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Activation of hepatic stellate cells (HSCs) is critical in the progression of liver fibrosis and is a promising target for anti-hepatic fibrosis drug development. Moreover, effective pharmacological interventions targeting this pathomechanism are scarce. Our study confirms the therapeutic value of β-sitosterol, a major constituent of Ranunculus ternatus Thunb, in hepatic fibrosis and identifies its underlying mechanisms. After treatment with β-sitosterol, CCL4-induced hepatic fibrosis was reversed in mice, while inflammatory and hepatic fibrosis indices were improved. Meanwhile, we explored the molecular mechanism of β-sitosterol treatment for hepatic fibrosis and, based on RNA-seq results, found that the ameliorative effect of β-sitosterol on hepatic fibrosis was associated with the MK3 and NF-κB signalling pathways. MK3, an important kinase in the MAPK pathway, plays a role in transmitting upstream and downstream signals, whereas the NF-κB signalling pathway has been shown to be associated with HSC activation. We verified the interaction between MK3 and IκB in HSC cells using endogenous Co-IP, whereas β-sitosterol reduced the binding of MK3 to IκB and the activation of the NF-κB signalling pathway. Our findings reveal the mechanism of β-sitosterol in the treatment of liver fibrosis, suggesting that β-sitosterol may be a promising drug for the treatment of liver fibrosis and deserves further investigation.

Ranunculus japonicus Thunb. is a traditional Chinese herb. Plants in the genus Ranunculus are generally rich in flavonoids, which have antibacterial, anti-infective, and other pharmacological effects. However, owing to the lack of reference genomes, little is known about the flavonoid biosynthetic pathway in R. japonicus. In this study, PacBio isoform sequencing (PacBio iso-seq) and DNA nanoball sequencing (DNB-seq) were combined to build a full-length transcriptome database for three different tissues of R. japonicus. A total of 395,402 full-length transcripts were obtained, of which 308,474 were successfully annotated. A Kyoto Encyclopedia of Genes and Genomes analysis identified 29 differentially expressed genes encoding nine key enzymes for flavonoid biosynthesis. Correlation analysis indicated that flavanone 3-hydroxylase and flavonol synthase genes might have key roles in the accumulation of flavonoid substances in the different tissues of R. japonicus. The structures of chalcone synthase and chalcone isomerase enzymes were spatially modeled. Reverse-transcription quantitative PCR was used to verify gene expression levels of key enzymes associated with flavonoid biosynthesis. In addition, 22 MYB transcription factors involved in flavonoid biosynthesis and phenylpropanoid biosynthesis were discovered. The reliable transcriptomic data from this study provide genetic information about R. japonicus as well as insights into the molecular mechanism of flavonoid biosynthesis. The results also provide a basis for developing the medicinal value R. japonicus., (© 2023 Scandinavian Plant Physiology Society.)

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation., (© 2022 Wiley Periodicals LLC.)

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Temperature stress can affect phenotypic plasticity, reproduction, and epigenetic profiles, which may affect acclimation and adaptation. Distribution patterns suggest that polyploidy seems to be advantageous under cold conditions. Nevertheless, whether temperature stress can induce gene expression changes in different cytotypes, and how the response is initialized through gene set pathways and epigenetic control remain vague for non-model plants. The perennial alpine plant Ranunculus kuepferi was used to investigate the effect of cold stress on gene expression profiles. Diploid and autotetraploid individuals were exposed to cold and warm conditions in climate growth chambers and analyzed via transcriptome sequencing and qRT-PCR. Overall, cold stress changed gene expression profiles of both cytotypes and induced cold acclimation. Diploids changed more gene set pathways than tetraploids, and suppressed pathways involved in ion/cation homeostasis. Tetraploids mostly activated gene set pathways related to cell wall and plasma membrane. An epigenetic background for gene regulation in response to temperature conditions is indicated. Results suggest that perennial alpine plants can respond to temperature extremes via altered gene expression. Tetraploids are better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps.

