Common Methods of Extraction and Determination of Phytomelatonin in Plants.

The presence of melatonin in plants, called phytomelatonin, has gained great interest in recent years. The determination of phytomelatonin levels in plant extracts for both physiological and plant foodstuff studies requires sophisticated techniques due to the low endogenous levels of this indolic compound with hormonal nature. This chapter presents the most common and advanced techniques in the determination of phytomelatonin, with special emphasis on the techniques of extraction, cleaning, separation, detection, identification, and quantification. Multiple examples and recommendations are presented for a clear overview of the pros and cons of phytomelatonin determinations in plant tissues, seeds, and fruits, mainly.

Chemical Characterization and Several Bioactivities of Cladanthus mixtus from Morocco.

The purpose of this work was to investigate, for the first time to our knowledge, the chemical composition and bioactivity of methanolic extracts (roots, stems, leaves, and flowers) from Cladanthus mixtus (L.) Chevall. that grows wild in northern Morocco (the Tangier-Tetouan-Al Hoceima region). The phenolic and flavonoid contents were determined by spectrophotometer methods, and the composition of derivatized methanolic extracts from C. mixtus using N-O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) was analyzed by gas chromatography-mass spectrometry (GC-MS). The antioxidant activity was carried out by applying the 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and DPPH (2,2-diphenyl-1-picrylhydrazyl) tests. The micro-dilution technique was chosen to investigate the antimicrobial activity of methanolic extracts against two bacterial strains and three fungal species. The results showed that the values of total phenolic and flavonoid contents were found to be higher in flower extracts (30.55 ± 0.85 mg of gallic acid equivalents (GAE)/g of dried weight (DW) and 26.00 ±1.34 mg of quercetin equivalents (QE)/g DW, respectively). Other groups of chemical compounds were revealed by GC-MS, such as carbohydrates (27.25-64.87%), fatty acids (1.58-9.08%), organic acids (11.81-18.82%), and amino acids (1.26-7.10%). Root and flower methanolic extracts showed the highest antioxidant activity using ABTS (39.49 mg of Trolox equivalents (TE)/g DW) and DPPH (36.23 mg TE/g DW), respectively. A positive correlation between antioxidant activity and polyphenol and flavonoid amounts was found. Antibacterial tests showed that the best activity was presented by the leaf extract against Staphylococcus aureus (minimum inhibitory concentration (MIC) = minimum bactericidal concentration (MBC) = 20 mg/mL) and Escherichia coli (MIC of 30 mg/mL and MBC of 35 mg/mL). S. aureus was more sensitive to the extracts compared to E. coli. All extracts showed antifungal activity against Trichophyton rubrum, with the best efficacy reported by the flower and leaf extracts (MIC = 1.25 mg/mL and minimum fungicidal concentration (MFC) = 2.5 mg/mL). In general, extracts of C. mixtus appeared less effective against Candida albicans and Aspergillus fumigatus.

Melatonin as a Possible Natural Anti-Viral Compound in Plant Biocontrol.

Melatonin is a multifunctional and ubiquitous molecule. In animals, melatonin is a hormone that is involved in a wide range of physiological activities and is also an excellent antioxidant. In plants, it has been considered a master regulator of multiple physiological processes as well as of hormonal homeostasis. Likewise, it is known for its role as a protective biomolecule and activator of tolerance and resistance against biotic and abiotic stress in plants. Since infections by pathogens such as bacteria, fungi and viruses in crops result in large economic losses, interest has been aroused in determining whether melatonin plays a relevant role in plant defense systems against pathogens in general, and against viruses in particular. Currently, several strategies have been applied to combat infection by pathogens, one of them is the use of eco-friendly chemical compounds that induce systemic resistance. Few studies have addressed the use of melatonin as a biocontrol agent for plant diseases caused by viruses. Exogenous melatonin treatments have been used to reduce the incidence of several virus diseases, reducing symptoms, virus titer, and even eradicating the proliferation of viruses such as Tobacco Mosaic Virus, Apple Stem Grooving Virus, Rice Stripe Virus and Alfalfa Mosaic Virus in tomato, apple, rice and eggplant, respectively. The possibilities of using melatonin as a possible natural virus biocontrol agent are discussed.

Melatonin from Microorganisms, Algae, and Plants as Possible Alternatives to Synthetic Melatonin.

