Dihydrochalcones: Methods of Acquisition and Pharmacological Properties—A First Systematic Review

Dihydrochalcones are a class of secondary metabolites, for which demand in biological and pharmacological applications is still growing. They posses several health-endorsing properties and, therefore, are promising candidates for further research and development. However, low content of dihydrochalcones in plants along with their low solubility and bioavailability restrict the development of these compounds as clinical therapeutics. Therefore, chemomicrobial and enzymatic modifications are required to expand their application. This review aims at analyzing and summarizing the methods of obtaining dihydrochalcones and of presenting their pharmacological actions that have been described in the literature to support potential future development of this group of compounds as novel therapeutic drugs. We have also performed an evaluation of the available literature on beneficial effects of dihydrochalcones with potent antioxidant activity and multifactorial pharmacological effects, including antidiabetic, antitumor, lipometabolism regulating, antioxidant, anti-inflammatory, antibacterial, antiviral, and immunomodulatory ones. In addition, we provide useful information on their properties, sources, and usefulness in medicinal chemistry.


Introduction
Improvement of knowledge about biologically active nonnutritive food components and their positive effect on human organism is one of the main reasons for the development of functional food. In addition, more and more attention is paid to designing structurally defined analogues of bioactive molecules, which is particularly important for their medical use. A wide range of applications of natural compounds and their derivatives have become possible due to the variety of methods of their acquisition, including isolation from natural sources, mainly from plants, transformations involving natural catalysts (microorganisms, plants, and enzymes), as well as classical chemical synthesis.
The possibility of using flavonoids as food additives as well as active ingredients of cosmetics and medicines is limited due to their poor solubility in nonpolar solvents [1,2] and, thus, to their weak ability to penetrate cell membranes. Therefore, attempts are being made to functionalize these compounds [3][4][5][6].
These compounds and other constituents of plants have been described many times. Much attention has been paid to natural flavones, flavanones, and chalcones, but in the literature, we have not found any comprehensive review of pro-health effects of dihydrochalcones and biotechnological methods of their synthesis, including biotransformations.

Dihydrochalcones from Natural Sources
The main natural source of dihydrochalcones are plants, in which they are biosynthesized by the phenylpropanoid pathway, which is a well-understood metabolic route [23]. In traditional medicine, they have been used for the treatment of liver cirrhosis, hypertension, diabetes, malaria, yellow fever, breast cancer, gastroenteric and inflammatory disorders, and dengue fever [24]. Initially, it was thought that the occurrence of dihydrochalcones was limited to only about 30 plant families. With the development of more sensitive analytical methods, it turned out that they are more widespread in nature [25]. The most common methods of dihydrochalcones isolation involve their extraction form leaves using such solvents as ethanol, diethyl ether, methanol, and hexane with dichloromethane or high-pressure extraction with supercritical CO 2 .
The richest source of natural dihydrochalcones in diet are apples. We can find these compounds also in apple leaves [26] and in hairy roots obtained through transgenesis [27]. Apple trees accumulate dihydrochalcones in very high concentrations-up to 14% of dry weight in leaves [28,29]. Their content in individual apple varieties is genetically determined [30]. Phloretin (1) is one of the best known apple dietary dihydrochalcones, characterized by the presence of 2,6-dihydroxyacetophenone pharmacophore. The composition of dihydrochalcones in various species and parts of apple trees is different. Flavonoid profile of apples also depends on the impact of pomace pretreatment and the extraction methods [31]. Some species of apples contain sieboldin (3-  human intestinal microbiota is its deglucosylation to hesperetin dihydrochalcone 4 -β-D-glucoside (7) and subsequently to the aglycon hesperetin dihydrochalcone (8) (Scheme 1). Next, the latter is hydrolyzed to the corresponding 3-(3-hydroxy-4-methoxyphenyl)propionic acid (9) and probably to phloroglucinol [65]. Scheme 1. Degradation pathway of neohesperidin dihydrochalcone by human intestinal microbiota [65].
In turn, Monge et al. [66] discussed the metabolism of phloretin (1) in rats and showed that both phloretin and phloridzin (2) were metabolised to phloretic acid and phloroglucinol. Courts and Williamson [67] noted that deglycosylation is not a prerequisite for C-glycosyl flavonoid absorption. The authors showed that flavonoid C-glycosides, like aspalathin (4), are methylated and glucuronidated in vivo in an intact form in humans (Scheme 2).
Kreuz et al. [68] performed the research on pigs, which were fed with rooibos tea extract for 11 days at an extremely high dose. Similarly, the results showed some intact metabolites in collected pig urine. Scheme 2. COMT-catalysed formation of two isomeric forms of O-methylated aspalathin metabolites.

