (AOC) is relevant to determine its quality. Usually AOC is performed by spectrophotometric methods, which lacks reproducibility and practicality. In this context, the electroanalytical methods offer higher simplicity and accuracy. Hence, the aim of this work was to use of electroanalytical tools and laccase based biosensor on the evaluation of AOC and total phenol content (TPC) of honey samples from different countries. The antioxidant power established by electrochemical index presented good correlation with the spectrophotometric FRAP (Ferric Reducing Ability of Plasma) and DPPH (2,2-Diphenyl-1-Picrylhydrazyl) radical scavenging assays. Also, TPC results obtained by the biosensor agreed with the Folin-Ciocalteu (FC) assay. In addition to the semi quantitative results, the electroanalysis offered qualitative parameters, which were useful to indicate the nature of major phenolic compounds.
The main treatment for cancer is by using chemotherapy and radiotherapy which themselves are toxic to other viable cells of the body. Recently, there are many studies focusing on the use of natural products for cancer prevention and treatment. Of these natural products, honey has been extensively researched. The mechanism of the anti-cancer activity of honey as chemopreventive and therapeutic agent has not been completely understood. The possible mechanisms are due to its apoptotic, antiproliferative, antitumor necrosis factor (anti-TNF), antioxidant, anti-inflammatory, estrogenic and immunomodulatory activities. We collate the findings of several studies published in the literature in order to understand the mechanism of its action.
Grape juices and wines are rich in numerous groups of polyphenolic compounds which require a dedicated separation technique for such complex samples. LC × LC is considered the best technique for the analysis of such samples as it can achieve better resolution and higher peak capacity compared to 1D LC. The ever-growing demand for protecting the environment necessitates reducing or eliminating hazardous solvents to improve the environmental friendliness of analytical procedures. In this study, propylene carbonate was used as an eco-friendly mobile phase component in comprehensive two-dimensional liquid chromatography to analyze phenolic compounds in grape juices and a dealcoholized wine sample. Novel green RPLC × RPLC-DAD and RPLC × RPLC-MS methods were developed for the first time to identify phenolic compounds in five samples (two red grape juice samples, two white grape juice samples, and one dealcoholized wine sample). Four different RPLC × RPLC systems were developed; three systems were connected to a diode array detector (RPLC × RPLC-DAD), while the fourth system was connected to DAD and MS detectors (RPLC × RPLC-DAD-ESI-MS). Solvent X (propylene carbonate:ethanol, 60:40) was adopted as a green organic modifier in the first dimension (1D) and methanol in the second dimension (2D). The practical peak capacity and the surface coverage were calculated as metrics to measure the separation performance of all proposed systems. The orthogonality values for the setups ranged from 0.64 to 0.92 when calculated by the convex hull method, and from 0.54 to 0.80 when calculated by the asterisk equations method. The practical peak capacity production rate ranged from 14.58 to 22.52 peaks/min. The results revealed that the phenolic compounds were separated efficiently with good coverage of the 2D separation space and high peak capacity. A total of 70 phenolic compounds were detected based on MS data and information from the literature.
Over the past decade, the measurement of phenolics and flavonoids in honey has received much attention, which is attributed to their broad spectrum of beneficial pharmacological activity and their potential to address issues related to the authenticity and botanical origin of honey. Due to their high variability in structural and physicochemical properties, the separation, detection and identification of these molecules in honey is challenging. Among all the chromatography-driven techniques, liquid chromatography (LC) is one of the most popular and powerful tools for the analysis of phenolics and flavonoids. In particular, the coupling of LC to various detectors, especially mass spectrometry, has enabled relatively fast tentative identification and accurate quantitative and qualitative analysis of polyphenolic compounds in honey. In this review, a survey of the recent considerations and developments in the application of extraction strategies and LC-related techniques for polyphenolics determination in honey during past decade is provided. The review will also feature future trends of LC-based polyphenolics analysis, with opportunities for high-resolution multidimensional LC analysis that offers significantly higher total peak capacities.
