Mechanism of Arnebia euchroma Against Melanoma： An Exploration Based on Network Pharmacology and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo preliminarily predict the active components， action targets， and signaling pathways of Arnebia euchroma in the treatment of melanoma based on network pharmacology and molecular docking， and to verify its possible mechanism of action in in vitro experiments. MethodThe active components and related targets of A. euchroma were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）SwissTargetPrediction and literature， and the targets related to melanoma from the GeneCards， Online Mendelian Inheritance in Man （OMIM）， and Comparative Toxicogenomics Database （CTD）. Following the construction of the protein-protein interaction （PPI） network of active components and related targets of A. euchroma and melanoma-related targets using STRING， Cytoscape 3.8.2 was used for screening and analyzing the nodes in the network of A. euchroma against melanoma. The intersections were subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis using DAVID 6.8. Acetyl alkannin， the active component in A. euchroma， was docked to the target by AutoDock Vina 1.1.2. The in vitro experiments were then carried out to verify the anti-melanoma effect of A. euchroma. ResultA total of 271 common targets of A. euchroma and melanoma were harvested， among which 23 were key targets， including matrix metalloproteinase-9 （MMP-9） and Janus kinase 2 （JAK2）. As revealed by KEGG enrichment analysis， A. euchroma mainly acted on Janus kinase/signal transduction and activator of transcription （JAK/STAT）， tyrosine kinase receptor （ErbB）， and vascular endothelial growth factor （VEGF） signaling pathways to resist melanoma. According to molecular docking， acetyl alkannin exhibited a good docking activity with JAK2， STAT3， VEGF， MMP-9， and E-cadherin receptors. The results of Western blot and Real-time quantitative polymerase chain reaction （Real-time PCR） showed that acetyl alkannin at different doses inhibited the protein and gene expression of JAK2， STAT3， VEGF， MMP-9， and E-cadherin in A375 cells （P<0.05）. ConclusionA. euchroma alleviates melanoma via multiple targets and multiple pathways， and it may exert the therapeutic effects by affecting the expression of such key target proteins as JAK2， STAT3， VEGF， MMP-9， and E-cadherin and inhibiting the invasion and metastasis of melanoma cells. This study has provided an experimental basis for the treatment of tumor with A. euchroma.

Cloning and functional analysis of caffeic acid and rosmarinic acid glycosyltransferases from Arnebia euchroma / 中国中药杂志

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.

Cloning and functional verification of carboxyl CoA ligases(AeCCLs) in Arnebia euchroma / 中国中药杂志

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.

Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence / 中国中药杂志

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

HPLC Fingerprint Establishment ，Chemistry Pattern Recognition Analysis and Content Determination of Arnebia euchroma / 中国药房

