Comparison of Three Species of Rhubarb in Inhibiting Vascular Endothelial Injury via Regulation of PI3K/AKT/NF-κB Signaling Pathway.

Background/Aim: Rhubarb, a traditional Chinese medicine derived from three species, is commonly used in the prescriptions for promoting blood circulation and removing blood stasis based on its traditional effects of removing blood stasis and dredging the meridians. It has been reported that rhubarb can protect blood vessels by reducing inflammation and inhibiting vascular endothelial injury (VEI), but the effective components and mechanism of rhubarb inhibiting VEI are still unclear. This study aimed to compare the differences in chemical compositions of the three species of rhubarb and their inhibitory effect on VEI, so as to explain the material basis and select the dominant species to inhibit VEI, and to elucidate the mechanism of rhubarb's inhibitory effect on VEI. Methods: Plant metabolomics was used to compare the chemical components of three species of rhubarb. The efficacy of three species of rhubarb in inhibiting VEI was compared through cell experiments in vitro. At the same time, combined with network pharmacology and molecular docking, the effective components and pathways of rhubarb involved in inhibiting VEI were screened. The mechanism of rhubarb inhibiting VEI was verified by molecular biology. Results: There were significant differences in the distribution of chemical components among the three species of rhubarb. We identified 36 different chemical components in the positive ion mode and 38 different chemical components in the negative ion mode. Subsequently, the results showed significant differences in inhibiting VEI among the three species of rhubarb based on the contents of inflammatory factors (such as IL-1ß, IL-6, and TNF-α), ROS, and NO and confirmed that R. tanguticum had the best inhibitory effect on VEI in the light of the comprehensive efficacy, compared with R. palmatum and R. officinale. Three species of rhubarb alleviated the inflammatory response in LPS-induced EA.hy926 cells by reducing the contents of inflammatory cytokines IL-6, IL-1ß, and TNF-α and decreasing expressions of PI3K, AKT, NF-κB p65, and STAT3 protein in the PI3K/AKT/NF-κB pathway and the inhibition of proteins phosphorylation. In addition, three species of rhubarb could lessen the contents of ROS and NO in EA.hy926 cells induced by LPS. All results indicated that the process of inflammation-induced cellular oxidative stress, which resulted in VEI, was obviously improved by three species of rhubarb. Conclusion: R. tanguticum was more effective among three species of rhubarb, and it had been proved that gallic acid, gallic-acid-O-galloyl-glucoside, procyanidin B-2,3,3'-di-O-gallatein, and other potential components could reduce the contents of inflammatory factors (such as IL-1ß, IL-6, and TNF-α), ROS, and NO by inhibiting the PI3K/AKT/NF-κB signaling pathway and protected the vascular endothelium and the blood vessels by improving the inflammation and oxidative stress reaction.

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.

Comparative Chloroplast Genome Analysis of Rhubarb Botanical Origins and the Development of Specific Identification Markers.

Rhubarb is an important ingredient in traditional Chinese medicine known as Rhei radix et rhizome. However, this common name refers to three different botanical species with different pharmacological effects. To facilitate the genetic identification of these three species for their more precise application in Chinese medicine we here want to provide chloroplast sequences with specific identification sites that are easy to amplify. We therefore sequenced the complete chloroplast genomes of all three species and then screened those for suitable sequences describing the three species. The length of the three chloroplast genomes ranged from 161,053 bp to 161,541 bp, with a total of 131 encoded genes including 31 tRNA, eight rRNA and 92 protein-coding sequences. The simple repeat sequence analysis indicated the differences existed in these species, phylogenetic analyses showed the chloroplast genome can be used as an ultra-barcode to distinguish the three botanical species of rhubarb, the variation of the non-coding regions is higher than that of the protein coding regions, and the variations in single-copy region are higher than that in inverted repeat. Twenty-one specific primer pairs were designed and eight specific identification sites were experimentally confirmed that can be used as special DNA barcodes for the identification of the three species based on the highly variable regions. This study provides a molecular basis for precise medicinal plant selection, and supplies the groundwork for the next investigation of the closely related Rheum species comparing and correctly identification on these important medicinal species.

The complete chloroplast genome sequence of the medicinal plant Rheum palmatum L. (Polygonaceae).

