Identification of Andrographis Herba and its common products using mini-barcode / 中国天然药物

Andrographis Herba, the aerial part of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), has a wide geographic distribution and has been used for the treatment of fever, cold, inflammation, and other infectious diseases. In markets, sellers and buyers commonly inadvertently confuse with related species. In addition, most Chinese medicinal herbs are subjected to traditional processing procedures, such as steaming and boiling, before they are sold at dispensaries; therefore, it is very difficult to identify Andrographis Herba when it is processed into Chinese medicines. The identification of species and processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA barcoding has received considerable attention as a new potential means to identify species and processed medicinal materials. In this study, 17 standard reference materials of A. paniculata, 2 standard decoctions, 27 commercial products and two adulterants were collected. Based on the ITS2 sequence, it could successfully identify A. paniculata and adulterants. Moreover, a nucleotide signature consisting of 71 bp was designed, this sequence is highly conserved and specific within A. paniculata while divergent among other species. Then, we used these new primers to amplify the nucleotide signature region from processed materials. In conclusion, the DNA barcoding method developed in the present study for authenticating A. paniculata is rapid and cost-effective. It can be used in the future to guarantee the quality of Andrographis Herba of each regulatory link for clinical use.

Research progress on mechanism of phytohormones in regulating flavonoid metabolism / 中国中药杂志

Phytohormones play an important role at all stages of plant growth, influencing plant growth and development and regulating plant secondary metabolism, such as the synthesis of flavone, flavonol, anthocyanin, and other flavonoids. Flavonoids, a group of important secondary metabolites ubiquitous in plants, have antioxidative, anti-microbial, and anti-inflammatory activities and thus have a wide range of potential applications in Chinese medicine and food nutrition. With the development of biotechnology, phytohormones' regulation on flavonoids has become a research focus in recent years. This study reviewed the research progress on the mechanism of common phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and salicylic acid, in regulating flavonoid metabolism, and discussed the molecular mechanism of the synthesis and accumulation of flavonoids, aiming at clarifying the key role of phytohormones in modulating flavonoid metabolism. The result is of guiding significance for improving the content of flavonoids in plants through rational use of phytohormones and of reference value for exploring the mechanism of hormones in regulating flavonoid metabolism.

Variation analysis of genomic methylation induced by high temperature stress in Pinellia ternata / 中国中药杂志

Pinellia ternata belongs to the Araceae family and is a medicinal herb. The tuber is the medicinal organ with antitussive, antiemetic and anti-tumor activities. It is easy to encounter high temperature environment during the growth periods, leading to decrease of tuber production. At present, the mechanism of response to high temperature stress in P. ternata is still unknown. DNA methylation plays a vital role in plant protection against adversity stress as a way of epigenetic regulation. In this study, P. ternata was used as material for treatment of high temperature stress at 0 h, 6 h and 80 h, and methylation sensitive amplification polymorphism(MSAP) analysis was conducted on the changes of DNA methylation in its genome. The results showed that 20 pairs of MSAP primers were selected from 100 MSAP primers with multiple clear and uniform bands, and 353, 355 and 342 loci were amplified from materials of P. ternata treated in the high temperature stress 0 h, 6 h and 80 h, respectively. Cytosine methylation levels of CCGG context in the above materials were characterized as 60.91%, 44.79% and 44.74%, respectively. And the full methylation ratios were 16.71%, 22.25% and 29.24, respectively. It demonstrated that high temperature stress significantly induced the down-regulation of DNA methylation level and up-regulation of the full methylation rate in P. ternata genome. This study provides a preliminary theoretical reference for analyzing the mechanism of P. ternata responding to high temperature stress from the epigenetic perspective.

