Topical rhubarb charcoal-crosslinked chitosan/silk fibroin sponge scaffold for the repair of diabetic ulcers improves hepatic lipid deposition in db/db mice via the AMPK signalling pathway.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is closely linked to metabolic syndrome, characterised by insulin resistance, hyperglycaemia, abnormal lipid metabolism, and chronic inflammation. Diabetic ulcers (DUs) comprise consequential complications that arise as a result of T2DM. To investigate, db/db mice were used for the disease model. The findings demonstrated that a scaffold made from a combination of rhubarb charcoal-crosslinked chitosan and silk fibroin, designated as RCS/SF, was able to improve the healing process of diabetic wounds in db/db mice. However, previous studies have primarily concentrated on investigating the impacts of the RSC/SF scaffold on wound healing only, while its influence on the entire body has not been fully elucidated. MATERIAL AND METHODS: The silk fibroin/chitosan sponge scaffold containing rhubarb charcoal was fabricated in the present study using a freeze-drying approach. Subsequently, an incision with a diameter of 8 mm was made on the dorsal skin of the mice, and the RCS/SF scaffold was applied directly to the wound for 14 days. Subsequently, the impact of RCS/SF scaffold therapy on hepatic lipid metabolism was assessed through analysis of serum and liver biochemistry, histopathology, quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blotting. RESULTS: The use of the RCS/SF scaffold led to an enhancement in the conditions associated with serum glucolipid metabolism in db/db mice. An assessment of hepatic histopathology further confirmed this enhancement. Additionally, the qRT-PCR analysis revealed that treatment with RCS/SF scaffold resulted in the downregulation of genes associated with fatty acid synthesis, fatty acid uptake, triglyceride (TG) synthesis, gluconeogenesis, and inflammatory factors. Moreover, the beneficial effect of the RCS/SF scaffold on oxidative stress was shown by assessing antioxidant enzymes and lipid peroxidation. Additionally, the network pharmacology analysis verified that the adenosine monophosphate-activated protein kinase (AMPK) signalling pathway had a vital function in mitigating non-alcoholic fatty liver disease (NAFLD) by utilizing R. officinale. The measurement of AMPK, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FASN), and acetyl CoA carboxylase (ACC) gene and protein expression provided support for this discovery. Furthermore, the molecular docking investigations revealed a robust affinity between the active components of rhubarb and the downstream targets of AMPK (SREBP1 and FASN). CONCLUSION: By regulating the AMPK signalling pathway, the RCS/SF scaffold applied topically effectively mitigated hepatic lipid accumulation, decreased inflammation, and attenuated oxidative stress. The present study, therefore, emphasises the crucial role of the topical RCS/SF scaffold in regulating hepatic lipid metabolism, thereby confirming the concept of "external and internal reshaping".

Herb pair of Rhubarb-Astragalus mitigates renal interstitial fibrosis through downregulation of autophagy via p38-MAPK/TGF-ß1 and p38-MAPK/smad2/3 pathways.

BACKGROUND: Chronic kidney disease (CKD) has a high incidence and poor prognosis; however, no effective treatment is currently available. Our previous study found that the improvement effect of the herb pair of Rhubarb-Astragalus on CKD is likely related to the inhibition of the TGF-ß1/p38-MAPK pathway. In the present study, a p38-MAPK inhibitor was used to further investigate the inhibitory effect of Rhubarb-Astragalus on the TGF-ß1/p38-MAPK pathway and its relationship with autophagy. METHODS: A rat model of unilateral ureteral obstruction (UUO) was established, and a subgroup of rats was administered Rhubarb-Astragalus. Renal function and renal interstitial fibrosis (RIF) were assessed 21 d after UUO induction. In vitro, HK-2 cells were treated with TGF-ß1 and a subset of cells were treated with Rhubarb-Astragalus or p38-MAPK inhibitor. Western blotting, immunohistochemistry, and qRT-PCR analyses were used to detect the relevant protein and mRNA levels. Transmission electron microscopy was used to observe autophagosomes. RESULTS: Rhubarb-Astragalus treatment markedly decreased the elevated levels of blood urea nitrogen, serum creatinine, and urinary N-acetyl-ß-D-glucosaminidase; attenuated renal damage and RIF induced by UUO; and reduced the number of autophagosomes and lysosomes in UUO-induced renal tissues. Additionally, Rhubarb-Astragalus reduced the protein and mRNA levels of α-SMA, collagen I, LC3, Atg3, TGF-ß1, p38-MAPK, smad2/3, and TAK1 in renal tissues of UUO rats. Rhubarb-Astragalus also reduced protein and mRNA levels of these indicators in vitro. Importantly, the effect of the p38-MAPK inhibitor was similar to that of Rhubarb-Astragalus. CONCLUSIONS: Rhubarb-Astragalus improves CKD possibly by downregulating autophagy via the p38-MAPK/TGF-ß1 and p38-MAPK/smad2/3 pathways.

