Dihydrochalcone Research Articles

Naringenin chalcone carbon double-bond reductases mediate dihydrochalcone biosynthesis in apple leaves.

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85-95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming.

Mechanism of Phloretin Accumulation in Malus hupehensis Grown at High Altitudes: Evidence from Quantitative 4D Proteomics.

Phloretin is a natural dihydrochalcone (DHC) that exhibits various pharmacological and therapeutic activities. Malus hupehensis Rehd. (M. hupehensis) is widely planted in the middle of China and its leaves contain an extremely high content of phloridzin, a glycosylated derivative of phloretin. In the present study, we observed a significant increase in phloretin content in the leaves of M. hupehensis planted at high altitudes. To investigate the mechanisms of phloretin accumulation, we explored changes in the proteome profiles of M. hupehensis plants grown at various altitudes. The results showed that at high altitudes, photosynthesis- and DHC biosynthesis-related proteins were downregulated and upregulated, respectively, leading to reduced chlorophyll content and DHC accumulation in the leaves. Moreover, we identified a novel phloridzin-catalyzing glucosidase whose expression level was significantly increased in high-altitude-cultivated plants. This work provided a better understanding of the mechanism of phloretin accumulation and effective and economic strategies for phloretin production.

Discovery of Loureirin analogues with colorectal cancer suppressive activity via regulating cell cycle and Fas death receptor

Chalcones and dihydrochalcones (DHCs) are important bioactive natural products (BNPs) isolated from traditional Chinese medicine. In this study, 13 chalcones were designed with the inspiration of Loureirin, a DHC extracted from Resina Draconis, and synthesized by classical Claisen-Schmidt reactions. Afterwards the reduction reactions were carried out to obtain the corresponding DHCs. Cytotoxicity assay indicated chalcones and DHCs possessed selective cytotoxicity against colorectal cancer (CRC) cells. The preliminary structure-activity relationships (SAR) of these compounds suggested the α, β-unsaturated ketone of the chalcones were crucial for the anticancer activity. Interestingly, compounds 3d and 4c exhibited selective anticancer activity against CRC cell line HCT116 with IC50s of 8.4 and 17.9 μM but not normal cell. Moreover, 4c could also inhibit the migration and invasion of CRC cells. Mechanism investigations showed 4c could induce cell cycle G2/M arrest by regulating cell cycle-associated proteins and could also up-regulate Fas cell surface death receptor. The virtual docking further pointed out that compounds 3d and 4c could nicely bind to the Fas/FADD death domain complex (ID: 3EZQ). Furthermore, silencing of Fas significantly enhanced the proliferation of CRC cells and attenuated the cytotoxicity induced by 4c. These results suggested 4c exerted its anticancer activity possibly regulating cell cycle and Fas death receptor. In summary, this study investigated the anticancer activity and mechanism of Loureirin analogues in CRC, suggesting these compounds may warrant further investigation as promising anticancer drug candidates for the treatment of CRC.

Exogenous Melatonin Enhances Dihydrochalcone Accumulation in Lithocarpus litseifolius Leaves via Regulating Hormonal Crosstalk and Transcriptional Profiling.

Dihydrochalcones (DHCs) constitute a specific class of flavonoids widely known for their various health-related advantages. Melatonin (MLT) has received attention worldwide as a master regulator in plants, but its roles in DHC accumulation remain unclear. Herein, the elicitation impacts of MLT on DHC biosynthesis were examined in Lithocarpus litseifolius, a valuable medicinal plant famous for its sweet flavor and anti-diabetes effect. Compared to the control, the foliar application of MLT significantly increased total flavonoid and DHC (phlorizin, trilobatin, and phloretin) levels in L. litseifolius leaves, especially when 100 μM MLT was utilized for 14 days. Moreover, antioxidant enzyme activities were boosted after MLT treatments, resulting in a decrease in the levels of intracellular reactive oxygen species. Remarkably, MLT triggered the biosynthesis of numerous phytohormones linked to secondary metabolism (salicylic acid, methyl jasmonic acid (MeJA), and ethylene), while reducing free JA contents in L. litseifolius. Additionally, the flavonoid biosynthetic enzyme activities were enhanced by the MLT in leaves. Multiple differentially expressed genes (DEGs) in RNA-seq might play a crucial role in MLT-elicited pathways, particularly those associated with the antioxidant system (SOD, CAT, and POD), transcription factor regulation (MYBs and bHLHs), and DHC metabolism (4CL, C4H, UGT71K1, and UGT88A1). As a result, MLT enhanced DHC accumulation in L. litseifolius leaves, primarily by modulating the antioxidant activity and co-regulating the physiological, hormonal, and transcriptional pathways of DHC metabolism.

