Functional specialization of two UDP-glycosyltransferases MpUGT735A2 and MpUGT743A1 in the liverworts Marchantia polymorpha.

Family 1 UDP-glycosyltransferases (UGTs) are known to glycosylate multiple secondary plant metabolites and have been extensively studied. The increased availability of plant genome resources allows the identification of wide gene families, both functional and organizational. In this investigation, two MpUGT isoforms were cloned and functionally characterized from liverworts marchantia polymorpha and had high glycosylation activity against several flavonoids. MpUGT735A2 protein, in particular, tolerates a wide spectrum of substrates (flavonols, flavanones, flavones, stilbenes, bibenzyls, dihydrochalcone, phenylpropanoids, xanthones, and isoflavones). Overexpression of MpUGT735A2 and MpUGT743A1 in Arabidopsis thaliana enhances the accumulation of 3-O-glycosylated flavonol (kaempferol 3-O-glucoside-7-O-rhamnose), consistent with its in vitro enzymatic activity. Docking and mutagenesis techniques were applied to identify the structural and functional properties of MpUGT735A2 with promiscuous substrates. Mutation of Pro87 to Ser, or Gln88 to Val, substantially altered the regioselectivity for luteolin glycosylation, predominantly from the 3'-O- to the 7-O-position. The results were elucidated by focusing on the novel biocatalysts designed for producing therapeutic flavonoids. This investigation provides an approach to modulate MpUGT735A2 as a candidate gene for diverse glycosylation catalysis and a tool to design GTs with new substrate specificities for biomedical applications.

Identification and Functional Characterization of UDP-Glycosyltransferases Involved in Isoflavone Biosynthesis in Astragalus membranaceus.

Isoflavones are rich natural compounds present in legumes and are essential for plant growth and development. Moreover, they are beneficial for animals and humans. Isoflavones are primarily found as glycoconjugates, including calycosin-7-O-ß-d-glucoside (CG) in Astragalus membranaceus, a legume. However, the glycosylation mechanism of isoflavones in A. membranaceus remains unclear. In the present study, three uridine diphosphate (UDP)-glycosyltransferases (UGTs) that may be involved in the biosynthesis of isoflavone were identified in the transcriptome of A. membranaceus. Enzymatic analysis revealed that AmUGT88E29 and AmUGT88E30 had high catalytic activity toward isoflavones in vitro. In addition, AmUGT88E29 and AmUGT88E30 could accept various flavones, flavanones, flavonols, dihydroflavonols, and dihydrochalcones as substrates. AmUGT71G10 was only active against phloretin and dihydroresveratrol. Overexpression of AmUGT88E29 significantly increased the contents of CG, an isoflavone glucoside, in the hairy roots of A. membranaceus. This study provided candidate AmUGT genes for the potential metabolic engineering of flavonoid compounds in plants and a valuable resource for studying the calycosin glycosides biosynthesis pathway.

Gastroprotective 2-(2-phenylethyl)chromone-sesquiterpene hybrids from the resinous wood of Aquilaria sinensis (Lour.) Gilg.

Six previously unprecedented 2-(2-phenylethyl)chromone-sesquiterpene hybrids, aquisinenins A-F (1 - 6), were isolated from the resinous wood of Aquilaria sinensis by a LC-MS-guided fractionation procedure. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1 - 6 were rare dimeric 2-(2-phenylethyl)chromone-sesquiterpene derivatives featuring 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone hybridized with different sesquiterpene (eudesmane/guaiane type) moieties via ester bond. Furthermore, all the isolated compounds were evaluated for their protective effects on taurocholic acid (TCA)-induced GES-1 cell injury. The most effective aquisinenin F (6) was used to elucidate the involved mechanism on protection against TCA-induced gastric mucosal damage. Our results indicated that 6 protected against gastric mucosal cell insult by downregulation of the ER stress triggered by TCA.

Xinjiang sambucus sibirica Nakai bark extract promotes fracture healing / 中国组织工程研究

BACKGROUND: Studies have found that Sambucus sibirica Nakai can promote osteoblast division, proliferation, and secretion of alkaline phosphatase to accelerate bone matrix calcification, promote new bone formation, and promote the absorption of necrotic bone. OBJECTIVE: To observe the effects of two extracts of Xinjiang Sambucus sibirica Nakai bark in promoting fracture healing in rats. METHODS: A total of 108 male Sprague-Dawley rats, 3 months old, were randomly divided into sham-operated group, model group, Zhong Hua Die Da Wan group (0.54 g/kg, negative control group), high-, middle- and low-dose ethanol extract groups (0.314, 0.157, 0.078 g/kg), high-, middle, and low-dose water extract groups (0.340, 0.170, 0.085 g/kg). Except for the sham-operated group, the middle segment of the femoral shaft was cut off to make a model of open femoral fracture. In the sham-operated group, only the femoral shaft was exposed and no osteotomy was performed. Intragastric administration began at 1 day after modeling and continued for 6 weeks (1 time/day). At 2, 4, 6 weeks after modeling, the effects of two extracts of Sambucus sibirica Nakai bark on serum-related biochemical indicators, X-ray imaging, biomechanical indicators and hematoxylin-eosin staining were observed. RESULTS AND CONCLUSION: (1) Serum biochemical indicators: The serum calcium level of the high-dose ethanol extract group in each phase was lower than that of the sham-operated group and the model group, indicating that a faster osteogenesis in the high-dose ethanol extract group, followed by middle-dose ethanol extract group, low-dose ethanol extract group, and high-dose water extract group. The serum phosphorus level of ethanol extract groups was higher than that of sham-operated group, model group, and Zhong Hua Die Da Wan group; the serum alkaline phosphatase level of high-dose ethanol extract group at each stage was significantly higher than the other groups, indicating that a stronger osteoblast activity in the high-dose ethanol extract group, followed by the high-dose water extract group. (2) X-ray film: Fracture healing was significant in the high-dose ethanol extract group, fair in the Zhong Hua Die Da Wan group, high-dose water extract, middle-dose water extract group, middle-dose ethanol extract group, and poor in the low-dose ethanol extract group and low-dose water extract group. (3) Three-point bending test: At 6 weeks, the maximum load and maximum deflection of the femurs of the two extract groups were higher than those in the model group. The maximum load and maximum deflection of the femurs increased significantly in the high-dose ethanol extract group followed by the high-dose water extract group, middle-dose water extract group and middle-dose ethanol extract group. (4) Hematoxylin-eosin staining: Fibrous epiphysis gradually formed in the fracture area, and the bone marrow cavity was communicated. Bone healing was better in the high-dose ethanol extract and high-dose water extract groups than in the other groups. To conclude, these two extracts of Xinjiang Sambucus sibirica Nakai bark are capable of improving fracture healing. Moreover, different extraction methods and dosages of Sambucus sibirica Nakai bark demonstrate various effects on fracture healing. Compared with water extracts, ethanol extracts are better to promote fracture healing, and high-dose extracts have better effects than middle- and low-dose extracts.