Neuroprotection of rhubarb extract against cerebral ischaemia-reperfusion injury via the gut-brain axis pathway.

BACKGROUND: The gut-brain axis (GBA) plays a central role in cerebral ischaemia-reperfusion injury (CIRI). Rhubarb, known for its purgative properties, has demonstrated protective effects against CIRI. However, it remains unclear whether this protective effect is achieved through the regulation of the GBA. AIM: This study aims to investigate the mechanism by which rhubarb extract improves CIRI by modulating the GBA pathway. METHODS: We identified the active components of rhubarb extract using LC-MS/MS. The model of middle cerebral artery occlusion (MCAO) was established to evaluate the effect of rhubarb extract. We conducted 16S rDNA sequencing and untargeted metabolomics to analyze intestinal contents. Additionally, we employed HE staining, TUNEL staining, western blot, and ELISA to assess intestinal barrier integrity. We measured the levels of inflammatory cytokines in serum via ELISA. We also examined blood-brain barrier (BBB) integrity using Evans blue (EB) penetration, transmission electron microscopy (TEM), western blot, and ELISA. Neurological function scores and TTC staining were utilized to evaluate neurological outcomes. RESULTS: We identified twenty-six active components in rhubarb. Rhubarb extract enhanced α-diversity, reduced the abundance of Enterobacteriaceae, and partially rectified metabolic disorders in CIRI rats. It also ameliorated pathological changes, increased the expressions of ZO-1, Occludin, and Claudin 1 in the colon, and reduced levels of LPS and d-lac in serum. Furthermore, it lowered the levels of IL-1ß, IL-6, IL-10, IL-17, and TNF-α in serum. Rhubarb extract mitigated BBB dysfunction, as evidenced by reduced EB penetration and improved hippocampal microstructure. It upregulated the expressions of ZO-1, Occludin, Claudin 1, while downregulating the expressions of TLR4, MyD88, and NF-κB. Similarly, rhubarb extract decreased the levels of IL-1ß, IL-6, and TNF-α in the hippocampus. Ultimately, it reduced neurological function scores and cerebral infarct volume. CONCLUSION: Rhubarb effectively treats CIRI, potentially by inhibiting harmful bacteria, correcting metabolic disorders, repairing intestinal barrier function, alleviating BBB dysfunction, and ultimately improving neurological outcomes.

Inulin increases the beneficial effects of rhubarb supplementation on high-fat high-sugar diet-induced metabolic disorders in mice: impact on energy expenditure, brown adipose tissue activity, and microbiota.

Consumption of prebiotics and plant-based compounds have many beneficial health effects through modulation of gut microbiota composition and are considered as promising nutritional strategy for the treatment of metabolic diseases. In the present study, we assessed the separated and combined effects of inulin and rhubarb on diet-induced metabolic disease in mice. We showed that supplementation with both inulin and rhubarb abolished the total body and fat mass gain upon high-fat and high-sucrose diet (HFHS) as well as several obesity-associated metabolic disorders. These effects were associated with increased energy expenditure, lower whitening of the brown adipose tissue, higher mitochondria activity and increased expression of lipolytic markers in white adipose tissue. Despite modifications of intestinal gut microbiota and bile acid compositions by inulin or rhubarb alone, combination of both inulin and rhubarb had minor additional impact on these parameters. However, the combination of inulin and rhubarb increased the expression of several antimicrobial peptides and higher goblet cell numbers, thereby suggesting a reinforcement of the gut barrier. Together, these results suggest that the combination of inulin and rhubarb in mice potentiates beneficial effects of separated rhubarb and inulin on HFHS-related metabolic disease and could be considered as nutritional strategy for the prevention and treatment of obesity and related pathologies.