The leaves of Ranunculus multifidus Forsk. are traditionally used for the treatment of malaria in several African countries. In the present study, 80% methanol (RM-M) and hydrodistilled (RM-H) extracts of fresh leaves from R. multifidus and its major constituent anemonin were tested for their in vivo antimalarial activity against Plasmodium berghei in mice. Anemonin was also tested for its in vitro antimycobacterial activity against Mycobacterium smegmatis and M. abscessus in a microbroth dilution assay, and bacterial growth was analyzed by OD measurement. The isolation of anemonin from RM-H was carried out using preparative thin layer chromatography (PTLC). The chemical structures of anemonin and its hydrolysis product were elucidated using spectroscopic methods (HR-MS; 1D and 2D-NMR). Results of the study revealed that both RM-M and RM-H were active against P. berghei in mice, although the latter demonstrated superior activity ( p < 0.001), as compared to the former. At a dose of 35.00 mg/kg/day, RM-H demonstrated a chemosuppression value of 70% in a 4-day suppressive test. In a 4-day suppressive, Rane's and prophylactic antimalarial tests, anemonin showed median effective doses (ED 50 s) of 2.17, 2.78 and 2.70 μM, respectively. However, anemonin did not inhibit the growth of M. smegmatis and M. abscessus .

Anthocyanin biosynthesis is one of the best studied secondary metabolisms. However, related pathways were generally concluded based on anthocyanin components; most studies focused on the backbone forming of anthocyanidins (cyanidin, delphinidin, and pelargonidin) of model or commercial plants, while anthocyanin modification was less discussed, and non-model plants with abundant colorations were less researched either. Ranunculus asiaticus L. has great diversity in flower colorations, not only indicating its value in researching anthocyanin biosynthesis but also implying it is unique in this regard. Based on transcriptome sequencing and gene annotation of three varieties (10 samples) of Ranunculus asiaticus L., 176 unigenes from 151,136 unigenes were identified as involved in anthocyanin biosynthesis, among which, 74 unigenes were related to anthocyanin modification; 61 unigenes were responsible for glycosylation at C3 and C5 with 3-monosaccharides of glucose, 3-biosides of rutinose, sophorose, or sambubiose to form 3Gly-, 3Gly5Gly-, 3Gly3'Gly-, 3Gly2''Gly-, 3Gly2''Xly-, 3Gly2''Rly-glycosylated anthocyanins, etc.; 2 unigenes transferred -CH 3 ; 11 unigenes of BAHD family catalyzd the aromatic or malonyl acylation at 6'' / 6''''position of 3/5-O-glucoside. Based on gene composition, a putative pathway was established. The pathway was validated by flower colorations, and gene expression patterns where F3H, F3'H, 3GT, 5GT, and FMT2 were highly expressed in varieties colored as lateritious and carmine, while variety with purple flowers had high expression of F3'5'H and 3MAT. In view of anthocyanin biosynthesis pathway of Ranunculus asiaticus L., great diversity in its flower colorations was illustrated via the complete branches (F3H, F3'H and F3'5'H) as well as complete modifications (glycosylation, methylation, and acylation), and besides, via the higher percentage of C3 glycosylation than C5 glycosylation.

Sampling for DUS test of flower colors should be fixed at the stages and sites that petals are fully colored, and besides, flower colorations are uniform among individuals and stable for a period of time to allow testers to get consistent results. It remains a problem since spatial and temporal flower colorations are reported a lot but their change traits are little discussed. In this study, expression state, uniformity and stability of color phenotypes, anthocyanin contents, and gene expression levels were taken into account based on measurements at 12 development stages and three layers (inner, middle, and outer petals) of two varieties of Ranunculus asiaticus L. to get their best sampling. Our results showed that, outer petals of L9⁻L10 (stage 9⁻stage 10 of variety 'Jiaoyan zhuanhong') and C5⁻C6 (stage 5⁻stage 6 of variety 'Jiaoyan yanghong') were the best sampling, respectively. For DUS test, it is suggested to track flower colorations continuously to get the best sampling as well as representative colors since different cultivars had different change traits, and moreover, full expression of color phenotypes came later and lasted for a shorter duration than those of anthocyanin contents and gene expressions. Our innovation exists in following two points. Firstly, a model of change dynamic was introduced to illustrate the change traits of flower colorations, anthocyanin contents, and gene expressions. Secondly, genes used for expression analysis were screened on account of tentative anthocyanins, which were identified based on comparison between liquid chromatography⁻mass spectrometry (LC⁻MS) results and molecular mass and mass fragment pattern (M²) of each putative anthocyanin and their fragments deduced in our previous study. Gene screening in this regard may also be interest for other non-model plant genera with little molecular background.

Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.

