Starch-digesting product analysis based on the hydrophilic interaction liquid chromatography coupled mass spectrometry method to evaluate the inhibition of flavonoids on pancreatic α-amylase.

An accurate hydrophilic interaction liquid chromatography coupled mass spectrometry (HILIC-MS) method is presented to characterize starch digestion by α-amylase and measure the inhibition properties of flavonoids against α-amylase in vitro. Eleven products were found as 1 â†’ 4 linkage glucose oligosaccharides with different degrees of polymerization (DPs) from 2 to 12. The products with DPs of 2, 3, 6, 7, and 9 had higher yields. The product with DP of 9 had the highest yields, which first increased and then decreased with the reaction time. Pelargonidin has the best inhibition activity on all enzyme products. The 3'-hydroxyl of B-ring enhanced the inhibition activity of flavonol and flavone but weakened that of anthocyanin. The C-ring 3-hydroxyl increased the inhibition effect of flavonol on maltose but decreased that on the products with higher DPs than flavone. The HILIC-MS method can provide more detailed information on enzyme products for the study of flavonoids inhibiting α-amylase.

Inhibitory properties of polyphenols in Malus "Winter Red" crabapple fruit on α-glucosidase and α-amylase using improved methods.

To explore the inhibitory activity of polyphenols on α-glucosidase and α-amylase, 16 polyphenols were isolated, identified, and quantified in an edible Malus "Winter Red" crabapple fruit. The limitations of two traditional methods for α-glucosidase and α-amylase activity assay in vitro were assayed. An improved method based on an HPLC assay for α-glucosidase and a colorimetric method coupled with a custom-made mini-column for α-amylase were established. Compared with positive controls, acarbose and miglito, most polyphenols, especially the four aglycones (cyanidin, quercetin, phloretin, and 3-hydroxyphloretin) showed higher inhibition rates on α-glucosidase. None of the polyphenols showed higher inhibition rates on α-amylase than acarbose, but most, especially the four aglycones, showed higher inhibition rates on α-amylase than miglito. The Malus Winter Red fruit has great potential for postprandial blood glucose management as a potential diet therapy for diabetic patients.

Competition between anthocyanin and kaempferol glycosides biosynthesis affects pollen tube growth and seed set of Malus.

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits of Malus. In Malus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flower Malus genotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set.

Relationships between Structure and Antioxidant Capacity and Activity of Glycosylated Flavonols.

The antioxidant capacity (AC) and antioxidant activity (AA) of three flavonols (FLV), aglycones and their glycosylated derivatives were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays in various solvents. Findings confirmed that the glycosylation at the 3-position (3-glycosylation) always decreased the AC under most conditions due to substitution of the 3-position hydroxyl group and glycoside disruption in the molecular planarity. The 7-glycosylated derivatives did not have the above effects, thus generally exhibited ACs similar to their aglycones. Glycosylation decreased the AA of kaempferol and isorhamnetin for both assays in methanol, 3-glycosylation inhibited quercetin AA in the ABTS assay. In the DPPH assay, the AA of 3-glycosylated quercetin was significantly higher than quercetin. Using LC-MS/MS analysis, we found that quercetin and quercetin-7-glucoside underwent dimerization during the antioxidant reaction, potentially leading to a decline in AAs. However, 3-glycoside substitution may have hindered dimer formation, thereby allowing the FLVs to retain strong free radical scavenging abilities.

Biosynthesis of the Dihydrochalcone Sweetener Trilobatin Requires Phloretin Glycosyltransferase2.

Epidemics of obesity and type 2 diabetes drive strong consumer interest in plant-based low-calorie sweeteners. Trilobatin is a sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain trilobatin's bitter positional isomer phloridzin. Variation in trilobatin content was mapped to the Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples. Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed protein purification and differential gene expression analysis in samples high in trilobatin but low in phloridzin. Markers developed for PGT2 cosegregated strictly with the Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4'-o-glycosylation of phloretin to trilobatin as well as 3-hydroxyphloretin to sieboldin. Transient expression of double bond reductase, chalcone synthase, and PGT2 genes reconstituted the apple pathway for trilobatin production in Nicotiana benthamiana Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing trilobatin, and for high amounts of this natural low-calorie sweetener to be produced via biopharming and metabolic engineering in yeast.