Transcriptome profiles of yellowish-white and fuchsia colored flowers in the Rheum palmatum complex reveal genes related to color polymorphism.

Flower color variation is ubiquitous in many plant species, and several studies have been conducted to elucidate the underlying molecular mechanism. There are two flower color variants (yellowish-white and fuchsia) in the Rheum palmatum complex, however, few studies have investigated this phenomenon. Here, we used transcriptome sequencing of the two color variants to shed light on the molecular and biochemical basis for these color morphs. Comparison of the two transcriptomes identified 9641 differentially expressed unigenes (DEGs), including 6477 up-regulated and 3163 down-regulated genes. Functional analyses indicated that several DEGs were related to the anthocyanin biosynthesis pathway, and the expression profiles of these DEGs were coincident with the qRT-PCR validation results, indicating that expression levels of structural genes have a profound effect on the color variation in the R. palmatum complex. Our results suggested that the interaction of transcription factors (MYB, bHLH and WRKY) also regulated the anthocyanin biosynthesis in the R. palmatum complex. Estimation of selection pressures using the dN/dS ratio showed that 1106 pairs of orthologous genes have undergone positive selection. Of these positively selected genes, 21 were involved in the anthocyanin biosynthetic pathway, indicating that they may encode the proteins for structural alteration and affect flower color in the R. palmatum complex.

Network pharmacology and molecular docking technology-based predictive study of the active ingredients and potential targets of rhubarb for the treatment of diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes and the main cause of end-stage renal failure. Rhubarb is a widely used traditional Chinese herb, and it has exhibited efficacy in reducing proteinuria, lowering blood sugar levels and improving kidney function in patients with DN. However, the exact pharmacological mechanism by rhubarb improves DN remain unclear due to the complexity of its ingredients. Hence, we systematically explored the underlying mechanisms of rhubarb in the treatment of DN. We adopted a network pharmacology approach, focusing on the identification of active ingredients, drug target prediction, gene collection, Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment. Molecular docking technology was used to verify the binding ability between the main active compounds and central therapeutic targets, and screen out the core active ingredients in rhubarb for the treatment of DN. Finally, molecular dynamics simulation was performed for the optimal core protein-ligand obtained by molecular docking using GROMACS software. The network analysis identified 16 active compounds in rhubarb that were linked to 37 possible therapeutic targets related to DN. Through protein-protein interaction analysis, TP53, CASP8, CASP3, MYC, JUN and PTGS2 were identified as the key therapeutic targets. By validation of molecular docking, finding that the central therapeutic targets have good affinities with the main active compounds of rhubarb, and rhein, beta-sitosterol and aloe-emodin were identified as the core active ingredients in rhubarb for the treatment of DN. Results from molecular dynamics simulations showed that TP53 and aloe-emodin bound very stably with a binding free energy of - 26.98 kcal/mol between the two. The results of the gene enrichment analysis revealed that the PI3K-Akt signalling pathway, p53 signalling pathway, AGE-RAGE signalling pathway and MAPK signalling pathway might be the key pathways for the treatment of DN, and these pathways were involved in podocyte apoptosis, glomerular mesangial cell proliferation, inflammation and renal fibrosis. Based on the network pharmacology approach and molecular docking technology, we successfully predicted the active compounds and their respective targets. In addition, we illustrated the molecular mechanisms that mediate the therapeutic effects of rhubarb against DN. These findings provided an important scientific basis for further research of the mechanism of rhubarb in the treatment of DN.