Analysis of Flavonoid Content and Key Gene Expressions in Sprout and Seedling of Tartary Buckwheat / 中国实验方剂学杂志

Objective:To reveal the dynamic changes of flavonoids secondary metabolites and relevant genes expressions in the process of germination of tartary buckwheat seeds by investigating the content of catechins，epicatechins，rutin，and quercetin，and the expressions of their relevant genes in tartary buckwheat sprouts and seedlings，in order to provide scientific basis for the selection of high-quality, high-nutrition tartary buckwheat sprouts.Method:Contents of catechin，epicatechin，rutin，and quercetin in tartary buckwheat sprouts and seedlings were detected by UPLC-ESI-QQQ-MS，and the expression levels of genes relating to flavonoids synthesis in tartary buckwheat sprouts and seedlings were detected by real-time quantitative PCR.Result:There were differences between tartary buckwheat sprouts and seedlings in the relative contents of catechin，epicatechin，rutin and quercetin，as well as the expressions of relevant genes in the synthesis pathway, including FtPAL，FtC4H，Ft4CL，FtCHS，FtCHI，FtF3H，FtF3'H，FtFLS，FtDFR，FtLAR，FtANS，FtANR. The contents of flavonoids and the expressions of relevant genes in tartary buckwheat sprouts were higher than those in tartary buckwheat seedlings.Conclusion:The higher accumulation of secondary metabolites and flavonoids in tartary buckwheat sprouts may be related to tartary buckwheat seeds' resistance to the external environment in the initial growth stage of germination. From the perspective of application，there are more flavonoids in tartary buckwheat sprouts than in tartary buckwheat seedlings, indicating that tartary buckwheat sprouts have a higher nutritional value.

Regulation of β-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells / 中国中药杂志

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

Analysis of shading on DNA methylation by MSAP in Pinellia ternata / 中国中药杂志

Pinellia ternata is a medicinal herb of Araceae, and its tubers are used as medicines. It is a common Chinese herbal medicine in China and has a large market demand. When exposing to strong light intensity and high temperature during the growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade can effectively delay sprout tumble formation and increase its yield, however the relevant regulation mechanism is unclear. DNA methylation, as a self-modifying response to environmental changes, is often involved in the regulation of plant growth and development. In this study, P. ternata grown under natural light and 90% shading were selected as the control group and the experimental group for genomic DNA methylation analysis by using methylate sensitive amplification polymorphism(MSAP). The results showed that a total of 617 loci were detected with 20 pairs of primers, of which 311 were in the natural light group and 306 in the shading group. The methylation sites in the light and shading groups accounted for 58.2% and 71.57%, respectively, and the methylation ratios in the methylation sites were 27.65% and 29.41%, respectively, indicating that shading significantly induced the genome DNA methylation of P. ternata. Compared to the natural light group, shading promoted 32.51% of the genes methylation, while inducing 16.25% gene demethylation. This study reveals the DNA methylation variation of P. ternata under shading conditions, which lays a preliminary theoretical foundation for further analysis of the mechanism of shading regulation of P. ternata growth from epigenetic level.

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation / 中国中药杂志

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

Suspension Culture and Alkaloid Content of Pinelliae Rhizoma Tuber Under Stimulation of Salicylic Acid / 中国实验方剂学杂志

Objective: To study the suspension culture of tuber and its alkaloid content based on the stimulation of salicylic acid. Method: The tubers of Pinelliae Rhizoma in suspension tube were treated with different concentrations of exogenous salicylic acid to analyze the growth status. The content of alkaloids in tuber was detected by HPLC. Test conditions:chromatographic column for Agilent Eclipse plus C18 column (4.6 mm×250 mm,5 μm),mobile phase of acetonitrile water(4:96),the column temperature was maintained at 35℃,detection wavelength for inosine 250 nm,guanosine 260 nm,volume flow rate 1.0 mL·min-1. Result: The results showed that the exogenous salicylic acid had a certain effect on the growth of suspension tuber of Pinelliae Rhizoma. When the salicylic acid concentration was 150 μmol·L-1,the culture lasted for 25 days and the fresh weight reached the maximum value of 7.483 8 g. It also accumulates a certain amount of alkaloids. The linear range of guanosine was 0.03-0.45 μg (R2=0.999 6). After 10-days cultivatation in the salicylic acid concentration of 50 μmol·L-1,guanosine content of Pinelliae Rhizoma tubers reached a maximum of 1.353 3 mg·g-1. The linear range of inosine 0.003-0.045 μg (R2=0.999 5). When the salicylic acid concentration was 200 μmol·L-1,cultured for 30 days,the content of inosine in Pinelliae Rhizoma tubers reached the maximum value of 0.149 8 mg·g-1. Conclusion: The results of this experiment provide a reference for the study of tissue culture and rapid propagation of Pinelliae Rhizoma tubers and regulation of alkaloids,which are of great significance for the development of Pinelliae Rhizoma industry.