The complete chloroplast genome of the medicinal plant Rheum palmatum L. (Polygonaceae) has been reconstructed from the whole-genome Illumina sequencing data. The genome is 161 541 bp in length, and exhibits a typical quadripartite structure of the large (LSC, 86 518 bp) and small (SSC, 13 111 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 30 956 bp each). The chloroplast genome contains 131 genes, including 84 protein-coding genes (78 PCG species), eight ribosomal RNA genes (four rRNA species) and 37 transfer RNA genes (28 tRNA species). Phylogenetic tree based on the maximum parsimony (MP) analysis of 65 chloroplast protein-coding genes for 13 taxa demonstrated a close relationship between R. palmatum and Fagopyrum esculentum subsp. ancestrale in Polygonaceae.

[Identification of different varieties of Rhei Radix et Rhizoma based on chemical analysis].

A HPLC method was established to determine the contents of the five anthraquinones and rhaponticin in the different varieties of Rhei Radix et Rhizoma. The difference existed in different varieties. The results showed that rhein and rhaponticin were marker substances which could be used to distinguish palm leaf groups rhubarb and wave leaf groups rhubarb. Authentic rhubarb didn't contain rhaponticin. Falsify rhubarb contains trace amounts of rhein. Rheum tanguticum contains abundant rhein. The ratio value of the content of rhein to chrysophanol could be used to distinguish R. tanguticum from the other two authentic varieties (R. palmatum and R. officinale). The content of rhaponticin varied largely in different varieties of wave leaf groups rhubarb.

[UPLC fingerprint of rhei radix et rhizoma from different habitats].

OBJECTIVE: To establish the UPLC fingerprint of Rhei Radix et Rhizoma and reference crude drugs, analyze the characteristics among fingerprints of three species of reference crude drugs and the common components of Rhei Radix et Rhizoma, and compare the application of different analysis methods. METHODS: UPLC procedure was performed on ACQUITY BEH C18 chromatographic column with mobile phase consisted of water (contained 0.1% phosphoric acid)-acetonitrile (gradient elution) at a flow rate of 0.21 mL/min. Detection wavelength was set at 260 nm and the column temperature was set at 30 degrees C. Fingerprints were analyzed by similarity evaluation, cluster analysis and principal component analysis. RESULTS: There were obvious characteristics among fingerprints of three species of reference crude drugs, 19 common chromatographic peaks were obtained from Rhei Radix et Rhizoma and 14 peaks were identified according to standard reference substances and by HPLC-MS. The cluster analysis and similarity evaluation showed the same result that 21 batches of sample were grouped into 5 categories and the result had no direct correlation with the botanical species. Both the contents of4 important ingredients suggested by principal component analysis and the whole fingerprint analysis were necessary in quality evaluation of Rhei Radix et Rhizoma. There was certain limitation in quality evaluation of multiple sources drug which analysis by similarity evaluation and cluster analysis. CONCLUSION: The method with good reproducibility and separation saves time and solvent, it can be used in identification of three species of reference crude drugs but can not be used in species identification of commercial Rhei Radix et Rhizoma.

Authentication of official Da-huang by sequencing and multiplex allele-specific PCR of a short maturase K gene.

Rhubarb (official Da-huang) is an important medicinal herb in Asia. Many adulterants of official Da-huang have been discovered in Chinese markets in recent years, which has resulted in adverse effects in medicinal treatment. Here, novel molecular markers based on a short maturase K (matK) gene were developed for authenticating official Da-huang. This study showed that all the species from official Da-huang were clustered together in one clade in the polygenetic trees based on short matK. Two highly conserved single nucleotide polymorphisms of short matK were mined in the species from official Da-huang. Based on these polymophisms, four improved specific primers of official Da-huang were successfully developed that generated reproducible specific bands. These results suggest that the short matK sequence can be considered as a favorable candidate for distinguishing official Da-huang from its adulterants. The established multiplex allele-specific PCR was determined to be simple and accurate and may serve as a preferable tool for authentication of official Da-huang. In addition, we suggest that short-sized specific bands be developed to authenticate materials used in traditional Chinese medicine.

[Sequence analysis and identification of a chloroplast matK gene in Rhei Rhizoma from different botanical origins].