Rhubarb free anthraquinones improved mice nonalcoholic fatty liver disease by inhibiting NLRP3 inflammasome.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases and has become a huge public health issue worldwide. Inhibition of nucleotide oligomerization domain-like receptors containing pyrin domain 3 (NLRP3) inflammasome is a potential therapeutic strategy for NAFLD. Currently, there are no drugs targeting NLRP3 inflammasome for clinical treatment of NAFLD. In this study, we explored the efficacy and mechanism of rhubarb free anthraquinones (RFAs) in treating NAFLD by inhibiting NLRP3 inflammasome. METHODS: First, NLRP3 inflammasome was established in mouse bone marrow-derived macrophages (BMDMs), Kuffer cells and primary hepatocytes stimulated by lipopolysaccharide (LPS) and inflammasome inducers to evaluate the effect of RFAs on inhibiting NLRP3 inflammasome and explore the possible mechanism. Further, Mice NAFLD were established by methionine and choline deficiency diet (MCD) to verify the effect of RFAs on ameliorating NAFLD by inhibiting NLRP3 inflammasome. RESULTS: Our results demonstrated that RFAs including rhein/diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone inhibited interleukin-1 beta (IL-1ß) but had no effect on tumor necrosis factor-alpha (TNF-α). Similar results were also showed in mouse primary hepatocytes and Kuffer cells. RFAs inhibited cleavage of caspase-1, formation of apoptosis-associated speck-like protein containing a CARD (ASC) speck, and the combination between NLRP3 and ASC. Moreover, RFAs improved liver function, serum inflammation, histopathological inflammation score and liver fibrosis. CONCLUSIONS: RFAs including rhein/diacerein, emodin, aloe emodin and 1,8-dihydroxyanthraquinone ameliorated NAFLD by inhibiting NLRP3 inflammasome. RFAs might be a potential therapeutic agent for NAFLD.

Regulation of the Bud Dormancy Development and Release in Micropropagated Rhubarb 'Malinowy'.

Culinary rhubarb is a vegetable crop, valued for its stalks, very rich in different natural bioactive ingredients. In commercial rhubarb stalk production, the bud dormancy development and release are crucial processes that determine the yields and quality of stalks. To date, reports on rhubarb bud dormancy regulation, however, are lacking. It is known that dormancy status depends on cultivars. The study aimed to determine the dormancy regulation in a valuable selection of rhubarb 'Malinowy'. Changes in carbohydrate, total phenolic, endogenous hormone levels, and gene expression levels during dormancy development and release were studied in micropropagated rhubarb plantlets. Dormancy developed at high temperature (25.5 °C), and long day. Leaf senescence and dying were consistent with a significant increase in starch, total phenolics, ABA, IAA and SA levels. Five weeks of cooling at 4 °C were sufficient to break dormancy, but rhizomes stored for a longer duration showed faster and more uniformity leaf growing, and higher stalk length. No growth response was observed for non-cooled rhizomes. The low temperature activated carbohydrate and hormone metabolism and signalling in the buds. The increased expression of AMY3, BMY3, SUS3, BGLU17, GAMYB genes were consistent with a decrease in starch and increase in soluble sugars levels during dormancy release. Moreover, some genes (ZEP, ABF2, GASA4, GA2OX8) related to ABA and GA metabolism and signal transduction were activated. The relationship between auxin (IAA, IBA, 5-Cl-IAA), and phenolic, including SA levels and dormancy status was also observed.

Bacterial Inoculant and Sucrose Amendments Improve the Growth of Rheum palmatum L. by Reprograming Its Metabolite Composition and Altering Its Soil Microbial Community.

