This study is to investigate the authenticity between COLD and HOT natural attribute in the famous Chinese medicine formulas--Zuojinwan (Coptis-Evodia 6 : 1) and Fanzuojinwan (Coptis-Evodia 1 : 6) based on mice temperature tropism, and establish an objective method to estimate the difference of two natural attribute by using a cold/hot plate differentiating technology. The results indicated that the COLD nature Zuojinwan could decrease significantly the remaining rate of HOT-symptom rat on warm pad (P < 0.05). That was not notable to COLD-symptom rat. The interference result of COLD-HOT temperature tropism to COLD/HOT symptom rat in Fanzuojinwan was the reverse with the COLD nature Zuojinwan. Meanwhile, biochemical indicators which are relative to energy metabolism such as ATPase enzyme activity and total anti-oxidant capability (T-AOC), had corresponding change in the organism. In the study, the COLD and HOT natural tendency in Zuojinwan and Fanzuojinwan which were composed by the same herbs with different proportion could be expressed qualitatively, quantitatively, objectively and directly with applying animal temperature tropism, and be verified to philosophical idea of treating disease theory with "expelling heat with cold herbs and cryopathy requiring warm prescription", not "expelling heat with heat herbs and cryopathy requiring cold prescription" in ancient traditional Chinese medicine, which brings a new approach in investigation of the nature theory of traditional Chinese medicine.

How to identify active constituents of traditional Chinese medicines (TCMs) and study their interactions are key problems in the development of TCMs. The inhibitory effect of six alkaloids from Rhizoma Coptidis (RC) on Shigella dysenteriae (S. dysenteria) growth had been investigated by microcalorimetry in this study. Main active constituents of RC were confirmed by comparing their contributions to the bacteriostatic effect, and the interactions among active constituents were further researched. According to the result, in 0.8 mg-mL-1 extract of RC, the contributions of six active alkaloids including berberine, coptisine, epiberberine, palmatine and the combination of jatrorrhizine and columbamine were 52.83%, 36.31%, 2.49%, 4.27% and 3.21%, respectively. Therefore, berberine and coptisine were the main active constituents of RC that inhibited the growth of S. dysenteria. The study of interactions among the six alkaloids indicated that, 1 there were some contstituents antagonizing the inhibitory effect of RC, 2 there was a synergy effect between berberine and coptisine, 3 there were additive effects between other four alkaloids and the main active constituents. These results may provide some useful references for the establishment of the quality standard for RC and the development of multi-component TCMs.

Objective To study the influence of long-term home noninvasive positive pressure ventilation (HNPPV) on respiratory muscle strength in patients with stable severe chronic obstructive pulmonary disease (COPD).Methods Sixty-four patients with stable severe COPD discharged from Huabei Oil-field Hospital,Renqiu,Hebei were divided into two groups,one (n=24) with HNPPV plus conventional therapy,and the other (n=40) with conventional therapy plus long-term oxygen therapy as controls.All parameters were followed-up for one-year and compared for the two groups,including maximal iuspiratory pressure (MIP),transdiaphragmatic pressure (Pdi),maximal transdiaphragmatic pressure (Pdimax),ratio of Pdi/Pdimax,arterial partial pressure of carbon dioxide (PaCO2),forced expiratory volume in one second (FEV1),6-min walking distance (6MWD),mortality and re-hospitalization rate.Results Age,gender,course of the disease,body mass index (BMI),arterial PaCO2,PaO2,MIP,Pdi,Pdiraax,ratio of Pdi/ Pdimax,FEV1,ratio of FEV1/FVC%,6MWD and re-hospitalization rate of the patients between the two groups were all comparable (P>0.05).In one-year follow-up,PaCO2averaged (52±8)mm Hg,MIP (64±7) cm H2O,Pdi (33±5) cm H2O,Pdimax (101±9) cm H2O,Pdi/Pdimax (0.31±0.04),FEV1 (35±4) %,FEV1/FVC% (44±4) %,6MWD (272±26) m and (2.6 ± 0.8) admissions per year in the HNPPV group,significantly different from those in the control group [ (57 ± 6) mm Hg,(59 ± 6) cm H2O,(31±4) cm H2O,(84±7) cm H2O,(0.35±0.05),(33±3)%,(41±4)%,(212±28) m,and (3.7±0.8) admissions per year] (P<0.05).One death was observed in the HNPPV group (1/24) and three in the control group (3/4 0) in one - year follow - up,with no statistically significant difference (X2=0.00,P>0.05).Conclusions Long-term use of HNPPV for patients with stable severe COPD could efficiently improve their respiratory muscle strength and endurance,thus improving their pulmonary ventilation and treatment efficcacy.