Melatonin dietary supplements are widely consumed worldwide, with developed countries as the largest consumers, with an estimated annual growth rate of approximately 10% until 2027, mainly in developing countries. The wide use of melatonin against sleep disorders and particular problems, such as jet lag, has been added to other applications, such as anti-aging, anti-stress, immune system activation, anticancer, and others, which have triggered its use, normally without a prescription. The chemical industry currently covers 100% of the needs of the melatonin market. Motivated by sectors with more natural consumption habits, a few years ago, the possibility of obtaining melatonin from plants, called phytomelatonin, arose. More recently, the pharmaceutical industry has developed genetically modified microorganisms whose ability to produce biological melatonin in bioreactors has been enhanced. This paper reviews the aspects of the chemical and biological synthesis of melatonin for human consumption, mainly as dietary supplements. The pros and cons of obtaining melatonin from microorganisms and phytomelatonin from plants and algae are analyzed, as well as the advantages of natural melatonin, avoiding unwanted chemical by-products from the chemical synthesis of melatonin. Finally, the economic and quality aspects of these new products, some of which are already marketed, are analyzed.

Role of Melatonin and Nitrogen Metabolism in Plants: Implications under Nitrogen-Excess or Nitrogen-Low.

Melatonin is a new plant hormone involved in multiple physiological functions in plants such as germination, photosynthesis, plant growth, flowering, fruiting, and senescence, among others. Its protective role in different stress situations, both biotic and abiotic, has been widely demonstrated. Melatonin regulates several routes in primary and secondary plant metabolism through the up/down-regulation of many enzyme/factor genes. Many of the steps of nitrogen metabolism in plants are also regulated by melatonin and are presented in this review. In addition, the ability of melatonin to enhance nitrogen uptake under nitrogen-excess or nitrogen-low conditions is analyzed. A model that summarizes the distribution of nitrogen compounds, and the osmoregulation and redox network responses mediated by melatonin, are presented. The possibilities of using melatonin in crops for more efficient uptake, the assimilation and metabolization of nitrogen from soil, and the implications for Nitrogen Use Efficiency strategies to improve crop yield are also discussed.

Effect of Melatonin in Broccoli Postharvest and Possible Melatonin Ingestion Level.

The post-harvest stage of broccoli production requires cold storage to obtain enough days of shelf life. It has been proved that melatonin is useful as a post-harvest agent in fruits and vegetables, including broccoli. In this study, the broccoli heads treated with melatonin have a longer shelf life than the control samples, which was reflected in parameters such as fresh weight, hue angle (expresses color quality), and chlorophyll and carotenoid contents. Treatments with 100 µM melatonin for 15 or 30 min seem to be the most appropriate, extending the broccoli's shelf life to almost 42 days, when it is normally around 4 weeks. In addition, a study on the possible impact that melatonin treatments in broccoli could have on melatonin intake in humans is presented. The levels of superficial melatonin, called washing or residual melatonin, are measured, showing the possible incidence in estimated blood melatonin levels. Our results suggest that post-harvest treatments with melatonin do not have to be a handicap from a nutritional point of view, but more research is needed.

Melatonin as a Possible Natural Safener in Crops.

Melatonin is a well-known animal hormone with relevant and multiple cellular and hormonal roles. Its discovery in plants in 1995 has led to a great diversity of molecular and physiological studies that have been showing its multiple actions also in plants. Its roles as a biostimulator and modulator agent of responses to abiotic and biotic stresses have been widely studied. This review raises the possible use of melatonin as a natural safener in herbicide treatments. Existing studies have shown excellent co-acting qualities between both the following agents: herbicide and melatonin. The presence of melatonin reduces the damage caused by the herbicide in the crop and enhances the stress antioxidant response of plants. In this area, a similar role is suggested in the co-action between fungicides and melatonin, where a synergistic response has been demonstrated in some cases. The possible reduction in the fungicide doses is proposed as an eco-friendly advance in the use of these pesticides in certain crops. Finally, future research and applied actions of melatonin on these pest control agents are suggested.

Melatonin in Brassicaceae: Role in Postharvest and Interesting Phytochemicals.

Brassicaceae plants are of great interest for human consumption due to their wide variety and nutritional qualities. Of the more than 4000 species that make up this family, about a hundred varieties of 6-8 genera are extensively cultivated. One of the most interesting aspects is its high content of glucosinolates, which are plant secondary metabolites with widely demonstrated anti-oncogenic properties that make them healthy. The most relevant Brassicaceae studies related to food and melatonin are examined in this paper. The role of melatonin as a beneficial agent in seedling grown mainly in cabbage and rapeseed and in the postharvest preservation of broccoli is especially analyzed. The beneficial effect of melatonin treatments on the organoleptic properties of these commonly consumed vegetables can be of great interest in the agri-food industry. Melatonin application extends the shelf life of fresh-cut broccoli while maintaining optimal visual and nutritional parameters. In addition, an integrated model indicating the role of melatonin on the organoleptic properties, the biosynthesis of glucosinolates and the regulatory action of these health-relevant compounds with anti-oncogenic activity is presented.