OBJECTIVE：To es tablish the HPLC fingerprint of Arnebia euchroma ，analyze them with chemical pattern recognition technology ， and determine the contents of 3 components. METHODS ： HPLC method was adopted. Using acetylshikonin as reference ，HPLC fingerprint of 34 batches of A. euchroma from different sources were drawn. Similarity Evaluation System for TCM Chromatographic Fingerprint （2012A edition ）was used to evaluate the similarity of the samples ，and common peaks were determined. SPSS 19.0 and SIMCA 14.1 statistical software was used for cluster analysis ，principle component analysis and orthogonal partial least squares-discriminate analysis. According to the standard of the variable importance in the project greater than 1，the differential markers affecting the quality difference of A. euchroma were screened. Meanwhile ，the contents of 3 components were determined by the same HPLC method. RESULTS ：There were 12 common peaks in HPLC fingerprints for 34 batches of A. euchroma . The similarity of other samples were more than 0.86，except that t he three （No.2016A3005-5） batches of medicinal herbs on the market were less than 0.72；3 common peaks were identified ， such as shikonin ，acetylshikonin， β ，β ′-dimethylacrylic acanine. These 34 batches of samples could be classified into two categories . S 1， qq.com S4-S6，S13，S15-S20，S22，S26-S34 were clustered into one category，and others clustered into the other category. By principal component analysis ，the contribution rates of three principle components were 52.834％ ，18.600％ and 8.387％ . Accumulative contribution rate was 79.821％ . Six constituents，such as shikonin，acetylshikonin and β，β'-dimethylacrylic acanine were screened as differential markers，representing the major differences of A. euchroma . The linear range of above three components were 0.72-90，2.05-410，2.50-500 µg/mL（r all more than 0.999）， respectively. The limits of quantification were 0.132，0.135，0.118 µg/mL，respectively. The limits of detection were 0.040，0.041， 0.036 µg/mL，respectively. RSDs of precision ，stability（24 h），reproducibility and durability tests were all lower than 3％. Recoveries were 95.959％-100.201％（RSD＝1.669％，n＝6），97.818％-102.698％（RSD＝1.788％，n＝6），95.831％-99.344％ （RSD＝1.600％，n＝6）. The contents of above three components were 0.002％-0.134％，0.025％-1.388％，0.022％-0.881％. CONCLUSIONS：Established HPLC fingerprint and content determination method are simple and stable ，can be used for quality evaluation and quantitative analysis of A. euchroma . Shikonin ，acetylshikonin and β，β'-dimethylacrylic acanine are different in the content and are differential markers of A. euchroma from different source.

Network Pharmacology Study of Arnebia euchroma in the Protective Effect of Heat Stroke / 中国药学杂志

OBJECTIVE: To predict the material basis of the “cooling blood” effect of Arnebia euchroma, and discusses its potential pathway and mechanism via network pharmacology. METHODS: The chemical constituents of Arnebia euchroma were collected based on database and literature search, the potential active components were filtered by admet SAR method, the targets of potential active ingredients were predicted by bSDTNBI method, the heat stroke related targets was retrieved from OMIM database. The STRING database was used to construct a "predicted target-disease target" protein interaction network, analyze and filter the key targets. A heat stroke model by heat stress-induced in mice to verify the efficacy of the key active ingredient (shikonin) for heat stroke protection was established. RESULTS: A total of 165 chemical constituents of Arnebia euchroma were filtered and 26 potential active ingredients were filtered out to act on 93 targets. Network analysis confirmed that naphthoquinone compounds were the main material basis of its "cooling blood" effect. Arnebia euchroma and heat stroke share multiple targets in the "predicted target-disease target" protein interaction network, suggesting that Arnebia euchroma may have protective effects on against heat stroke(HS). Forty-six key targets were mainly involved in biological processes and pathways such as inflammation, neurotransmitter conduction, redox and coagulation. Pharmacodynamic studies showed that shikonin could significantly prolong the survival time of mice with HS, reduce acute reactions and improve tissue organ damage. CONCLUSION: In summary, Arnebia euchroma may inhibit the inflammatory responses, promote blood circulation, protect the central nervous system, thereby improving multiple organ damage, and play a protective role in against HS.

Simultaneous determination of six anthraquinone components in Arnebia euchroma by QAMS / 中草药

Objective: To establish a new method for the quantitative analysis of multi-components by single-marker (QAMS) to simultaneous determine six naphthoquinone components in Arnebia euchroma. Methods: The chromatographic peaks of the main naphthoquinone components in A. euchroma were identified by high resolution LC-MS. The acetyl Shikonin was used as internal marker to calculate the relative correlation factors (RCF) of deoxyshikonin, isobutyrylshikonin, β-acetoxyisovalerate shikonin, and β,β'-dimethylacryloyl shikonin by HPLC, and examine the durability and reproducibility of the RCF. The external standard method and QAMS were compared to determine the six components in A. euchroma. Results: The repeatability of RCF was good. The results calculated with QAMS were consistent with the results by the external standard method. Conclusion: The QAMS method for simultaneously measuring the content of six components is accurate and reliable to evaluate the quality of A. euchroma.

