Study on Optimization of Ethanol Reflux Extraction of Phenolic Acids from Salvia miltiorrhiza

The extraction technology of phenolic acid compounds from Salvia miltiorrhiza by ethanol reflux was studied. In this experiment, salvianolic acid B standard was used to make the standard curve. Single factor experiment and orthogonal experiment were used to study the extraction of different ethanol concentrations, reflux times and material-to-liquid ratios. The OD value of salvianolic acid compounds was measured with a spectrophotometer. The extraction rate of phenolic acid compounds under different extraction conditions was calculated through a regression equation, so as to obtain the optimal conditions for the ethanol reflux extraction process of Salvia miltiorrhiza. The experimental data can provide a reference for the ethanol reflux extraction process of salvianolic acids in the industry. According to the experiment, the extraction rate of phenolic acids in Salvia miltiorrhiza was the highest when the ethanol concentration was 60%, the reflux time was 1.5 hours, and the ratio of material-to-liquid was 1:10.

Improving the antioxidant activity and enriching salvianolic acids by the fermentation of Salvia miltiorrhizae with Geomyces luteus

The antioxidant activities and total phenolic content of fermented Salvia miltiorrhiza with fungus Geomyces luteus were investigated. The results revealed that G. luteus fermentation could significantly improve the antioxidant activity and total phenolic content of S. miltiorrhiza. The main antioxidant constituents were characterized by spec- troscopic analysis as salvianolic acids. High-performance liquid chromatography （HPLC） quantification also showed the enhanced content of salvianolic acid B after fermentation. The present study suggests that G. luteus fermentations are effective in the S. miltiorrhiza salvianolic acids＇ enrichment process.

Determination of Chemical Composition of Salvia miltiorrhiza Cultivated in Jizhou

[Objectives] This study aimed to evaluate the quality of Salvia miltiorrhiza cultivated in Jizhou. [Methods] The contents of chemical ingredients( tanshinone ⅡA,tanshinone Ⅰ and salvianolic acid B) in S. miltiorrhiza were determined by high-performance liquid chromatography( HPLC),and the chemical composition of S. miltiorrhiza planted in Jizhou and purchased from market was compared.[Results]The contents of tanshinone ⅡA,tanshinone I and salvianolic acid B in S. miltiorrhiza planted in Jizhou were 0. 014%,0. 421 2%and 3. 018%,respectively,higher than the standards of the Chinese Pharmacopoeia of the 2015 edition. [Conclusions]The chemical composition of S. miltiorrhiza planted in Jizhou meets the standards of the Chinese Pharmacopoeia of the 2015 edition.

Effects of LaCl_3 on photosynthesis and the accumulation of tanshinones and salvianolic acids in Salvia miltiorrhiza seedlings

The effects of LaCl3 on the growth,photosynthetic gas-exchange characteristics,chlorophyll fluorescence,and the accumulation of tanshinones and salvianolic acids in Salvia miltiorrhiza seedlings were investigated. The results showed that the increase in photosynthesis induced by LaCl3 might be attributed to the enhanced stomatal conductance of the leaves and the increased level of the photochemical efficiency of PS II. The accumulation of tanshinone IIA and cryptotanshinone was markedly increased with the application of LaCl3 at 20 and 60 mg/L,while tanshinone I was only slightly increased. The content of salvianolic acid B was,however,decreased with the treatment of LaCl3 at 200 mg/L.

Insights into salvianolic acid B biosynthesis from chromosome-scale assembly of the Salvia bowleyana genome