Microbiological and Enzymatic Hydrogenation of Chalcones
The chemical synthesis of dihydrochalcones from their corresponding flavones, flavanone glycosides, or chalcones is not economical because it requires a two-step synthesis, isolation of the corresponding product intermediate, and complicated hydrogenation procedure, utilizing high pressure.
An alternative to the chemical method of obtaining dihydrochalones is the use of whole cells of microorganisms. The majority of living organisms is able to hydrogenate the double bond. Biotransformation of chalcones results in similar yields as the chemical synthesis, under relatively mild process conditions. Whole cells of microorganisms can also catalyse the reduction of simpler aromatic ketones, aldehydes, imines, and unsaturated structures containing C=C double bond in the vicinity of strongly polar groups such as nitro, carbonyl, or hydroxyl ones. Additionally, these microbially synthesized products can be used as natural flavouring substances in accordance with the European Flavouring Regulation.
Due to the high selectivity of natural biocatalysts, production of dihydrochalcones by microbiological processes is a useful tool for the synthesis of chiral synthons that are used in the pharmaceutical industry. The obtained dihydrochalcones can be also used in food industry for production of multicomponent low-calorie sweeteners of natural origin as well as in pharmaceutical industry for production of natural flavonoid compounds showing anticancer, anti-inflammatory, antimalarial, and antioxidant properties.
In another study, chalcone derivatives containing either nitro or alkoxy groups in aromatic rings (16)(17)(18)(19)(20)(21)(22)(23) were studied. The products of highly efficient chemoselective microbiological hydrogenation in the presence of Saccharomyces cerevisiae industrial yeast strains were obtained. Biotransformations were carried out in a water-organic solvent two-phase system (Scheme 4) [70]. The team of Pathan et al. [71] used baker's yeast Saccharomyces cerevisiae for stereoselective reduction of chalcones (24 and 25), β-diketones, and their derivatives containing various substituents in ring A (Scheme 5). They optimized the reaction conditions, including substrate concentration, temperature, pH, biomass amount, reaction time, and solvents. It has been proven that whole microbial cells reduce substrates independently of regeneration of the carbonyl reductase cofactor (NADPH). The reaction was most effective in the two-phase systems: water-strongly polar organic solvent. In similar experiments carried out with chalcone derivatives, live culture of the fungus Aspergillus flavus [72] was used. Products of microbiological hydrogenation of olefinic bond in chalcones were also obtained by transformation of flavones: apigenin and luteolin, using Clostridium orbiscindens strain (Scheme 6) [73]. Scheme 6. Pathways of quercetin (26), apigenin (30), and luteolin (31) degradation by C. orbiscindens [73].
In turn, a biotransformation of 2 -hydroxychalcone (38) and its halogenated derivatives (42)(43)(44)(45)(46)(47)(48) in the marine-derived fungus Penicillium raistrickii CBMAI 931 led to the regio-and chemoselective hydrogenation (Scheme 8) to give the products with good conversion (78-99%) and isolated yields between 31 and 65%. The formation of flavanones was low (3-14%). 2 -Hydroxydihydrochalcone (39) with 72% conversion and R-flavanone with 18% conversion were obtained after 7 days of reaction in phosphate buffer (pH 7) [75]. The hydroxychalcones containing a fluorine atom were hydrogenated with higher yields than the analogous 2 -hydroxychalcones with bromine or chlorine atoms in the same positions of the aromatic B ring. It may be related to the strong hydrogen bonds that fluorine-containing compounds can form with the amino acids, such as histidine and asparagine residues, which are present at the active sites of the ene-reductases that are responsible for catalyzing the reaction (Scheme 9). Scheme 9. Reaction sequence proposed for the chemoselectve reduction of C=C of 2 -hydroxychalcone (38) mediated by ene-reductases [75].
Yeast Rhodotorula possesses specific enzymes capable of hydrogenating chalcones in good yield (Scheme 10) [76]. The strain Rhodotorula rubra AM 82 biotransformed chalcones into dihydrochalcones and respective secondary alcohols. In addition, some other species of yeast like Saccharomyces and Yarrowia posses a high ability to reduce the double bond in chalcones and their derivatives, which was described recently [77]. The substrates in these reactions included predominantly hydroxy-and methoxyderivatives of chalcones and compounds containing furan and thiophene substituents. Such an activity has also been reported in relation to cyanobacteria (Scheme 11) [78]. The bioconversion of 2-hydroxychalcone (54) proceeded with high efficiency. Most of the tested cyanobacterial strains converted this compound into its hydrogenated derivative (55) in nearly 100% yield. Scheme 11. Biotransformation of 2-hydroxychalcone (54) by cyanobaceria [78].
In turn, Klingel et al. [81] modified and applied a recombinant dextransucrase from Lactobacillus reuteri TMW 1.106 to glucosylate various flavonoids and flavonoid glycosides. An efficient glucosylation was achieved for quercetin (26) and its glycosides-quercetin-3-O-β-glucoside and rutin-whereas the efficiency of glucosylation for neohesperidin dihydrochalcone was low. Additionally, it was noted that the glycosyl substituents such as β-glucose, rutinose, or neohesperidose were glucosylated at varying positions.
Lin et al. [82] developed a new methodology for production of natural sweetener trilobatin in a two-step enzymatic process, in which naringin was hydrogenated into naringin dihydrochalcone, which was followed by removal of the rhamnosyl group of naringin dihydrochalcone by enzymatic hydrolysis using immobilised α-l-rhamnosidase as the catalyst. The optimized conditions involved using a very low loading of the α-l-rhamnosidase catalyst at 60 • C in a neutral aqueous buffer solution within 2 h. The overall yield of 96% was achieved.