Simultaneous Determination of Shikonin, Acetylshikonin and β, β-dimethylacrylshikonin in Arnebia euchroma by RP-HPLC / 中国药房

OBJECTIVE: To establish a method for the simultaneous determination of shikonin, acetylshikonin and β, β-dimethylacrylshikonin in Arnebia euchroma. METHODS: RP-HPLC method was adopted. The determination was performed on Kromasil 100-5 C18 column with mobile phase consisted of acetonitrile-0. 1％ formic acid solution (80: 20, V/V) at the flow rate of 1. 0 mL/min. The detection wavelength was set at 516 nm, column temperature was 25 ℃, and sample size was 10 μL. RESULTS: The linear ranges of shikonin, acetylshikonin and β, β-dimethylacrylshikonin were 0. 404-10. 100 μg/mL(r=0. 999 8), 5. 350-107. 000 μg/mL(r=0. 999 6), 2. 035-40. 700 μg/mL(r=0. 999 8), respectively. The limit of quantitation was 0. 40, 2. 91, 1. 34 μg/mL, and the limit of detection was 0. 12, 0. 87, 0. 40 μg/mL. RSDs of precision, stability and reproducibility tests were all lower than 2. 0％ (n=6). The recovery rate were 99. 12％-104. 18％ (RSD=1. 85％, n=6), 96. 51％-100. 21％ (RSD=1. 43％, n=6), 98. 11％-102. 51％ (RSD=1. 42％, n=6), respectively. CONCLUSIONS: The method is simple, precise, stable and reproducible. It can be used for simultaneous determination of shikonin, acetylshikonin and β, β-dimethylacrylshikonin in A. euchroma.

Secondary metabolic responses to treatment of 2-aminoindan-2-phosphonic acid in cell lines from Arnebia euchroma / 中国中药杂志

The accumulation of rosmarinic acid, acetylshikonin, deoxyshikonin, β, β'-dimethylacrylshikonin and isovalerylshikonin was investigated in cell suspension cultures of Arnebia euchroma (Royle) Johnst under the influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), and of the effector methyl jasmonate (MeJA). The results showed that methyl jasmonate promoted the accumulation of rosmarinic acid and shikonin derivatives. Conversely, 2-aminoindan-2-phosphonic acid suppressed the formation of rosmarinic acid, which indicated that AIP, indeed, was able to inhibit the phenylpropanoid pathway in A. euchroma. Meanwhile, the content of total shinkonins and other four kinds of shikonin derivatives, though varied in degrees, was also inhibited. And the inhibition was dose-dependent and time-dependent. Acetylshikonin responsed most rapidly to the treatment of AIP, the content reduced after 24 h of treatment and decreased to only half of those untreated control 48 h after teratment. β, β'-Dimethylacrylshikonin, difffer from acetylshikonin, responded much slowly to the treatment, inhibition could only be observed 96 h later. These suggest that phenylpropanoid pathway plays an important role in the shikoninsbiosynthesis, and this study provides a reference for the further research in metabolic regulation of producing shikonins by cell culture technology and biosynthesis pathways of shikonin derivatives. Still, shikonins biosynthesis pathways is complicated, the exact dose- and time-effect relationship of AIP and interaction between AIP and other effectors like MeJA need further research.

