Problem Analysis and Standard Establishment of Grade Evaluation Method of Traditional Chinese Medicine Decoction Pieces / 中国实验方剂学杂志

High quality is the premise for the implementation of high quality and good price for decoction pieces， and grade is the most direct manifestation of high quality of decoction pieces. However， there is still a lack of scientific and reasonable methods for evaluating the grade of decoction pieces， and it is urgent to establish a widely recognized and unified standard for the grade of decoction pieces to ensure the quality of the decoction pieces and guarantee the safety and efficacy of clinical use. Based on this， this paper focused on analyzing the problems of the current grade evaluation methods， such as unclear distinction between quality standards and grade standards， unreasonable selection of grade evaluation indicators， and inaccurate application of mathematical statistical methods. Based on the analysis of the grade evaluation of decoction pieces， this paper proposed four criteria for establishing the grade evaluation method of decoction pieces， namely universality， comprehensiveness， reliability and convenience， in order to establish a more reasonable and unified grade standard for decoction pieces and promote the quality improvement of decoction pieces and the development of the industry.

Construction of ATMT System for Clonostachys rosea and Its Conditional Optimization of Colonization into Glycyrrhiza uralensis Seeds / 中国实验方剂学杂志

ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.

Effects of LASIK on Biomechanical Properties of Human Cornea / 医用生物力学

Objective To study the changes in biomechanical properties of human cornea after laser in situ keratomileusis (LASIK) and predict corneal stiffness after the LASIK surgery. Methods According to the measurement results from corneal visualization scheimpflug technology (Corvis ST), the corneal tangent stiffness coefficient (STSC) and energy absorbed area (Aabsorbed) were calculated. The change patterns of corneal stiffness and viscosity after refractive surgery were analyzed. Results The difference of corneal STSC and Aabsorbed before and after LASIK had a statistical significance (P<0.05). The obtained formula for predicting corneal stiffness after refractive surgery was: Sbefore surgery =1.055bIOPbefore surgery + 0.015CCTbefore surgery，Safter surgery =0.937Sbefore surgery +0.019CCTafter surgery. Conclusions LASIK surgery not only changes corneal thickness, but also reduces corneal stiffness and viscosity. Prediction of corneal stiffness after surgery can provide guidance for the design of clinical surgery and improve the safety of surgery.

Effect of CRRT on the clearance of antibiotics in the treatment of sepsis / 中华危重病急救医学

Sepsis should be defined as life-threatening organ dysfunction caused by a dys-regulated host response to infection. Continuous renal replacement therapy (CRRT) is one of the methods for the clinical treatment of sepsis. For patients undergoing CRRT, rational antimicrobial therapy is very important for the control of patient's infection. However, during CRRT, there is no clear guideline for the dose adjustment of antibiotics. In this paper, we analyzed the effect of CRRT combined with antibiotics on sepsis treatment in China and abroad, and discussed its effect on antibiotic clearance, and provided reference for clinical work.

The antiviral and antimicrobial activities of licorice, a widely-used Chinese herb

Licorice is a common herb which has been used in traditional Chinese medicine for centuries. More than 20 triterpenoids and nearly 300 flavonoids have been isolated from licorice. Recent studies have shown that these metabolites possess many pharmacological activities, such as antiviral, antimicrobial, anti-inflammatory, antitumor and other activities. This paper provides a summary of the antiviral and antimicrobial activities of licorice. The active components and the possible mechanisms for these activities are summarized in detail. This review will be helpful for the further studies of licorice for its potential therapeutic effects as an antiviral or an antimicrobial agent.

Mechanism of genuineness of Glycyrrhiza uralensis based on SNP of β-Amyrin synthase gene / 药学学报

β-Amyrin synthase (β-AS) genes of Glycyrrhiza uralensis from 6 different regions were analyzed by PCR-SSCP and sequenced, then the correlationship between β-AS SNP and regions of Glycyrrhiza uralensis were determined. According to the 1 coding single nucleotide polymorphism on the first exon of β-AS gene at 94 bp site, Glycyrrhiza uralensis could be divided into 3 genotypes. In these genotypes, the percentage of 94A type in genuine regions was much higher, and it had significant differences with the percentage in non-genuine regions (P < 0.001). The results of the experiment proved that different β-AS genotypes at 94 bp site from different regions may be one of the important reasons to result in the genuineness of Glycyrrhiza uralensis.