Salvia bowleyanais a traditional Chinese medic-inal plant that is a source of nutritional supple-ments rich in salvianolic acid B and a potentialexperimental system for the exploration ofsalvianolic acid B biosynthesis in the Labiatae.Here,we report a high‐quality chromosome‐scalegenome assembly of S.bowleyana covering 462.44 Mb,with a scaffold N50 value of 57.96 Mband 44,044 annotated protein‐coding genes.Evolutionary analysis revealed an estimateddivergence time betweenS.bowleyanaand its close relativeS.miltiorrhiza of~3.94 millionyears.We also observed evidence of a whole‐genome duplication in theS.bowleyanagenome.Transcriptome analysis showed that SbPAL1(PHENYLALANINE AMMONIA‐LYASE1)ishighlyexpressed in roots relative to stem and leaves,paralleling the location of salvianolic acid Baccumulation.The laccase gene family in S.bowley anaoutnumbered their counterparts inbothS.miltiorrhiza and Arabidopsis thaliana,suggesting that the gene family has undergoneexpansion inS.bowleyana.Several laccasegenes were also highly expressed in roots,wheretheir encoded proteins may catalyze the oxida-tive reaction from rosmarinic acid to salvianolicacid B.Thesefindings provide an invaluable ge-nomic resource for understanding salvianolicacid B biosynthesis and its regulation,and will beuseful for exploring the evolution of the Labiatae.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid(RA) and lithospermic acid B(LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase(HPPR) gene, Sm HPPR, which plays an important role in the biosynthesis of RA. Sm HPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant Sm HPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid(p HPP) to 4-hydroxyphenyllactic acid(p HPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. Sm HPPR expression could be induced by various treatments, including SA, GA_3, Me JA and Ag^+, and the changes in Sm HPPR activity were correlated well with hydrophilic phenolic acid accumulation. Sm HPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing Sm HPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Effects of Water─soluble Components Isolated from Salviamilltiorrhiza on Oxygen Free Radical Generation and LipidPeroxidation

The effects of water─soluble components isolated from Salvia miltiorrhiza on oxygen free radical prodction and lipid peroxidation were estimated. Five components(10 μmol/L)were shown to inhibit superoxide anion generation by xanthine─xanthine oxidase system. It was also demonstrated that six components( 100 μmol/L) prevented H_2O_2─induced hemolysis and MDA fonnation in mouse erythrocytes.The effects of Sal A, Sal B, Sal C and Ros A were dose dependent. In Langendorff rat heart, pretreatment with Sal A 20μmol/L significantly prevented MDA production induced by 30 min reoxygenation after 45 min anoxia.The results indicated that there were also other potent antioxidant componentts in Salvia milliorrhiza besides Dphl and Pal. The protection of Sal A against myocardial anoxiareoxygenation inury may be mainly ascribed to its oxygen free radical scavenging activity.

HPLC法同时测定乙肝扶正胶囊中丹酚酸B、隐丹参酮和丹参酮Ⅱ_(A)的含量

目的:建立同时测定乙肝扶正胶囊中丹酚酸B、隐丹参酮和丹参酮Ⅱ_(A)含量的高效液相色谱法。方法:采用Agilent ZORBAX-SB C 18色谱柱(4.6 mm×250 mm,5μm),以乙腈-0.1%磷酸为流动相,梯度洗脱,检测波长为270 nm。结果:丹酚酸B、隐丹参酮和丹参酮Ⅱ_(A)分别在7.82~313.01μg·mL^(-1)(r 2=0.9998)、0.087~17.324μg·mL^(-1)(r^(2)=1.0000)、0.067~13.468μg·mL^(-1)(r^(2)=1.0000)范围内线性关系良好,平均回收率分别为103.99%、97.76%、100.88%,RSD均≤1.00%。测定样品83批次,丹酚酸B、隐丹参酮和丹参酮IIA的含量分别为0.16~4.06、0.00~103.53、0.00~126.49μg·粒^(-1)。结论:本方法为提升乙肝扶正胶囊的质量控制手段提供了理论依据和有效的技术支撑。

高效液相色谱法测定丹参中丹酚酸B

目的 :建立丹参中丹酚酸B高效液相色谱测定方法。方法 :75 %甲醇回流提取 ,色谱柱YWGC18,流动相甲醇 5 %乙酸 (35∶6 5 ) ,检测波长 2 81nm。结果 :丹酚酸B 0 .17～ 1.70 μg线性关系良好 (r =0 .9997) ,回收率98.9% ,RSD1.82 %。结论

UV-B辐射和干旱对丹参生长和叶片中酚酸类成分的影响

在3种UV-B辐射强度以及3种干旱的单因子以及复合作用下,采用盆栽法对丹参植株生物量、叶面积、株高等形态指标和光合参数以及叶片中丹酚酸B、迷迭香酸、咖啡酸和丹参素含量的变化进行研究。旨在探索西北干旱、半干旱地区强光和干旱对丹参生物量和叶片中酚酸类含量的影响,为丹参的深入开发利用提供依据。结果表明:干旱和UV-B辐射单因子作用均对丹参生长指标有抑制作用,而对酚酸积累有促进作用;相比于干旱,UV-B辐射的影响更加显著。UV-B辐射显著引起植株矮化、叶面积减小、生物量降低、净光合速率降低以及叶片中酚酸类物质含量增加,两种因子复合作用对酚酸类物质积累的效果表现为复杂的加强或交互抗性作用。两种因子单独和复合作用处理下,适宜水分和重度UV-B辐射复合处理(75%T2)最有利于丹参叶片中酚酸类物质积累。