Pharmacological Properties of Dihydrochalcones
Dihydrochalcones have beneficial therapeutic effects on the human body, proven in vitro and in vivo (Table 1). Studies on their pharmacological potential include antidiabetic, immonomodulatory, antiviral, cardio, and hepatoprotective activity. Some studies already spotted the significance of chalcones and dihydrochalcones as antimicrobial agents. However, the literature data concerning the antimicrobial activity of this group of compounds are still scarce.
Many data show that flavonoids, including dihydrochalcones, are excellent antioxidants that can scavenge free radicals through a variety of mechanisms.
On the other hand, anticancer and antimetastatic effects have also been ascribed to these metabolites. They may influence the development of cancer, as a result of several important mechanisms: regulation of angiogenesis, modification of the antitumor response, and direct influence on tumour cell proliferation and apoptosis. Additionally, a correlation was found between total polyphenolic content of crabapple fruit extracts and their antioxidant and antiproliferative effects.
However, the biological activity of dihydrochalcones depends mainly on their chemical structure and the presence of sugar residues attached to the molecule.
Some similarities in the structures of phloterin's analogue phlorizin and 17-β-estradiol, both of which have two aromatic nuclei and hydroxyl substitution in the ring, may be the reason for the fact that phlorizin shows both estrogenic or antiestrogenic effects and can bind to estrogen receptors. This property may be used in anticancer therapy, for example, in the case of breast cancer, where cell proliferation may be induced by estrogen-like substances, and in other estrogen-related diseases [83]. There is also some evidence that the anticancer effects are enhanced by the antioxidant and antiinflammatory properties of dihydrochalcones.
Moreover, anti-hyperglycaemic and anticholinesterase effects of dihydrochalcones have been reported. According to the latest information, some of the natural dihydrochalcones may be potentially used as safe nutraceuticals to protect against metabolic disease-related complications.
Dihydrochalcones may be also good candidates for drugs against Alzheimer's disease, which was confirmed by the virtual screening method (computer simulation and experiment) [84].
The interest in natural sweeteners is constantly growing among nutrition and food production specialists as well as among doctors and nutritionists. This is due to the fact that consumption of synthetic sweeteners causes many civilization diseases, such as atherosclerosis, obesity, and diabetes.
Dihydrochalcones are a part of our daily diet. We can find them in plants, or we can synthesize them by chemical and enzymatic methods. Naringin dihydrochalcone and neohesperidin dihydrochalcone, which may be also chemically synthesized from citrus flavanones, are low-calory sweeteners with one and twenty times the sweetness of saccharin on a molar basis, respectively [85]. Neohesperidin dihydrochalcone was also approved as a food additive in Europe (E959 code) [86].
Bearing in mind the demand from the pharmaceutical and medical industry for preparations based on natural compounds that are effective in prevention and therapy of various diseases, further research on pharmacokinetic and pharmacodynamic aspects is encouraged to improve new production and delivery methods and to achieve feasible dihydrochalcone-based clinical formulations. In vitro, MKN45 (human gastric cancer) In vivo, BALB/C nude mice [92] conjugates of phloretin (Pht) and phlorizin (Phl) with gold nanoparticles (AuNPs) Fourty-five-fold increase in the efficacy in the antineoplastic activity of Pht-AuNPs over pure phloretin and 4.49-fold increase in efficacy of Phl-AuNP over pure phlorizin.

60-100 µg/mL
Induces cancer cellapoptosis via a mitochondrial-dependent pathway. Downregulates the antiapoptosis-associated molecule B-cell lymphoma 2 (bcl-2) and increases the levels of the apoptosis-associated molecules bcl-2-like protein 4 and tumor protein p53. Affects the expression of apoptotic protease activating factor-1.
In vivo, type-2 diabetic mouse model [107] complications associated with hepatic insulin resistance and metabolic disease-relate Plays a significant role in the maintenance of hormonal homeostasis. Influences the steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid, and androgen pathways, thus possibly contributes to the alleviation of negative effects arising from elevated glucocorticoid levels.