Influence factors of microdialysis linear probe recovery for in vivo and in vitro cyclophosphamide calibration by HPLC-MS/MS / 国际药学研究杂志

Objective To establish an optimal linear probe recovery correction method for in vivo and in vitro cyclophosphamide. Methods with ringer solution as the perfusion, retrodialysis gain and loss method were used to calibrate the linear probe. The impact of different perfusion flow rates on microdialysis relative recovery for cyclophosphamide was investigated by HPLC-MS/MS. Results When the perfusion was 1.0μl/min, in vitro recovery results were in the range of (24.11±1.75)%to (50.93±0.91)%for the gain method, and(26.19±0.76)% to(53.31±1.23)% for the loss method;recovery results in vivo were(26.19±0.76)%. Conclusion Cyclophosphamide is suitable for microdialysis sampling. This experiment calibration method is simple and effective.

Dimethyl sulfoxide acts as a protective agent to perfuse rabbit amputated limbs:the relative recovery of local drug concentrations / 中国组织工程研究

BACKGROUND:The effect of dimethyl sulfoxide cryoprotectants has been got a lot of verification in the low-temperature medical applications. But there is no literature addressing microdialysis detection of dimethyl sulfoxide cryoprotectants. OBJECTIVE:To investigate the microdialysis relative recovery of different concentrations of dimethyl sulfoxide cryoprotectants used for limb reattachment. METHODS:In vitro linear probe relative recovery of different concentrations of dimethyl sulfoxide (2%, 5%, 8%) was detected by retrodialysis gain and loss method. The correction in vivo experiment was done to estimate dimethyl sulfoxide relative recovery in rabbit amputated limbs. RESULTS AND CONCLUSION:The relative recoveries of different concentrations of dimethyl sulfoxide (2%, 5%, 8%) were (49.49±3.56)%, (46.30±1.48)%, (52.66±2.54)%using retrodialysis gain method and (50.99±6.89)%, (43.86±1.35)%, (50.67±0.75)%using retrodialysis loss method. The average recoveries were (49.48±3.18)%and (48.51±4.03)%, respectively. There was no difference in the relative recovery of dimethyl sulfoxide detected using two methods. The change of dimethyl sulfoxide concentration could not influence the retrodialysis gain and loss method calibration results. The recovery was (15.45±4.8)%in vivo. These findings indicate that the microdialysis technology is suitable for dimethyl sulfoxide sampling in vivo that has no obvious influence on the relative recovery.

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing-decreasing-increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.

Application of microdialysis in tumor / 国际肿瘤学杂志

Microdialysis is an automatic biological sampling technique that is directed into the target tissue. Microdialysis is used to monitor the changes of drug and related substances associated with pathological physiology of tumor in the target tissue at the molecular level. Microdialysis is applied to evaluate the pharmaco-kinetics of drugs in the target tissue of tumor,optimize the combination therapy,explore molecular targeted therapy and study the early diagnosis and prognosis of tumor. Microdialysis provides a new idea and method for the prevention and cure research of tumor.

Enhancing the accumulation of beta-amyrin in Saccharomyces cerevisiae by co-expression of Glycyrrhiza uralensis squalene synthase 1 and beta-amyrin synthase genes / 药学学报

Glycyrrhiza uralensis Fisch. ex DC is widely used in traditional Chinese medicine (TCM). Among its various active components, glycyrrhizic acid is believed to be the marker component. Squalene synthase (SQS) and beta-amyrin synthase (beta-AS) are key enzymes in the biosynthetic pathway of glycyrrhizic acid in G uralensis. To reveal the effects of co-expression of SQS1 and beta-AS genes on this pathway, 7 yeast expression vectors harboring different SQS1 variants and beta-AS were constructed and expressed in Saccharomyces cerevisiae as fusion proteins. TLC and GC-MS results showed that co-expression of SQS1 and beta-AS enhanced the accumulation of beta-amyrin. The effects of SQS12 were more obvious than the other two SQS1 variants. This study is significant for further investigations concerned with exploring the biosynthesis of glycyrrhizic acid in vitro and strengthening the efficacy of G. uralensis by means of increasing the content of glycyrrhizic acid.

Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis / 药学学报

The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

Sequence analysis and identification of a chloroplast matK gene in Rhei Rhizoma from different botanical origins / 药学学报

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.

Detection method and pattern recognition of ginseng and American ginseng pieces by electronic nose / 中国中药杂志

<p><b>OBJECTIVE</b>To distinguish the ginseng and American ginseng pieces accurately and rapidly by electronic nose technology and principal component analysis (PCA) method.</p><p><b>METHOD</b>The optimum conditions of electronic nose for ginseng and American ginseng pieces, such as sample size and volume, headspace volume, incubation time and temperature were determined by the orthogonal test, the data were processed by the normalization method and the preprocessed data were analyzed PCA.</p><p><b>RESULT</b>The detection methods of ginseng and American ginseng pieces was established by electronic nose, and the odor fingerprint figures of ginseng and American ginseng pieces were obtained, and ginseng and American ginseng pieces were distinguished by PCA recognition pattern.</p><p><b>CONCLUSION</b>A new accurate and rapid method to distinguish ginseng and American ginseng pieces was established by electronic nose detection.</p>

Establishment of detection system of CNVs HMGR, SQS1, beta-AS synthase gene of Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a stabilized and reliable detection system of CNVs of HMGR, SQS1, beta-AS gene of Glycyrrhiza uralensis.</p><p><b>METHOD</b>Real time PCR was used to detect the CNVs of HMGR, SQS1, beta-AS gene of G. uralensis.</p><p><b>RESULT</b>In the quantitative detection experiments of HMGR, SQS1, beta-AS gene of G. uralensis, the change of value of C(t) was 25.82-25.88, 29.01-29. 08, 15.52-15.56, 19.06-19.08 respectively, the alue of SD was 0.033, 0.032, 0.024, 0.011 respectively, and the value of CV was 0.12%, 0.22%, 0.16%, 0.06% respectively.</p><p><b>CONCLUSION</b>The repeatability of detection system of Real time PCR was stabilized and reliable, and the method could be used to detect the CNVs of HMGR, SQS1, beta-AS gene of G. uralensis.</p>

Preliminary study in glycyrrhizin content and its influencing factors of wild and cultivated in different region of China / 中国中药杂志

<p><b>OBJECTIVE</b>In order to understand the glycyrrhizin content range in the wild and cultivated Glycyrrhiza uralensis in China and to find the related influencing factors of glycyrrhizin content.</p><p><b>METHOD</b>The glycyrrhizin content of 165 wild and 1 013 cultivated G. uralensis samples from 37 countries in 9 provinces was determined by HPLC, and the effects of the producing region, medicinal parts, cultivation years, soil type and texture on the glycyrrhizin content were analyzed.</p><p><b>RESULT AND CONCLUSION</b>The average glycyrrhizin content was (4.43 +/- 1.32)% in the wild G. uralensis population, and (1.51 +/- 0.49)% in the cultivated and the glycyrrhizin content in the cultivated was less than the minimum sandards in the Chinese Pharmacopoeia. The glycyrrhizin content was significant different in the wild and cultivated G. uralensis in different producing regions, respectively. The glycyrrhizin content in roots and rhizome of the wild G. uralensis had no significant difference, it had no significant difference in the cultivated G. uralensis from 1 to 4 years and it increased rapidly after 5 years, and the effects of the soil types and texture on it were significant.</p>

Mechanism of genuineness of liquorice Glycyrrhiza uralensis based on CNVs of HMGR, SQS1 and beta-AS gene / 药学学报

Abstract: This study is to reveal the correlation between CNVs of HMGR, SQS1, beta-AS gene and genuineness of liquorice. Real-time PCR was used to detect the copy number of HMGR, SQS1, beta-AS gene of liquorice. According to the results, the range of the copy number variation of HMGR gene was between 1 and 3, the copy number of SQS1 gene was 1 or 2, and the copy number of beta-AS gene was only 1. On the basis of the copy number of HMGR, SQS1 and beta-AS gene, there were five groups, type A (2 + 1 + 1), type B (1 + 1 + 1), type C (3 + 2 + 1), type D (2 + 2 + 1) and type E (3 + 1 + 1). There were two types, type A and type B, in Hangjinqi of Inner Mongolia, and the ratio of A to B was 1:1.3. There were also two types, type A and type B, in Chifeng of Inner Mongolia, and the ratio of A to B was 3:1. There were four types, type A, type B, type C and type D, in Yanchi of Ningxia province, and the ratio of A to B was 1:5.1. There were three types, type A, type B and type E, in Minqin of Gansu province, and the ratio of A to B was 2:1. So CNVs mainly existed in the liquorice from Ningxia and Gansu provinces. While the genetic background of liquorice from Hangjinqi of Inner Mongolia was stabilized. The results of the experiment proved that the correlation between CNVs and origins was one of the reasons of genuineness of liquorice.

