Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome / 中国中药杂志

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

Effect of psammosilene gavage on the expression of apoptosis-related proteins in chondrocytes of a rabbit osteoarthritis model / 中国组织工程研究

BACKGROUND: Psammosilene Capsule is a traditional Miao medicine formula that consists of Psammosilene tunicoides and Schefflera kwangsiensis. Psammosilene Capsule has been shown to hold protective effect on cartilage, but the underlying mechanism remains unclear. OBJECTIVE: To explore the possible mechanism of Psammosilene gavage in the treatment of osteoarthritis. METHODS: Ten of the 40 rabbits were randomly selected as the blank control group. The remaining 30 rabbits were used to establish the osteoarthritis model in the right hind knee of the rabbit by the modified Hulth method, and then were randomly divided into model (n=10), Psammosilene gavage (n=10), and p38 inhibitor (n=10) groups. At 7 days after modeling, the Psammosilene gavage group was fed with Psammosilene (57.5 mg/kg per day, p38 inhibitor group was injected with p38 inhibitor into the right hind knee joint (SB203580, 10 μmol/L, 0.5 mL), once weekly. The blank control and model groups were given the same amount of clean water, once daily. After 8 weeks, all animals were sacrificed to remove the right hind knee femoral condyle and tibial plateau. Severity of cartilage injury was evaluated by Pelletier score. Cartilage degeneration was examined by hematoxylin-eosin staining, saffron O staining and Mankin score. The expression levels of P-p38 and Cleaved Caspase-3 protein in cartilage tissue were detected by western blot assay. The expression levels of Bcl-2 and Bax mRNA in cartilage tissues were detected by quantitative real-time PCR. RESULTS AND CONCLUSION: (1) Compared with the Psammosilene gavage group, the model and p38 inhibitor groups had significantly increased Pelletier and Mankin scores (P < 0.05). (2) Compared with the Psammosilene gavage group, the expression levels of Bax mRNA and Cleaved Caspase-3 protein in the cartilage tissue in the model and p38 inhibitor groups were significantly increased (P < 0.05). (3) Compared with the Psammosilene gavage group, the expression level of Bcl-2 mRNA in the cartilage tissue in the model and p38 inhibitor groups was significantly decreased (P < 0.05). (4) Compared with the Psammosilene gavage group, the expression level of P-p38 protein in the model and p38 inhibitor groups was significantly increased (P < 0.05). (5) Our findings suggest that Psammosilene can inhibit the expression of apoptosis-related proteins and P-p38 protein in cartilage tissue, and then delay the degeneration of articular cartilage in the rabbit osteoarthritis model.

Two new maltol glycosides from roots of Psammosilene tunicoides / 中草药

Objective: To investigate the chemical constituents from the roots of Psammosilene tunicoides. Methods: Chemical constituents were separated by column chromatography over silica gel, Sephadex LH-20, and reverse-phase silica gel (ODS), and chemical structures were determined by analysis of HR-ESI-MS, 1D, and 2D NMR spectroscopic data. Results: Two new maltol glycosides were isolated from the 80% aq. Ethanol extract from the roots of P. tunicoides, and their structures were determined as maltol-3-O-[6-O-(4-O-α-L-rhamnopyranosyl)-Z-p-coumaroyl]-β-D-glucopyranoside (1) and maltol-3-O-[6-O-(4-O-α-L- rhamnopyranosyl)-E-p-coumaroyl]-β-D-glucopyranoside (2). Conclusion: Compounds 1 and 2 are identified as new maltol glycosides, and named as tunicosides A and B, respectively.

Analysis on structure and phylogeny of complete chloroplast genomes in Psammosilene tunicoides / 中草药

Objective: To obtain the structural characteristics of chloroplast genome in Psammosilene tunicoides and analyze its phylogenetic position within the family of Caryophyllaceae. Methods: High-through-put sequencing technology and bioinformatic analysis softwares were used to analyze the structures of complete chloroplast genome of P. tunicoides. RAxML 8.2.11 software was utilized to reconstruct the Caryophyllaceae phylogentic tree with Amaranthus hypochondriacus as outgroup. Results: The complete chloroplast genome was 153 977 base pairs (bp) in size, including two inverted repeat (IR, 26 033 bp) regions separated by one large singe copy region (LSC, 84 385 bp) and one small singe copy region (SSC, 17 526 bp). The genome encoded 125 genes, of which 109 were unique, including 75 protein-coding genes (PCGs), 30 tRNA genes and 4 rRNA genes. The overall GC content of P. tunicoides was 36.5%, while those of IR regions (42.4%) were higher than LSC (34.2%) and SSC (30.1%) regions. The phylogenetic tree showed that P. tunicoides and Dianthus longicalyx were sister groups with 100% bootstrap value. All nodes of the phylogenetic tree of Caryophyllaceae were of high supports, and the ML tree had good resolution to reflect the phylogeny relationship among the Caryophyllaceae. Conclusion: The complete chloroplast genome of P. tunicoides and the phylogenetic relationship within Caryophyllaceae were analyzed in this study. The results will provide effective molecular information for further studies on evaluation of germplasm and molecular phylogeny of Caryophyllaceae.

