Larvicidal and antibiofilm potential of three mountain plants:Centaurea ensiformis,Origanum hypericifolium,Paeonia turcica

Background:Plants are known to produce a diverse group of natural metabolites with different biological activities.Centaurea ensiformis P.H.Davis,Origanum hypericifolium O.Schwartz&P.H.Davis,and Paeonia turcica Davis&Cullen are endemic plant species that grow on mountains in select regions in Türkiye and have been used in traditional Turkish medicine for various ailments.Methods:As first,we evaluated the larvicidal and antibiofilm activities of ethanol,ethyl acetate,acetone,and water extracts obtained from these plants.Antioxidant activities of the extracts were also investigated.Results:All tested extracts were effective at concentrations>25 ppm on Aedes aegypti larval mortality with the lethal concentration 50(LC_(50))values ranging between 32.82-48.35 ppm and LC90 between 46.26-63.2 ppm.O.hypericifolium was the most effective plant,ethanol extracts presented LC50 values of 32.82 ppm.Extracts demonstrated varying degrees of antibiofilm activity depending on the dose and bacterial species.Origanum hypericifolium extracts notably inhibited biofilms of Staphylococcus aureus(up to 98%inhibition),while P.turcica showed moderate efficacy against the same bacterial species.Pseudomonas aeruginosa biofilms displayed high resistance to all extracts.Conclusion:The results indicated that these endemic Turkish plants possess promising larvicidal and antibiofilm potential,particularly Origanum hypericifolium.Extracts analyzed by liquid chromatography-tandem mass spectrometry contained caffeic acid,myricetin,cinnamic acid,quercetin,gallic acid,epicatechin,and ascorbic acid.Further research should explore their potential applications in mosquito control and biofilm-related infections.

Genetic diversity and population genetic structure of Paeonia suffruticosa by chloroplast DNA simple sequence repeats(cpSSRs)

Paeonia suffruticosa Andr.is an endemic shrub flower in China with 2n=10.This study used 228 cultivars from four populations,i.e.,Jiangnan,Japan,Northwest,and Zhongyuan,as materials to explore the genetic diversity levels among different populations of tree peony varieties.The results showed that 34 bands were amplified using five pairs of cp SSR primers,with an average of 6.8 bands per primer pair.The average number of different alleles(N_(a)),effective alleles(N_(e)),Shannon's information index(I),diversity(H),and polymorphic information content(PIC)were 3.600,2.053,0.708,0.433,and 0.388,respectively.The PIC value was between 0.250 and 0.500,indicating a moderate level of polymorphism for the five cp SSR primer pairs.The genetic diversity levels of peony cultivars varied among different populations,with the Northwest population showing relatively lower levels(I=0.590,H=0.289,and PIC=0.263).A total of 52 haplotypes were identified in the four examined populations,and the number of haplotypes per population ranged from 11 to 22.Forty-four private haplotypes were detected across populations,and the Northwest population exhibiting the highest count of private haplotypes with 17.The mean number of effective number of haplotypes(N_(eh)),haplotypic richness(R_h),and diversity(H)were 8.351,6.824,and 0.893,respectively.Analysis of molecular variance indicated that genetic variation within tree peony germplasm was greater than that between germplasm resources,and the main variation was found within individuals of peony germplasm.Cluster analysis,principal coordinate analysis,and genetic structure analysis classified tree peonies from different origins into two groups,indicating a certain degree of genetic differentiation among these four tree peony cultivation groups.This study provides a theoretical basis for the exploration,utilization,and conservation of peony germplasm resources,as well as for research on the breeding of excellent varieties.

