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In order to analyze the similarities and differences of chemical compositions between the roots and stems and leaves of Isodon japonicus(IJ), this study utilized UPLC-Q-TOF-MS technology to systematically characterize its chemical compositions, analyzed and identified the structure of its main compounds, and established a method for simultaneous determination of its content by refe-rence substance. A total of 34 major compounds in IJ, including 14 reference compounds, were identified or predicted online. Moreover, an UPLC-UV content determination method was developed for 11 compounds [danshensu, caffeic acid, vicenin-2,(1S,2S)-globoidnan B, rutin,(+)-rabdosiin,(-)-rabdosiin,(1S,2S)-rabdosiin, shimobashiric acid C, rosmarinic acid, and pedalitin]. The method exhibited excellent separation, stability, and repeatability, with a wide linear range(0.10-520.00 μg·mL~(-1)) and high linearity(R~2>0.999). The average recovery rates ranged from 94.72% to 104.2%. The principal component analysis(PCA) demonstrated a clear difference between the roots and stems and leaves of IJ, indicating good separation by cluster. Furthermore, the orthogonal partial least squares discriminant analysis(OPLS-DA) model was employed, and six main differentially identified compounds were identified: rosmarinic acid, shimobashiric acid C, epinodosin, pedalitin, rutin, and(1S,2S)-rabdosiin. In summary, this study established a strategy and method for distinguishing different parts of IJ, providing a valuable tool for quality control of IJ and a basis for the ratio-nal utilization and sustainable development of IJ.

Moutan Cortex(MC) residues produced after the extraction of MC can be re-extracted for active components and used to produce organic fertilizer and animal feed. However, they are currently disposed as domestic waste, which pollutes the environment. This study analyzed the chemical composition of the medicinal material, residues, and residue compost of MC by UPLC-UV-Q-TOF-MS. Furthermore, the nutrient composition of MC residues and the residue compost was analyzed. The results showed that:(1)MC residues had lower content of chemicals than the medicinal material, and content of paeonol, gallic acid, and galloylglucose in MC residues were about 1/3 of that in the medicinal material. The content of chemicals were further reduced after residue composting, and the quantitative compounds were all below the limits of detection.(2)Compared with MC residues, the residue compost showed the total nitrogen, total phosphorus, total potassium, and organic matter content increasing by 122.67%, 31.32%, 120.39%, and 32.06%, respectively. Therefore, we concluded that the MC residues can be used to re-extract active compounds such as paeonol, gallic acid, and galloylglucose. The MC residue compost is a high-quality organic fertilizer containing minimal content of chemicals and can be widely used in the cultivation of Chinese medicinal herbs.

This study aimed to characterize and identify the non-volatile components in Pogostemonis Herba by using ultra-perfor-mance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS) combined with UNIFI and an in-house library. The chemical components in 50% methanol extract of Pogostemonis Herba were detected by UPLC-Q-TOF-MS in both positive and negative MS~E continuum modes. Then, the MS data were processed in UNIFI combined with an in-house library to automatically characterize the metabolites. Based on the multiple adduct ions, exact mass, diagnostic fragment ions, and peak intensity of compounds and the fragmentation pathways and retention behaviors of reference substances, the structures identified by UNIFI were further verified and those of the unidentified compounds were tentatively elucidated. A total of 120 compound structures were identified or tentatively identified, including flavonoids, phenylpropanoids, phenolic acids, terpenes, fatty acids, alkaloids, and phenylethanoid glycosides. Sixteen of them were accurately identified by comparison with reference substances, and 53 compounds were reported the first time for Pogostemonis Herba. This study systematically characterized and identified the non-volatile compounds in Pogostemonis Herba for the first time. The findings provide a scientific basis for revealing the pharmacodynamic material basis, establishing a quality control system, and developing products of Pogostemonis Herba.

Codonopsis Radix is a traditional tonic medicine commonly used in China, which has the effects of strengthening the spleen and tonifying the lung, as well as nourishing blood and engendering liquid. The chemical constituents of Codonopsis species are mainly polyacetylenes, alkaloids, phenylpropanoids, lignans, terpenoids and saponins, flavonoids, steroids, organic acids, saccharides, and so on. Modern pharmacological studies showed that Codonopsis Radix also has a variety of pharmacological effects such as enhancing body immunity, protecting gastrointestinal mucosa and resisting ulcers, promoting hematopoietic function, regulating blood sugar, and delaying aging. In this paper, the chemical constituents of Codonopsis species and the pharmacological effects of Codonopsis Radix were summarized, and on this basis, the quality markers of Codonopsis Radix were analyzed. It was predicted that lobetyolin, tangshenoside I, codonopyrrolidium A, and the oligosaccharides were the possible Q-markers of Codonopsis Radix. This paper will provide scientific references for the quality evaluation and profound research and the development of Codonopsis Radix.