Phytomelatonin: an unexpected molecule with amazing performances in plants.

Phytomelatonin, a multifunctional molecule that has been found to be present in all plants examined to date, has an important role in plants as a modulatory agent (a biostimulator) that improves plant tolerance to both biotic and abiotic stress. We present a review of phytomelatonin that considers its roles in plant metabolism and in particular its interactions with plant hormone network. In the primary metabolism of plants, melatonin improves the rate and efficiency of photosynthesis, as well related factors such as stomatal conductance, intercellular CO2, and Rubisco activity. It has also been shown to down-regulate some senescence transcription factors. Melatonin up-regulates many enzyme transcripts related to carbohydrates (including sucrose and starch), amino acids, and lipid metabolism, optimizing N, P, and S uptake. With respect to the secondary metabolism, clear increases in polyphenol, glucosinolate, terpenoid, and alkaloid contents have been described in numerous melatonin-treated plants. Generally, the most important genes of these secondary biosynthesis pathways have been found to be up-regulated by melatonin. The great regulatory capacity of melatonin is a result of its control of the redox and plant hormone networks. Melatonin acts as a plant master regulator, up-/down-regulating different plant hormone levels and signalling, and is a key player in redox homeostasis. It has the capacity to counteract diverse critical situations such as pathogen infections and abiotic stresses, and provide plants with varying degrees of tolerance. We propose possible future applications of melatonin for crop improvement and post-harvest product preservation.

A Phytomelatonin-Rich Extract Obtained from Selected Herbs with Application as Plant Growth Regulator.

The animal hormone melatonin (N-acetyl-5-methoxytryptamine) is a pleiotropic molecule with multiple and various functions. Phytomelatonin is the melatonin from plants and was discovered in 1995 in some species. Phytomelatonin is considered an interesting molecule in the physiology of plants, as it seems to be involved in many actions, such as germination, growth, rooting and parthenocarpy, including fruit set and ripening; it also seems to play a role during postharvest. It has been studied in processes such as primary and secondary metabolism, photosynthesis and senescence, as well as in the nitrogen and sulfur cycles. Phytomelatonin up- and down-regulates many relevant genes related to plant hormones and key genes related to the above-mentioned aspects. One of the most decisive aspects of phytomelatonin is its relevant role as a bioprotective and alleviating agent against both biotic and abiotic stressors, which has opened up the possibility of using melatonin as a phytoprotector and biostimulant in agriculture. In this respect, using material of plant origin to obtain extracts rich in phytomelatonin instead of using synthetic melatonin (thus avoiding unwanted by-products) has become a topic of discussion. This work characterized the phytomelatonin-rich extracts obtained from selected herbs and determined their contents of phytomelatonin, phenols and flavonoids; the antioxidant activity was also measured. Finally, two melatonin-specific bioassays in plants were applied to demonstrate the excellent biological properties of the natural phytomelatonin-rich extracts obtained. The herb composition and the protocols for obtaining the extracts rich in phytomelatonin are in the process of registration for their legal protection.

Melatonin and Carbohydrate Metabolism in Plant Cells.

Melatonin, a multifunctional molecule that is present in all living organisms studied, is synthesized in plant cells in several intercellular organelles including in the chloroplasts and in mitochondria. In plants, melatonin has a relevant role as a modulatory agent which improves their tolerance response to biotic and abiotic stress. The role of melatonin in stress conditions on the primary metabolism of plant carbohydrates is reviewed in the present work. Thus, the modulatory actions of melatonin on the various biosynthetic and degradation pathways involving simple carbohydrates (mono- and disaccharides), polymers (starch), and derivatives (polyalcohols) in plants are evaluated. The possible applications of the use of melatonin in crop improvement and postharvest products are examined.

Melatonin as a plant biostimulant in crops and during post-harvest: a new approach is needed.