Light shading improves the yield and quality of seed in oil-seed peony(Paeonia ostii Feng Dan)

Tree peony seed is unique for its super-high content of unsaturated fatty acid and is thus considered as an important source of woody oil. However, photosynthetic production is greatly reduced under high light intensity and air temperature during the seed filling period, which negatively affects seed yield and quality. The objective of this study was to determine if appropriate shading improves yield and quality of seed in oilseed peony. In this study, oilseed peony trees were shaded by different density polyethylene nets from four weeks after flowering to harvest stages to form light, moderate, and severe shadings, equivalent to about 80, 40, and 20% of full solar exposure, respectively. The effects of different shadings on some physiological parameters, yield and yield components, and nutritional composition of seed were examined. Averaged across two years, light shading increased the actual net photosynthetic rate（P_n） by 16.8%, the maximum net photosynthetic rate（P_（max）） by 81.4%, chlorophyll（Chl） content by 52.8%, auxin（IAA） content by 38.1%, and gibberellic acid（GA_3） content in leaves by 6.3%; it decreased the accumulation of H_2O_2 in leaves by 24.8%, malondialdehyde（MDA） by 22%, and endogenous abscisic acid（ABA） by 8.8%, indicating that leaf senescence in late season was considerably delayed. Light shading increased seed yield, and contents of crude fat and unsaturated fatty acids by 9.7, 5.6, and 9.6%, respectively, while moderate or severe shading significantly reduced all the three parameters. Light shading increased seed weight, but moderate or severe shading reduced seed weight or follicle density. The improved seed yield under light shading was mainly due to increased seed weight, while the reduced seed yield under moderate or severe shading was mainly attributed to reduce follicle density and seed weight. The improved seed weight and content of unsaturated fatty acids under light shading was possibly due to the delayed leaf senescence. The overall results indicated that light shading is beneficial to yield and quality parameters of seed in oilseed peony. Cultivating oilseed peony under a light shading environments such as partially closed forests would better increase total output and income per unit land area than that under full solar exposure.

Effect of exogenous GA3 on flowering quality, endogenous hormones, and hormone-and flowering-associated gene expression in forcing-cultured tree peony（Paeonia suffruticosa）

Gibberellins(GAs)promote flowering in the forcing-cultured tree peony(Paeonia suffruticosa),however,the mechanism of regulating flowering is not fully understood.In this study,exogenous GA3 was applied to five-year-old Luoyang Hong plants to explore responses in terms of endogenous hormones,flowering quality,and the hormone-and flowering-associated gene expression.Exogenous GA3 application significantly promoted flower bud development and new branch growth,as well as improved flowering quality.Exogenous GA3 application also stimulated the synthesis of endogenous GA3 and indole-3-acetic acid(IAA)but reduced abscisic acid(ABA)levels.To further elucidate the regulatory mechanism,eight genes for GA biosynthesis and signaling,including PsCPS,PsKS,PsGA3ox,PsGA2ox,PsGID1b,PsGID1c,PsDELLA,and PsGID2 were cloned for the first time,and sequence analysis was also performed.The results suggested that all the cloned genes have conserved structure as each homologous gene reported in the other species.Phylogenetic trees constructed by the each cloned gene showed that the phylogenetic evolutionary relationship of P.suffruticosa was closely related to Vitis vinifera.The expression patterns of the above genes,and genes for ABA and IAA biosynthetic and signaling,and the flowering time were also investigated.Most of the above genes showed higher expression in the control buds than those in the GA3 treated buds at six developmental stages,whereas the expression levels of PsSOC1 and PsSPL9 were up-regulated by GA3 treatment.The results also showed that the GA-biosynthetic and signaling pathways are conserved in tree peony,and the PsCPS,PsGA3ox,PsGA2ox,PsGID1,PsDELLA,and PsGID2 genes are necessary for feedback regulation of GAs.Furthermore,hormone changes promoted PsSOC1 and PsSPL9 expression,and repressed PsSVP expression,which contributed to the improvement flowering quality in tree peony of forcing culture.

Development and application of microsatellite markers within transcription factors in flare tree peony(Paeonia rockii)based on next-generation and single-molecule long-read RNA-seq