Investigation and optimization on ability of enzymatic hydrolysis of Mori Cortex residue / 中国中药杂志

Residue of Mori Cortex was studied to optimize its enzymatic hydrolysis process, and explore its potential as a carbon source for biochemistry and biofuel production. The cellulose content of diluted acid pretreated (DAP) and non-pretreated from Mori Cortex were measured in this study, and the results showed that the cellulose content of DAP and non-pretreated from Mori Cortex were 52.5% and 47%, respectively. This higher cellulose content indicated that residue of Mori Cortex had the potential to act as a carbon source for biochemistry and biofuel production. Enzymatic hydrolysis of pretreated and non-pretreated from Mori Cortex was conducted under different enzyme loading amount. 40 FPU·(g DW）⁻¹ enzyme loading was determined as the optimal amount by comparing the yield of sugar and the rate of enzymolysis. Under this condition, the concentrations of glucose, xylose, arabinose sugar were 23.82, 4.84, 3.6 g·L⁻¹, and the corresponding enzymatic hydrolysis rate was 45.33% which was 2.3 times higher than that of non-pretreated from Morus alba residues. Fed-batch enzymatic hydrolysis was conducted finally to get higher sugar yield, and the final glucose concentration reached up to 38 g·L⁻¹ with the enzymatic hydrolysis rate of 36.19%. The results indicated that Mori Cortex residue had higher cellulose and hemicellulose contents, so it had the potential to become a carbon source to produce the bio-chemicals and biofuels. Through enzymatic hydrolysis, it can be converted into microbial available monosaccharides; and through fermentation, it can be converted into high value-added chemicals, biofuels, etc., to solve the problem of residue pollution, and achieve the sustainable development and greening of Chinese pharmaceutical production process.

Influence of light on gene expression of key synthesis enzyme genes FtANR and FtLAR about proanthocyanidin in seeds of homologous plant of food and medicine Fagopyrum tataricum / 中国中药杂志

Tartary buckwheat Fagopyrum tataricum is an important medicinal and functional herb due to its rich content of flavonoids in the seeds. F.tataricum exhibited good functions for free radicals scavenging， anti-oxidation， anti-aging activities. Although much genetic knowledge of the synthesis， regulation， accumulation of rutin， the genetic basis of proanthocyanidins(PAs) in tartary buckwheat and their related gene expression changes under different lights(blue， red， far red， ultraviolet light) remain largely unexplored. In this study， we cloned one anthocyanidin reductase gene(ANR) and two leucocyanidin reductase gene(LAR) named FtANR，FtLAR1，FtLAR3 involved in formation of(+)-catechin and(-)-epicatechin precusor proanthocyanidin by digging out F. tataricum seed transcriptome data. The expression data showed that the opposite influence of red light on these gene transcript level compared to others lights. The expression levels of FtANR and FtLAR1 decreased and FtLAR3 appeared increment after exposed in the red light， while the expression levels of those genes appeared opposite result after exposed in the blue and far red light.

Identification of bear bile powder and its adulterants by using DNA barcoding technology / 中国中药杂志

To identify the precious bile powder and its adulterants by DNA barcoding, and establish its standard experimental process to ensure the safe and effective utilization. Total twelve sequences from samples of bear bile powder which come from Ursus thibetanus for DNA extraction, PCR(polymerase chain reaction) and sequence, then using CodonCode Aligner V 7.0.1 shear primer region to obtain COI sequence. The COI sequences of U. arctos and their adulterants were obtained from GenBank. MEGA7.0 software was applied for analyzing mutation, calculating intraspecific and interspecific K2P(Kimura 2-Parameter) genetic distance and constructing the Neighbor-joining tree(NJ). The results showed that the maximum K2P genetic distance of bear bile powder of U. thibetanus and U. arctos are far less than minimum K2P genetic distance within its adulterants species, and the results of NJ tree demonstrated that each species could be distinguished from the counterfeits obviously. DNA barcoding is a safe, convenient and reliable technique for species identification, and it is important to establish the standard sequence of COI sequences for animal medicines.