A great amount of data covering a wide variety of plant species and experimental conditions has demonstrated the beneficial actions that melatonin exerts on many aspects of plant development, including germination, photosynthesis and water economy. Melatonin behaves especially well as a plant biostimulator against biotic and abiotic stressors, increasing stress tolerance. The present contribution sets out possible future multidisciplinary studies, in which the impact of using melatonin with respect to agriculture, food technology, human nutrition and the environment needs to be clearly established. In crops, the effective dose and best formulations for individual plant species and cultivation conditions should be studied. As regards post-harvest, the focus should be on the half-life time of melatonin in fruits and water-residue treatments. Detailed studies are lacking on the human intake of phytomelatonin in different diets. Studies on the metabolization of phytomelatonin and the combined effect with other phytonutrients such as carotenoids, chlorophylls, flavonoids, fibers, etc., would also be of interest. In soils, the possible interaction between melatonin and microbiome and non-vertebrate animals is of primordial interest. In terms of the environment, although melatonin is classified as a non-hazardous agent, its limitations as a possible animal hormone disruptor have been suggested. Specific studies on the permanence of melatonin in plant tissues, plant by-products, soil, freshwater and honeybees, amongst others, are proposed to obtain crucial information. © 2021 Society of Chemical Industry.

Melatonin Against Environmental Plant Stressors: A Review.

Harsh or extreme environmental conditions largely determine the vegetative and reproductive development of plants. In the case of cultivated plants, their growth and yield are clearly diminished if they are exposed to severe conditions such as drought, waterlogging, extreme heat or cold, UV radiation, or toxic substances in the soil such as salts, heavy metals and pesticides. Melatonin has been studied for decades as a molecule capable of reducing the negative effects of abiotic stressors by increasing tolerance to these adverse growth conditions. This work presents a review of the most outstanding studies with various plant species in each of the above-mentioned stress situations, including proteomic and post-translational studies. Melatonin mediates plant responses to abiotic stress, generally inducing an antioxidative response, and also regulating a complex gene response adapted to individual stressors. Plants are able to increase their endogenous melatonin levels through the application of exogenous melatonin or through the inductive mechanism of endogenous melatonin biosynthesis. In such ways, plants are able to cope with the stressful situation at hand, accommodating their metabolism, morphology and physiology in order to increase overall survival and induce greater tolerance to stress. The agronomic implications of the use of melatonin are discussed.

Development of a Phytomelatonin-Rich Extract from Cultured Plants with Excellent Biochemical and Functional Properties as an Alternative to Synthetic Melatonin.

Melatonin is a pleiotropic molecule with multiple and various functions. In recent years, there has been a considerable increase in the consumption of melatonin supplements for reasons other than those related with sleep (as an antioxidant, for anti-aging, and as a hunger regulator). Although the chemical synthesis of melatonin has recently been improved, several unwanted by-products of the chemical reactions involved occur as contaminants. Phytomelatonin, melatonin of plant origin, was discovered in several plants in 1995, and the possibility of using raw plant material as a source to obtain dietary supplements rich in phytomelatonin instead of synthetic melatonin, with its corresponding chemical by-products was raised. This work characterizes the phytomelatonin-rich extract obtained from selected plant material and determines the contents in phytomelatonin, phenols, flavonoids, and carotenoids. Additionally, the antioxidant activity was measured. Finally, a melatonin-specific bioassay in fish was carried out to demonstrate the excellent biological properties of the natural phytomelatonin-rich extract obtained.

A colorimetric method for the determination of different functional flavonoids using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and peroxidase.

In many occasions it is necessary to use fast and simple methods, different to the chromatographic techniques, for the quantification of biomolecules such as flavonoids. Also, the flavonoid levels in some foodstuffs can be influenced by industrial extraction processes such as pressing and squeezing, resulting in modification of their functional value. For this purpose, we have developed a rapid method to analyze flavonoids, based on a coupling reaction between ABTS and flavonoid mediated by peroxidase. The present method can be used to detect and measure flavonoids with hydroxyl moieties on A- or B-rings, not adjacent to methoxy or oxo substitutions. The visible spectrum of the ABTS-flavonoid complex, the calibration curve (within the range 5-50 µM) and the molar absorption coefficients for isosakuranetin, isonaringin, rhoifolin, hyperoside, rutin, hesperetin, quercetin, kaempherol and naringenin are given. The method has been applied to complex culture media and is sensitive, accurate, quick and easy to apply. This method can be used in laboratories that do not have sophisticated and expensive techniques such as liquid chromatography and also as a quick, simple and inexpensive technique for student practice laboratories.

Melatonin: A New Plant Hormone and/or a Plant Master Regulator?