Tree peonies native to China are a precious crop with ornamental,medicinal and edible oil properties,of which flare tree peony(Paeonia rockii)is one of the most significant germplasms in Paeonia.The development and application of expressed sequence tag-simple sequence repeat(EST-SSR)markers are very valuable for genetic and breeding applications,but EST-SSR resources for the genus Paeonia are still limited.In this study,we first reported the development of SSRs within transcription factors(TFs)in P.rockii based on next-generation sequencing(NGS)and single-molecule long-read sequencing(SMLRS).A total of 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield,with an average of one SSR per 5.83 unigenes.In total,102(61.45%)pairs of primers produced amplification products in the two RNA-seq cultivars.Among them,58(56.86%)pairs of primers from 18 gene families(AP2,b HLH,HSF,etc.)were identified to be polymorphic both in the parents of a linkage mapping population and in eight randomly selected accessions of P.rockii.Further,the 58 EST-SSRs indicated a high level of informativeness with PIC values ranging from 0.32 to 0.91(mean 0.70)after assessment in 37 tree peony accessions.Transferability studies indicated that the amplification ratio of the 58 pairs of primers ranged from 89.66 to 100%across seven species of Paeonia.In addition,a genetic relationship study was performed in 62 accessions.Cluster analysis using the neighbour-joining(NJ)tree demonstrated that major clusters corresponded to the known pedigree trees.Taken together,these newly developed EST-SSRs have a potential use in the conservation of tree peony germplasm and marker-assisted selection(MAS)breeding.

Molecular characterization of chalcone isomerase (CHI) regulating flower color in herbaceous peony (Paeonia lactiflora Pall.)

Chalcone isomerase （CHI） is a key enzyme that converts yellow chalcone to colorless naringenin, playing an important regulatory role in color formation of ornamental flowers. We determined the coding sequence of CHI in herbaceous peony using rapid-amplification of cDNAends （RACE） technology, and subsequently detected the expression pattern of CHI in the inner and outer petals at different developmental stages using qRT-PCR. We cloned the upstream promoter sequences of CHI using genome walking technology and predicted the location of CpG islands and 5＇ truncation. In addition, we con- structed five dual-luciferase reporter gene carriers and detected the promoter activities of different fragments. Our results showed that the full-length cDNA sequence of CHI was 898 bp, and the 5＂-upstream core promoter was located at -1 651 to -2050 bp region, where contained one CpG island （-1 897 to -2010 bp） and several important binding sites of transcription factor, such as Spl, serum response factor （SRF）, activating protein （AP）-2alpha and CCAAT/enhancer binding protein （C/ EBP）alpha. Expression results showed that the expression of CHI at different developmental stages was generally higher in inner petals than those in outer petals, and the maximum at the bud stage （S1）. Thus, this study will provide theoretical basis for an in-depth study of CHI gene function and expression regulation.

Identification of differentially expressed genes associated with bud dormancy release in tree peony(Paeonia suffruticosa) by suppression subtractive hybridization

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different mo- lecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Flavonoid content and expression analysis of flavonoid biosynthetic genes in herbaceous peony(Paeonia lactiflora Pall.)with double colors

Herbaceous peony（Paeonia lactiflora Pall.） is a famous flower with medicinal values, and its flowers have a number of medicinal constituents, especially flavonoids. In this study, a P. lactiflora cultivar with double colors including white outer-petal and yellow inner-petal was used as the experimental materials to perform the qualitative and quantitative analysis of flavonoids by high-performance liquid chromatograph-electrospray ionization-mass spectrometry（HPLC-ESI-MSn） and investigate the expression patterns of flavonoid biosynthetic genes using real-time quantitative polymerase chain reaction（Q-PCR）. The results showed that the colors of both petals gradually weakened with flower development. Moreover, one main anthocyanin composition（peonidin 3,5-di-O-glucoside） and five main anthoxanthin compositions（kaempferol di-hexoside, kaempferol-3-O-malonylglucoside-7-O-glucoside, quercetin-3-O-galactoside, luteolin-7-O-glucoside and isorhamnetin-3-O-glucoside） were found in the both, differing significantly in their peak areas only. Total anthocyanin, anthoxanthin and flavonoid contents in white outer-petal and yellow inner-petal gradually decreased during flower development, and were consistently higher in white outer-petal. Furthermore, the expression patterns of nine structural genes in P. lactiflora flavonoid biosynthetic pathway showed that the expression levels of phenylalanine ammonialyase gene（Pl PAL）, chalcone synthase gene（PlC HS）, flavanone 3-hydroxylase gene（PlF 3H）, anthocyanidin synthase gene（PlA NS） and UDP-glucoside： flavonoid 5-O-glucosyltransferase gene（Pl F5GT） in two petals basically presented declined tendencies, and transcription levels of Pl PAL, Pl CHS, Pl ANS, Pl F3 GT and Pl F5 GT also tended to be higher in white outer-petal, which was correlated with their flavonoid contents. These results would lay a solid foundation for the exploration and utilization of flavonoid resources in P. lactiflora flowers.