Rheum palmatum L. is an important traditional Chinese medicinal herb now in demand worldwide. Recently, the theoretical framework suggested that sucrose triggers colonization of PGPM (plant growth-promoting microbes) in the rhizosphere, but their interactions on the plant remain largely unknown. Here, we applied three concentrations of both Bacillus amyloliquefaciens EZ99 inoculant (1.0 × 105, 1.0 × 106, and 1.0 × 107 colony-forming units (CFU)/mL, denoted as LB, MB, and HB, respectively) and sucrose (0.15, 1.5, and 15 g/L, denoted as LS, MS, and HS, respectively) to investigate their co-effects on R. palmatum in a field experiment. The results showed that LB + MS (1.0 × 105 CFU/mL Bacillus + 1.5 g/L sucrose) and LB + LS (1.0 × 105 CFU/mL Bacillus + 0.15 g/L sucrose) treatments significantly increased root fresh weight (p ≤ 0.05). Metabolite analysis revealed that the treatment LB + LS significantly increased the relative content of major active components in rhubarb, namely anthraquinones and phenolic compounds, by 1.5% and 2.3%. Although high sucrose addition increased the activities of certain soil enzymes, the LB + LS treatment significantly increased total potassium (TK), whereas it decreased available potassium (AK), which facilitated the potassium utilization in rhizosphere soil. Furthermore, rhizosphere microbiomes revealed that fungal diversity was augmented in LB + LS treatment, in which the common causative fungal pathogen Fusarium spp. showed an effective suppression. Additionally, the redundancy analysis and Spearman correlations revealed a positive relationship of Sphingomonas associated with change in potassium bioavailability. Altogether, our findings suggest that the combined application of a bacterial inoculant and sucrose can improve the growth and quality of R. palmatum, and stimulate uptake of plant nutrients that contribute to alter the microbial community for biocontrol potential. Hence, this work not only has broad application prospects across economical plants, but also emphasizes agroecological practices for sustainable agriculture.

Rhubarb Enema Increasing Short-Chain Fatty Acids that Improves the Intestinal Barrier Disruption in CKD May Be Related to the Regulation of Gut Dysbiosis.

The incidence of CKD seriously endangers people's health. Researchers have proposed that improving the intestinal barrier damage in CKD may be an effective target for delaying the progression of CKD. Rhubarb can effectively improve the intestinal barrier and renal fibrosis, which may be related to the regulation of gut dysbiosis, but the mechanism needs to be further studied. Short-chain fatty acids (SCFAs) are important metabolites of the gut microbiota and play an important role in maintaining the intestinal barrier. The purpose of this study was to investigate whether rhubarb enema regulates the production of short-chain fatty acid-related gut microbiota and improves the intestinal barrier damage of CKD. 5/6 nephrectomy rats were used as the animal model, sevelamer was used as the positive control group, and the sham operation rats were used as the control group. After 4 weeks of enema treatment, the general clinical indicators, short-chain fatty acid levels, renal pathology, intestinal tissue pathology, intestinal tight junction protein, and changes in gut microbiota were detected. The results showed that rhubarb enema can increase the level of short-chain fatty acids in the 5/6 nephrectomy model rats, improve the intestinal barrier damage, inhibit the decrease of intestinal tight junction proteins, reduce inflammation levels, improve kidney pathology, reduce blood creatinine levels, and regulate the intestinal tract, the abundance, and composition of the flora. Further correlation analysis showed that rhubarb enema increased the level of short-chain fatty acids in 5/6 nephrectomy model rats, which may be related to the 7 strains that may regulate the production of short-chain fatty acids. This study indicated that rhubarb enema can improve the intestinal barrier damage of 5/6 nephrectomy model rats and improve CKD, which may be related to the regulation of short-chain fatty acid-producing gut microbiota.

Investigation of the Effect of Rhubarb stalks extracts on Mice Exposed to Oxidative Stress.