This study investigated the growth response of Ranunculus sceleratus to pollution and its capacity to accumulate trace metals for its use as a phytoremediator in Lake Maruit, Egypt. Three basins (main basin, fish farm, and southwestern basin) representing the natural distribution of the plant as well as the pollution loads in the lake, were chosen for collecting plant and sediment samples. In each basin ten quadrats (0.5 m × 0.5 m), distributed equally along two sites, were selected for measuring growth parameters, nutrients, and trace metal concentration. The highest biomass of the shoot and root (610 and 236 g m -2 ) was recorded in the main basin and fish farm, respectively. R. sceleratus accumulated high concentrations of Cu and Pb (27.7 and 9.9 mg kg -1 ), while toxic concentration of Mn (2508.0 mg kg -1 ) in their roots compared to shoots. The bioaccumulation factor (BF) for the investigated metals was greater than one, and in the decreasing order: Ni (27.1) > Zn (20.0) > Cd (16.4) > Cu (7.7) > Mn (3.9) > Pb (3.6). The translocation factor of all analyzed trace metals was less than one. The ability of R. sceleratus to accumulate Mn, Ni, Cu, and Pb in its roots indicates the potential use of this species for phytostabilization of these metals (mainly Mn) in contaminated water bodies.

Buttercup ( Ranunculus spp.) flowers are exceptional because they feature a distinct gloss (mirror-like reflection) in addition to their matte-yellow coloration. We investigated the optical properties of yellow petals of several Ranunculus and related species using (micro)spectrophotometry and anatomical methods. The contribution of different petal structures to the overall visual signal was quantified using a recently developed optical model. We show that the coloration of glossy buttercup flowers is due to a rare combination of structural and pigmentary coloration. A very flat, pigment-filled upper epidermis acts as a thin-film reflector yielding the gloss, and additionally serves as a filter for light backscattered by the strongly scattering starch and mesophyll layers, which yields the matte-yellow colour. We discuss the evolution of the gloss and its two likely functions: it provides a strong visual signal to insect pollinators and increases the reflection of sunlight to the centre of the flower in order to heat the reproductive organs., (© 2017 The Author(s).)

Foliar ionome, photosystem II activity, and leaf growth parameters of Ranunculus acris L., a potential biomonitor of trace element (TE) contamination and phytoavailability, were assessed using two riverbank soil series. R. acris was cultivated on two potted soil series obtained by mixing a TE (Cd, Cu, Pb, and Zn)-contaminated technosol with either an uncontaminated sandy riverbank soil (A) or a silty clay one slightly contaminated by TE (B). Trace elements concentrations in the soil-pore water and the leaves, leaf dry weight (DW) yield, total leaf area (TLA), specific leaf area (SLA), and photosystem II activity were measured for both soil series after a 50-day growth period. As soil contamination increased, changes in soluble TE concentrations depended on soil texture. Increase in total soil TE did not affect the leaf DW yield, the TLA, the SLA, and the photosystem II activity of R. acris over the 50-day exposure. The foliar ionome did not reflect the total and soluble TE concentrations in both soil series. Foliar ionome of R. acris was only effective to biomonitor total and soluble soil Na concentrations in both soil series and total and soluble soil Mo concentrations in the soil series B.

Adaptation to aquatic habitats is a formidable challenge for terrestrial angiosperms that has long intrigued scientists. As part of a suite of work to explore the molecular mechanism of adaptation to aquatic habitats, we here sequenced the transcriptome of the submerged aquatic plant Ranunculus bungei, and two terrestrial relatives R. cantoniensis and R. brotherusii, followed by comparative evolutionary analyses to determine candidate genes for adaption to aquatic habitats. We obtained 126,037, 140,218 and 114,753 contigs for R. bungei, R. cantoniensis and R. brotherusii respectively. Bidirectional Best Hit method and OrthoMCL method identified 11,362 and 8,174 1:1:1 orthologous genes (one ortholog is represented in each of the three species) respectively. Non-synonymous/synonymous (dN/dS) analyses were performed with a maximum likelihood method and an approximate method for the three species-pairs. In total, 14 genes of R. bungei potentially involved in the adaptive transition from terrestrial to aquatic habitats were identified. Some of the homologs to these genes in model plants are involved in vacuole protein formation, regulating 'water transport process' and 'microtubule cytoskeleton organization'. Our study opens the door to understand the molecular mechanism of plant adaptation from terrestrial to aquatic habitats.

