Illuminating the biosynthesis pathway genes involved in bioactive specific monoterpene glycosides in Paeonia veitchii Lynch by a combination of sequencing platforms.

BACKGROUND: Paeonia veitchii Lynch, a well-known herb from the Qinghai-Tibet Plateau south of the Himalayas, can synthesize specific monoterpene glycosides (PMGs) with multiple pharmacological activities, and its rhizome has become an indispensable ingredient in many clinical drugs. However, little is known about the molecular background of P. veitchii, especially the genes involved in the biosynthetic pathway of PMGs. RESULTS: A corrective full-length transcriptome with 30,827 unigenes was generated by combining next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRT) of six tissues (leaf, stem, petal, ovary, phloem and xylem). The enzymes terpene synthase (TPS), cytochrome P450 (CYP), UDP-glycosyltransferase (UGT), and BAHD acyltransferase, which participate in the biosynthesis of PMGs, were systematically characterized, and their functions related to PMG biosynthesis were analysed. With further insight into TPSs, CYPs, UGTs and BAHDs involved in PMG biosynthesis, the weighted gene coexpression network analysis (WGCNA) method was used to identify the relationships between these genes and PMGs. Finally, 8 TPSs, 22 CYPs, 7 UGTs, and 2 BAHD genes were obtained, and these putative genes were very likely to be involved in the biosynthesis of PMGs. In addition, the expression patterns of the putative genes and the accumulation of PMGs in tissues suggested that all tissues are capable of biosynthesizing PMGs and that aerial plant parts could also be used to extract PMGs. CONCLUSION: We generated a large-scale transcriptome database across the major tissues in P. veitchii, providing valuable support for further research investigating P. veitchii and understanding the genetic information of plants from the Qinghai-Tibet Plateau. TPSs, CYPs, UGTs and BAHDs further contribute to a better understanding of the biology and complexity of PMGs in P. veitchii. Our study will help reveal the mechanisms underlying the biosynthesis pathway of these specific monoterpene glycosides and aid in the comprehensive utilization of this multifunctional plant.

[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.

New Insights into the Impact of Ecological Factor on Bioactivities and Phytochemical Composition of Paeonia veitchii.

Paeonia veitchii has been widely distributed in China under different ecological types. Its roots contain diverse phytochemical constituents, which possess very high bioactivities. However, the influence of ecological factors on activities and ingredients of P. veitchii roots still remains unknown. The purpose of this research was to analyze the variation in bioactivities and phytochemical composition of P. veitchii roots upon exposure to various ecological factors. Seven P. veitchii populations collected from different regions in China were evaluated. The results of correlation analysis suggested that four major ecological factors, including average annual temperature, elevation, total potassium, and organic matter, had a strong correlation with the bioactivities of P. veitchii roots. Further, the major ecological factors were also highly correlated with the contents of naringin, gallic acid, benzoylpaeoniflorin, and paeoniflorin. The principal component analysis results supported four major metabolites as the main contributing ingredients. All populations were classified into three groups, G1, G2, and G3, through hierarchical cluster analysis. G1 showed more significant advantages in the above-mentioned four ecological factors, four active ingredients, and bioactivities compared to the other two groups. P. veitchii roots growing at lower average annual temperature, high elevation, rich total potassium and organic matter in the soils were presumed to have relatively higher bioactivities. These data expand the study on the bioactivities and phytochemical composition of P. veitchii roots and have a guiding significance for the ecological factor selection during the cultivation process of this herbaceous peony species.

Monoterpene glycosides from Paeonia veitchii.

The EtOH extract of the roots of Paeonia veitchii afforded two new monoterpene glycosides paeonidanin I (1) and paeonidanin J (2), and a new dimeric monoterpene glycoside paeonidanin K (3). Their structures were elucidated on the basis of spectroscopic means and hydrolysis products.

Data mining-guided alleviation of hyperuricemia by Paeonia veitchii Lynch through inhibition of xanthine oxidase and regulation of renal urate transporters.

BACKGROUND: Hyperuricemia (HUA) is a metabolic disease characterized by a high level of uric acid (UA). The extensive historical application of traditional Chinese medicine (TCM) offers a range of herbs and prescriptions used for the treatment of HUA-related disorders. However, the core herbs in the prescriptions and their mechanisms have not been sufficiently explained. PURPOSE: Our current investigation aimed to estimate the anti-HUA effect and mechanisms of Paeonia veitchii Lynch, an herb with high use frequency identified from data mining of TCM prescriptions. METHODS: Prescriptions for HUA/gout treatment were statistically analyzed through a data mining approach to determine the common nature and use frequency of their composition herbs. The chemical constituents of Paeonia veitchii extract (PVE) were analyzed by UPLC-QTOF-MS/MS, while its UA-lowering effect was further evaluated in adenosine-induced liver cells and potassium oxonate (PO) and hypoxanthine (HX)-induced HUA mice. RESULTS: A total of 225 prescriptions involving 246 herbs were sorted out. The properties, flavors and meridians of the appearing herbs were mainly cold, bitter and liver, respectively, while their efficacy was primarily concentrated on clearing heat and dispelling wind. Further usage frequency analysis yielded the top 20 most commonly used herbs, in which PVE presented significant inhibitory activity (IC50 = 131.33 µg/ml) against xanthine oxidase (XOD), and its constituents showed strong binding with XOD in a molecular docking study and further were experimentally validated through XOD enzymatic inhibition and surface plasmon resonance (SPR). PVE (50 to 200 µg/ml) dose-dependently decreased UA levels by inhibiting XOD expression and activity in BRL 3A liver cells. In HUA mice, oral administration of PVE exhibited a significant UA-lowering effect, which was attributed to the reduction of UA production by inhibiting XOD activity and expression, as well as the enhancement of UA excretion by regulating renal urate transporters (URAT1, GLUT9, OAT1 and ABCG2). Noticeably, all doses of PVE treatment did not cause any liver injury, and displayed a renal protective effect. CONCLUSIONS: Our results first comprehensively clarified the therapeutic effect and mechanisms of PVE against HUA through suppressing UA production and promoting UA excretion with hepatic and renal protection, suggesting that PVE could be a promising UA-lowering candidate with a desirable safety profile for the treatment of HUA and prevention of gout.