丹酚酸B对缺氧损伤心脏微血管内皮细胞细胞间粘附分子表达的影响

目的 :探讨丹参有效成分丹酚酸B在心脏微血管内皮细胞 (CMEC)缺氧 /复氧损伤中的保护作用。方法 :通过心脏微血管内皮细胞体外培养技术 ,建立缺氧预适应 (HPC)模型并给予丹酚酸B ,应用酶联免疫方法检测细胞间粘附分子 1(ICAM 1)表达。结果 :在CMEC缺氧 3h ,复氧 6h后ICAM 1表达升高 ;缺氧预适应和应用丹酚酸B对CMEC进行预处理则能有效降低CMECICAM 1的表达。结论 :丹酚酸B在CMEC缺氧 /复氧损伤中可产生与缺氧预适应类似的保护效果。

丹参氮、磷、钾积累分配特点及其与干物质、丹酚酸B积累的关系

田间条件下研究了丹参的干物质和丹酚酸B积累与植株氮、磷、钾积累的特点及其相互关系。结果表明,丹参干物质积累总量随氮、磷、钾积累总量的增加呈直线增加趋势,相关系数达显著水平。丹参的叶、茎的干物质积累在整个生育期内呈先上升后下降的趋势;根部干物质积累呈上升趋势。在整个生育期内,丹酚酸B的积累趋势与丹参干物质积累趋势相似。不同时期,丹参对氮、磷、钾的吸收量不同。在移栽后30 d内对氮、磷、钾的吸收较少,分别占总吸收量的2.68%,2.23%,2.20%;移栽后60～120 d吸收量迅速增加,氮、磷、钾的吸收量占全部吸收量的58.57%,39.92%,50.18%;移栽120 d后又有所下降。总的来说,植株对氮的吸收量最多,钾次之,磷最少,整个生育期内对氮、磷、钾的吸收比例为1∶0.12∶0.46。

丹参水提液中丹酚酸B湿热降解动力学研究

目的:研究丹参水提液中丹酚酸B的湿热降解动力学。方法:以HPLC测定不同温度不同加热时间丹参水提液中丹酚酸B湿热降解动力学相关参数。结果:丹参水提液中丹酚酸B湿热降解符合一级动力学。结论:缩短丹参水提、浓缩、干燥时间,尽快使丹参水提液固体化可减少丹酚酸B损失。

不同产地丹参药材中丹参酮ⅡA和丹酚酸B含量比较

目的:测定和比较不同大规模栽培产地丹参药材中丹参酮ⅡA和丹酚酸B含量。方法:采用高效液相色谱法,选用Synchropak PR100-5色谱柱,检测波长分别为270nm和286nm。结果:丹参酮ⅡA和丹酚酸B含量均较高的是四川中江栽培品。山西芮城栽培品丹参酮ⅡA含量尽管最低,但丹酚酸B含量却最高。结论:丹参中丹参酮ⅡA和丹酚酸B的含量与产地关系较大,此研究结果可作为丹参药材采购及临床选用的参考依据。

丹参酮ⅡA和丹酚酸B在丹参根组织中的分布特征研究

对生长在粘土、沙土和马骨石土上1～2年生丹参不同根组织的分析表明，丹参酮ⅡA主要分布在木栓层，丹酚酸B分布在木质部和韧皮部．1年生丹参根木栓层的丹参酮ⅡA含量较2年生丹参为高，而1年生丹参根木质部和韧皮部中的丹酚酸B含量与2年生丹参没有显著差异．粘土地栽培丹参根中的丹参酮ⅡA含量较沙土、马骨石土地栽培丹参为高，但不同类型土壤上丹参根中丹酚酸B含量的差异不显著．

丹参酮IIA与丹酚酸B在丹参药材中的分布研究

目的对丹参酮IIA与丹酚酸B在丹参药材中的分布规律进行研究,为丹参药材的GAP栽培与采收加工提供科学依据。方法在丹参药材的采收季节,分别在不同的主产地采集药材样品,按实验需要分成不同部分,晒干后,以HPLC法测定药材中活性成分丹参酮IIA和丹酚酸B的含量。结果研究表明,丹参酮IIA在皮层含量最高,木质部含量甚微,根的下端含量高于上端,须根的含量高;丹酚酸B在皮层的含量高于木质部,根下端含量高于上端,同样以须根的含量高。结论脂溶性的丹参酮IIA与水溶性的丹酚酸B的在丹参根中的分布有一定的规律;这两个成分在丹参根中的横向分布以皮层为高,纵向分布均以根的上端高于下端,须根含量最高。