Normal blood lipid levels have a crucial role in lowering cardiovascular mortality. This study was designed to investigate the effect of aqueous rhubarb extract on serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde, glutathione, and ceruloplasmin levels, as well as glutathione and malondialdehyde levels in the liver, kidney, and heart tissue in mice exposed to oxidative stress. 40 Balb/c mice were randomly allocated into 8 groups (n=5). Group 1: The control group was left eating feed and water without treatment for (15) days. Group 2: A group exposed to oxidative stress by giving hydrogen peroxide at a rate of (0.5%) with drinking water for 15 days. Group 3: A group exposed to oxidative stress induced by hydrogen peroxide at a rate of (0.5%) for 15 days with injecting on the seventh day, daily for a week, with insulin subcutaneously (15) units/kg. Group (4-8): the Groups were exposed to the oxidative stress induced by hydrogen peroxide (0.5%) for 15 days with injecting on the seventh day into the peritoneal cavity with both the cold aqueous and nonprotein extract, the extract of flavonoids at a dose of 400, 400, 0.4, 8.8, 1.96 mg/kg body weight, respectively. All animals were anesthetized on the last day of the experiment, blood samples were obtained for biochemical testing, and tissue samples from the livers were collected for research. The results revealed that the cold crude aqueous, non-proteinous extracts, flavonoids, proteinous precipitate, and proteinous compound caused a significant decrease (P<0.05) in serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde levels in kidney, liver, and heart. The recorded data showed a significant increase (P<0.05) in serum glutathione and ceruloplasmin in serum and glutathione levels in liver, kidney, and heart tissues in male mice exposed to oxidative stress. The results showed that all Rhubarb extracts have an antioxidant effect in mice exposed to oxidative stress.

Growth Cessation and Dormancy Induction in Micropropagated Plantlets of Rheum rhaponticum 'Raspberry' Influenced by Photoperiod and Temperature.

Dormancy development in micropropagated plantlets at the acclimatization stage and early growth ex vitro is undesirable as it lowers their survival rate and restricts the efficient year-round production of planting material. Thus far, little is known about the factors and mechanisms involved in the dormancy development of micropropagated herbaceous perennials, including rhubarb. This study determined physiological and molecular changes in the Rheum rhaponticum (culinary rhubarb) 'Raspberry' planting material in response to photoperiod and temperature. We found that the rhubarb plantlets that were grown under a 16-h photoperiod (LD) and a temperature within the normal growth range (17-23 °C) showed active growth of leaves and rhizomes and did not develop dormancy. Rapid growth cessation and dormancy development were observed in response to a 10-h photoperiod (SD) or elevated temperature under LD. These morphological changes were accompanied by enhanced abscisic acid (ABA) and starch levels and also the upregulation of various genes involved in carbohydrate synthesis and transport (SUS3, AMY3, BMY3, BGLU17) and ABA synthesis and signaling (ZEP and ABF2). We also found enhanced expression levels of heat shock transcription factors (HSFA2 and HSFA6B), heat shock proteins (HSP22, HSP70.1, HSP90.2 and HSP101) and antioxidant enzymes (PRX12, APX2 and GPX). This may suggest that dormancy induction in micropropagated rhubarb plantlets is a stress response to light deficiency and high temperatures and is endogenously coordinated by the ABA, carbohydrate and ROS pathways.

Global analysis of protein lysine 2-hydroxyisobutyrylation (Khib) profiles in Chinese herb rhubarb (Dahuang).

BACKGROUND: Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. RESULTS: In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. CONCLUSIONS: A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.

Comparative transcriptome analysis of Rheum australe, an endangered medicinal herb, growing in its natural habitat and those grown in controlled growth chambers.

Rheum australe is an endangered medicinal herb of high altitude alpine region of Himalayas and is known to possess anti-cancerous properties. Unlike many herbs of the region, R. australe has broad leaves. The species thrives well under the environmental extremes in its niche habitat, therefore an understanding of transcriptome of R. australe to environmental cues was of significance. Since, temperature is one of the major environmental variables in the niche of R. australe, transcriptome was studied in the species growing in natural habitat and those grown in growth chambers maintained at 4 °C and 25 °C to understand genes associated with different temperatures. A total of 39,136 primarily assembled transcripts were obtained from 10,17,74,336 clean read, and 21,303 unigenes could match to public databases. An analysis of transcriptome by fragments per kilobase of transcript per million, followed by validation through qRT-PCR showed 22.4% up- and 22.5% down-regulated common differentially expressed genes in the species growing under natural habitat and at 4 °C as compared to those at 25 °C. These genes largely belonged to signaling pathway, transporters, secondary metabolites, phytohormones, and those associated with cellular protection, suggesting their importance in imparting adaptive advantage to R. australe in its niche.