Dihydrochalcones in Malus inhibit bacterial growth by reducing cell membrane integrity.

In recent years, increasing research has evaluated the use of natural products as antimicrobial food additives. In this study, antibacterial activity was evaluated for six dihydrochalcone compounds from Malus. Phloretin and 3-hydroxyphloretin exhibited antibacterial effects on both Gram-positive and Gram-negative bacteria, and the antibacterial capacity of these compounds was greater than that of their glycosylated derivatives. Within a certain range, dihydrochalcone hydrophobicity was positively correlated with antibacterial activity. Additionally, glycosylation at the 2'-position of the A-ring and hydroxyl group at the 3-position of the B-ring played a key role in the antibacterial activity of dihydrochalcones. Phloretin and 3-hydroxyphloretin caused damage to bacterial cells by significantly increasing protein and inorganic phosphate leakage. Compared to phloretin, 3-hydroxyphloretin exhibited a smaller effect on Gram-positive Micrococcus luteus and a greater effect on Gram-negative Klebsiella pneumoniae, suggesting different antibacterial mechanisms. At a low dihydrochalcone concentration, the respiration of M. luteus did not change, while membrane permeability increased significantly. These results indicate that the antibacterial mechanism of M. luteus was primarily damage to the cell membrane. However, damage to respiration and the cell membrane might occur simultaneously in K. pneumoniae, suggesting that the antibacterial mechanism of dihydrochalcones also depends on strain type. This study demonstrated the broad-spectrum antibacterial properties of dihydrochalcone compounds commonly found in the genus Malus to foodborne pathogens and elucidated the antibacterial mechanisms. It provides theoretical guidance for future research and application of dihydrochalcones in the food industry.

Structure-antioxidant capacity relationship of dihydrochalcone compounds in Malus.

The antioxidant capacity (AC) of six dihydrochalcone compounds was evaluated using DPPH and ABTS assays. In water-based solution 3-hydroxyphlorizin exhibited the highest AC among all dihydrochalcones. In acetone and acidic solutions (pH = 2.5 or 2.0), presence of an o-dihydroxyl at the B-ring increased AC, whereas glycosylation at the A-ring decreased AC of dihydrochalcones. By comparing the AC of dihydrochalcones with similar structures, it was found that the o-dihydroxyl at the B-ring and 2'-hydroxyl group at the A-ring were critical for maintaining the AC of dihydrochalcones by promoting hydrogen atom transfer or single electron transfer mechanism. Sequential proton-loss electron transfer commonly occurred during free radical scavenging in water-based solution. Moreover, we report a unique phenomenon in which glycosylation at the 2'-position enhanced the dissociation ability of the 4'-hydroxyl group and increased the AC of dihydrochalcones containing o-dihydroxyl. We speculate that this increase in AC might occur through intramolecular electron transfer.

Extraction, identification, and antioxidant and anticancer tests of seven dihydrochalcones from Malus 'Red Splendor' fruit.

Five dihydrochalcone compounds, including phlorizin, trilobatin, 3-hydroxyphlorizin, sieboldin and phloretin 2'-xyloglucoside, were isolated from ornamental Malus 'Red Splendor' fruit. The chemical structures of these compounds were elucidated by LC-ESI-MS and NMR. Phloretin and 3-hydroxyphloretin were produced by hydrolysis. The antioxidant capacities of these seven compounds were examined by DPPH and ABTS assays, while their cytotoxicity to five cancer cell lines were evaluated by the MTT assay. The results showed that the DPPH assay mainly reflected the antioxidant capacity of the B ring, whereas the ABTS assay was mostly related to the A ring of the dihydrochalcone molecule. Moreover, 3-hydroxyphloretin was the best antioxidant among the seven compounds. Both glycosylation of the A ring and the ortho phenolic hydroxyl groups of the B ring were important for the cytotoxicity of dihydrochalcone molecules. Sieboldin and 3-hydroxyphlorizin exhibited better cytotoxicity than other dihydrochalcone compounds. Dihydrochalcones from Malus may benefit human health.