Capillary Electrophoresis Fingerprint of Arnebia Euchroma (Royle) Johnst / 分析化学

A method of capillary electrophoresis fingerprint was developed for evaluation of the quality of Arnebia euchroma ( Royle) Johnst. The samples were separated on a 50 μm × 40 cm uncoated capillary separation column at separation voltage of 25 kV with 100 mmol/ L borate buffer ( pH 8. 0) containing 25 mmol/ L SDS and 20% (V/ V) dehydrated alcohol as running buffer. The injection volume of sample was 0. 5 psi ×5 s and the detection wavelength was 214 nm. The results indicated that the samples of Arnebia euchroma (Royle) Johnst were well separated and detected in 35 min. With Shikonin peak as reference peak, 6 characteristic peaks of standard Arnebia euchroma (Royle) Johnst were determined. The quality discriminant analyses were accomplished for different kinds of samples named Arnebia euchroma ( Royle) Johnst that purchased from eight sources by means of characteristic fingerprint peak analysis, similarity evaluation and cluster analysis. This method had good reproducibility, and could be used for the quality control of Arnebia euchroma (Royle) Johnst.

Simultaneous determination of eight hydroxyl naphthoquinones in different parts of Arnebiae euchroma by HPLC / 中国中药杂志

This present study is to develop an HPLC method for simultaneous determination of eight hydroxyl naphthoquinones, shikonin, β-hydroxy-isovalerylshikonin, acetylshikonin, β-acetoxy-isovalerylshikonin, deoxyshikonin, isobutyrylshikonin, β,β'-dimethylacrylshikonin and isovalerylshikonin. The eight constituents were measured on a Waters Xbridge C18 column (4.6 mm×250 mm，5 μm) with isocratic elution of acetonitrile-0.05% formic acid solution (70∶30) at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was 275 nm and the column temperature was 30 ℃. The results of content determination indicated that significant differences of the eight compounds exist in every part of Arnebia euchroma，in which the highest part was the root bark, followed with the root, then the stem residues. The content of the xylem of root and aerial part was lower than the above parts. The results provided scientific basis for the medicinal parts of A. euchroma.

Transcriptome-based gene mining and bioinformatics analysis of p-hydroxybenzoate geranyltransferase genes in Arnebia euchroma / 中国中药杂志

The p-hydroxybenzoate geranyltransferases(PGT) play an important role in the biosynthesis pathways of shikonin derivatives. Six PGTs were obtained from transcriptome datebase of Arnebia euchroma by using bioinformatics methods and the proteins＇physiochemical properties they encoded were predicted. The result of protein domain prediction showed all of the six protein sequences contained the conserved domain of Ubia prenyltransferase family and possessed the motif NDxxDxxxD for prenyldiphosphate binding and a GX(K/Y)STAL sequence for putative aromatic ring binding. The phylogenetic tree showed that PGT and p-hydroxybenzoate polyprenyltransferase(PPT) belonged to two different clades. The results of gene expression analyses showed that the expression levels in the red shikonin-proficient line and the overground part of A. euchroma that could produce shikonin derivatives was much higher than the white shikonin-deficient line and the underground part, which suggested a positive correlation between the expression levels of PGT genes and shikonin production. This study aims to lay a foudation for further understanding of the function and enzymatic properties of PGT and provide a basis for the biosynthesis pathways and metabolic regulation of shikonin derivatives.

Determination of five naphthaquinones in Arnebia euchroma by quantitative analysis multi-components with single-marker / 中国中药杂志

This study is to determine five naphthaquinones (acetylshikonin, β-acetoxyisovalerylalkannin, isobutylshikonin, β,β'-dimethylacrylalkannin,α-methyl-n-butylshikonin） by quantitative analysis of multi-components with a single marker (QAMS). β,β'-Dimethylacrylalkannin was selected as the internal reference substance, and the relative correlation factors (RCFs) of acetylshikonin, β-acetoxyisovalerylalkannin, isobutylshikonin and α-methyl-n-butylshikonin were calculated. Then the ruggedness of relative correction factors was tested on different instruments and columns. Meanwhile, 16 batches of Arnebia euchroma were analyzed by external standard method (ESM) and QAMS, respectively. The peaks were identifited by LC-MS. The ruggedness of relative correction factors was good. And the analytical results calculated by ESM and QAMS showed no difference. The quantitative method established was feasible and suitable for the quality evaluation of A. euchroma.