RAD-sequencing improves the genetic characterization of a threatened tree peony(Paeonia ludlowii)endemic to China:Implications for conservation

Compared with traditional genetic markers,genomic approaches have proved valuable to the conservation of endangered species.Paeonia ludlowii having rarely and pure yellow flowers,is one of the world’s most famous tree peonies.However,only several wild populations remain in the Yarlung Zangbo Valley(Nyingchi and Shannan regions,Xizang)in China due to increasing anthropogenic impact on the natural habitats.We used genome-wide single nucleotide polymorphisms to elucidate the spatial pattern of genetic variation,population structure and demographic history of P.ludlowii from the fragmented region comprising the entire range of this species,aiming to provide a basis for conserving the genetic resources of this species.Unlike genetic uniformity among populations revealed in previous studies,we found low but varied levels of intra-population genetic diversity,in which lower genetic diversity was detected in the population in Shannan region compared to those in Nyingzhi region.These spatial patterns may be likely associated with different population sizes caused by micro-environment differences in these two regions.Additionally,low genetic differentiation among populations(Fst=0.0037)were detected at the species level.This line of evidence,combined with the result of significant genetic differentiation between the two closest populations and lack of isolation by distance,suggested that shared ancestry among now remnant populations rather than contemporary genetic connectivity resulted in subtle population structure.Demographic inference suggested that P.ludlowii probably experienced a temporal history of sharp population decline during the period of Last Glacial Maximum,and a subsequent bottleneck event resulting from prehistoric human activities on the Qinghai-Tibet Plateau.All these events,together with current habitat fragment and excavation might contribute to the endangered status of P.ludlowii.Our study improved the genetic characterization of the endangered tree peony(P.ludlowii)in China,and these genetic inferences should be considered when making different in situ and ex situ conservation actions for P.ludlowii in this evolutionary hotspot region.

Effects of Waterlogging Stress on the Physiological Characteristics and Secondary Metabolites of Herbaceous Peony (<i>Paeonia lactiflora</i>Pall.)

Herbaceous peony is an ornamental plant with medicinal properties. Waterlogging can affect its yield and quality as it grows and matures. In this study, we subjected “Taohuafeixue”, “Yangfeichuyu” and “Hongxiuqiu” herbaceous peony varieties to a simulated waterlogging stress treatment and investigated the effects of waterlogging on their physiological characteristics and the secondary metabolite contents in their leaves and roots. Short-term waterlogging caused the leaves to turn yellow or red and the roots to turn black. The stele and the cell wall of the endothelial cells thickened, and the cortical cells enlarged. Waterlogging did not significantly change plant height, leaf length, and leaf area;however, it significantly decreased the root-shoot ratio of “Yangfeichuyu” and “Hongxiuqiu” varieties. The activity of antioxidant enzymes and the content of osmotic regulators increased under waterlogging. After short-term waterlogging stress treatment, the content of paeoniflorin and albiflorin increased in the roots of “Taohuafeixue” and “Yangfeichuyu”, and the content of benzoylpaeoniflorin increased in the root of “Hongxiuqiu”. The content of gallic acid and total flavonoids increased in the leaves of “Taohuafeixue” and “Yangfeichuyu”. After the waterlogging, paeoniflorin and benzoylpaeoniflorin increased in the autumn root of “Hongxiuqiu”. This study expands our knowledge about the medicinal properties of herbaceous peony and informs about its production and cultivation under waterlogged conditions.