Cloning and sequence analysis of squalene synthase gene and cDNA in Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To clone and sequence the open reading frame and genomic sequence of squalene synthase (SQS) from Glycyrrhiza uralensis.</p><p><b>METHOD</b>The primers were designed according to cDNA sequence of SQS from G. glabra reported by Hiroaki HAYASHI, SQS cDNA was cloned with total RNA extracted from roots of G. uralensis. Specific fragments were amplified by RT-PCR and then were cloned and sequenced. SQS DNA was cloned with total DNA extracted from roots of G. uralensis. Specific fragments were amplified by PCR and then were cloned and sequenced.</p><p><b>RESULT</b>GuSQS1 (GenBank accession number: GQ266154) was 1 242 bp in length encoding proteins with 412 amino acid. NCBI Blast x search results showed GuSQS1 had the highest amino acid similarity to the corresponding proteins from G. uralensis. The identities of GuSQS1 with the two proteins were 98. 55% and 88. 62%. SQS (GenBank accession number: GQ180932) gene with 4 484 bp containing 13 exons and 12 introns was then amplified by PCR with genomic DNA extracted from roots of G. uralensis.</p><p><b>CONCLUSION</b>These findings of cloning and sequencing the open reading frame and genomic sequence of squalene synthase (SQS) from G. uralensis brought some new clues for the further exploration of SmSQS function in sterol and terpenes biosynthesis.</p>

Correlation between expression level of functional genes and tanshinones' accumulation in Salvia miltiorrhiza from different areas / 中国中药杂志

<p><b>OBJECTIVE</b>To study the relationship between functional gene expression in Salvia miltiorrhiza from different producing areas and active principles, which might provide scientific basis for the gene regulation of tanshinones.</p><p><b>METHOD</b>The quantitative determination of cryptotanshinone and tanshinone II A was carried out by using HPLC method, expression level of 3 functional genes of SmAACT, SmCMK and SmIPPI were investigated by real-time PCR method.</p><p><b>RESULT</b>The content of active principles together with expression level of SmAACT and SmCMK were higher in S. miltiorrhiza from genuine producing areas including Henan and Shanxi, but lower in samples from Beijing which was non-genuine producing area.</p><p><b>CONCLUSION</b>Expression level of SmAACT and SmCMK had close relationships involving tanshinones' accumulation, but the SmIPPI gene had not.</p>

New analysis of EST-SSR distribution and development of EST-SSR markers in Salvia miltiorrhiza / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the new EST-SSR markers for analyzing the genetic variation of different population of Salvia miltiorrhiza.</p><p><b>METHOD</b>It was dealt with ESTs newly downloaded from Genbank and that of acquired from HMPL lab EGassembler software, and then carried out SSR loci search and SSR type analysis by SSRIT software. After that, it was designed the EST-SSR primer pairs for PCR amplification condition optimization.</p><p><b>RESULT</b>Abundant and high coverage of SSR loci distribution were found in S. miltiorrhiza with having one SSR per 5.8 kb ESTs. Among them, the occurrences of different repeat units were mainly the di- (63.0%) and tri- (35.5%). The CT/AG was the most frequent motif in dinucleotide motif type and the GAA/TCC was the most frequent motif in trinucleotide repeats. Out off 36 primer pairs, 29 primer pairs (80.5%) were successfully amplified in all samples of S. miltiorrhiza while the rest failed to give PCR products at various annealing temperature and Mg2+ concentrations. The selected primer pairs also showed the polymorphism in samples from different S. miltiorrhiza populations.</p><p><b>CONCLUSION</b>The newly establishment of EST-SSR markers showed high SSR loci coverage and genetic polymorphisms in S. miltiorrhiza population. It could be used for genetic variation analysis.</p>