Construction of core collection of Psammosilene tunicoides based on polymorphism of β-AS gene / 中国中药杂志

Psammosilene tunicoides is one of the main ingredients of the "Yunnan Baiyao". P. tunicoides is an endangered species included in the secondary protection list in China Plant Red Data Book as well as the endemic species in Southwest China. Its natural resources could not meet the needs of pharmaceutical production. Construction of core collection of P. tunicoides will lay the foundation for germplasm improvement and molecular breeding. The sequence variation of the key enzymes gene locus (β-AS) were carried out to survey the population structure and population history of the species. Among the 11 populations across its geographical range, 36 haplotypes were identified. The levels of haplotype diversity (Hd=0.905) were high, while the levels of population differentiation (GST=0.280) were low. Analysisof molecular variance (AMOVA) indicated that a significantly greater proportion of total genetic variationpartitioned among populations thanwithin populations (values of 77.43% and 22.57%, respectively). These results in combination with the star-like phylogenetic network analysis indicate that Hap1 as an ancestral haplotypewas shared in four populations, Hap2, Hap4, Hap15 and Hap16 are occurred in two populations, the remains as private haplotype only distributed in single population. The strategy of core collection was constructed in order to maximumpreserve genetic diversity of P. tunicoides.

EST-SSR analysis of genetic diversity of Psammosilene tunicoides in Yunnan- Guizhou provincial region / 中草药

Objective To analyze the genetic diversity and variation of Psammosilene tunicoides in Yunnan-Guizhou provincial region. Methods The genetic diversity of different populations of P. tunicoides with high polymorphism was analyzed by using EST-SSR primers developed from transcriptomic sequencing technique. Results A total of 17 530 SSR-containing EST sequences were obtained by transcriptomic sequencing, 14 pairs of polymorphism EST-SSR primers were used to analyze the genetic diversity of 17 populations of P. tunicoides in Yunnan-Guizhou region, the results showed that the P. tunicoides in different populations had a high level of genetic diversity with the polymorphic information content (PIC) in the range from 0.350 0 to 0.795 0 in locus level; and had a lower value in group level with percentage of polymorphic bands (PPB) of 64.29%-100%. And the range of Nei’s genetic similarity coefficient was from 0.188 2 to 0.477 7 with mean value of 0.323 2. The gene flow in P. tunicoides in Yunnan-Guizhou region was small with Nm mean value of 0.302 0, and there is a large genetic differentiation between groups with Fst mean value of 0.452 9. Conclusion Transcriptomic sequencing enriched P. tunicoides EST database. The genetic diversity of P. tunicoides might be related to the long evolutionary history of reproductive pattern and distribution area. And there was a highly genetic diversity among the populations of P. tunicoides in Yunnan-Guizhou region, it might be that the geographic barrier cut off the genes exchange among different populations.

A Comparative Study on Quality of Different Populations of Wild Herbs ofPsammosilene tunicoides / 世界科学技术-中医药现代化

This article was aimed to carry a comparative study on the quality of 16 populations of wild herbs of rare and endangered plant ofPsammosilene tunicoidesin order to provide a basis for its domestication, GAP and selective breeding. Methods for determination of moisture content, total ash and extract content were taken in this comparative study. The results showed that different populations had different moisture content, total ash and extract content. The moisture content and the total ash ofXiang-Shan population were the lowest; and the extract content was the highest. The total ash ofLiu-De population was the highest; and the extract content was the lowest. The moisture content ofShun-Zhou population was the highest. It was concluded that different populations had different quality among 16 populations of wild herbs. Therefore, there was a probability to choose the best population.