Identification of two key genes involved in flavonoid catabolism and their different roles in apple resistance to biotic stresses.

Biosynthesis of flavonoid aglycones and glycosides is well established. However, key genes involved in their catabolism are poorly understood, even though the products of hydrolysis and oxidation play important roles in plant resistance to biotic stress. Here, we report on catabolism of dihydrochalcones (DHCs), the most abundant flavonoids in domesticated apple and wild Malus. Two key genes, BGLU13.1 and PPO05, were identified by activity-directed protein purification. BGLU13.1-A hydrolyzed phlorizin, (the most abundant DHC in domesticated apple) to produce phloretin which was then oxidized by PPO05. The process differed in some wild Malus, where trilobatin (a positional isomer of phlorizin) was mainly oxidized by PPO05. The effects of DHC catabolism on apple resistance to biotic stresses was investigated using transgenic plants. Either directly or indirectly, phlorizin hydrolysis affected resistance to the phytophagous pest two-spotted spider mite, while oxidation of trilobatin was involved in resistance to the biotrophic fungus Podosphaera leucotricha. DHC catabolism did not affect apple resistance to necrotrophic pathogens Valsa mali and Erwinia amylovara. These results suggest that different DHC catabolism pathways play different roles in apple resistance to biotic stresses. The role of DHC catabolism on apple resistance appeared closely related to the mode of invasion/damage used by pathogen/pest.

Dihydrochalcone glycoside biosynthesis in Malus is regulated by two MYB-like transcription factors and is required for seed development.

Dihydrochalcones (DHCs) including phlorizin (phloretin 2'-O-glucoside) and its positional isomer trilobatin (phloretin 4'-O-glucoside) are the most abundant phenylpropanoids in apple (Malus spp.). Transcriptional regulation of DHC production is poorly understood despite their importance in insect- and pathogen-plant interactions in human physiology research and in pharmaceuticals. In this study, segregation in hybrid populations and bulked segregant analysis showed that the synthesis of phlorizin and trilobatin in Malus leaves are both single-gene-controlled traits. Promoter sequences of PGT1 and PGT2, two glycosyltransferase genes involved in DHC glycoside synthesis, were shown to discriminate Malus with different DHC glycoside patterns. Differential PGT1 and PGT2 promoter activities determined DHC glycoside accumulation patterns between genotypes. Two transcription factors containing MYB-like DNA-binding domains were then shown to control DHC glycoside patterns in different tissues, with PRR2L mainly expressed in leaf, fruit, flower, stem, and seed while MYB8L mainly expressed in stem and root. Further hybridizations between specific genotypes demonstrated an absolute requirement for DHC glycoside production in Malus during seed development which explains why no Malus spp. with a null DHC chemotype have been reported.

SFC and CE—A Comparison of Two Orthogonal Methods for the Analysis of Dihydrochalcones in Apple Leaves

In recent years the analysis of natural products has been carried out using a range of approaches, but mainly utilizing liquid chromatography (LC) or gas chromatography (GC). However, alternative approaches with orthogonal selectivity like capillary electrophoresis (CE) and supercritical fluid chromatography (SFC) have increasingly been employed as well, even though they are often considered niche techniques only. In this study, we intended to confirm and compare their suitability as reliable state-of-the-art methods for the analysis of bioactive compounds by developing CE and SFC for the analysis of dihydrochalcones (DHCs) in apple leaves. The analytes were chosen as they have shown interesting pharmacological effects, such as anti-inflammatory, anti-tumor and immunomodulatory activities, and also present an interesting analytical challenge due to their structural similarity and polarity range. Both methods were well capable to separate the five standard compounds within short separation times and fulfilling the demands for an environmentally friendly “green” technology. CE as well as the SFC assay were validated for linearity, sensitivity, accuracy and precision according to ICH guidelines and met all respective requirements. Using the optimized methods, several Malus sp. samples were analyzed whereby a significant difference in the qualitative as well as quantitative DHC profile was revealed, with overall DHC concentrations ranging from 5.47% to 17.24%.