Rapid identification of Gekko gecko by loop-mediated isothermal amplification based on 12S rRNA sequence / 中国中药杂志

Gekko gecko (Tokay Gecko) is a valuable traditional Chinese medicine. In this study, the loop-mediated isothermal amplification (LAMP) technique was introduced for visual rapid identification of G. gecko from adulterants. A total of sixty-five 12S rRNA sequences of fourteen species of G. gecko and its adulterants were obtained. The results showed that G. gecko could be identified from its adulterants through BLAST analysis based on 12S rRNA regions. The 12S rRNA sequences of ten batches of G. gecko were conserved. There were only two haplotypes and three variation sites in the available regions for primers design. Six specific LAMP primers were successfully designed online based on 12S rRNA sequences. The visual rapid detection of G. gecko could be achieved with the optimized conditions (64 °C for 1 h and 80 °C for 5 min). And the required minimal template concentration was 5 μg·L⁻¹ while conventional PCR with 0.5 mg·L⁻¹. Consequently, the LAMP method established from this study was rapid, specific, highly sensitive, and simple. It could be applied to detect G. gecko from its adulterants efficiently.

Recent advances of synthetic biology for production of functional ingredients in Chinese materia medica / 中国中药杂志

The functional ingredients in Chinese materia medica are the main active substance for traditional Chinese medicine and most of them are secondary metabolites derivatives. Until now,the main method to obtain those functional ingredients is through direct extraction from the Chinese materia medica. However, the income is very low because of the high extraction costs and the decreased medicinal plants. Synthetic biology technology, as a new and microbial approach, can be able to carry out large-scale production of functional ingredients and greatly ease the shortage of traditional Chinese medicine ingredients. This review mainly focused on the recent advances in synthetic biology for the functional ingredients production.

Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an efficient genetic transformation system of Pinellia ternata.</p><p><b>METHOD</b>With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata.</p><p><b>RESULT AND CONCLUSION</b>The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.</p>

Study on optimization of induction system of test-tube tuberous roots from leaves of Rehmannia glutinosa / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of sucrose and plant growth substances of different concentrations on the induction of test-tube tuberous roots of Rehmannia glutinosa, in order to establish an efficient system for the induction of test-tube tuberous roots from leaves of R. glutinosa.</p><p><b>METHOD</b>Leaves from test-tube seedlings of 85-5 R. glutinosa were used as explants. After rooting induction, they were transferred to medium with orthogonal design for inducing test-tube tuberous roots of R. glutinosa.</p><p><b>RESULT AND CONCLUSION</b>NAA played a significant role in induction of test-tube tuberous roots of R. glutinosa, followed by sucrose and 6-BA. With leaves from test-tube seedlings as the explants, the optimal medium for inducing test-tube tuberous roots of R. glutinosa was MS + BA 3.0 mg x L(-1) + NAA 0.1 mg x L(-1) + sucrose 7%. The study provides an efficient induction system for studies on artificial seeds and secondary metabolism with test-tube tuberous roots of R. glutinosa.</p>

Study on optimization of SRAP-PCR reaction system for Pinellia ternata in Suzhou / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the optimization system of SRAP-PCR molecular marker technology in the analysis on Pinellia ternata.</p><p><b>METHOD</b>SRAP-PCR reaction system for P. ternata was optimized by L16 (5(4)) orthogonal design with five elements (dNTPs, Mg2+, the template DNA, primers, Taq enzyme) and four standards.</p><p><b>RESULT</b>The most suitable forward primer for SRAP for Pinellia ternata was 5'-TGAGTCCAAACCGGAAG-3', while the reverse primer was 5'-GACTGCGTACGAATTACG-3'. The optimized reaction system contained 70 ng DNA template, 0.9 micromol x L(-1) primer, 0.20 mmol x L(-1) dNTP s, 1.5 - 2.0 mmol x L(-1) Mg2+, and 2 U Taq enzyme.</p><p><b>CONCLUSION</b>SRAP-PCR system for P. ternata is established to lay a foundation for future construction of SRAP genetic map of P. ternata.</p>