Metabolomics analysis of peony root using NMR spectroscopy and impact of the preprocessing method for NMR data in multivariate analysis.

Peony root is an important herbal drug used as an antispasmodic analgesic. To evaluate peony roots with different botanical origins, producing areas, and post-harvest processing, 1H NMR-based metabolomics analysis was employed. Five types of monoterpenoids, including albiflorin (4), paeoniflorin (6), and sulfonated paeoniflorin (25), and six other compounds, including 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (18), benzoic acid (21), gallic acid (22), and sucrose (26) were detected in the extracts of peony root samples. Among them, compounds 4, 6, 18, and total monoterpenoids including 21 were quantified by quantitative 1H NMR (qHNMR). Compound 25 was detected in 1H NMR spectra of sulfur-fumigated white peony root (WPR) extracts indicating that 1H NMR was a fast and effective method for identifying sulfur-fumigated WPR. The content of 26, the main factor affecting extract yield, increased significantly in peony root after low-temperature storage for one month, whereas that in WPR did not increase due to the boiling treatment after harvesting. We investigated the impact of preprocessing methods to such analysis for NMR data from commercial samples, resulting that the data matrix transformed from qHNMR spectra and normalized to internal standard were optimum for multivariate analysis. The multivariate analysis demonstrated that among commercial samples derived from P. lactiflora, peony root samples in Japanese market (PR) had high contents of 18 and 22, and red peony root (RPR) samples had high content of monoterpenoids represented by 6; and among RPR samples, those derived from P. veitchii showed higher contents of 18 and 22 than those from P. lactiflora. The 1H NMR-based metabolomics method coupled with qHNMR was useful for evaluation of peony root and would be applicable for other crude drugs.

Shade effects on growth, photosynthesis and chlorophyll fluorescence parameters of three Paeonia species.

Insufficient light intensity inhibits the growth of cultivated herbaceous peony and decreases its economic value. Owing to the increased demand for shade-tolerant herbaceous peony, the selection of appropriate parents for hybridization is essential. Paeonia anomala, Paeonia intermedia and Paeonia veitchii can grow under shade conditions in their natural habitats; however, their photosynthetic capacities under shade have not been studied. In this study, we simulated low light intensity (30% sunlight) and evaluated the morphological, photosynthetic and chlorophyll fluorescence parameters of these three species. Moreover, the shade tolerance of these species as well as two common cultivars (Paeonia lactiflora 'Da Fugui', which is suitable for solar greenhouse cultivation, and P. lactiflora 'Qiao Ling', which is not suitable for solar greenhouse cultivation) was evaluated. The results showed that under shade, the leaf area of P. anomala and P. intermedia increased, the single flowering period of P. intermedia and P. veitchii was prolonged, and the flower color of P. veitchii faded. With respect to P. anomala, P. intermedia and P. veitchii, shade eliminated the photosynthetic 'lunch break' phenomenon and decreased photoinhibition at midday. Furthermore, the maximum photochemical efficiency (Fv/Fm) and maximum primary photochemical yield (Fv/Fo) of photosystem II (PSII) in the three species improved significantly, and their changes in light dissipation were different. The shade tolerance of the tested accessions was in the order P. veitchii > P. intermedia > P. anomala > 'Da Fugui' > 'Qiao Ling', showing that the three wild species were better adapted to low light intensity than the cultivars. Thus, P. anomala, P. intermedia and P. veitchii could potentially be used in the development of shade-tolerant herbaceous peony cultivars.

Paeoveitols A-E from Paeonia veitchii.

Paeoveitols A-E (1-5), involving three monoterpenes and two benzofuran constituents, were isolated from Paeonia veitchii. Their structures were determined based on extensive spectral analyses (IR, UV, MS, 1D and 2D NMR), and the absolute configuration of compound 1 was confirmed by single-crystal X-ray diffraction. By the agitating human melatonin receptor 1 (MT1) assay on HEK293 cell line in vitro, compound 4 showed weak activity with the agitation rate of 22.52% at the concentration of 1.79 mM.