Kinetic and Mechanistic Studies of the Selective Hydrogenation of (E)‐Chalcones in Biomass‐Derived γ‐Valerolactone Catalyzed by Rh−PPh3 Complexes

Abstractγ‐Valerolactone as a renewable, polar aprotic solvent was evaluated in the selective hydrogenation of carbon‐carbon double bond of chalcones from both catalytic and mechanistic aspects. By using triphenylphosphine‐modified cationic rhodium catalyst systems reasonably high activities (TOFinitial∼1200 h−1) were achieved in the selective hydrogenation of (E)‐chalcone (CHL), as a common model substrate for α,β‐unsaturated ketones, to dihydrochalcone (DHC). Various derivatives of CHL, which were substituted on the styryl phenyl groups also showed exclusive selectivity for the hydrogenation of the C=C double bond. The optimal PPh3/Rh ratio was between 2.5 to 3, but higher ratios were advantageously also tolerated, which is beneficial for the process chemistry. The solvent GVL proved to stable enough under the reaction conditions for waste‐free recycling. Kinetic studies and NMR spectra from the analogous deuteration of chalcone by using the cationic Rh‐PPh3 system clearly show the olefin insertion as the rate‐determining step with definite irreversibility. By investigating the substituent effect on the phenyl ring of the styryl moiety of chalcone, a quasi‐linear correlation was found between the electronic parameters of the para‐substituents and the activity with electron donor groups being favorable for the reaction rate. In summary, an environmentally friendly and feasible method is presented for the production of valuable DHC intermediates in view of their pharmaceutical importance.

Molecular Modeling Identification of Key Secondary Metabolites from Xylopia aethiopica as Promising Therapeutics Targeting Essential Measles Viral Proteins.

This study computationally screened three key compounds (vanillin (VAN), oxophoebine (OPB), and dihydrochalcone (DHC)) derived from Xylopia aethiopica (Guinea pepper), a medicinal plant with known antiviral activity, against key druggable measles virus (MV) proteins (fusion protein (FUP), haemagglutinin protein (HMG), and phosphoprotein (PSP)). Each molecular species was subjected to a 100 ns molecular dynamics (MD) simulation following docking, and a range of postdynamic parameters including free binding energy and pharmacokinetic properties were determined. The docking scores of the resulting OPB-FUP (-5.4 kcal/mol), OPB-HMG (-8.1 kcal/mol), and OPB-PSP (-8.0 kcal/mol) complexes were consistent with their respective binding energy values (-25.37, -28.74, and -40.68 kcal/mol), and higher than that of the reference standard, ribavirin (RBV) in each case. Furthermore, all the investigated compounds were thermodynamically compact and stable, especially HMG of MV, and this observation could be attributed to the resulting intermolecular interactions in each system. Overall, OPB may possess inhibitory properties against MV glycoproteins (FUP and HMG) and PSP that play important roles in the replication of MV and measles pathogenesis. While OPB could serve as a scaffold for the development of novel MV fusion and entry inhibitors, further in vitro and in vivo evaluation is highly recommended.

De novo transcriptome assembly and functional analysis reveal a dihydrochalcone 3-hydroxylase(DHC3H) of wild Malus species that produces sieboldin in vivo.

Sieboldin is a specialised secondary metabolite of the group of dihydrochalcones (DHC), found in high concentrations only in some wild Malus species, closely related to the domesticated apple (Malus × domestica L.). To date, the first committed step towards the biosynthesis of sieboldin remains unknown. In this study, we combined transcriptomic analysis and a de novo transcriptome assembly to identify two putative 3-hydroxylases in two wild Malus species (Malus toringo (K. Koch) Carriere syn. sieboldii Rehder, Malus micromalus Makino) whose DHC profile is dominated by sieboldin. We assessed the in vivo activity of putative candidates to produce 3-hydroxyphloretin and sieboldin by de novo production in Saccharomyces cerevisiae. We found that CYP98A proteins of wild Malus accessions (CYP98A195, M. toringo and CYP98A196, M. micromalus) were able to produce 3-hydroxyphloretin, ultimately leading to sieboldin accumulation by co-expression with PGT2. CYP98A197-198 genes of M. × domestica, however, were unable to hydroxylate phloretin in vivo. CYP98A195-196 proteins exerting 3-hydroxylase activity co-localised with an endoplasmic reticulum marker. CYP98A protein model from wild accessions showed mutations in key residues close to the ligand pocket predicted using phloretin for protein docking modelling. These mutations are located within known substrate recognition sites of cytochrome P450s, which could explain the acceptance of phloretin in CYP98A protein of wild accessions. Screening a Malus germplasm collection by HRM marker analysis for CYP98A genes identified three clusters that correspond to the alleles of domesticated and wild species. Moreover, CYP98A isoforms identified in M. toringo and M. micromalus correlate with the accumulation of sieboldin in other wild and hybrid Malus genotypes. Taken together, we provide the first evidence of an enzyme producing sieboldin in vivo that could be involved in the key hydroxylation step towards the synthesis of sieboldin in Malus species.