Protein tyrosine phosphatase 1B(PTP1B) inhibitors from Arnebia euchroma / 中国药学杂志

OBJECTIVE: To study the chemical constituents of the extract of Arnebia euchroma (Royle) Johnst. and screen natural protein tyrosine phosphatase 1B (PTP1B) inhibitors. METHODS: Silica gel, MCI gel, ODS gel, and Sephadex LH-20 chromatographic techniques were used to study the chemical constituents of A. euchroma, the chemical structures were elucidated by analysis of physico-chemical and spectral data, and the inhibitory activity on PTP1B enzyme was tested in vitro. RESULTS: Eight compounds were obtained and their structures were identified as deoxyshikonin (1), shikonin (2), acetylshikonin (3), β, β'-dimethylacrylalkannin (4), quercetin (5), kaempferol (6), kaempferide (7), and β-sitosterol (8). Compounds 1-4 exhibited inhibitory activities on PTP1B with IC50 values of (0.80±0.16), (4.42±0.37), (1.02±0.13), and (0.36±0.08) μmol·L-1, respectively. The study of structure-activity relationship showed that the ring of naphthoquinone might be the key skeleton structure for the inhibition activity on PTP1B, and the 2-substituted long lipo-chain of naphthoquinone significantly affected the activity of these compounds: the increase in the polarity of the lipo-chain led to the reduction of activity, while the increase in the terminal double bond of the lipo-chain led to the enhancement of activity. CONCLUSION: Shikonin derivatives with 1, 4-naphthoquinone skeleton is a new type of leading compounds for the treatment of diabetes.

Effects of low temperature stress on secondary metabolites of Arnebia euchroma suspension cells / 中草药

Objective: By examining the secondary metabolites content changes of the low temperature stressed Arnebia euchroma suspension cells to explore the effects of unique environmental factors in genuine regional areas on A. euchroma in non-genuine regional areas. Methods: After stressing A. euchroma suspension cells for 24 h at 4 ℃, rosmarinic acid, lithospermum acid, shikonofuran A, shikonofuran E, acetylshikonin, deoxyshikonin, β,β'-dimethylacryl shikonin, isovalerylshikonin, and total shikonin compounds were measured by HPLC on six time points, one of which was before the stress and counted as 0 h, and others were in 12, 24, 48, 72, and 168 h after the stress. Results: The results showed that the different time points after low temperature stress, similar compounds had the same consistent change trends basically. However, the contents had significant differences. In addition to DAS, the maximum value of all the chemical ingredients were in low stress group. Conclusion: Low temperature stress can promote the accumulation of secondary metabolites in A. euchroma suspension cells, and have an important role in revealing the mechanism of the formation of its genuineness.

Quality Control of Arnebia euchroma inZizhu Ointment / 中国中医药信息杂志

Objective To establish a method for quality control of Arnebia euchroma inZizhu Ointment.Methods The TLC was used to make qualitative identification to Arnebia euchroma in Zizhu Ointment. The content of shikonin was targeted as the evaluation index and the method of orthogonal design was used to optimize the extraction of shikonin. UV-visible spectrophotometry was used for the determination of shikonin.Results Main spots of TLC were clear, and negative control had no interference. The optimum extraction of shikonin was extracted for three times, 30 min for each time, NaOH volume fraction of 1%, NaOH amount of 50 mL. The absorbance of shikonin showed a good linear relationship in the range of 0.010 2-0.035 7 mg (r2=0.999 7), and the average recovery was 103.27%, RSD=4.17%.Conclusion This method was simple and feasible, and can be used for the quality control and the evaluation ofZizhu Ointment.