Comprehensive analyses of the proteome and ubiquitome revealed mechanism of high temperature accelerating petal abscission in tree peony

Tree peony(Paeonia suffruticosa Andrews)is a well-known ornamental plant with high economic value,but the short fluorescence is a key obstacle to its ornamental value and industry development.High temperature accelerates flower senescence and abscission,but the associated mechanisms are poorly understood.In this study,the tandem mass tag(TMT)proteome and label-free quantitative ubiquitome from tree peony cut flowers treated with 20℃for 0 h(RT0),20℃or 28℃for 60 h(RT60 or HT60)were examined based on morphological observation,respectively.Totally,6970 proteins and 1545 lysine ubiquitinated(Kub)sites in 844 proteins were identified.Hydrophilic residues(such as glutamate and aspartate)neighboring the Kub sites were in preference,and 36.01%of the Kub sites were located on the protein surface.The differentially expressed proteins(DEPs)and Kub-DEPs in HT60 vs RT60 were mainly enriched in ribosomal protein,protein biosynthesis,secondary metabolites biosynthesis,flavonoid metabolism,carbohydrate catabolism,and auxin biosynthesis and signaling revealed by GO and KEGG analysis,accompanying the increase of endogenous abscisic acid(ABA)accumulation and decrease of endogenous indoleacetic acid(IAA)level.Additionally,the expression patterns of six enzymes(SAMS,ACO,YUC,CHS,ANS and PFK)putatively with Kub modifications were analyzed by proteome and real-time quantitative RT-PCR.The cell-free degradation assays showed PsSAMS and PsACO proteins could be degraded via the 26 S proteasome system in tree peony flowers.Finally,a working model was proposed for the acceleration of flower senescence and abscission by high temperature.In summary,all results contributed to understanding the mechanism of flower senescence induced by high temperature and prolonging fluorescence in tree peony.

Study on the Response and Recovery Characteristics of Different Herbaceous Peony (<i>Paeonia lactiflora</i>Pall.) Varieties to Waterlogging Stress

The root of herbaceous peony (Paeonia lactiflora Pall.) is fleshy, and different varieties have different tolerance to waterlogging stress. In order to explore its response and recovery characteristics to waterlogging stress, six varieties of herbaceous peony with strong, medium and weak waterlogging tolerance and high ornamental value were selected as experimental materials. After the vegetative growth of each variety was completed, the field simulated waterlogging stress experiment was carried out by the semi-flooded (the water surface is half the height of the flowerpot) pot method. Changes in photosynthetic parameters, chlorophyll fluorescence parameters, relative water content (RWC), relative conductivity (REC), chlorophyll content, lutein cycle, and leaf microstructure were analyzed during recovery from waterlogging stress and stress relief. The results showed that the time of reaching the most significant difference between CK and tested varieties was different. From the beginning of stress to 60% of the leaves with symptoms, the varieties with strong and moderate waterlogging tolerance experienced longer time;the proportion of palisade tissue in leaves was larger;the maximum photochemical quantum yield (Fv/Fm) was reduced less;it could increase xanthophyll cycle and heat dissipation (NPQ) to consume excess light energy, and maintain a higher net photosynthetic rate (Pn) for normal growth of plants in a short period of time. The REC in leaves of varieties with weak waterlogging tolerance increased more, and the damage of cell membrane was more serious. After the stress was removed, all indexes recovered to different degrees. Based on our comprehensive analysis, the comprehensive waterlogging resistance of the experimental materials followed the pattern: “Lihong” > “Yangfeichuyu” > “Taohuafeixue” > “Dafugui” > “Qihualushuang” > “Hongxiuqiu”. It is suggested that the variety “Lihong” and “Yangfeichuyu”, with strong comprehensive waterlogging tolerance, can be selected for propagation and cultivation in areas prone to waterlogging. In the later stage, it is still necessary to further expand the number and scale of varieties, combined with the in-depth study of waterlogging-resistance genes, so as to provide a theoretical reference for the cultivation and production of new waterlogging-resistant varieties.