Synthesis of Biobased Phloretin Analogues: An Access to Antioxidant and Anti-Tyrosinase Compounds for Cosmetic Applications.

The current cosmetic and nutraceutical markets are characterized by a strong consumer demand for a return to natural products that are less harmful to both the consumers and the environment than current petrosourced products. Phloretin, a natural dihydrochalcone (DHC) found in apple, has been widely studied for many years and identified as a strong antioxidant and anti-tyrosinase ingredient for cosmetic formulations. Its low concentration in apples does not allow it to be obtained by direct extraction from biomass in large quantities to meet market volumes and prices. Moreover, its remarkable structure prevents its synthesis through a green process. To overcome these issues, the synthesis of phloretin analogs appears as an alternative to access valuable compounds that are potentially more active than phloretin itself. Under such considerations, 12 chalcones (CHs) and 12 dihydrochalcones (DHCs) were synthesized through a green Claisen–Schmidt condensation using bio-based reagents. In order to evaluate the potential of these molecules, radical scavenging DPPH and anti-tyrosinase tests have been conducted. Moreover, the UV filtering properties and the stability of these analogs towards UV-radiations have been evaluated. Some molecules showed competitive antioxidant and anti-tyrosinase activities regarding phloretin. Two compounds in particular showed EC50 lower than phloretin, one chalcone and one dihydrochalcone.

Anti-Microbial Properties of C-benzylated Dihydro Chalcone Derivatives: 2’, 4’-Dihydroxy-3’,5’-( 2’’, 2’’’- Dihydroxy Dibenzyl) - 6’-Methoxy Dihydro Chalcone, 2’,4’-Dihydroxy-3’- ( 2’’- Hydroxy Benzyl) –6’- Methyl Dihydrochalcone and 2’, 4’, 6’ – Tri Hydroxy- 3’- ( 2’’-Hydroxy Benzyl) Dihydro Chalcone Isolated from Stem Bark of uvaria chamae P. Beav

Anti-Microbial Properties of C-benzylated Dihydro Chalcone Derivatives: 2’, 4’-Dihydroxy-3’,5’-( 2’’, 2’’’- Dihydroxy Dibenzyl) - 6’-Methoxy Dihydro Chalcone, 2’,4’-Dihydroxy-3’- ( 2’’- Hydroxy Benzyl) –6’- Methyl Dihydrochalcone and 2’, 4’, 6’ – Tri Hydroxy- 3’- ( 2’’-Hydroxy Benzyl) Dihydro Chalcone Isolated from Stem Bark of uvaria chamae P. Beav

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature’s treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)—a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17β-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Antioxidant activity and mechanism of dihydrochalcone C-glycosides: Effects of C-glycosylation and hydroxyl groups

Dihydrochalcones (DHCs), an important subgroup of flavonoids, have recently received much attention due to their diverse biological activities. In contrast to their O-glycosides, understanding of the antioxidant property and mechanism of DHC C-glycosides remains limited. Herein, the free radical scavenging activity and mechanism of two representative C-glycosyl DHCs, aspalathin (ASP) and nothofagin (NOT) as well as their aglycones, 3-hydroxyphloretin (HPHL) and phloretin (PHL) were evaluated using the density functional theory (DFT) calculations. The results revealed the crucial role of sugar moiety on the conformation and the activity. The o-dihydroxyl in the B-ring and the 2′,6′-dihydroxyacetophenone moiety were found significant in determining the activity. Our results showed that hydrogen atom transfer (HAT) is the dominant mechanism for radical-trapping in the gas and benzene phases, while the sequential proton loss electron transfer (SPLET) is more preferable in the polar environments. Also, the results revealed the feasibility of the double HAT and double SPLET as well as the SPLHAT mechanisms, which provide alternative pathways to trap radical for the studied DHCs. These results could deepen the understanding of the antiradical activity and mechanism of DHCs, which will facilitate the design of novel efficient antioxidants.

Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants

Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and chemical methods to make products of naringin DC by hydroxylation reactions have not been developed. Here, an enzymatic strategy for the efficient synthesis of potentially valuable products from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. The major product was identified to be neoeriocitrin DC by NMR and LC-MS analyses. Sixty-seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min−1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h−1, which corresponds to 0.65 g L−1 h−1. In this study, we achieved a regioselective hydroxylation of naringin DC to produce neoeriocitrin DC.

Erratum to "Antibacterial Activity of Chalcone and Dihydrochalcone Compounds from Uvaria chamae Roots against Multidrug-Resistant Bacteria".

[This corrects the article DOI: 10.1155/2018/1453173.].

Linkage and association analysis of dihydrochalcones phloridzin, sieboldin, and trilobatin in Malus

Dihydrochalcones (DHCs) are a distinctive characteristic of Malus species, with phloridzin as the major DHC in most Malus species, including cultivated apple. DHCs in apple have unique chemical properties with commercial and nutritional value and may yield important insights into the evolution and physiology of apple. A few species produce sieboldin and trilobatin instead of phloridzin, and interspecific hybridization produce offspring with combinations of phloridzin, sieboldin, and trilobatin. Using Malus prunifolia PI 89816 as a common male parent, five F1 populations were developed to understand the genetic basis of these DHCs in Malus. We measured DHC content in each population and observed segregation into five distinct DHC profiles, which fit a model for three independently segregating loci. QTL associated with DHC content were identified on linkage groups 7 and 8 of the Malus genome using linkage analysis with a cross of NY-152 by M. prunifolia PI 589816 and association mapping with a Malus germplasm collection. In addition to DHC segregation, we observed variation in the relative proportions of phloridzin, sieboldin, and trilobatin. The QTL identified represent a critical step in understanding the genetic controllers of DHC content in Malus.

Identification of apple fruits rich in health-promoting dihydrochalcones by comparative assessment of cultivated and wild accessions

Abstract Apple (Malus sp.) is the most important dietary source of dihydrochalcones (DHCs) that serve as valuable antioxidants, antidiabetics, and sweeteners. Although the technologies of DHC extraction and metabolic engineering are being developed and perfected, natural dietary sources are still significant. Therefore, to identify apples with high levels of these health-promoting DHCs, we conducted a large-scale investigation of 140 accessions and examined their DHCs profiles, concentrations of total phenolics and total flavonoids, and antioxidant capacity in the fruit (peel and flesh). Our results indicated that DHCs are major phenolics and significantly contribute to antioxidant capacity. Furthermore, these DHCs could be further enriched by an independent pathway. The great diversity and wide range of DHCs found in these accessions provide abundant natural materials for further efforts toward the breeding and utilization of apple fruits rich in these phenolics.

Same ammo, different weapons: Enzymatic extracts from two apple genotypes with contrasted susceptibilities to fire blight (Erwinia amylovora) differentially convert phloridzin and phloretin in vitro

The necrogenic bacterium Erwinia amylovora responsible for the fire blight disease causes cell death in apple tissues to enrich intercellular spaces with nutrients. Apple leaves contain large amounts of dihydrochalcones (DHCs), including phloridzin and its aglycone phloretin. Previous work showed an important decrease in the constitutive DHCs stock in infected leaves, probably caused by transformation reactions during the infection process. At least two flavonoid transformation pathways have been described so far: deglucosylation and oxidation. The aim of the present study was to determine whether DHCs are differentially converted in two apple genotypes displaying contrasted susceptibilities to the disease. Different analyses were performed: i) enzymatic activity assays in infected leaves, ii) identification/quantification of end-products obtained after in vitro enzymatic reactions with DHCs, iii) evaluation of the bactericidal activity of end-products. The results of the enzymatic assays showed that deglucosylation was dominant over oxidation in the susceptible genotype MM106 while the opposite was observed in the resistant genotype Evereste. These data were confirmed by LC-UV/Vis-MS analysis of in vitro reaction mixtures, especially because higher levels of o-quinoid oxidation products of phloretin were measured by using the enzymatic extracts of Evereste infected leaves. Their presence correlated well with a strong bactericidal activity of the reaction mixtures. Thus, our results suggest that a differential transformation of DHCs occur in apple genotypes with a potential involvement in the establishment of the susceptibility or the resistance to fire blight, through the release of glucose or of highly bactericidal compounds respectively.

Neohesperidine dihydro chalcone is an activator of mammalian alpha-amylase

Neohesperidine dihydro chalcone is an activator of mammalian alpha-amylase