UPLC和HPLC方法对丹参药材中丹酚酸B含量测定结果的比对

目的:比对超高效液相色谱与高效液相色谱方法测定丹参药材中丹酚酸 B 的含量结果。方法:采用中国药典2005年版(一部)丹参药材含量测定项下有关标准对原高效液相色谱方法进行转换并优化为超高效液相色谱方法。原高效液相色谱方法色潜柱为 Waters X-Bridge^(TM)BEH C_(18)柱(4.6 mm×250 mm,5μm),流动相:乙腈-甲醇-水-甲酸(10:30:59:1),流速:1mL·min^(-1),检测波长:286 nm,柱温:30℃;超高效液相色谱方法色潜柱为 Waters ACQUITY UPLC^(TM)。BEH C_(18)柱(2.1 mm×50mm,1.7μm),流动相:乙腈-甲醇-水-甲酸(8:25:66:1),流速:0.4 mL·min^(-1),检测波长:286 nm,柱温:30℃。结果:2种方法所得含量测定结果一致。结论:超高效液相色谱方法能够替代高效液相色谱分析方法测定丹参药材中丹酚酸 B 含量,既加快了分析速度,达到了样品分析的高通量,减少有机溶剂的使用,又得到更高的分析灵敏度。

丹参和丹酚酸B对抗泼尼松致大鼠皮肤衰老作用的观察

目的:探讨丹参(Salvia Miltiorrhiza)和丹酚酸B(Salvianolic acid B,Sal B)对用泼尼松引起的大鼠皮肤衰老作用的影响。方法:32只SD雄性大鼠随机分为4组(n=8):正常对照组,激素模型组(GC)、激素加用丹参组(GC+DS),激素加用丹酚酸B组(GC+Sal B)。正常对照组每天灌胃生理盐水,后3组每天分别灌胃泼尼松3.5mg.kg-1.d-1造模,GC+DS组再给予丹参水提液5g.kg-1.d-1;GC+Sal B组再给予丹酚酸B原料药125mg.kg-1.d-1。喂养90d后处死大鼠取皮肤进行组织病理学检查,并用计算机图像分析系统定量分析表皮厚度、弹力纤维的面积;另外以高效液相色谱法对药品进行丹酚酸B含量测定。结果:GC组可见大鼠皮肤表皮显著变薄,弹力纤维减少(P<0.01),胶原纤维减少,断裂等皮肤衰老样的表现,GC+DS、SalB组可使大鼠表皮增厚、弹力纤维总面积增大(P<0.01),皮肤衰老样改变明显减轻;丹参水提液、丹酚酸B原料药分别检测出相当含量的丹参水溶性成份丹酚酸B。结论:长期灌胃给药泼尼松可使大鼠出现皮肤衰老样改变,丹参和丹酚酸B对其有较好的对抗作用,丹酚酸B可能是丹参对皮肤作用的重要有效成分。

高速逆流色谱法分离制备丹酚酸B

采用高速逆流色谱法分离纯化丹参水溶性成分丹酚酸类物质,制备丹酚酸B化学对照品。分离采用的溶剂系统为正己烷-乙酸乙酯-水-甲醇(1.5:5:5:1.5),上相做固定相,下相做流动相,流速为1.7 mL/min,仪器转速850 rpm,进样量80 mg,纯度用HPLC方法测定。结果表明:一次分离可制备63.4 mg丹酚酸B,其纯度为98.6%。该方法操作简单,可作为高纯度丹酚酸B化学对照品的制备分离方法。

丹酚酸B的体外抗氧化活性

为考察丹酚酸B的体外抗氧化活性,应用比色法测定丹酚酸B对Fenton体系产生的.OH、邻苯三酚自氧化体系产生的O2-.、DPPH.的清除能力及其还原能力、抗脂质过氧化能力,并考查丹酚酸B与抗氧化活性之间的量效关系。结果表明,丹酚酸B有很强的.OH、O2-.、DPPH.清除能力、还原能力和抗脂质过氧化能力,且其抗氧化活性随丹酚酸B浓度的增大而增强,呈明显的量效关系。