Objective: To establish an HPLC fingerprint analysis method for Atractylodis Macrocephalae Rhizoma (warm dried)., Methods: Took methanol extract of Atractylodis Macrocephalae Rhizoma as sample by ultrasonic processing, HPLC analysis was carried out on Sinochrom ODS-BP (4.6 mm x 250 mm, 5 microm) chromatographic column with mobile phase of acetonitrile (A)-water (B), gradient elution with a flow at 1.0 mL/min, ultraviolet detection wavelength at 242 nm and column temperature at 30 degrees C., Results: An HPLC fingerprint analysis method for Atractylodis Macrocephalae Rhizoma was developed. Eight common peaks were obtained and three peaks were identified., Conclusion: The method is accurate and credible which can be used for quality control of Atractylodis Macrocephalae Rhizoma (warm dried).

Objective: To study the distribution laws of the Chinese medicine syndrome patterns and its correlated symptoms in patients with polycystic ovarian syndrome (PCOS), and the possible correlation between Chinese medicine syndrome patterns and PCOS associated parameters, thus to provide a guidance for selecting proper indices in curative effectiveness assessment., Methods: Using clinical epidemiological methods and mathematical statistics, the Chinese medicine syndrome patterns were studied in 228 PCOS patients. The distribution features of Chinese medicine syndrome patterns were summarized., Results: Shen-deficiency blood-stasis syndrome was the most frequently seen in PCOS patients, followed by Pi-deficiency phlegm-dampness syndrome, Pi-Shen yang-deficiency syndrome, and Shen-yin deficiency syndrome. Positive correlation existed between serum levels of follicle stimulating hormone (FSH) and Pi-Shen yang-deficiency syndrome. Positive correlation existed between fasting blood sugar (FBS), waist to hip ratio (WHR), body mass index (BMI), fasting insulin (FIN), and Hirsutism score and Pi-deficiency phlegm-dampness syndrome. Positive correlation existed between serum levels of prolactin (PRL), estradiol (E2 ) and qi stagnancy and blood stasis syndrome. Positive correlation existed between serum progesterone (PRG) level, FSH, FIN, BMI, acne score and Gan stagnancy and blood heat syndrome. Positive correlation existed between luteinizing hormone (LH) and Shen-deficiency blood-stasis syndrome. Besides, LH/FSH >3 was possibly more frequently seen in Pi-deficiency phlegm-dampness syndrome and Pi-Shen yang-deficiency syndrome. Family heritability could be seen in each syndrome patterns. Among them, female heritability was more often seen in Shen-deficiency blood-stasis syndrome, while male heritability was more often seen in Shen-yin deficiency syndrome., Conclusions: Shen-deficiency blood-stasis syndrome, Pi-deficiency phlegm-dampness syndrome, Pi-Shen yang-deficiency syndrome, and Shen-yin deficiency syndrome were most frequently seen in PCOS patients. The sex hormones (including 6 items), FBS, FIN, WHR and BMI, etc. were correlated with each Chinese medicine syndrome pattern to various extents, which could be taken as reference in Chinese medicine syndrome differentiation.

To understand the genetic diversity of buffalo Y chromosome, 14 microsatellite markers specific for cattle Y chromosomes were used to analyze 31 male buffaloes of three populations (i.e., HH, BN, and PR) originating from southeastern Yunnan Province, P. R. China. The aim of this study was to examine the practicability of cattle Y chromosome-specific microsatellite markers in studying genetic diversity of buffalo Y chromosome. Three markers (INRA008, UMN0103, and UMN0504) produced single allele, and three (UMN1113, UMN0304, and BC1.2) produced three alleles. But these markers were all monomorphic. Another three markers (UMN0920, UMN0307, and UMN3008) amplified irregular ladder-like bands. These markers were not suitable for investigating the swamp buffalo Y chromosome genetic diversity. The remaining five microsatellites (INRA124, INRA189, BM861, PBR1F1, and UMN2001) were polymorphic, which can be used to study the swamp buffalo Y chromosome genetic diversity. The mean number of alleles (Na), expected heterozygosity (He), genetic diversity (GD), polymorphism information content (PIC), and Shannon's information index (SI) across these five polymorphic loci in the buffalo population samples studied were 2.8000, 0.3998, 0.4144, 0.3245, and 0.5849, respectively, demonstrating a moderate degree of polymorphism in the Y chromosome of the buffalo population.

