Search

Introduction: Due to its remarkable anti-inflammatory pharmacological activity, Farfarae Flos has gained extensive usage in the treatment of various inflammatory diseases such as bronchitis, pneumonia, prostatitis and colitis. And Farfarae Flos come in two color types depending on the color of the flowers: yellowish-white (YW), and purplish-red (PR). However, the difference in anti-inflammatory activity and metabolic profiles between the two flower colors remains unexplored. Methods: This study aims to explore the difference in the anti-inflammatory potential between YW and PR variants of Farfarae Flos and unravel the mechanisms responsible for the observed differences in anti-inflammatory activity through an integrated approach encompassing untargeted metabolomics and in vivo / vitro experimental studies. Initially, we verified the contrasting effects of YW and PR on the inhibition of the inflammatory factors interleukin-6 (IL-6) and nitric oxide (NO) by utilizing an in vitro RAW 264.7 cell inflammation model. Subsequently, a comprehensive evaluation of the systemic inhibitory capacity of YW and PR on IL-6, Interleukin-10 (IL-10), and tumor necrosis factor- α (TNF- α) was conducted using a validated whole-body mouse model, followed by the analysis of inflammatory factors and histological examination of collected serum, liver, and spleen after 7 days. Furthermore, non-targeted metabolomics profiling was employed to analyze the metabolite profiles of Farfarae Flos with different colors, and quantitative analysis was conducted to identify differential metabolites between YW and PR. The correlation between the anti-inflammatory activities of differentially accumulated metabolites (DAMs) and Farfarae Flos was investigated, resulting in the identification of 48 compounds exhibiting significant anti-inflammatory activity. Additionally, KEGG pathway enrichment analysis was performed to elucidate the underlying mechanisms. Results: Our findings demonstrate that both YW and PR possess anti-inflammatory abilities, with PR exhibiting significantly superior efficacy. The integration of in vivo / vitro experiments and non-targeted metabolomics confirmed the exceptional anti-inflammatory potential of PR and solidified its classification as the "purplish-red better" of Farfarae Flos. Discussion: This study provides valuable insights into the breeding and medical transformation of Farfarae Flos varieties, along with a scientific basis for the establishment of quality standards and the development of new drugs utilizing Farfarae Flos. [ABSTRACT FROM AUTHOR]

The Cytochrome P450 (CYP450) family is the largest enzyme protein family in plants, distributed across various organs and involved in significant catalytic activities in primary and secondary metabolic processes. In this study, we cloned the PtCYP721A57 gene, characterized its open reading frame (ORF), and conducted comprehensive analyses including physicochemical properties, evolutionary relationships, subcellular localization, prokaryotic expression, and correlation between the relative expression of different parts and the content of tenuifolin, hormones, and abiotic stress response associated with the encoded protein. The ORF of PtCYP721A57 was 1,521 bp, with a secondary structure predominantly composed of α-helices and random coils. Subcellular localization experiments confirmed the presence of PtCYP721A57 in the endoplasmic reticulum. For prokaryotic expression, we constructed the recombinant plasmid pET28a-PtCYP721A57 using pET28a as the vector, which was then transformed into BL21(DE3). Induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at temperatures of 16 and 25 °C and varying concentrations (0.1, 0.2, 0.5, 1, 2 mM) resulted in the formation of inclusion bodies, with higher expression observed at 25 °C. Our qPCR analyses revealed that PtCYP721A57 exhibited the highest expression in the cortex of Polygala tenuifolia, followed by roots and xylem, correlating with the observed tenuifolin content distribution. Induction with abscisic acid (ABA) and chitosan (CHT) initially decreased PtCYP721A57 expression followed by a subsequent increase, peaking at 48 h. Similarly, drought stress induced a gradual increase in PtCYP721A57 expression, also peaking at 48 h. NaCl treatment for 6 h significantly upregulated PtCYP721A57 expression. In conclusion, our study provides foundational insights into the PtCYP721A57 gene in Polygala tenuifolia, laying the groundwork for further exploration of its role in the biosynthesis pathway of triterpenoid saponins. [ABSTRACT FROM AUTHOR]

Polygala tenuifolia Willd. is a commonly used clinical traditional Chinese medicine with a variety of biological activities. Hairy root cultures of medicinal plants are considered as a source of active constituents for pharmaceutical industries. In this study, P. tenuifolia hairy root cultures (PTHRCs) were established by induction of four Rhizobium rhizogenes , including ATCC15834, LBA9402, C58C1 and ACCC10060. The contents of active ingredients such as polygalaxanthone III, 3′,6-disinapoylsucrose, onjisaponin B, onjisaponin F, polygalacic acid and senegenin were detected in the hairy roots by high performance liquid chromatography (HPLC). Forty-five day-old leaf disc of P. tenuifolia sterile seedlings were prepared for explants, the highest hairy root transformation rate reached 85.83 % by using R. rhizogenes C58C1 co-cultured for 3 d with an optical density of 1.5 at 600 nm, coupled with 200 µM acetosyringone added in the solid culture medium. Meanwhile, the conditions of PTHRCs were optimized for the xanthones, saponins, and oligosaccharides components production. The results showed that the dry weight of the hairy roots reached its maximum of 0.44 g/flask (9.26 fold than that of the control) under the optimal culture conditions: 6,7-V medium, 3 % sucrose (m/v), pH 5.8, and 6 week-old PTHRCs, and the six signature ingredients in the hairy roots accumulated massively under the same condition simultaneously. Furthermore, trehalose and brassinolide were found to significantly promote the biomass and active ingredients biosynthesis in the hairy root cultures. The content of polygalaxanthone III was 8.92 folds higher than the description in the Chinese Pharmacopoeia (2020 year version) induced by 10 µM trehalose, while 1 × 10−12 M brassinolide treatment promoted onjisaponin F accumulation at 33.63 mg/g (dry weight) in PTHRCs. In addition, total phenolics, flavonoids, and antioxidant activity of PTHRCs extracts were significantly increased induced by trehalose (5 µM) and brassinolide (1 × 10−10 M), respectively. This is the first report on the systematic construction of PTHRCs and trehalose and brassinolide as effective elicitors for polygalaxanthone III, 3′,6-disinapoylsucrose, onjisaponin B, onjisaponin F, polygalacic acid and senegenin production in PTHRCs. The results demonstrated that PTHRCs can be used to produce active constituents in pharmaceutical and nutraceutical fields. • An efficient hairy root induction and culture protocol of Polygala tenuifolia Willd. was developed. • Rhizobium rhizogenes species, bacterial density, acetosyringone and liquid media influenced the hairy root induction and growth. • Polygalaxanthone III, 3′,6-disinapoylsucrose, onjisaponin B, onjisaponin F, polygalacic acid and senegenin were enhanced by trehalose and brassinolide. • Trehalose and brassinolide induced hairy root extracts have possessed higher total phenolic, total flavonoid accumulation and good antioxidant activities. [ABSTRACT FROM AUTHOR]