OBJECTIVETo clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene.

METHODThe CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR.

RESULTThe full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control.

CONCLUSIONThe CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.

Calmodulin ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Eleutherococcus ; classification ; genetics ; metabolism ; microbiology ; Endophytes ; physiology ; Fungi ; physiology ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo detect the influence of compatibility of rhubarb with different traditional Chinese medicines (TCM) on extracted quantities of AQs, and to provide scientific basis for the clinical code for rhubarb preparation.

METHODThe influence of compatibility of rhubarb with different traditional Chinese medicines (saponin, alkaloids, flavonoids TCMs, animal medicines and mineral medicines) on decocting volume of anthraquinone substance was detected using ultra performance liquid chromatography.

RESULTIn comparable conditions, more AQs were extracted from mixed decoction of rhubarb and saponin medicinal materials (Astragali Radix, Notoginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, Polygalae Radix, Ginseng Radix et Rhizoma) than single decocting of rhubarb. The mixed decoction of rhubarb and alkaloid medicinal materials (Coptidis Rhizoma, Sophorae Flavescentis Radix, Prepared Aconiti Lateralis Radix Praeparata, Phellodendri Chinensis Cortex, Aconiti Lateralis Radix) caused a remarkable decrease in extracted quantities of AQs. And the mixed decoction of rhubarb and mineral medicines (Natrii Sulfas, Gypsum Fibrosum, Ostreae Concha, Alumen) also resulted in less extracted quantities of AQs to varying degrees. Besides, more rhubarb AQs were extracted from mixed decoction with Curcuma than single decoction. But less rhubarb AQs were observed in mixed decoction with Lonicerae Flos, Rehmanniae, Artemisiae Herb and Forsythiae Fructus than single decoction to varying degrees. In the study, the maximum extracted quantities of AQs is 2. 3-fold higher than the minimum, the largest difference existed in the extracted quantity of physcion which was 13.5 times.

CONCLUSIONIn compatibility between rhubarb and different TMCs, mixed decoction and single decoction show different influences on extracted quantity of rhubarb AQs. It is proved that more AQs may be extracted from mixed decoction between rhubarb and saponin medicinal materials, whereas less AQs may be observed in mixed decoction between rhubarb and alkaloid medicinal materials.

Alkaloids ; chemistry ; Animals ; Anthraquinones ; analysis ; isolation & purification ; Chromatography, High Pressure Liquid ; methods ; Drug Compounding ; methods ; standards ; Flavonoids ; chemistry ; Humans ; Medicine, Chinese Traditional ; methods ; standards ; Plants, Medicinal ; chemistry ; Reference Standards ; Reproducibility of Results ; Rheum ; chemistry ; Saponins ; chemistry

OBJECTIVETo clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus.

METHODTotal RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy.

RESULTSequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices.

CONCLUSIONThe two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Eleutherococcus ; enzymology ; genetics ; Isoenzymes ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Squalene Monooxygenase ; classification ; genetics

OBJECTIVETo analyze the effect of endophytic fungi on expression amount of key enzyme genes SS (squalene synthase gene), SE (squalene epoxidase gene) and bAS (beta-amyrin synthase gene) in saponin biosynthesis and saponins content in Eleutherococcus senticosus.

METHODWound method was used for back meeting the endophytic fungi to E. senticosus. With GAPDH as internal control gene, the expression of key enzyme genes was detected by real time PCR method. E. senticosus saponins content was measured by spectrophotometry method.

