Enhancement of solubility and dissolution rate of cryptotanshinone, tanshinone I and tanshinone IIA extracted from Salvia miltiorrhiza

Cryptotanshinone, tanshinone I and tanshinone IIA are three major components in the extract of Salvia miltiorrhiza with pharmacological significance. However, their effective utilization is limited due to poor water solubility and bioavailability. Solid dispersion (SD) of the extract of Salvia miltiorrhiza was prepared to enhance solubility and dissolution of the three major components. Various carriers were screened for SD preparation by conventional solvent method. Dissolution of the components from selected SD systems was compared with commercial tablets of the extract from Salvia miltiorrhiza. The solubility of three components viz., cryptotanshinone, tanshinone I and tanshinone IIA, after forming SD with either of povidone K-30 (PVP K-30) or poloxamer 407, exhibited enhanced solubility in pH 6.8 buffer. Dissolution test revealed that the amount of three components released was higher from SD tablets as compared to the commercial tablets. Pharmacokinetic profile was evaluated using cryptotanshinone as a representative compound. AUC of cryptotanshinone was significantly increased when administered as a solid dispersion.     

参草通脉颗粒丹参酮ⅡA提取工艺研究

目的参草通脉颗粒在临床中治疗慢性心衰疗效确切,有效率达到90%以上,为了进一步明确中药的疗效成分便于推广研究。我们探讨其主要药物丹参中丹参酮ⅡA的最佳醇提取工艺条件,为丹参药材的综合利用提供实验依据。方法采用正交试验法,以丹参酮ⅡA的提取量及浸膏得率为考察指标,优选回流法提取的最佳工艺条件。结果最佳提取工艺为:以10倍量80%乙醇回流提取3次,每次1h。结论优选的提取工艺设计合理,丹参酮ⅡA提取率较高。     

一测多评法测定白花丹参中丹参酮Ⅱ_A、隐丹参酮和丹参酮Ⅰ的含量

目的《山东省中药材标准》中收载的白花丹参项下只控制了丹参酮Ⅱ_A一种成分,现参考《中国药典》(2010年版)丹参项下丹参酮类测定方法,通过实验验证该方法在白花丹参药材中应用的准确性和可行性,达到质量可控的目的。方法测定白花丹参药材中3个有效成分,将其作为指标性成分,利用公式计算确立3种成分的相对校正因子,得到隐丹参酮和丹参酮Ⅰ的含量,达到一测多评的目的。同时以外标法测得数据,进行比较。结果线性关系良好,方法精密度,重复性符合要求。结论该方法适用于白花丹参的含量测定,重现性良好,具备可行性。     

Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge

Osteoclasts, multinuclear cells specialized for bone resorption, differentiate from the monocyte/macrophage lineage of hematopoietic cells. Intervention in osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone diseases involving osteoclasts. In this study, we found that tanshinone IIA, originating from Salvia miltiorrhiza Bunge, inhibited the differentiation of osteoclasts. Addition of tanshinone IIA to the osteoclast precursor culture caused a significant decrease in the level of calcitonin receptor, c-Src, and integrin beta3 mRNA, which are normally upregulated during the osteoclast differentiation dependent on RANKL (receptor activator of nuclear factor kappa B ligand). RANKL activated the ERK, Akt, and NF-kappaB signal transduction pathways in osteoclast precursor cells, and tanshinone IIA suppressed this activation. Tanshinone IIA also inhibited the bone resorptive activity of differentiated osteoclasts, which was accompanied with the disruption of the actin ring. Thus, tanshinone IIA has the potential to ameliorate bone-resorption diseases in vivo by reducing both the number and activity of osteoclasts.     

丹参毛状根中多种丹酚酸类化合物的鉴定与生物合成(英文)

应用发根农杆菌9402侵染丹参叶片获得了一株可以稳定产生多种丹酚酸类化合物的丹参毛状根。除迷迭香酸和丹酚酸B外还含有丹酚酸K、丹酚酸L、乙基丹酚酸B、甲基丹酚酸B以及一个分子量为538的未知化合物(化合物538)。LC-MS对这些化合物进行了鉴定。对茉莉酸甲酯(MeJA)和酵母诱导子对几种主要化合物积累的影响进行了分析:MeJA促进丹酚酸B、迷迭香酸、丹酚酸K和化合物538的积累,分别从干重4.21%、2.48%、0.29%和0.01%提高到7.11%、3.38%、0.68%和0.04%;酵母诱导子提高迷迭香酸含量,从干重2.83%上升到5.71%,抑制了丹酚酸B、丹酚酸K和化合物538的生物合成。实验结果表明所获得的丹参毛状根可以作为生产丹酚酸B、迷迭香和丹酚酸K以及化合物538的替代资源。对这些酚酸类化合物对诱导子的生物累积效应变化趋势进行分析推测:丹参毛状根中丹酚酸K和化合物538可能是从迷迭香酸合成丹酚酸B的中间产物。     

