Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum-ethyl acetate-methanol-water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3mg of cryptotanshinone, 16.2mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3mg of miltirone were obtained from 400mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.     

SmMYB98b positive regulation to tanshinones in Salvia miltiorrhiza Bunge hairy roots

Plant Cell, Tissue and Organ Culture (PCTOC) - Tanshinones are major secondary metabolites in Salvia miltiorrhiza Bunge, the traditional Chinese medicinal plant Danshen. Increasing the production...     

Insulin-sensitizing activities of tanshinones,diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1–10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents.     

丹参晚期胚胎蛋白基因SmLEA的克隆及表达分析

对丹参EST序列进行Blast分析,发现一条序列与晚期胚胎丰富蛋白(late embryogenesis abundant)基因有较高的相似性,在此基础上设计引物,分别从cDNA和gDNA水平克隆到该基因的全长(Genbank注册号:AY725206),命名为SmLEA.该序列全长739 bp,无内含子,包含1个长为495 bp的开放阅读框,编码164个氨基酸.序列比对结果显示,该序列与番茄的晚期胚胎丰富蛋白Lemmi9有较高的相似性(69%),推测该编码蛋白属于晚期胚胎蛋白LEA14家族成员.生物信息学显示,SmLEA所编码蛋白SmLEA的相对分子质量为17.34 kD,理论等电点为4.51,富含天冬氨酸及AS、IP、KV、VS、TIP肽段,定位于细胞质中,为稳定类蛋白.实时荧光定量PCR结果显示,该基因在丹参的根、茎、叶中均有表达,为组成型表达基因.     

Plant DNA extraction using silica

Described here is a method that uses silicon dioxide (silica) to extract whole genomic plant DNA of high molecular weight. The protocol is presented in a microcentrifuge format, and yields were approximately 2–4 μg per 200 mg of plant leaf tissue. The method involves fewer steps than many previous extraction protocols and, as shown here for 4 taxonomically distant angiosperms, produces DNA suitable for digestion with restriction endonucleases. The use of commercial kits is not required; the silica costs are comparatively inexpensive (<$0.03 per tube); and CTAB, rather than the more expensive guanidine thiocyanate salt, is used.     

Selective extraction of hemicelluloses from spruce using switchable ionic liquids

Switchable ionic liquids (SILs) made from alcohols, either hexanol or butanol, and CO₂ together with an amidine (1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU)) were investigated as dissolution/fractionation solvents for wood material. Both native spruce (Picea abies), and pre-extracted spruce were treated with either butanol SIL (SIL1) or hexanol SIL (SIL2) for 5 days at 55 °C under normal pressure. The SILs were formed by bubbling CO₂ through an equimolar mixture of either 1-hexanol or 1-butanol and DBU. The viscosity of the mixture increased from 7.1 mPa s to 2980 mPa s for SIL2 and 5.1 to 1600 mPa s for SIL1. Melting points of the SILs 1 and 2 were at 8 and 14 °C, respectively. After the treatment time (5 days), the undissolved fraction contained 38 wt.% less hemicelluloses compared to native spruce. There was an increase in the glucose content of the milled spruce treated with both SILs, since the milling step reduced the cellulose crystallinity of the wood and facilitated an easier SIL access into the wood. The solvents were very neutral in terms of lignin removal. Consequently, only about 2% of the lignin was removed from native wood. Moreover, a priori removal of the wood extractives did not influence the lignin removal.     

Solid-phase extraction of liquiritin and glycyrrhizin from licorice using porous alkyl-pyridinium polymer sorbent

Introduction – Liquiritin and glycyrrhizin are valuable components of licorice. An effective separation and determination procedure is needed to separate the liquiritin and glycyrrhizin from the licorice extract. Methodology – A polymer‐confined, ionic liquid sorbent was developed using a process involving polymerisation and modification. The obtained porous particles were used as a sorbent in a solid‐phase extraction process to isolate liquiritin and glycyrrhizin from licorice with different washing and elution solvents. The porous alkyl‐pyridinium polymer sorbent was compared with the C₁₈ sorbent. Results – A simple and convenient method was established to the selectively separate and determinate of liquiritin and glycyrrhizin using a porous ionic liquid‐based polymer coupled with HPLC. Additionally, this study evaluated the application of this sorbent for the detection of these two compounds in commercial medicines. Conclusion – This method was a viable tool that was compatible with the existing HPLC methods and was used to separate and analyse the content of liquiritin and glycyrrhizin in licorice. Copyright © 2010 John Wiley & Sons, Ltd.     