The isolation of secondary metabolites from Rheum ribes L. and the synthesis of new semi-synthetic anthraquinones: Isolation, synthesis and biological activity.

Rheum ribes L. (Rhubarb) is one of the most important edible medicinal plants in the Eastern Anatolia region and is called "Iskin" by local people. Resveratrol and 6-O-methylalaternin were isolated from the Rhubarb for the first time in addition to well-known secondary metabolites including emodin, aloe-emodin, ß-sitosterol and rutin. The new semi-synthetic anthraquinone derivatives with the NαFmoc-l-Lys and ethynyl group were synthesized from the isolated anthraquinones emodin and aloe-emodin of Rhubarb to increase the bioactivities. Aloe-emodin derivative with NαFmoc-l-Lys shows the highest inhibition values by 94.11 ± 0.12 and 82.38 ± 0.00% against HT-29 and HeLa cell lines, respectively, at 25 µg/mL. Further, modification of the aloe-emodin with both the ethynyl and the NαFmoc-l-Lys groups showed an antioxidant activity-enhancing effect. From molecular docking studies, the relative binding energies of the emodin and aloe-emodin derivatives to human serum albumin ranged from -7.30 and -10.62 kcal/mol.

Identification and quantification of target metabolites combined with transcriptome of two rheum species focused on anthraquinone and flavonoids biosynthesis.

Rheum emodi is a perennial herb and an important medicinal plant, with anthraquinones and flavonoids as its main bioactive compounds. However, there is little knowledge about the biosynthetic pathway of anthraquinones in rhubarbs. In this study, we qualitatively and quantitatively assessed 62 pharmacological metabolites in rhubarb using dynamic multiple reaction monitoring (dMRM) of triple-quadrupole mass spectrometry (QqQ-MS), including 21 anthraquinones, 17 flavonoids, 6 stilbenes, 12 gallate esters, 3 tannins, and 3 others. Besides, the metabolomics results showed significant differences among all the 60 metabolites, except for gallic acid and piceatannol-O-ß-glucoside. The combined transcriptome data of R. palmatum L. (RPL) and R. officinale Baill. (ROB) showed that 21,691 unigenes were annotated in the metabolic pathways. Taken together, 17 differentially expressed genes (DEGs) were associated with the anthraquinone biosynthetic pathway. Additionally, a significant correlation between anthraquinone peak intensity and DEG expression level existed, validating that DEGs contribute to the anthraquinone biosynthetic pathway. RT-qPCR results showed that the cluster-14354.38156 gene may catalyze the O-methylation of emodin to produce physcion. This study provides a useful resource for further studies on secondary metabolism in rhubarb and the combination analysis of transcriptome and metabolome, which can help with the discovery of enzyme genes involved in metabolite biosynthesis.

Characterization and Comparative Analysis of Two Rheum Complete Chloroplast Genomes.

Rheum species present a significant economic value. Traditional Chinese medicine rhubarb is an important medicinal material in China. It has a long history of use, with a record of use as early as two thousand years ago. Here, we determined the complete chloroplast genome sequences of Rheum nobile and Rheum acuminatum and comprehensively compared them to two other available Rheum cp genomes at the genome scale. The results revealed cp genomes ranging in size from 159,051 to 161,707 bp with a similar typical quadripartite and circular structure. The genome organization, gene numbers, gene order, and GC contents of these four Rheum cp genomes were similar to those of many angiosperm cp genomes. Repeats and microsatellites were detected in the R. nobile and R. acuminatum cp genomes. The Mauve alignment revealed that there were no rearrangements in the cp genomes of the four Rheum species. Thirteen mutational hotspots for genome divergence were identified, which could be utilized as potential markers for phylogenetic studies and the identification of Rheum species. The phylogenetic relationships of the four species showed that the members of Rheum cluster into a single clade, indicating their close relationships. Our study provides valuable information for the taxonomic, phylogenetic, and evolutionary analysis of Rheum.