Rhei Rhizoma is a Chinese medicine with multiple botanical origins. There is a problem to identify it with conventional methods. To compare the characteristics of chloroplast matK gene sequences of different Rheum species and authenticate inspected species, the matK gene sequences of different species from different origins were amplified, cloned, and sequenced. Genomic DNA of Rheum plants was extracted using modified DNA extracted Kit and matK gene sequences were analyzed by ContingExpress, DNAman and MEGA5.0. The length of matK gene sequences of Rheum palmatum, R. tanguticum and R. officinale were 1 518 bp containing 57 variable loci. According to the mutation sites, R. palmatum, R. tanguticum and R. officinale were divided into different genotypes separately. Based on the established method according to the loci 587, 707, 838, we successfully identified the genuine Rheum species from its adulterants.

Genetic diversity of the endemic and medicinally important plant Rheum officinale as revealed by Inter-Simpe Sequence Repeat (ISSR) Markers.

Rheum officinale Baill., an important but endangered medicinal herb, is endemic to China. Inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 12 populations of R. officinale. Thirteen selected primers yielded 189 bright and discernible bands, with an average of 14.54 per primer. The genetic diversity was low at the population level, but pretty high at the species level (H = 0.1008, I = 0.1505, PPB = 28.95% vs. H = 0.3341, I = 0.5000, PPB = 95.24%, respectively) by POPGENE analysis. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly among populations (74.38%), in line with the limited gene flow (N(m) = 0.2766) among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.5381, P = 0.002), indicating the role of geographic isolation in shaping the present population genetic structure. Both Bayesian analysis and UPGMA cluster analysis demonstrated the similar results. Our results imply that the conservation efforts should aim to preserve all the extant populations of this endangered species, and cultivation is proposed in this study.

Chemical-based species classification of rhubarb using simultaneous determination of five bioactive substances by HPLC and LDA analysis.

INTRODUCTION: Rhubarb is a traditional Chinese medicine derived from the rhizome of three species: Rheum tanguticum, Rheum palmatum and Rheum officinale. There are several species that are often misidentified as rhubarb. Taxonomical identification of these various species can be challenging. We have developed an HPLC-based species classification to identify rhubarb. OBJECTIVE: The objective of this study was to develop a simple HPLC method for the simultaneous determination of bioactive compounds and identification of medicinal rhubarb rhizome and non-medicinal species. METHODOLOGY: Quantitative analysis was performed on a C18-column using 0.05 M aqueous phosphoric acid and acetonitrile as the mobile phase under gradient conditions with ultraviolet detection at 280 nm. The method was validated with respect to linearity, accuracy, precision, and recovery. Statistical analysis was used to classify different groups of species. RESULTS: All calibration curves showed good linearity (r ≥ 0.9995). The method showed good repeatability with intra- and inter-day standard deviations of less than 1.13% and 1.32%, respectively. The accuracy and recovery of all marker compounds were in the ranges of 98.0 to 102.6% and 99.21 to 102.04%, respectively. Seventeen peaks were selected, and 39 known and 57 unknown samples were classified into five species based on linear discriminant analysis with an accuracy of 100%. CONCLUSION: A chemical-based species classification method of rhubarb using simultaneous determination of bioactive compounds by HPLC was developed with 39 known samples of five different species and successfully applied to identify 57 unknown samples collected from Korea and China.

[Study on genetic relationship of official Rheum by SRAP].

OBJECTIVE: To determine the genetic relationship of three species of official Rheum in molecular level. METHOD: Twelve samples from three species of official Rheum were employed to be analyzed by the approach of sequence-related amplified polymorphism (SRAP). Systematic relationships were constructed based on the UPGMA method by TREECONW software. RESULT: A total of 272 bands were scored and 199 bands of them were polymorphic, which were up to 73.2% polymorphic ratio. Genetic similarity coefficient was changed from 0.578 4 to 0.941 6. The results indicated that there was abundant genetic diversity among the tested materials. The clustering analysis revealed that the results between SRAP marker and the traditional morphological characteristics was almost the same. CONCLUSION: SRAP marker is suitable for variety identification and genetic relationship research in official Rheum.

[Study of quality on rhubarb from different habitats in Qinghai Province].

OBJECTIVE: To investigate the effect of different habitats on rhubarb quality. METHOD: The rhubarb samples from various parts of Qinghai province were analysed by fingerprint. RESULTS AND CONCLUSION: The distribution and quality of wild Tangute rhubarb is better than Palmate sorrel rhubarb. The quality of high altitude sampes are better than low altitudes. The quality which from the grassland of plateau is better than chestnut soil area's, and the wild is better than that of planting.