Introduction: The copper bioaccumulation by the floating Lemna minor and by the completely submerged Ranunculus tricophyllus as a function of exposure time and copper concentration was studied, with the aim of proposing these species as environmental biosensors of the water pollution., Results: The results show that both these aquatic angiosperms are good indicators of copper pollution because the copper uptake is the only function of metal concentration (water pollution)., Conclusion: Uptake behavior is reported as a function of the time and concentration, based on the results of a 3-year study. Kinetic evaluations are proposed.

Growing evidence suggests that the freely accessible pollen of some plants is chemically protected against pollen-feeding flower visitors. For example, a diet of pollen from buttercup plants (Ranunculus) recently was shown to have a deleterious effect on developing larvae of several bee species not specialized on Ranunculus. Numerous Ranunculus species contain ranunculin, the glucosyl hydrate form of the highly reactive and toxic lactone protoanemonin, that causes the toxicity of these plants. We tested whether the presence of ranunculin is responsible for the lethal effects of R. acris pollen on the larvae of two bee species that are not Ranunculus specialists. To investigate the effect on bee larval development, we added ranunculin to the pollen provisions of the Campanula specialist bee Chelostoma rapunculi and the Asteraceae specialist bee Heriades truncorum, and allowed the larvae to feed on these provisions. We quantified ranunculin in pollen of R. acris and in brood cell provisions collected by the Ranunculus specialist bee Chelostoma florisomne. We demonstrated that although ranunculin was lethal to both tested bee species in high concentrations, the concentration in the pollen of R. acris was at least fourfold lower than that tolerated by the larvae of C. rapunculi and H. truncorum in the feeding experiments. Ranunculin concentration in the brood cells of C. florisomne was on average even twentyfold lower than that in Ranunculus pollen, suggesting that a mechanism different from ranunculin intoxication accounts for the larval mortality reported for bees not specialized on Ranunculus pollen.

The biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and their stability have been reported in this paper. The aqueous catclaw buttercup was used as mild reducing agent for gold nanoparticles synthesis from HAuCl4 solutions. The influence of reaction time, temperature and mass ratio of HAuCl4/catclaw buttercup were evaluated to investigate their effects on gold nanoparticles synthesis. Under the optimized reaction parameters, the gold nanoparticles obtained are characterized by UV-vis spectrum, X-ray diffraction (XRD), EDAX technique (EDX), high resolution transmission electron microscopy (HRTEM), FTIR spectrum, anthrone-sulfuric colorimetric method, and plus Improved-Lowry Protein Assay Kit. The HRTEM images showed that the biosynthesized gold nanoparticles are mostly spherical with size range from 9-24 nm. Furthermore, it was found that the biosynthesized gold nanoparticles possessed outstanding colloid stability in aqueous solutions as a function of category and concentration of monovalent salt and pH value of the solution when compared with chemosynthetic ones with the similar size. Anthrone-sulfuric colorimetric method revealed that there is no sugar in the biosynthesized gold colloid. While Improved-Lowry tests results demonstrated that the existence of much protein in the biosynthesized gold colloid, which may played an important role in stabilization of it. Owing to their stability, biocompatibility, lower cost and so on, gold nanoparticles synthesized by this biosynthesis method show potential application prospect in optoelectronic and biomedicine.

The significance of total glutathione content was investigated in two alpine plant species with highly differing antioxidative scavenging capacity. Leaves of Soldanella alpina and Ranunculus glacialis incubated for 48 h in the presence of buthionine-sulfoximine had 50% lower glutathione contents when compared with leaves incubated in water. The low leaf glutathione content was not compensated for by activation of other components involved in antioxidative protection or electron consumption. However, leaves with normal but not with low glutathione content increased their ascorbate content during high light (HL) treatment (S. alpina) or catalase activity at low temperature (LT) (R. glacialis), suggesting that the mere decline of the leaf glutathione content does not act as a signal to ameliorate antioxidative protection by alternative mechanisms. CO(2)-saturated oxygen evolution was not affected in glutathione-depleted leaves at various temperatures, except at 35°C, thereby increasing the high temperature (HT) sensitivity of both alpine species. Leaves with low and normal glutathione content were similarly resistant to photoinhibition and photodamage during HL treatment at ambient temperature in the presence and absence of paraquat or at LT. However, HL- and HT-induced photoinhibition increased in leaves with low compared to leaves with normal glutathione content, mainly because the recovery after heat inactivation was retarded in glutathione-depleted leaves. Differences in the response of photosystem II (PSII) activity and CO(2)-saturated photosynthesis suggest that PSII is not the primary target during HL inactivation at HT. The results are discussed with respect to the role of antioxidative protection as a safety valve for temperature extremes to which plants are not acclimated., (Copyright © Physiologia Plantarum 2011.)