Solid-phase extraction of phospholipids from hemoglobin solutions using Empore styrene–divinylbenzene disks

Styrene–divinylbenzene Empore disks were investigated for the extraction of phospholipids from red blood cells or aqueous solutions of hemoglobin as a means to reduce the time and solvent use required in sample preparation. Red blood cells are the source for hemoglobin used in the preparation of a hemoglobin-based oxygen carrier which is being developed to replace blood in transfusion therapy. Phospholipids are a major component of the membrane of red blood cells, and are toxic when administered directly into the vasculature. Sensitive analytical methods are required to detect phospholipids to ensure that concentrations in purified hemoglobin are well below toxic levels. This requires isolation from large volumes of purified hemoglobin solutions. The method described utilizes Empore disks to extract phospholipids from 30 ml of stroma free Hb preparations. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and sphingomyelin were recovered with an average of 92% yield. The recovery of phosphatidylserine was 65%. The use of solvent and time required for sample preparation were reduced by an average of 80% relative to liquid–liquid extraction. The capacity of the 47-mm disk for the total of five phospholipids exceeds 0.3 mg. The method has been used for quantitation of phospholipids in red blood cells and stroma free hemoglobin solutions.     

丹参肉桂酰辅酶A还原酶基因克隆与生物信息学分析

分析丹参转录组数据库,获得一条新的肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase,CCR)基因,命名为SmCCR-2(GenBank注册号为JF784010)。该基因包含一个长为966 bp的完整开放读码框,编码321个氨基酸残基。生物信息学分析显示,SmCCR-2编码的蛋白具有NWYCY基序,属于NABD_Rossmann超家族,相对分子量为35.80 kD;预测SmCCR-2为中性亲水的稳定蛋白,存在跨膜结构域。实时荧光定量PCR结果表明,SmCCR-2基因在丹参各组织都有表达,茎中表达量最高。其表达受到病原菌的影响,表明SmCCR-2基因可能与植物防御反应有关。     

丹参抗肝纤维化有效部位化学成分研究

从丹参抗肝纤维化有效部位提取物中分离得到7个化合物,经物理常数测定、波谱分析等鉴定为,4、5、4’、5’-四羟基1、2双苯醚(1),原二茶醛(2),原二茶酸(3),异阿魏酸(4),咖啡酸(5),丹参甲素(6),丹酚酸D(7),其中化合物1为首次从丹参中分得的化合物。     

Magnetic molecularly imprinted polymers for improved extraction of tanshinones from herbs via integrated extraction and cleanup system

A novel separation technology at room temperature for traditional Chinese medicines was proposed in this work by adding magnetic molecularly imprinted polymers (M-MIPs) into extraction solution and sample matrix. The M-MIPs show a more adsorption capacities and higher selectivity for the template than magnetic non-molecularly imprinted polymers (M-NMIPs) without the specific binding sites. Addition of the M-MIPs to the extraction solution provides one-step extraction and cleanup, the improvement of extraction rate and extraction yields of three tanshinones (from 0.40, 0.23 and 0.12 mg g⁻¹ in 240 min by solvent extraction to 0.52, 0.27 and 0.19 mg g⁻¹ in 5 min by 200 mg sorbent), and reusability of extraction solvent. The extraction yields of three tanshinones by this technology at room temperature in 5 min were higher than those by ultrasonic extraction in 30 min, by heat reflux extraction in 45 min and by solvent extraction at room temperature in 4 h. The integrated technology has the advantages of one-step extraction and cleanup, high extraction efficiency, low solvent consumption and room temperature.     