Objective: To investigate the biological functions of TTG1A in liver fibrosis., Methods: Yeast two-hybrid system was used to screen proteins associated with TTG1A. Briefly, the coding sequence of TTG1A was cloned into pGBKT7 vector, and the recombinant plasmid was transformed into yeast cells AH109 ( a type), then these cells were mated with yeast cells Y187 (a type) transformed with human leukocyte cDNA library plasmid pACT2. The obtained diploid yeast cells were plated on synthetic dropout nutrient medium containing X-alpha-gal for double selection. The plasmids from positive colonies were transformed into E.coli and sequenced., Results: The recombinant yeast expression vector pGBKT7-TTG1A was successfully constructed. Nineteen TTG1A binding proteins, including Homo sapiens major histocompatibility complex, class II DP beta 1 (HLA-DPb1), Homo sapiens ribosomal protein L30 (RPL30), Homo sapiens nucleophosmin Homo sapiens nucleobindin 2 (NUCB2), Homo sapiens ash2, variant Gaucher disease and variant metachromatic leukodystrophy, MORF4L1, Homo sapiens ubiquitin-conjugating enzyme E2L3 (UBE2L3), APOA1, Homo sapiens lectin, and galectin 1, were identified., Conclusions: This study may help to elucidate the molecular function of TTG1A.

Objective: To explore the osteogenic potential of the nano-hydroxyapatite/collagen/calcium alginate composite implanted in animals., Methods: Eighteen 3-month-old New Zealand white rabbits were adopted to prepare 15 mm segmental defect model at the middle part of radius. Rabbit models were randomly divided into experimental group and blank control group. Nano-hydroxyapatite/collagen/calcium alginate was implanted into the defects of experimental group. Four, 8, and 12 weeks after operation, all specimens were examined by X-ray and histological methods., Results: All the 18 rabbit models entered the final analysis. X-ray showed that osteotylus was seen in the whole defect area in the experimental group 12 weeks after operation, during which osteogenesis was more obvious than in weeks 4 and 8 and the bridge grafting of defect area was obviously visible. In the blank control group, osteotylus was only observed at the two ends of the defects, and no osteogenesis was found in the central part of the defect area. Histological examination showed that new osteoid formation was seen in internal porous zone in the experimental group in weeks 4 and 8; in week 12, more woven bone-like tissues were visible and trabecular-like structure was formed., Conclusion: The nano-hydroxyapatite/ collagen/calcium alginate has good osteogenic potential.

The main function of ZAG gene is to enable the decomposition of fat, and reduced fat content. In this study, polymorphisms of four loci (Z1, Z2, Z3, Z4) at the coding region of the bovine ZAG gene were detected in 145 Jiaxian red cattle, and polymorphisms were found on Z1, Z3, Z4 loci. The fragments showing different SSCP patterns were sequenced, nd a total of six SNPs (C115T, A3257G, A4013G, T4027C, C4032T, and T4120C) were found. The Z3 locus of ZAG gene was at Hardy-Weinberg equilibrium, while Z1 and Z4 loci of ZAG gene were not at Hardy-Weinberg disequilibrium. The association analysis of different genotypes with some of the growth and development traits showed that the individuals with genotype AC had a larger body length, heart girth, circumference of cannon bone, body weight than the individuals with genotypes AA and AB (P<0.05 or P<0.01, respectively) in Z4 locus. It is suggest that Z4 locus may be one of the marker-assisted selection used as growth traits in Jiaxian red cattle.

Objectives: To construct a cDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1., Methods: mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction twice it then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search., Results: The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC was constructed successfully. The amplified library contained 146 positive clones, which contained 200-1000 bp of inserts. Randomly, thirty clones were analyzed by sequencing and bioinformatics, consisting of 28 known genes and 2 unknown genes., Conclusions: The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC using SSH technique was constructed successfully. Some gene coding proteins are those involved in cell growth regulation, protein synthesis, signal transduction, extracellular matrix metabolism, and anti-lipid peroxidative, which gives us some new clues for the study of the mechanism of liver fibrosis.