Osmotic Regulation,Antioxidant Enzyme Activities and Photosynthetic Characteristics of Tree Peony(Paeonia suffruticosa Andr.)in Response to High-Temperature Stress

Tree peony(Paeonia suffruticosa Andr.)is a traditional Chinese flower,which prefers cool weather.However,high temperature in summer in the middle and lower reaches of the Yangtze River restricts its growth and development.In this study,osmotic regulation,antioxidant enzyme activities,and photosynthetic characteristics of tree peony in response to high-temperature stress were investigated.The results showed that high-temperature stress had destroyed the cell membrane,manifested as the increased relative electrical conductivity and malondialdehyde content.Moreover,high-temperature stress led to excessive accumulation of reactive oxygen species,thereby,activating antioxidant enzyme activities.Also,photosynthetic parameters and chlorophyll fluorescence parameters directly reflected the damage to the photosystem II reflection center under high-temperature stress.In addition,high-temperature stress led to stomatal closure and chloroplast damage.This study revealed the physiological responses of tree peony to high-temperature stress,laying a foundation for the promotion of tree peony in high-temperature areas and the improvement of high-temperature resistance.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony(Paeonia lactiflora Pall.),as a high-end cut flower in the international market,has high ornamental and medicinal values.But in Northern China,drought is a major environmental factor influencing the growth and development of P.lactiflora.Quantitative real-time polymerase chain reaction(qRT-PCR)can evaluate gene expression levels under different stress conditions,and stable internal reference is the key for qRT-PCR.At present,there is no systematic screening of internal reference for correcting gene expressions of P.lactiflora in response to drought stress.In this study,10 candidate genes[ubiquitin(UBQ2),UBQ1,elongation factor 1-α(EF-1α),Histidine(His),eukaryotic initiation factor(eIF),tubulin(TUB),actin(ACT),UBQ3,ACT2,RNA polymerase II(RNA Pol II)]were chosen,and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress.Due to the difference of operation methods,the results of different analysis were distinct,and the final comprehensive analysis indicated that EF-1αwas a relatively stable internal reference gene for P.lactiflora under drought stress.Also,UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis.This study will lay a foundation for screening the key genes of P.lactiflora in response to drought stress.

Age-Related Alterations in DNA Methylation and APETALA2(AP2)Levels in Herbaceous Peony(Paeonia lactiflora Pall.)

The ornamental and commercial values of herbaceous peony(Paeonia lactiflora Pall.)are directly related to its flower pattern.However,the molecular mechanisms underlying the type formation of P.lactiflora flowers have not been studied in great detail.Previous studies identified,using integrated multipleomics analysis,revealed that APETALA2(AP2)is an important candidate gene that modulates type formation of P.lactiflora flowers.To further reveal the expression mechanism of AP2 in P.lactiflora petals,we examined the profile of AP2 expression in the inner and outer petals of‘ZiFengyu’at various developmental stages using qRT-PCR and BSP+Miseq methylation analysis.Based on our data,the AP2 levels in the outer petals were obviously increased,relative to the inner petals.In addition,the S3 levels at the bloom stage were significantly higher than at the flower-bud stage S1,thereby promoting bloom stage S2,while declining stage S4.Using chromosome walking,the 2000 bp of the 5′-end upstream promoter region was achieved.This region harbored a CpG island(−665∼−872 bp),with multiple essential transcription factor binding sites(TFBS)such as NF-kappa B,GATA-1,Sp1,and C/EBP.Methylation sequencing revealed 7 methylated CpG sites in the CpG island region of the AP2 promoter,thereinto,the methylation ratio of the CpG-3 site in the inner petals was significantly higher than in the outer petals.Correlation analysis revealed a negative association between the level of methylation(CpG-3,CpG-6),and AP2 mRNA expression.CpG-3 was located on the Sp1 transcription factor binding site.Thus,we speculated that the CpG-3 methylation may inhibit transcription factor Sp1 binding to the gene promoter,thereby regulating AP2 expression.Herein,we examined the role of AP2 in the determination of flower patterns in P.lactiflora.Our conclusion will provide theoretical guidance for the molecular breeding of the flower pattern in P.lactiflora.