Distribution of tanshinones in the genus Salvia (family Lamiaceae) from China and its systematic significance

Salvia L.(family Lamiaceae),a large genus of over 1000 species,is widely distributed throughout tropical and temperate regions of the world.Eight-four species are native to China.However,there are many taxonomic uncertainties at the sub-generic level.Diterpenoids are a class of secondary metabolites with a large variety of structures that have been used as chemotaxonomic markers at infra-and suprageneric levels.For the sake of further chemotaxonomic understanding of Salvia,the present study investigated the...     

丹参晚期胚胎富集蛋白基因SmLEA的表达特征研究

利用DNA walking的方法克隆到了丹参SmLEA基因 5′ 端上游调控序列,约1 038bp。经生物信息学预测分析,该区域含有多种与逆境胁迫、ABA、种子特异表达相关的顺式作用元件。实时荧光定量PCR检测结果表明,100 μmol/L ABA,200mmol/L NaCl,低温（4℃）及脱水处理后,SmLEA基因的表达均得到了明显的提高,与启动子序列分析的结果相符合。表明SmLEA基因可对盐、脱水和低温胁迫以及 ABA做出响应。     

Binding mode prediction of biologically active compounds from plant Salvia Miltiorrhiza as integrase inhibitor

Integrase (IN), an essential enzyme for HIV-1 replication, has been targeted in antiretroviral drug therapy. The emergence of HIV-1 variants clinically resistant to antiretroviral agents has lead to the development of alternative IN inhibitors. In the present work, binding modes of a high potent IN inhibitor, M₅22 and M₅32, within the catalytic binding site of wild type (WT) IN were determined using molecular docking calculation. Both M₅22 and M₅32 displayed similar modes of binding within the IN putative binding pocket and exhibited favorable interactions with the catalytic Mg²⁺ ions, the nearby amino acids and viral DNA through metal-ligand chelation, hydrogen bonding and π-π stacking interactions. Furthermore, the modes of action of these two compounds against the mutated Y212R, N224H and S217H PFV IN were also predicted. Although the replacement of amino acid could somehow disturb inhibitor binding mode, almost key interactions which detected in the WT complexes were fairly conserved. Detailed information could highlight the application of M₅22 and M₅32 as candidate IN inhibitors for drug development against drug resistant strains.     

丹参胁迫诱导基因SmSIP1的表达特征分析

从丹参EST库中筛选到一个胁迫诱导蛋白基因,命名为SmS1P1,其序列全长296bp,编码80个氨基酸。生物信息学预测表明SmS1P1是一个亲水的,不具有跨膜结构域,包含一个N-端信号肽和多个可能的磷酸化位点的蛋白。实时荧光定量PCR分析显示,SmS1P1在根中的表达量高于茎和叶,并且受ABA和干旱的诱导,推测其可能参与根部的胁迫应答反应。     

丹参生物工程的研究与开发

中药材的品质改良是中药现代化进程中的一个重要问题,现代生物工程技术是进行中药材品质改良的可行途径之一。对丹参的组织培养、毛状根诱导培养及基因工程等方面的最新进展作一综述,以期为利用生物工程技术遗传改良丹参品质提供一些参考。     

Solid-phase extraction of N-linked glycopeptides

Protein glycosylation is a common post-translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. N-linked glycosylation is prevalent in proteins on the extracellular membrane, and many clinical biomarkers and therapeutic targets are glycoproteins. Here, we describe a protocol for solid-phase extraction of N-linked glycopeptides and subsequent identification of N-linked glycosylation sites (N-glycosites) by tandem mass spectrometry. The method oxidizes the carbohydrates in glycopeptides into aldehydes, which can be immobilized on a solid support. The N-linked glycopeptides are then optionally labeled with a stable isotope using deuterium-labeled succinic anhydride and the peptide moieties are released by peptide-N-glycosidase. In a single analysis, the method identifies hundreds of N-linked glycoproteins, the site(s) of N-linked glycosylation and the relative quantity of the identified glycopeptides.