PCR-SSCP and DNA sequencing methods were conducted to detect single nucleotide polymorphism of alpha-lactalbumin (LALBA) gene in 452 Inner Mongolian white cashmere goats (IMWC). Correlations between SNP of goat LALBA gene and economic traits, e.g., cashmere yield, cashmere thickness, length and weight, were analyzed. The SSCP in P2 primer locus, which was caused by the point mutation M63868:g.1897T>C in the exon 3 of LALBA gene was detected. At this locus, the genotype TT and allele T were predominant in the IMWC population, which agreed with Hardy-Weinberg equilibrium. Moreover, there was a significant correlation between polymorphism of goat M63868:g.1897 locus and cashmere yield of IMWC (P=0.017). The individuals with genotype TC had more cashmere yield than those with geontype TT. Hence, genotype TC of LALBA gene can be used as a molecular marker for breeding superior cashmere yield in goat marker-assisted selection.

The aim of the present study was to identify and characterize polymorphisms within the coding region and the 3' flanking region of the bovine serotonin receptor 1B gene among different cattle breeds. Four DNA fragments were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. The fragments showing different SSCP patterns were sequenced. And a total of six SNPs (G205T, C507T, C546G, C744T, G816A and G942A) were detected. The SNPs were at Hardy-Weinberg equilibrium except C507T and C546G in all genetic population. The frequencies of allele 205T of Holstein were much higher than that of the other six beef cattle populations. Almost the PIC of all SNPs were not more than 0.10 except that of G205T in Holstein cows, which indicated the bovine HTR1B gene was conserved.

In this paper PCR-SSCP technique was used to analyze the polymorphisms of the tenth exon of GHR gene in Qinchuan cattle. The results showed that PCR products demonstrated polymorphisms in Qinchuan cattle and the population was at Hardy-Weinberg equilibrium (P > 0.05). There were three allele gene A, B, C in this locus with the corresponding frequencies of 0.5956, 0.2905 and 0.1140. The amplified fragments of homozygosis genotype were sequenced. The result showed that there were two mutation in these fragments. Then the relationship between genotypes and productive performances was analyzed by the Least Squire Method (LSM). The results showed that some productive performances of the indi-viduals with genotype AB were higher than those of the other genotypes, and the derivation was significant among geno-types. It suggested that GHR gene may be a candidate gene responsible for growth trait in Qinchuan cattle.

Objective: To explore the impact of HBeAg positivity/negativity and HBV DNA loads on the prognosis of chronic severe hepatitis B., Methods: 206 patients with chronic severe hepatitis B hospitalized in Beijing Ditan Hospital from July 2002 to Dec. 2004 were analyzed. HBeAg positivity/negativity, HBV DNA loads and other factors relating to the prognosis of the patients were studied with univariate and multivariate analyses., Results: Chi2 univariate analysis showed that there was no significant difference in the prognosis between different HBeAg groups (chi2 = 0.440, OR = 0.777, 95% CI 0.424-1.425, P = 0.50). But there was a significant difference in the prognosis between different HBV DNA load groups: the prognosis of patients with lower HBV DNA loads was better than those with higher loads (chi2 = 9.806, OR = 3.055, 95% CI 1.554-6.007, P = 0.002), and the improving rates of the two groups were 53.1% and 27.0% respectively. Using multivariate logistic regression analysis, 9 screened factors showed great impact on the prognosis of chronic severe hepatitis B. Cirrhosis, hepatorenal syndrome, hepatic encephalopathy, PTA < 20%, TBil > 513 mmol/L, Alb < 30 g/L, CHO < 1.6 mmol/L, PLT < 5 x 10(9)/L, and higher HBV DNA loads (HBV DNA > 3 x 10(4) copies/ml in HBeAg negative patients and > 1 x 10(5) copies/ml in HBeAg positive patients) were shown to be associated with a poor prognosis. Coefficients of regression of the above factors were 1.539, 21.356, 1.398, 1.650, 2.440, 2.266, 1.738, 2.631 and 2.656 respectively. The coefficients of regression of HBV DNA loads were: B = 2.656, Wald = 7.768, P = 0.005, EXP(B) = 14.235, and 95.0% CI for EXP(B) = 2.199-92.133., Conclusions: Our results indicate that the HBV DNA loads were one of the most important factors influencing the prognosis of the chronic severe hepatitis B patients, the importance is only next to hepatorenal syndrome and over grade II hepatic encephalopathy. HBeAg positivity/negativity has no influence on the prognosis, but HBV DNA loads are important; the lower the viral loads, the better the prognosis.