Analysis of the Volatile Oil from Lithospermum erythrorhizon and Arnebia euchroma by GC-MS / 中国药房

OBJECTIVE:To analyze the volatile oil extracted from the root of Lithospermum erythrorhizon and Arnebia euchroma.METHODS:Volatile oil were extracted from L.erythrorhizon and A.euchroma by steam distillation and determined by GC-MS.RESULTS:34 components and 36 components of volatile oil extracted from the root of L.erythrorhizon and A.euchroma were separated and identified respectively,accounting for 70.16% and 74.79% of the total peak area.There were 4 kinds of common peak among these components.The main components were 2,6-di-tert-butyl-p-Cresol(9.29%),2,4-di-t-butyl-1,3-Pentadiene(8.17%),and trans-Nerolidol(6.05%) in L.erythrorhizon and muscalure(16.28%),3-Allyl-2-methoxyphenol(6.20%)and camphor(5.82%) in A.euchroma.CONCLUSION:The study can provide scientific basis for the further development of Radix Arnebiae.

Content Determination of Shikonin in Arnebia euchroma by HPCE / 中国药房

OBJECTIVE:To determine the content of shikonin in Arnebia euchroma by HPCE-UV.METHODS:The separation was performed on uncoated fused silica capillary column (53 cm?50 ?m ID,46 cm effective length).2.0 mmol?L-1 H3BO3,6.0 mmol?L-1 triethylamine buffer solution and 5.0 mmol?L-1?-CD was used as background electrolyte,14 kV separation voltage and time of gravity injection 15 s (25 cm).The detection wavelength was set at 516 nm.RESULTS:The linear range of shikonin was 7.2～36.0 ?g?mL-1(r=0.999 0) with an average recovery of 100.15%(RSD=1.68%,n=6).CONCLUSION:The method is convenient,rapid,accurate and reliable for the content determination of shikonin in A.euchroma.

Establishment of asexual multiplication system of Arnebia euchroma / 中草药

Object In order to promote the artificial cultivation of Arnebia euchroma (Royle) Johnst , and save the wild herb from relieving the lack of resources, the asexual multiplication system of A euchroma was established by tissue culture technique Methods Dedifferentiation in many kinds of explants of A euchroma by inducement succeeded After the embryos were multiplied, there were a lot of germinated transplants in media After callus were multiplied, many adventitious buds had formed in the media, then they were rooting; the transplants formed by inducement and the plants survived after being cultured in soil Results During the dedifferentiation, 2,4 D was essential, the effect of KT was better than BA The shoot sections were dedifferentiated in the most rapid way among the explants The callus from them were in very good quality It is of benefit to the callus multiplication and redifferentiation after reducing the concentration of 2,4 D Both KT and BA can induce adventitious buds, KT was also suitable for the formation of globular shaped embryos In initial period of sphere embryos culture, it was necessary to add the plant growth regulators in media for multiplying sphere embryos and promoting their development Proper temperature difference between day and night (10 ℃-28 ℃) and natural light were helpful for the formation of strong sprouts and survival of transplants The survival rate was 13 2% with 31 plantlets survived among 236 transplants cultured in soil Conclusion The asexual multiplication system of A euchroma established through tissue culture is stable and available

Optimization of Microemulsion Preparation of Fufang Zicao Oil / 中药新药与临床药理

Objective To optimize the microemulsion preparation of Fufang Zicao Oil(FZO).Methods The extraction conditions of microemulsion preparation of FZO were optimized by orthogonal experiment;the kinds and contents of surfactants and cosurfactants determined by pseudo-ternary phase diagram were used for the optimization of the prescription of FZO;centrifugation method was used for detecting the stability of FZO.Results The optimum extract conditions were as follows:10-mesh coarse powder of medicinal material,four times amount of solvent(sesame oil),extraction for 25 minutes.With Tween 80 and propylene glycol(7:3)as the surfactant and cosurfactant,and mixing with the oils in the proportion of 20:10,a steady microemulsion of FZO can be obtained after centrifugation by 13000 r? min-1 for 30 min.Conclusion Under the above achieved conditions,the obtained microemulsion of FZO is clear,transparent and steady.