The main goal of this article is to present an overview of the analytical methodologies employed in recent years (2015-2021) to determine several honey constituents, and, specifically, those with health-promoting effects and nutritional value, like phenolic compounds, sugars, amino acids and proteins, vitamins, lipids, minerals, and organic acids. The review is structured according to the different families of compounds, and they will be discussed along with the main extraction and analytical techniques used for their determination. Phenolic compounds, sugars and amino acids have been the main compounds determined in honey. The analytical methods (sample treatment and determination techniques) are strongly dependent on the compound. Nevertheless, it can be concluded that high-performance liquid chromatography was predominantly selected for determining honey constituents; while, in relation to the sample treatment, the preferred option was a dilution of the honey with water or a buffer.
Comprehensive two-dimensional liquid chromatography is a well-established method for the unraveling of very complex real-world samples. With regard to food and natural products such a technique turned out to be a very promising approach due to its high resolving power and improved identification capability, especially in combination with mass spectrometry. In this context, polyphenols comprise a particular complex class of bioactive compounds, due to their nature and content in commonly consumed foodstuffs, making their analysis challenging. The present contribution shows an overview of the two commonly employed approaches used for polyphenol analysis, viz. RP-LC × RP and HILIC × RP-LC. Furthermore, the latest implementations as well as limitations and future perspectives are critically reported.
The phenolic compound profiles of 393 raw mono- and polyfloral Latvian honey samples were investigated using a targeted ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) method. This strategy allowed simultaneous determination of the content of 11 phenolic acids, 18 flavonoids, 2 plant hormones, and 3 water-soluble vitamins in honey samples of botanically diverse origin, including buckwheat (Fagopyrum esculentum), heather (Calluna vulgaris), linden (Tilia cordata), and rapeseed (Brassica napus). Honey samples were collected directly from the beekeepers and were analyzed in their natural, raw form. A selective high-throughput analysis was implemented using sugaring-out assisted liquid-liquid extraction (SULLE) combined with the pentafluorophenyl (PFP) stationary phase. The robustness of the method was evaluated by using the design of experiments (DoE) approach and validated. Pooled quality control (QC) samples allowed monitoring of the method performance across the analysis sequences and principal components analysis (PCA) of QC samples resulted in a small amount of total variance in the total projection, indicating that the data were of good quality. The acquired occurrence data were compiled and compared with other studies. To the best of our knowledge, this study is the first of this scale and purpose, providing unique insights into the phenolic compound composition of fresh, regional European honey.
Not all honey are made equal. Honey is a natural substance from a mixture of flower nectars or the secretions of plant-sucking insects that are collected and digested by honey bees. There are 300 known types of honey, produced by over 20,000 honey bee species, that vary in flavor, color, and health effects. For this reason, honey type and composition vary with geography, and angiosperm and honey bee species distribution. In this chapter, we discuss the different types of honey and their associated health benefits. We also provide insights on the future of honey production in the context of functional food advocacy, climate change and honey bee species decline.
Comprehensive two-dimensional liquid chromatography represents a valuable and powerful tool for the analysis of very complex samples. This review article illustrates how the coupling of two single LC “orthogonal” separations can be of valid aid for ensuring a deep chemical investigations of foods and food products. Specifically, in the first part, considerations about method optimization and novel modulation interfaces are reported whereas in the second part selected applications to polyphenols, lipids and carotenoids in various real-world samples are reported and discussed.
The development of a new comprehensive two-dimensional liquid chromatographic method is described, to obtain the profiles of polyphenolic compounds present in olive (Olea europaea L.) leaves and pulps from different genetic origin. Optimisation of the stationary phase nature, particle size, column length and internal diameter, as well as other separation conditions, was performed. Along the study, three stationary phases (C18, PFP and phenyl) in the first dimension (¹D), and five (C18, amide, cyano, phenyl and PFP) in the second dimension (²D) were combined to obtain the maximal number of resolved peaks. The optimised method successfully characterised the presence of 26 and 29 common polyphenols in olive leaves and pulp extracts, respectively. Peak volume ratios were used to develop linear discriminant analysis models able to distinguish olive leaves and pulp extracts among seven cultivars from several Spanish regions. The results demonstrate that polyphenolic profiles were characteristic of each cultivar.