Efficient method for extraction of high quality RNA from microtubers of Pinellia ternata in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To extract RNA from Pinellia ternata and lay a foundation for studying the formation mechanism of P. ternata.</p><p><b>METHOD</b>By modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds, a simple and convenient method for extraction of total RNA from the tubers, stems and leaves of P. ternate containing abundant polyphenols and polysaccharides was established. High concentrated p-mercaptoethanol was added in the RNA extracted buffer to remove polyphenols, phenol and chloroform were used to eliminate proteins, and isopropanol and sodium acetate were used to precipitate polysaccharides.</p><p><b>RESULT</b>The A260/A230 value of RNA extracted with improved method were all over 2.0 and the values of A260/A280 were between 1.7 and 2.0. The electrophoresis bands were cleared on agarosegel and integrity of RNA was good.</p><p><b>CONCLUSION</b>The results showed that RNA obtained from the tubers, stems and leaves of P. ternate with this method had high purity and quality and could be used in molecular biological research, as DDRT-PCR and reverse Northern blotting analysis directly. This method is simple, economic, stable performance, and has a good repeatability as well as is suitable for extracting total RNA of medicinal plants with high concentrations of phenolics and polysaccharides.</p>

Application of orthogonal design in tissue culture of Dioscorea opposite / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of different factors on buds and microtuber. These factors included plant growth substances and sucrose.</p><p><b>METHOD</b>stems were selected as explants. The effects of three kinds of factors were studied by orthogonal design method including sucrose, 6-BA, NAA on the buds and microtuber induction. The data were analyzed with range analysis and vadance analysis. RESULT AND CONDUSION: The optimal media to induce many buds from stems were MS + 6-BA 1 mg x L(-1) + NAA 1 mg x L(-1) + sucrose 3%, the effect of the three factors was in sequence of sucrose >6-BA > NAA. The optimal media to induce microtuber from stems were MS+6-BA 1.5 mg x V1 +NAA 1.5 mg x L(-1) + sucrose 5%, the effect of the three factors was in sequence of sucrose >6-BA > NAA.</p>

Study on bulblet induction of Fritillaria anhuiensis in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To study the optimal condition for bulb induction of Fritillaria anhuiensis in vitro.</p><p><b>METHOD</b>Effects of sucrose, salicylic acid, active carbon and 5 degrees C pre-incubation on bulb formation in vitro were investigated by adopting the number of bulb and fresh weight as indexes.</p><p><b>RESULT</b>The number of bulb and fresh weight in medium added with 50 g x L(-1) sucrose were significantly higher than those with other treatments. The medium with different concentrations of salicylic acid showed no significant promotion on callus differentiation. However, 5 g x L(-1) active carbon (AC) treatment was better than other treatments. Meanwhile, a large number of plantlets formed after 5 degrees C pre-incubation for 30-40 days was suitable for bulb formation and growth.</p><p><b>CONCLUSION</b>Bulblet formation was optimal in MS + KT 2 mg x L(-1) + NAA 2 mg x L(-1) + 50 g x L(-1) sucrose +5 g x L(-1) AC after callus pre-incucation at 5 degrees 1 for 30-40 days.</p>

Effects of shading on photosynthetic characteristics of Pinellia ternata leaves / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of shading on photosynthetic physiology and chlorophyll fluorescence of Pinellia ternata.</p><p><b>METHOD</b>Plant growth, chlorophyll content, net photosynthetic rate (P(n)) and chlorophyll fluorescence in P. ternata were investigated under different shading treatments (0%, 70% and 90%) when it grew about 15 cm high.</p><p><b>RESULT</b>The results showed that fresh weight of a tuber, height, leaf length, width, leaf area, specific leaf area (SLA) and contents of chlorophyll content were enhanced after shaded, and chlorophyll a/b rate declined. Compared with control, net photosynthetic rate, light compensation point (LCP) and light saturation point (LSP) of P. ternata decreased after shading, but apparent quantum yield (AQY) increased; quantum yield of PS II (PhiPS II), minimal fluorescence (F(o)), maximal fluorescence (F(m)), intrinsic photochemical efficiency of PS II (F(v)/F(m)) and photochemical quenching coefficient (qP) were enhanced.</p><p><b>CONCLUSION</b>Compared with control, all data indicated that there were distinctive differences between the height, SLA, chlorophyll content, P(n) and chlorophyll fluorescence characteristics under the shading treatments (70% and 90%), the fresh weight of a tuber increased after 70% shading, and provided better environmental conditions for the growth of P. ternata.</p>