RESULTWhen wound method back meeting P116-1a and P116-1b after 30 d, the expression content of SS improved significantly (P < 0.05), however the back meeting of P109-4 and P312-1 didnt change the expression of SS. After that SS expression showed reduction-equality-reduction varying trend. Thirty days after back meeting P312-1, the expression content of SE improved significantly (P < 0.05). Ninty days after back meeting P116-1b and P312-1, the expression content of SE improved significantly to 130%,161%, respectively (P < 0.05). After 120 d, back meeting four endophytic fungi, the expression of SE were significantly higher than the control (P < 0.05). Back meeting four endophytic fungi form 60 d to 120 d, the expression of bAS was significantly higher than the control (P < 0.05). The back meeting four endophytic fungi improved E. senticosus saponins content significantly (P < 0.05).

CONCLUSIONEndophytic fungi P116-1a, P116-1b, P1094 and P312-1 significantly effected the expression of key enzyme genes SS, SE and bAS and then affected E. senticosus saponins content. Among the genes, bAS was key target gene.

Eleutherococcus ; chemistry ; metabolism ; microbiology ; Endophytes ; physiology ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; Fungi ; physiology ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Intramolecular Transferases ; genetics ; Saponins ; analysis ; biosynthesis ; Squalene Monooxygenase ; genetics

OBJECTIVETo clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene.

METHODThe FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR.

RESULTThe full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05).

CONCLUSIONThe FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; Conserved Sequence ; Eleutherococcus ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Geranyltranstransferase ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Conformation

OBJECTIVE To establish the quality standard of Kuipingning gastric floating tablets. METHODS Kuipingning gastric floating tablets were prepared and investigated in terms of property ，weight difference and friability. Crydalis yanhusuo was identified qualitatively by thin layer chromatography （TLC）method. High performance liquid chromatography method was used to determine the content of total anthraquinones in Rheum palmatum ，and set the content limit of total anthraquinones. The floating performance and release degree of the preparation were investigated ，and the release kinetic process was fitted. RESULTS Kuipingning gastric floating tablets prepared in this study were gray white to gray tablets with slight smell and bitter taste ；the weight difference and friability were all in line with relevant regulations ；the established TLC method possessed strong specificity and could accurately identify C. yanhusuo . The average content of total anthraquinones in R. palmatum was 17.95 mg/tablet，and its content limit would not be less than 14.36 mg/tablet. The initial floating time of the preparation was no more than 10 s，and the holding time was more than 8 h. The release kinetics process accorded with the Retger-Peppas release model. CONCLUSIONS The method established in this study shows good reliability ，stability and feasibility ，and can effectively control the quality of Kuipingning gastric floating tablets.

Uncovering the functionally essential variations related to tumorigenesis and tumor pro-gression from cancer genomics data is still challenging due to the genetic diversity among patients, and extensive inter-and intra-tumoral heterogeneity at different levels of gene expression regulation, including but not limited to the genomic, epigenomic, and transcriptional levels. To minimize the impact of germline genetic heterogeneities, in this study, we establish multiple primary cultures from the primary and recurrent tumors of a single patient with hepatocellular carcinoma (HCC). Multi-omics sequencing was performed for these cultures that encompass the diversity of tumor cells from the same patient. Variations in the genome sequence, epigenetic modification, and gene expression are used to infer the phylogenetic relationships of these cell cultures. We find the discrepancy among the relationships revealed by single nucleotide variations (SNVs) and transcriptional/epigenomic pro-files from the cell cultures. We fail to find overlap between sample-specific mutated genes and differ-entially expressed genes (DEGs), suggesting that most of the heterogeneous SNVs among tumor stages or lineages of the patient are functionally insignificant. Moreover, copy number alterations (CNAs) and DNA methylation variation within gene bodies, rather than promoters, are significantly correlated with gene expression variability among these cell cultures. Pathway analysis of CNA/DNA methylation-related genes indicates that a single cell clone from the recurrent tumor exhibits distinct cellular characteristics and tumorigenicity, and such an observation is further confirmed by cellular experiments both in vitro and in vivo. Our systematic analysis reveals that CNAs and epigenomic changes, rather than SNVs, are more likely to contribute to the phenotypic diversity among subpop-ulations in the tumor. These findings suggest that new therapeutic strategies targeting gene dosage and epigenetic modification should be considered in personalized cancer medicine. This culture model may be applied to the further identification of plausible determinants of cancer metastasis and relapse.