[Study on the selection of crude drugs of rhubarb dispensing granule].

OBJECTIVE: To research and choose the best crude drugs of Rhubarb dispensing granule. METHODS: According to standard method from the eighth edition of China pharmacopoeia, determined and compared the contents of TLC, water, total ash, acid insoluble ash, extractives, total anthraquinones, rhein, emodin and chrysophanol of Rhubarb in diffrent producing areas and species. RESULTS: The Contents of Rheum palmatum from Rhubarb GAP base in Ganzhi state were higher than others and the drug yield was large. CONCLUSION: This species are chosen to be the crude drugs for Rhubarb dispensing granule. The preliminary study provides the scientific grounds for next study about establishment of quality criteria.

[Comparative studies on purgative potency among three spieces of certified rhubarb].

OBJECTIVE: To investigate the difference and causes of purgative activity in three species of certified rhubarb, so as to lay steady foundations of further research on assessing purgative activity impersonally by using measurable indexes. METHOD: The potencies of three species were comparied with purgative ED, of mice as quantitative index which were calculated, and activities of Na+ K + -ATP ase in mouse colonic epithelial cell membrane were also investigated . The related purgative contents (conjunct and free rhein, chrysophanol, chrysophanic acid, sennoside A) were detected by HPLC and contents (total anthraquinones, anthraglucosennin; conjunct and free anthraquinones) were detected by UV. RESULT: There were different purgative activities among three spieces of certified rhubarb. Each purgative ED, of mice was Rheum tanguticum ( ED50 = 0. 37 g x kg (-1)) , R. officinale ( ED50 = 0. 99 g x kg(-1) ) and R. palrnatum from Gansu (ED50 = 1. 83 g x kg(-1)) , the ratio of potency of those was 4. 94: 1. 85: 1. In the meanwhile, the difference of the inhibitory effect on Na+ -K + -ATP ase in mouse colonic epithelial cell membrane and relative purgative components also existed in the three species of certified rhubarb. CONCLUSION: It disclosed that there was notable diference of purgative activity and components among three spieces of certified Rhubarb, which probably resulted in the ultimate diference in clinical prescription and the production of Chinese patent medicines.

[Radial basis function networks and IR spectrometry applied for identification of official rhubarb samples].

The Fourier transform infrared spectrometry (FTIRS) and radial basis function neural network (RBF-NN) have been applied to develop classification models for identifying official and unofficial rhubarb samples. The original data were compressed from 775 variables to 49 variables by using wavelet transformation method. The compressed spectra with reduced variables maintain the characteristics of the IR spectra and speed up the network training process. The effects of network parameters including error goal and spread constant, were investigated. The rate of correct classification is up to 97.78% at optimized conditions. Results show that the combination of IRS and ANN can be used as fast and convenient tool for identification of Chinese herbal samples.

[Identification of official rhubarb samples based on IR spectra and neural networks].

The Fourier transform infrared (IR) spectrometry and neural networks have been used to identification of official and un-official rhubarb samples in the present work. The IR spectra were compressed by using wavelet transform and then were normalized prior to network training. Spectra with 700 data points were compressed to 44 variables, therefore, the training process of neural networks were speed up. 52 rhubarb samples in which 25 official and 27 unofficial rhubarb samples are included have been used to network modeling. The effects of neuron number in hidden layer and momentum parameter on classification have been investigated. Results showed that about 98% rhubarb samples could be identified correctly when optimized parameters were used. This method can be useful for quality control in rhubarb-contained Chinese medicine production.

Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification.

Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.

[Identification of official rhubarb samples based on NIR spectra and neural networks].

Rhubarb is one of the most widely used Chinese medicinal herbs in China. Fast and accurate identification of official and unofficial rhubarb samples is most critical for quality control of Chinese medicine production. In the present paper near-infrared reflectance spectrometry (NIRS) and artificial neural network (ANN) were combined to develop classification models for identifying 52 official and unofficial rhubarb samples. The measured spectra were compressed by wavelet transformation (WT) and then the ANN classification models were trained with the reduced-variables spectral data. The rate of correct classification was over 96%. The effects of neurons in hidden layer and the momentum were also discussed. Owing to its fast and nondestructive properties, NIRS is a promising approach to identifying Chinese medicinal herbs.

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences.

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.