Protective mechanism of Paeoniae Radix Alba against chemical liver injury based on network pharmacology,molecular docking,and in vitro experiments

Objective:To explore and validate the potential targets of Paeoniae Radix Alba(P.Radix,Bai Shao)in protecting against chemical liver injury through network pharmacology,molecular docking technology,and in vitro cell experiments.Methods:Network pharmacology was used to identify the common potential targets of P.Radix and chemical liver injury.Molecular docking was used to fit the components,which were subsequently verified in vitro.A cell model of hepatic fibrosis was established by activating hepatic stellate cell(HSC)-LX2 cells with 10 ng/mL transforming growth factor-β1.The cells were exposed to different concentrations of total glucosides of paeony(TGP),the active substance of P.Radix,and then evaluated using the cell counting kit-8 assay,enzyme-linked immunosorbent assay,and western blot.Results:Analysis through network pharmacology revealed 13 key compounds of P.Radix,and the potential targets for preventing chemical liver injury were IL-6,AKT serine/threonine kinase 1,jun protooncogene,heat shock protein 90 alpha family class A member 1(HSP90AA1),peroxisome proliferator activated receptor gamma(PPARG),PTGS2,and CASP3.Gene Ontology(GO)enrichment analysis indicated the involvement of response to drugs,membrane rafts,and peptide binding.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis revealed that the main pathways involved lipid and atherosclerosis and chemical carcinogenesis-receptor activation.Paeoniflorin and albiflorin exhibited strong affinity for HSP90AA1,PTGS2,PPARG,and CASP3.Different concentrations of TGP can inhibit the expression of COL-I,COL-III,IL-6,TNF-a,IL-1β,HSP-90a,and PTGS2 while increasing the expression of PPAR-γand CASP3 in activated HSC-LX2 cells.Conclusion:P.Radix primarily can regulate targets such as HSP90AA1,PTGS2,PPARG,CASP3.TGP,the main active compound of P.Radix,protects against chemical liver injury by reducing the inflammatory response,activating apoptotic proteins,and promoting the apoptosis of activated HSCs.

基于响应面法对皇冠牡丹(Paeonia×lemoinei‘Yellow Crown’)增殖培养基大量元素配方的优化

增殖系数低一直是制约牡丹微繁殖技术推广应用的障碍之一,但目前的研究多集中于植物生长调节剂的使用与组合,较少关注培养基配方对牡丹试管苗增殖效果的影响。本研究以皇冠牡丹(Paeonia×lemoinei‘Yellow Crown’)为试验材料,使用Design-Expert响应面法设计优化试验,探究WPM中5种大量元素[K_(2)SO_(4)、MgSO_(4)·7H_(2)O、KH_(2) PO_(4)、NH_(4) NO_(3)、Ca(NO_(3))_(2)·4H_(2)O]对皇冠牡丹试管苗增殖效果的影响,并建立数学模型分析预测最佳优化培养基配方。单因素效应分析结果表明,KH_(2) PO_(4)、NH_(4) NO_(3)与Ca(NO_(3))_(2)·4H_(2)O对牡丹试管苗的增殖效果有显著或极显著影响,而MgSO_(4)·7H_(2)O与K_(2)SO_(4)对牡丹试管苗的增殖效果无明显影响。交互作用分析结果表明,K_(2)SO_(4)与KH_(2) PO_(4)的交互作用对株高、茎长有显著或极显著影响,K_(2)SO_(4)与MgSO_(4)·7H_(2)O、KH_(2) PO_(4)与NH_(4) NO_(3)的交互作用对茎长有显著或极显著影响。根据试验数据构建模型进行分析预测,结果显示,5种大量元素优化配方为:K_(2)SO_(4)2257 mg/L、MgSO_(4)·7H_(2)O 907 mg/L、KH_(2) PO_(4)481 mg/L、NH_(4) NO_(3)200 mg/L与Ca(NO_(3))_(2)·4H_(2)O 1963 mg/L。在该条件下开展验证试验,获得试管苗的增殖系数、株高、茎长与叶片数分别为2.43、4.62 cm、1.30 cm、6.75张,略高于Design-Expert试验得出的预测值,显著优于对照组,说明本研究成功优化了皇冠牡丹的专用增殖培养基配方。研究结果不仅能大幅提高皇冠牡丹的增殖效率,也为其他牡丹品种增殖培养体系的构建与改良提供重要的技术参考。

