[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

Efficient and Selective Extraction of Rhamnogalacturonan-I-Enriched Pectic Polysaccharides from Tartary Buckwheat Leaves Using Deep-Eutectic-Solvent-Based Techniques.

Tartary buckwheat green leaves are considered to be among the most important by-products in the buckwheat industry. Although Tartary buckwheat green leaves are abundant in pectic polysaccharides, their potential applications in the food industry are quite scarce. Therefore, to promote their potential applications as functional or fortified food ingredients, both deep-eutectic-solvent-assisted extraction (DESE) and high-pressure-assisted deep eutectic solvent extraction (HPDEE) were used to efficiently and selectively extract pectic polysaccharides from Tartary buckwheat green leaves (TBP). The results revealed that both the DESE and HPDEE techniques not only improved the extraction efficiency of TBP but also regulated its structural properties and beneficial effects. The primary chemical structures of TBP extracted using different methods were stable overall, mainly consisting of homogalacturonan and rhamnogalacturonan-I (RG-I) pectic regions. However, both the DESE and HPDEE methods could selectively extract RG-I-enriched TBP, and the proportion of the RG-I pectic region in TBP obviously improved. Additionally, both the DESE and HPDEE methods could improve the antioxidant and anti-glycosylation effects of TBP by increasing its proportion of free uronic acids and content of bound polyphenolics and reducing its molecular weight. Moreover, both the DESE and HPDEE methods could partially intensify the immunostimulatory effect of TBP by increasing its proportion of the RG-I pectic region. These findings suggest that DES-based extraction techniques, especially the HPDEE method, can be promising techniques for the efficient and selective extraction of RG-I-enriched TBP.

Impacts of ultrasound-assisted Fenton degradation and alkaline de-esterification on structural properties and biological effects of pectic polysaccharides from Tartary buckwheat leaves.

Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) leaf has abundant rhamnogalacturonan-I enriched pectic polysaccharides, which exert various health-promoting effects. Nevertheless, the potential relationship between the chemical structure and the biological function of pectic polysaccharides from Tartary buckwheat leaves (TBP) remains unclear. Therefore, to bridge the gap between the chemical structure and the biological function of TBP, the impacts of ultrasound-assisted Fenton degradation (UFD) and mild alkaline de-esterification (MAD) on structural properties and biological effects of TBP were systematically studied. Compared with the native TBP (molecular mass, 9.537 × 104 Da), the molecular masses of degraded TBPs (TBP-MMW, 4.811 × 104 Da; TBP-LMW, 2.101 × 104 Da) were significantly reduced by the UFD modification, while their primary chemical structures were overall stable. Besides, compared with the native TBP (esterification degree, 22.73 %), the esterification degrees of de-esterified TBPs (TBP-MDE, 14.27 %; TBP-LDE, 6.59 %) were notably reduced by the MAD modification, while their primary chemical structures were also overall stable. Furthermore, the results revealed that both UFD and MAD modifications could significantly improve the antioxidant, antiglycation, and immunostimulatory effects of TBP. Indeed, TBP's biological effects were negatively correlated to its molecular mass and esterification degree, while positively linked to its free uronic acids. The findings demonstrate that both UFD and MAD modifications are promising techniques for the structural modification of TBP, which can remarkedly promote its biological effects. Besides, the present results are conducive to better understanding TBP's structure-bioactivity relationship.

Pectic polysaccharides from Tartary buckwheat sprouts: Effects of ultrasound-assisted Fenton treatment and mild alkali treatment on their physicochemical characteristics and biological functions.

Buckwheat sprouts are rich in pectic polysaccharides, which possess numerous health-improving benefits. However, the precise structure-activity relationship of pectic polysaccharides from Tartary buckwheat sprouts (TP) is still scant, which ultimately restricts their applications in the food industry. Hence, both ultrasound-assisted Fenton treatment (UAFT) and mild alkali treatment (MATT) were utilized for the modification of TP, and then the effects of physicochemical characteristics of original and modified TPs on their bioactivities were assessed. Our findings reveled that the UAFT treatment could precisely reduce TP's molecular weight, with the levels decreased from 8.191 × 104 Da to 0.957 × 104 Da. Meanwhile, the MATT treatment could precisely reduce TP's esterification degree, with the values decreased from 28.04 % to 4.72 %. Nevertheless, both UAFT and MATT treatments had limited effects on the backbone and branched chain of TP. Moreover, our findings unveiled that the UAFT treatment could notably promote TP's antioxidant, antiglycation, and immunostimulatory effects, while remarkedly reduce TP's anti-hyperlipidemic effect, which were probably owing to that the UAFT treatment obviously reduced TP's molecular weight. Additionally, the MATT treatment could also promote TP's immunostimulatory effect, which was probably attributed to that the MATT treatment significantly decreased TP's esterification degree. Interestingly, the MATT treatment could regulate TP's antioxidant and antiglycation effects, which was probably attributed to that the MATT treatment simultaneously reduced its esterification degree and bound phenolics. Our findings are conducive to understanding TP's structure-activity relationship, and can afford a scientific theoretical basis for the development of functional or healthy products based on TPs. Besides, the UAFT treatment can be a promising approach for the modification of TP to improve its biological functions.

Potential structure-function relationships of pectic polysaccharides from quinoa microgreens: Impact of various esterification degrees.

Pectic polysaccharides are one of the most vital functional ingredients in quinoa microgreens, which exhibit numerous health-promoting benefits. Nevertheless, the detailed information about the structure-function relationships of pectic polysaccharides from quinoa microgreens (QMP) remains unknown, thereby largely restricting their applications as functional foods or fortified ingredients. Therefore, to unveil the possible structure-function relationships of QMP, the mild alkali de-esterification was utilized to modify QMP, and then the correlations of esterification degrees of native and modified QMPs to their biological functions were systematically investigated. The results showed that the modified QMPs with different esterification degrees were successfully prepared by the mild alkali treatment, and the primary chemical structure (e.g., compositional monosaccharides and glycosidic linkages) of the native QMP was overall stable after the de-esterified modification. Furthermore, the results revealed that the antioxidant capacity, antiglycation effect, prebiotic potential, and immunostimulatory activity of the native QMP were negatively correlated to its esterification degree. In addition, both native and modified QMPs exerted immunostimulatory effects through activating the TLR4/NF-κB signaling pathway. These results are conducive to unveiling the precise structure-function relationships of QMP, and can also promote its applications as functional foods or fortified ingredients.