Kidney diseases aspalathin and nothofagin
Induce renal damage. Inhibits nuclear factor (NF)-κB activation and reduces the induction of NO synthase and excessive production of nitric acid. Reduce the plasma levels of NO, TNF-α, and IL-6; increases lipid peroxidation; and markedly enhances the antioxidant defence system by restoring the levels of superoxide dismutase, glutathione peroxidase, and catalase.

3-hydroxyphloretin 3-hydroxyphlorizin
Presence of an o-dihydroxyl group in the B-ring increased activity, whereas glycosylation in the A-ring decreased antioxidant potential of dihydrochalcones. Glycosylation at the 2 -position enhanced the dissociation ability of the 4 -hydroxyl group and increased the antioxidant activity of dihydrochalcones containing o-dihydroxyl.
FRAP, DPPH, ABTS, and superoxide radical scavenging assays [125] aspalathin, nothofagin Some of the flavonoid monoglucosides showed significant improvement in the antioxidant activity. DPPH, FRAP, and ORAC assays [127] flavanones and dihydrochalcones Dihydrochalcones exhibited higher antioxidant activities than the corresponding flavanones. The hydroxyl group at the 2'-position in dihydrochalcone A ring is an essential pharmacophore for its radical scavenging potential.

Dihydrochalcones as the Scaffolds for Drug Development
Dihydrochalcones are naturally occurring α,β-unsaturated carbonyl compounds, which are the key precursors for the biosynthesis of other flavonoids and for the synthesis of many biologically valuable heterocyclic compounds such as benzothiazepines; pyrazolines; derivatives containing other ring type of N-, O-, and S-heterocycles; and flavones [142]. The biological potential of chalcone derivatives may be increased by the presence of nitrogen, oxygen, or sulfur-containing heterocyclic scaffolds.
In turn, the biotransformation products, such as 2'-hydroxydihydrochalcone, may be used as substrates for synthesis of antiarrhythmic drugs used for restoring normal heart rhythm, e.g., propafenone, which additionally shows high anticancer activity [143].
Synthesis of a series of 2 -hydroxy chalcones and aurones containing isoxazole and pyrazole functions and biological evaluation of their antioxidant and antibacterial activity were performed by Irshad et al. [144].
A novel prenylated dihydrochalcone named adunchalcone was extracted from Piper aduncum L. (Piperaceae) by Dal Picolo et al. [145]. In further perspectives, this molecule may be used as a scaffold for the design of novel antileishmanial drugs for skin diseases.
Chakraborty et al. [146] described the multifunctional neuroprotective activity of neohesperidin dihydrochalcone as a potential novel scaffold for therapeutics against neurodegenerative disorders, such like Alzheimer's disease. Jin et al. [147] noted that some natural dihydrochalcones might be good drug candidates to treat Alzheimer's disease. It has been shown that a compound extracted from Daemonorops draco tree inhibited amyloid-β peptide aggregation and stopped production of the toxic fibrillary aggregates, which are responsible for the formation of neurofibrillary tangles, one of the main symptoms of dementia [146]. There are also some reports on synthesis of dihydrochalcone derivatives as potential sweeteners [148].
Some reports show that the glucuretic effect resulting from SGLT2 inhibition contributes to reduction of hyperglycaemia and also assists weight loss and blood pressure reduction. This phenomenon was later used for the development of synthetic antidiabetic drugs [149], three of which have been approved by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) [150].
Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in kidneys is a relatively new strategy for treating type-2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition.
In other studies, Jesus et al. [150] synthesized a library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors and tested them as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. These results point toward the discovery of structures that will be potent and highly selective inhibitors of SGLT2. Due to the high potential of dihydrochalcones, research in this area should be continued. The SGLT2 inhibitors such as dapagliflozin, canagliflozin, and tofogliflozin are also available in many countries as pharmaceutical drugs to treat diabetes.

Conclusions
Recently, much attention has been paid to the search for new compounds that show properties conducive to maintaining health and to proper functioning of the body. Flavonoids are examples of substances that may be included in nutraceuticals. Microbial transformations of flavonoid compounds can be a natural alternative to chemical synthesis of dihydrochalcones. The biotransformation processes may be useable to modify the compound structures in order to improve their biological properties and to increase lipophilicity and bioavailability. Another natural method of obtaining of these compounds is their isolation from plants.
This review article contains sections with general information about the biological activity of dihydrochalcones as well as documented results confirming their potential usefulness in alternative medicine. This review can be a guide for researchers dealing with innovative treatment methods that use natural dietary components as well as for planning new methods of obtaining more effective molecules containing the dihydrochalcone skeleton. Funding: This work received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.