Cloning, prokaryotic expression, and functional identification of β-amyrin synthase cDNA of Psammosilene tunicoides / 中草药

Objective: To clone, express, and characterize the full-length cDNA of β-amyrin synthase provided an important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins and secondary metabolism engineering applied to Psammosilene tunicoides. Methods: The full-length cDNA fragment of P. tunicoides was isolated by the method of RT-PCR and rapid amplification of cDNA ends (RACE). The fragment was transformed into Escherichia coli expression strain BL21, which was induced by IPTG, and the crude recombinant enzyme was purified from E. coli cell. In the presence of 2, 3-oxidosqualene and other substances, 2, 3-oxidosqualene was converted into β-amyrin efficiently. The catalytic product of P. tunicoides β-amyrin synthase was detected by high-performance liquid chromatography (HPLC) and identified as β-amyrin. Results: The full-length cDNA fragment of P. tunicoides is 2 882 bp and contains an open reading frame of 2 284 bp nucleotides, which codes for 760 amino acids. Conclusion: The full-length cDNA can produce β-amyrin synthase by prokaryotic expression. The expression product has the catalytic activity of 2, 3-oxidosqualene into β-amyrin, which would provide an important basis for the secondary metabolism engineering to P. tunicoides.

Identification of Psammosilene Radix and Its Adulterants by ITS2 Sequence / 世界科学技术-中医药现代化

Psammosilene Radix is a famous miao national herb and it is rare and endangered nowadays. However, it is often confused with the root of Silene viscidula. In this study, the ITS2 regions was used to identify Psam-mosilene Radix and its adulterants. All DNA samples of Psammosilene Radix and its adulterants were extracted. The ITS2 regions were amplified and sequenced, and the final sequences were assembled using the CondonCode Aligner. The genetic distances were calculated by kimura 2-parameter (K2P) model and the Neighbor-Joining(NJ) phylogenetic trees were constructed using MEGA5.1.BLAST 1, the nearest distance and phylogenetic tree (NJ-tree)methods were used to assess the identification efficiency of the ITS2 region. Results indicated that the length of ITS2 sequences of Psammosilene Radix w 229 bp, the identification effficiency of ITS2 region using BLAST 1 was 100%;and the maximum intra-specific K2P distance were lower than the minimum inter-specific K2P distance. Additionally, the NJ tree based on ITS2 sequence indicated that Psammosilene Radix and its adul-terants could be distinguished clearly . In conclusion , the ITS2 region as DNA barcodes could identify Psammosi-lene Radix and its adulterants stably and accurately. Furthermore, the application of ITS2 barcode in the identifi-cation of TCM has a good prospective .

Study on biological characteristic and conservation of tuniclike psammosilene root / 中华中医药杂志

Objective: To study the endangered mechanism of tuniclike psammosilene root and put forward the conservation ways,so as to provide basis for resources conservation.Methods: Wild on-site inspection was used to study the natural distribution,living condition,biological characteristic,growing adaptability of tuniclike psammosilene root.Results: Tuniclike psammosilene root can grow well in barren,dry and cold circumstances but wetting.The population quantity of tuniclike psammosilene root was reduced largely for destroy of biogeocoenosis.Conclusion: To conserve resources,the exploitation of tuniclike psammosilene root should follow the pattern of planting and finish machining.

Genetic diversity analysis of germplasm resources of Psammosilene tunicoides / 中草药

Objective To analyze the genetic diversity of Psammosilene tunicoides,an endangered and endemic medicinal plant,in southwest China.Methods The genetic diversity of seven representational populations of P.tunicoides including 137 individuals had been investigated by amplified fragment length polymorphisms(AFLP)maker technique.Results The genetic diversity had been revealed as follow:the Nei's genetic diversity index(He),Shannon's information index(I),percentage of polymorphic loci(PPB)were 0.243 4?0.179 1,0.373 5?0.248 5,and 82.30%,respectively at the species level;and 0.091 8?0.161 0,0.140 2?0.236 2,and 30.48%,respectively at population level.The genetic differentiation index(Gst)was 0.624 6 and genetic differentiation coefficient by Shannon's diversity(Ist)was 0.624 6.The result of dendrogram of seven populations indicated that Lijiang and Gejiu of Yunnan populations shared the maximum genetic identity,though they distributed in a relatively great geographical distance;Kunming population of Yunnan had the greater genetic distance from other populations.Nine characteristic fingerprint bands that can distinguish the different populations had been acquired.Conclusion The genetic diversity of P.tunicoides is relatively higher at the species levels,while lower within population levels,and a significant degree of genetic differentiation occurrs among the populations.There is little relativity between the relationship of populations and geographical distance.The combination of characteristic fingerprint bands between intraspecies and populations provide important basis data for germplasm resources diagnostics and plant breeding by AFLP maker technique.