Melatonin is a pleiotropic molecule with many diverse actions in plants. It is considered primarily an antioxidant with important actions in the control of reactive oxygen and nitrogen species (ROS and RNS), among other free radicals, and harmful oxidative molecules present in plant cells. In addition, plant melatonin is involved in multiple physiological actions, such as growth, rooting, seed germination, photosynthesis, and protection against abiotic and/or biotic stressors. The recent identification of the first plant melatonin receptor opened the door to this regulatory molecule being considered a new plant hormone. However, due to the diversity of its actions, melatonin has also been proposed as a plant master regulator. Here, we discuss the most recent data in respect to both perspectives.

Stability of biomarkers of oxidative stress in canine serum.

The purpose of this study was to determine the effect of storage time and temperature on the measured concentrations of a profile of biomarkers of oxidative stress in canine serum: Trolox equivalent antioxidant capacity (TEAC) measured by two different methods, using acidic medium (TEACA), and horseradish peroxidase (TEACH), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), total thiol, Paraoxonase 1 (PON1), total oxidant status (TOS), ferrous oxidation xylenol orange (FOX), advanced oxidation protein products (AOPP), reactive oxygen species (ROS), and thiobarbituric reactive species (TBARS). Six serum pools from healthy dogs were divided in various aliquots and stored at 4 °C and 25 °C for a period of 6, 24 and 72 h, and at -20 °C and - 80 °C for a period of 7, 14, 30, 60, 180, and 360 days. In addition, the effect of repeated freeze-thaw cycle was evaluated. For the short-term storage of biomarkers of oxidative stress in canine serum, 4 °C is preferred to room temperature. In case of long-term storage, the optimal stability was achieved when samples were stored at -80 °C. Most of the biomarkers were affected after two freeze-thaw cycles. In addition, as individual oxidative biomarkers can show differences in stability, it is important to consider the stability of each different analyte when it is measured in stored samples and avoid the use of samples stored in conditions in which the analyte is not stable.

The Potential of Phytomelatonin as a Nutraceutical.

Phytomelatonin (plant melatonin) is chemically related to the amino acid tryptophan and has many diverse properties. Phytomelatonin is an interesting compound due to its outstanding actions at the cellular and physiological level, especially its protective effect in plants exposed to diverse stress situations, while its vegetable origin offers many opportunities because it is a natural compound. We present an overview of its origin, its action in plants in general (particularly in plant species with high levels of phytomelatonin), and its possibilities for use as a nutraceutical with particular attention paid to the beneficial effects that it may have in human health. The differences between synthetic melatonin and phytomelatonin, according to its origin and purity, are presented. Finally, the current market for phytomelatonin and its limits and potentials are discussed.

Serum antioxidant capacity and oxidative damage in clinical and subclinical canine ehrlichiosis.

The objective of this study was to evaluate and compare the antioxidant response and the products of oxidative damage analysed by various assays in clinical and subclinical canine monocytic ehrlichiosis (CME). For this purpose, four assays to measure the total serum antioxidant capacity (TAC), such as the cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC) using acidic medium (TEACA), and the TEAC using the horseradish peroxidase (TEACH) were used. In addition, the serum thiol concentrations were analysed. Reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), and ferrous oxidation-xylenol orange (FOX) were measured to determine the concentrations of free radical and the products of oxidative damage as result of the disease. All antioxidant markers were significantly lower in the dogs on clinical ehrlichiosis when compared with healthy dogs; however only the CUPRAC, FRAP and thiol were significantly lower in subclinical CME compared with healthy dogs. TBARS and FOX showed no significant differences between dogs with CME and healthy dogs; however, a significant increased ROS concentration was observed in dogs with clinical and subclinical CME when compared with healthy dogs. Results showed that in CME there is a state of oxidative stress with significant changes in markers of antioxidant defence and in concentrations of free radicals. However, the detection of these changes would depend of the assay used.

Analytical validation of an automated assay for ferric-reducing ability of plasma in dog serum.

We performed analytical validation of an automated ferric-reducing ability of plasma (FRAP) assay in the serum of dogs. Intra- and interassay precision, accuracy, detection limit, and effects of hemolysis and lipemia were evaluated. Intra- and interassay coefficients of variation were <1% and <13%, respectively. The assay showed a high correlation with a FRAP assay described previously, and results were linear when serial sample dilutions were tested. The detection limit was lower than the values observed in sera from healthy dogs; decreased serum FRAP was found in dogs with leishmaniosis. Lipemia and hemolysis caused a significant increase in the results of the assay.