The development of efficient analytical methods for the identification and quantification of polyphenols in natural products is needed. The present study was aimed to optimize the polyphenols by UFLC using two different Ascentis express columns. Chromatographic separation was performed using UFLC-DAD connected with Ascentis Express Biphenyl column (system I) and Ascentis Express F5 pentafluorophenylpropyl (PFP) column (system II). Multistep gradient elution program was used in both analytical instruments to detect 34 compounds of different classes (phenolic acids, flavonoids, isoflavones, flavanols, flavonols and flavanones). The detection wavelength, linear calibration range, theoretical plate, tailing factor, limits of detection and of quantification were determined. The results were validated and the optimized method was proven to be precise, revealing good separation in the UFLC system II using Ascentis Express F5 PFP column. In conclusion, this study shows analytical preciseness and is useful in determining polyphenols in natural products.
Plant origin, physicochemical parameters and composition were analysed to characterize the avocado honey (Persea americana Mill.) from Andalusia (Southern, Spain). Ashes content, total polyphenol, and electrical conductivity corresponded to these of a typical dark honey (>80 mm scale Pfund). Regarding mineral elements, K was predominant, followed by P and Mg. Antioxidant and invertase activities presented some desirable values. In the 20 analysed samples, 48 pollen types corresponding to 33 families were identified. Avocado pollen was found in high variability (13–58%). At least a 20% was suggested to guarantee the authentic avocado honey. Perseitol, sugar-alcohol identified only in avocado honey, fundamentally contributes to distinguish this kind of honey. The content varied between 0.31 and 1.56 g/100 g. The correlation between perseitol and avocado pollen was found to be significant. A minimum concentration of 0.30 g/100 g of perseitol is suggested to characterize the proposed monofloral avocado honey.
This work aimed to characterise four seaweed species: nori (Phorphyra), kombu (Laminaria), wakame (Undaria) and sea spaghetti (Himanthalia elongata). Their nutritional composition, total phenolic compounds (TPC), antioxidant capacity, oil and water holding capacity (OHC and WHC), and swelling capacity (SC) were determined. Wakame and nori exhibited the highest proteins contents, rich in essential amino acids and in those related to umami flavour. All the samples had a low lipid content and high ash content values. High fibre levels were observed, especially in kombu. The TPC content and antioxidant capacity of sea spaghetti was significantly higher than in the other samples. The OHC, WHC and SC of the seaweeds demonstrated their potential influence on texture of food products. The incorporation of these seaweeds into different foodstuffs could entail an improvement of the nutritional quality and texture properties, and could also reduce the use of Na and synthetic additives.
Honey is a precious natural product that is marketed with a wide range of nutritional and medicinal properties. However, it is also a product subjected to frequent adulteration through mislabeling and mixing with cheaper and lower-quality honeys and various sugar syrups. In that sense, honey authentication regarding its genuine botanical and geographical origins, as well as the detection of any adulteration, is essential in order to protect consumer health and to avoid competition that could create a destabilized market. Various analytical techniques have been developed to detect adulterations in honey, including measuring the ratios of stable isotopes (mostly (13)C/(12)C) and the use of different spectroscopic, chromatographic, and electrochemical methods. This review aims to provide a cross-section of contemporary analytical methods used for the determination of honey authenticity in order to help the scientific community engaged in the field of honey chemistry make appropriate choices and select the best applications that should lead to improvements in the detection and elimination of fraudulent practices in honey manufacturing.