Physicochemical and bioactive properties of edible methylcellulose films containing Rheum ribes L. extract.

Methylcellulose (MC) polymer was used to prepare the edible films. Then, the Rheum ribes L. ethanol extract (RE) was added to the films in order to act as a natural antimicrobial agent. All the methylcellulose films were characterized for bioactivity and the physicochemical analysis were carried out to define functional group interactions between the polymer and RE. The strongest antimicrobial effect was obtained with MC films enriched with 2% (w/w) RE against to Listeria monocytogenes with 20.3 ±â€¯2.5 mm zone diameter. In the presence of RE, the tensile strength of film decreased, whereas the extract concentration significantly increased. The elongation at break and the water vapor permeability (WVP) values of the films were found to have decreased significantly in parallel with the increase in RE content. Moreover, the increase in the concentration of RE caused an increment in the hydrophilic properties, especially contact angle values of the films.

Phytochemical and Antioxidant Studies on a Rare Rheum cordatum Losinsk. Species from Kazakhstan.

An optimisation of extraction towards an increased antioxidant capacity and the study on the extracts' composition by HPLC-ESI-Q-TOF-MS were performed on different organs of a rarely studied plant: Rheum cordatum Losinsk (Polygonaceae) growing in Kazakhstan. More than 20 compounds from anthraquinones and phenolics were identified in an optimised method. The plant was proven to contain a wide variety of phenolic compounds (catechins, flavonoids, and their glucosides and phenolic acids) in contrast to the anthraquinone composition, which was mainly represented by emodin and its analogues. The results of the studies could determine the plant as a rich source of pharmacologically precious polyphenols. It was evidenced that the extracting solvents, the time of collection, and the organs tested affected both the chemical content and the antioxidant potential of the extracts. Ethanol : water (50 : 50 v/v) was selected as the most beneficial extractant for all metabolites, and based on the principal component analysis of raw data, the radical scavenging potential of the plant was strictly related to the presence of epicatechin gallate (ECG), kaempferol glucoside, and epigallocatechin gallate (EGCG) occurring in this extract at the concentration of 1.69-5%, 0.16-0.47%, and 0.001-2.93%, respectively.

Apoptotic effects of rhein through the mitochondrial pathways, two death receptor pathways, and reducing autophagy in human liver L02 cells.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a major component of many medicinal herbs such as Rheum palmatum L. and Polygonum multiflorum. Despite being widely used, intoxication cases associated with rhein-containing herbs are often reported. Currently, there are no available reports addressing the effects of rhein on apoptosis in human liver L02 cells. Thus, the aim of this study is to determine the cytotoxic effects and the underlying mechanism of rhein on human normal liver L02 cells. In the present study, the methyl thiazolyl tetrazolium assay demonstrated that rhein decreased the viability of L02 cells in dose-dependent and time-dependent ways. Rhein was found to trigger apoptosis in L02 cells as shown by Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit and cell mitochondrial membrane potential (MMP) assay, with nuclear morphological changes demonstrated by Hoechst 33258 staining. Detection of intracellular superoxide dismutase activity, lipid oxidation (malondialdehyde) content, and reactive oxygen species (ROS) levels showed that apoptosis was associated with oxidative stress. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was presumably via the death receptor pathway and the mitochondrial pathway, as illustrated by upregulation of TNF-α, TNFR1, TRADD, and cleaved caspase-3, and downregulation of procaspase-8, and it is suggested that rhein may increase hepatocyte apoptosis by activating the increase of TNF-α level. Meanwhile, rhein upregulates the expression of Bax and downregulates the expression of procaspase-9 and -3, and it is suggested that the mitochondrial pathway is activated and rhein-induced apoptosis may be involved. In addition, we also want to explore whether rhein-induced apoptosis is related to the autophagic changes induced by rhein. The results showed that rhein treatment increased P62 and decreased LC3-II and beclin-1, which means that autophagy was weakened. The results of our studies indicated that rhein induced caspase-dependent apoptosis via both the Fas death pathway and the mitochondrial pathway by generating ROS, and meanwhile the autophagy tended to weaken.