响应面法优化牡丹花果胶提取工艺及其抗氧化活性

以丹凤牡丹花为原料,以果胶提取率为指标,在单因素试验基础上,采用响应面法优化牡丹花果胶提取工艺,并检测牡丹花果胶的体外抗氧化活性,结果显示最优工艺为:提取温度为95℃,pH值为1.5,提取时间2 h,溶剂倍数20倍,在该工艺条件下牡丹花果胶提取率为(19.06±0.18)%,与预测值几乎相当。牡丹花果胶对DPPH自由基和ABTS自由基清除率的IC50分别为(3.44±0.12)、(2.13±0.11)mg/mL,对铁离子的还原能力为(4.23±0.10)mmol TE/g。

Paeonia suffruticosa Andrews的界定,兼论栽培牡丹的分类鉴定问题

栽培牡丹一直称为PaeoniasuffruticosaAndrews。在把作丹皮用的栽培牡丹 (凤丹 )描述为新种P .ostii后 ,观赏的栽培牡丹仍然被统称为P .suffruticosa。但在P .suffruticosa复合体内 ,P .rockii,P .jis hanensis,P .qiui和P .suffruticosassp .yinpingmudan等野生类型相继被描述并得到承认。于是 ,有人提出了栽培牡丹多元发生的观点 ,同时也出现了P .suffruticosa“是牡丹组数种植物复合体”的说法。本文在详细论述P .suffruticosa复合体分类历史的基础上 ,根据我们自己对野生类型和栽培类型的考察研究 ,认为观赏栽培牡丹的形成是多元的 ,并且包含几个物种 ;P .suffruticosa有明确的概念 ,它是观赏栽培牡丹的组成部分 ,而不代表栽培牡丹的全部 ,更不是数种植物的复合体。我们认为应根据形态特征正确鉴定观赏栽培牡丹 ,不应把它们一概鉴定为P .suffruticosa。通过文献和标本引证 ,文中界定了P .suf fruticosa的范围。此外 ,把P .yinpingmudan (D .Y .Hong,K .Y .Pan&Z .W .Xie)B .A .Shen作为P .suffruticosassp.yinpingmudanD .Y .Hong,K .Y .Pan&Z .W .Xie的异名 ,并且认定P .yinpingmudan (D .Y .Hong ,K .Y .Pan&Z .W .Xie)B .A .Shenssp .henanensis(D .Y .Hong,K .Y .Pan&Z .W .Xie)B .A .Shen是不?

凤丹(Paeonia ostii T.)自毒物质的检测及其作用机制

药用牡丹的种植中存在明显的连作障碍现象,为探讨其机制,利用已萌发长根的凤丹(Paeonia ostii)种子对凤丹根际土壤和根的水浸提液进行了自毒化感检测;采用高效液相色谱法(HPLC)对种植4a的凤丹根际土壤及其根皮中的化感自毒物质进行了定性和定量分析;在此基础上,利用外源法研究了盆栽凤丹幼苗对5种检出物质(阿魏酸、肉桂酸、香草醛、香豆素和丹皮酚)及其混合物在3种不同浓度下对植株生长的影响和生理响应。结果表明:(1)连续种植4a的凤丹根际土壤和根浸提液对已萌发种子根的生长有显著的抑制作用(p<0.05),显示凤丹根分泌物具有自毒化感的潜势;(2)HPLC分析表明在凤丹根际土壤中存在5种以上的酚酸类物质(阿魏酸、肉桂酸、香草醛、香豆素和丹皮酚),其中阿魏酸、肉桂酸、香草醛、香豆素为前人已报道的化感物质;(3)在实验室条件下,所设浓度范围内,5种物质及其混合物对凤丹幼苗的高度、根长及地下和地上生物量均有明显影响,尤其对根长和地下生物量的影响最为明显(p<0.05),此结果与连作移栽幼苗时,根部首先发黑死亡的现象相一致;(4)凤丹幼苗的根系活力和叶绿素含量在各处理中表现出相似性,即低浓度时根系活力和叶绿素含量高,高浓度时根系活力和叶绿素含量低,低浓度的阿魏酸和香草醛对幼苗根系活力和叶绿素合成有促进作用。此结果显示自毒物质可能是通过影响根系酶活性和叶绿素合成来影响群体中其它个体的生长。本实验的结果显示栽培4a的凤丹土壤水提液具有抑制自身种子根系生长的活性,此提示栽培凤丹连作障碍可能主要与自身分泌到土壤中的酚酸类物质的自毒作用有关。