Thirty-three commercial thyme honeys (Thymus spp.) were characterized and classified according to geographical origin based on quality parameters, total phenolic content and radical scavenging activity. Quality parameter analysis included the determination of: pH, free, lactonic and total acidity, electrical conductivity, moisture, ash, hydroxymethylfurfural (HMF) content and diastase activity, using official methods of analysis. Finally, total phenolic content and % radical scavenging activity (%RSA) were estimated using the Folin–Ciocalteu and the 1,1-diphenyl-2-picryl-hydrazil [DPPH·] assay, respectively. Results showed that thyme honeys analyzed: (1) met all the quality criteria set by the EU; (2) showed variations in total phenolic content and radical scavenging activity; (3) possessed a special food character since total phenolic content was positively correlated with radical scavenging activity and (4) were successfully classified (correct prediction rate 93.9%) according to geographical origin applying chemometric analyses to the data collected. The present study highlights quality criteria and bioactive properties of Greek commercial thyme honeys, produced in different regional departments in Greece. Such parameters determined, give an enhanced functional food character to this product which can lead to consumers' awareness on nutritional foods of specific regional origin. In addition, the use of chemometric analyses on quality parameters, total phenolic content and radical scavenging activity proved to be a strong key for correct geographical classification of Greek commercial thyme honey.
A novel analytical procedure based on solid-phase extraction, HPLC-DAD and HPLC-MS2 analyses is here presented for the study of seven different monofloral honey types from Tuscany (Italy) in terms of their phenolic content. In particular, an embedded polar group RP-Amide stationary phase is employed in order to achieve the separation and quantitation of 24 different polyphenols. Extraction recoveries and dynamic linear ranges are determined, and limits of detection as low as 1 ng/g are obtained. Quantitation of the polyphenol content revealed total concentrations in the order of 100–102 μg/g, with chestnut honeys showing the highest results. Further analyses with HPLC-MS2 proved invaluable in the identification of unknown compounds found in the honeys. Processing of the chromatographic data via Principal component analysis divided the sample pool in three groups. Each group showed distinctive traits that can be used to distinguish some of the honey types from the others.
In the last years, the consumption of soy-based foods has increased due to the health benefits related to soy bioactives like phenolic compounds. Thus, in the present study, a new chromatographic method using reverse-phase high performance liquid chromatography coupled to diode array detection (RP-HPLC/DAD) was developed using a fused core pentafluorophenyl (PFP) column. The established method allowed the determination of twenty-one free phenolic compounds and eleven bound phenolics in a soy isoflavone concentrate. The method was validated in terms of precision and recovery. Intra and inter-day precision were less than 5% (% RSD) and the recovery was between 97.4% and 103.6%. Limits of quantification (LOQs) ranged between 0.093 and 0.443μg/mL. Because of that, PFP stationary phase can be easily applied for routine determination of phenolic compounds in soy based foods.
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Different monofloral honeys from Castilla-La Mancha (Spain) have been studied in order to determine their main functional and biological properties. Thyme honey and chestnut honey possess the highest antioxidant capacity, which is due to their high vitamin C (in thyme honey) and total polyphenolic content (in chestnut honey). On the other hand, chestnut honey showed high antimicrobial activity against Staphylococcus aureus and Escherichia coli, whilst others had no activity against S. aureus and showed very small activity against E. coli. Moreover it was found that the antimicrobial activity measured in chestnut honey was partly due to its lysozyme content. In addition the angiotensin I-converting enzyme (ACE) inhibitory activity was measured, and the ACE inhibition is one mechanism by which antihypertensive activity is exerted in vivo. All the types of honey showed some activity but chestnut honey had the highest ACE inhibitory activity.
A great many tools have been developed for supervised classification, ranging from early methods such as linear discriminant analysis through to modern developments such as neural networks and support vector machines. A large number of comparative studies have been conducted in attempts to establish the relative superiority of these methods. This paper argues that these comparisons often fail to take into account important aspects of real problems, so that the apparent superiority of more sophisticated methods may be something of an illusion. In particular, simple methods typically yield performance almost as good as more sophisticated methods, to the extent that the difference in performance may be swamped by other sources of uncertainty that generally are not considered in the classical supervised classification paradigm.
A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.