Identification of toxic or harmful agents continues to be a key goal in agricultural chemistry. This paper reports a metabolomic analysis of Ranunculus repens and related species, which were recently postulated to be cocausative agents in the etiology of equine grass sickness (EGS). Specifically, samples collected at EGS sites were compared with those from non-EGS sites. Furthermore, interspecific and seasonal variations and the species' response to edaphic and climatic factors were investigated. (1)H NMR spectroscopy in combination with multivariate data analysis was applied to the crude methanol extracts of the Ranunculus samples, as well as their chloroform fractions. Samples from EGS sites were significantly different from control samples. The metabolite composition varied greatly between different Ranunculus species. No significant changes could be observed between samples collected in different seasons. This work provides strong evidence that Ranunculus is involved in the etiology of EGS and has implications for agricultural management of pastures.

Very little is known about the primary carbon metabolism of the high mountain plant Ranunculus glacialis. It is a species with C3 photosynthesis, but with exceptionally high malate content in its leaves, the biological significance of which remains unclear. 13C/12C-isotope ratio mass spectrometry (IRMS) and 13C-nuclear magnetic resonance (NMR) labelling were used to study the carbon metabolism of R. glacialis, paying special attention to respiration. Although leaf dark respiration was high, the temperature response had a Q10 of 2, and the respiratory quotient (CO2 produced divided by O2 consumed) was nearly 1, indicating that the respiratory pool is comprised of carbohydrates. Malate, which may be a large carbon substrate, was not respired. However, when CO2 fixed by photosynthesis was labelled, little labelling of the CO2 subsequently respired in the dark was detected, indicating that: (i) most of the carbon recently assimilated during photosynthesis is not respired in the dark; and (ii) the carbon used for respiration originates from (unlabelled) reserves. This is the first demonstration of such a low metabolic coupling of assimilated and respired carbon in leaves. The biological significance of the uncoupling between assimilation and respiration is discussed.

The annual developmental cycle of tuberous roots of Ranunculus asiaticus was studied with respect to structure and content of their cells, to understand how these roots are adapted to desiccation, high temperature and rehydration. Light microscopy, histochemical analysis, and protein analyses by SDS-PAGE were employed at eight stages of annual root development. During growth and maturation of the roots, cortical cells increased in size and their cell walls accumulated pectin materials in a distinct layer to the inside of the primary walls, with pits between adjoining cells. The number of starch granules and protein bodies also increased within the cells. Several discrete proteins accumulated. Following quiescence and rehydration of the roots there was a loss of starch and proteins from the cells, and cell walls decreased in thickness. The resurrection geophyte R. asiaticus possesses desiccation-tolerant annual roots. They store carbon and nitrogen reserves within their cells, and pectin within the walls to support growth of the plant following summer quiescence and rehydration., (Copyright New Phytologist (2005).)

[1-14C]Linolenic acid was incubated with homogenates of leaves from the aquatic plants Ranunculus lingua (greater spearwort) or R. peltatus (pond water-crowfoot). Analysis by reversed-phase high-performance liquid radiochromatography demonstrated the formation of a new divinyl ether FA, i.e., 12-[1'(E),3'(Z)-hexadienyloxyl-9(Z), 11 (Z)-dodecadienoic acid [11 (Z)-etherolenic acid] as well as a smaller proportion of omega5(Z)-etherolenic acid previously identified in terrestrial Ranunculus plants. The same divinyl ethers were formed upon incubation of 13(S)-hydroperoxy-9(Z),11 (E),15(Z)-octadecatrienoic acid, a lipoxygenase metabolite of linolenic acid, whereas the isomeric hydroperoxide, 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid, was not converted into divinyl ethers in R. lingua or R. peltatus. Incubation of [1-14C]linoleic acid or 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid produced the divinyl ether 12-[1'(E)-hexenyloxyl-9(Z),11(Z)-dodecadienoic acid [11(Z)-etheroleic acid] and a smaller amount of omega5(Z)-etheroleic acid. The experiments demonstrated the existence in R. lingua and R. peltatus of a divinyl ether synthase distinct from those previously encountered in higher plants and algae.