Random microsatellite amplify polymorphic DNA (RMAPD) is a new method of molecular marker. It can reveal genome DNA polymorphisms of organism only when random primer and microsatellite forward or reverse primer are combined together as a pair of primers by the PCR reaction system with Taq DNA polymerase, MgCl2, dNTPs and contemplate DNA. The core question of RMAPD is validity of the primers used. A lot of experiments of RMAPD in 69 Xinong Saanen dairy goats showed that RMAPD primers were effective. Comparison of RMAPD, microsatellite and RAPD markers demonstrated that RMAPD was different from each other in primers, amplification protocol and repetition. RMAPD is not equal to RAPD, but a extendable RAPD. Therefore, RMAPD is regarded as a new method of molecular marker. As it has many characteristics of DNA marker, RMAPD has a widest potential application in animal breeding and genetics, such as analysis of genetic structure and relationship and MAS.

The genetic polymorphism of Yangxian artificially reared 43 crested ibises (Nipponia nippon) was firstly investigated by RMAPD (random microsatellite amplify polymorphic DNA) marker. The results showed that RMAPD was more stable and polymorphic than RAPD. 2147 bands were amplified by 12 pairs of primers,of which 93 bands had polymorphisms. The band sharing frequency and the genetic diversity index were 0.718 and 3.664, respectively, indicating that the genetic structure of crested ibis population was simple and poor genetic variations existed in crested ibis population in Yangxian. It is imperious to protect the genetic diversity of crested ibis population.

PCR-RFLP technique was applied to analyze correlation between the polymorphisms of CSN1 S2 (alpha(s2) casein), CSN3 (kappa casein) and beta-lg (beta-lactoglobulin) genes and milk performance in 69 individuals of Xinong Saanen dairy goat. The results showed that there was significant correlation between different genotypes of CSN1 S2 locus and milk yield:average milk yield of individuals with genotype FF was less than that of genotype NN (P < 0.05); the second lactation milk yield of individuals with genotype NN was over 90 kg higher than that with genotype FF (P < 0.01). This indicates that allele F of CSN1 S2 locus probably has significant negative effect on milk yield. The results of CSN3 gene digested with endonuclease Hind III cleavage showed that no significant difference of milk yield between genotype DE and genotype EE was detected in first, second, third and fourth lactation milk yield and average milk yield (P > 0.05). The results of CSN3 gene with endonuclease Taq I cleavage showed that no significant difference of milk yield among individuals with genotype TT, TC and CC was detected (P > 0.05). No polymorphism was detected in PCR products of CSN3 gene digested with endonuclease Hae III. The analysis of beta-lg gene's 5' flanking region (710 bp) by PCR-RFLP in Xinong Saanen dairy goat showed that milk yield of individuals with genotype AA was higher than that with genotype AB in second, third lactation milk yield and average milk yield (P < 0.05). The results implied that allele A of beta-lg gene's 5' flanking region is probably related to high milk yield.

PCR-RFLP was applied to analyze the polymorphism of CSN1S2 gene in 170 goats that comprised of five goat breeds, namely Xinong Saanen dairy goat, Guanzhong dairy goat, Shaannan white goat, Angora goat and Boer goat. A 310 bp -long PCR product was digested with Alw26I and demonstrated polymorphism in five goat populations that were all at Hardy-Weinberg equilibrium (P>0.05). For Xinong Saanen dairy goat, Guanzhong dairy goat, Shaannan white goat, Angora goat and Boer goat, gene heterozygosity/effective allele gene number/Shaanon information entropy /Polymorphism information content were 0.1589/1.1889/0.2955/0.1463, 0.4114/1.6981/0.6017/0.5171, 0.1653/1.1980/0.3046/0.1516, 0646/1.0691/0.1463/ 0.0625, 0.0541/1.0572/0.1270/ 0.0526, respectively. According to the heredity diversity indexes described above of the five goat breeds, Guanzhong dairy goat had the most abundant heredity diversity and showed high polymorphism, and Xinong Saanen dairy goat and Shaannan white goat were inferior, while Angora goat and Boer goat had the lowest genetic variability.