Role of P-glycoprotein in the intestinal absorption of tanshinone IIA, a major active ingredient in the root of Salvia miltiorrhiza Bunge

The extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9-3.4% in rats, with non-linear pharmacokinetics when its dosage increased. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSA disappearance from the luminal perfusates (P(lumen)) were 6.2- to 7.2-fold higher (P < 0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The P(blood), but not P(lumen), was significantly increased when co-perfused with verapamil, or quinidine (both P-gp inhibitors). The uptake and efflux of TSA in confluent Caco-2 cells were significantly altered in the presence of verapamil, quinidine, MK-571, or probenecid. The transport of TSA across Caco-2 monolayers was pH-, temperature- and ATP-dependent. Furthermore, the transport from the apical (AP) to basolateral (BL) side of the Caco-2 monolayers was 3.3- to 8.5-fold lower than that from the BL to AP side, but such a polarized transport was attenuated by co-incubated verapamil or quinidine. A polarized transport was also observed in the control MDCKII cells and more apparent in MDR1-MDCKII monolayers, with the P(app) values of TSA in the BL-AP direction being 7- to 9-fold higher in MDR1-MDCKII monolayers than those in the control MDCKII cells. Moreover, TSA significantly inhibited P-gp-mediated transport of digoxin in P-gp-overexpressing membrane vesicles with an IC(50) of 2.6 microM, but stimulated vanadate-sensitive P-gp ATPase activity with estimated K(m) and V(max) values of 10.70 +/- 0.69 microM and 67.65 +/- 1.31 nmol/min/mg protein, respectively. TSA was extensively metabolized to tanshinone IIB (TSB), and two other oxidative metabolites in rat liver microsomes, but the formation rate of TSB in rat intestinal microsomes was only about 1/10 of that in liver microsomes. These findings indicate that TSA is a substrate and reversing agent for P-gp; and P-gp-mediated efflux of TSA into the gut lumen and the first-pass metabolism contribute to the low oral bioavailability. Further studies are needed to explore the role of other drug transporters and first-pass metabolism in the low bioavailability of TSA.     

Inhibitory activity of diacylglycerol acyltransferase by tanshinones from the root of Salvia miltiorrhiza

The inhibitory activity of tanshinones from Salvia miltiorrhiza was tested on rat liver diacylglycerol acyltransferase (DGAT). Cryptotanshinone (1) and 15,16-dihydrotanshinone I (3) exhibited potent DGAT inhibitory activities dose-dependently with IC50 values of 10.5 microg/ml and 11.1 microg/ml. However, tanshinone IIA (2) and tanshinone I (4) showed very weak inhibition (IC50 value: > 250 microg/ml). A dihydrofuran moiety was seemed to be responsible for the stronger inhibitory activity.     

丹参在炮制大生产过程中丹酚酸B的损失情况研究

目的考察丹参药材炮制大生产过程中指标性成分丹酚酸B的含量变化及损耗情况。方法进行10批次丹参饮片炮制生产,应用HPLC法,在生产过程各主要工艺点进行跟踪取样,测定丹酚酸B的含量并计算损耗;同时在其中一个批次药材炮制大生产过程中,检测每个工艺环节对丹酚酸B的含量的影响。结果原药材、水洗药材、润透药材和切片4个取样点丹酚酸B的平均含量分别为6.83%,4.72%,4.46%和3.55%,相对原药材的损失情况分别为30.84%,34.59%和47.99%。水洗工艺直接导致丹酚酸B损失22.11%,最终损失高达47.48%。结论严格控制(优化)水洗工艺,可有效保留丹酚酸B的含量,提高丹参的炮制效率。     

Role of ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB,one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration–time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p?P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0–24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.     

Role of ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C(max) (T(max)) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSB disappearance from the lumen (P(lumen)) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P(app) values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC(0-24hr) in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.     

Anti-allergic effects of salvianolic acid A and tanshinone IIA from Salvia miltiorrhiza determined using in vivo and in vitro experiments

Salvia miltiorrhiza root has been used in Asian traditional medicine for the treatment of cardiovascular diseases, asthma, and other conditions. Salvianolic acid B from S. miltiorrhiza extracts has been shown to improve airway hyperresponsiveness. We investigated the effects of salvianolic acid A, tanshinone I, and tanshinone IIA from S. miltiorrhiza in allergic asthma by using rat RBL-2H3 mast cells and female Balb/c mice. Antigen-induced degranulation was assessed by measuring β-hexosaminidase activity in vitro. In addition, a murine ovalbumin-induced allergic asthma model was used to test the in vivo efficacy of salvianolic acid A and tanshinone IIA. Tanshinone I and tanshinone IIA inhibited antigen-induced degranulation of mast cells, but salvianolic acid A did not. Administration of salvianolic acid A and tanshinone IIA decreased the number of immune cells, particularly eosinophils in allergic asthma-induced mice. Histological studies showed that salvianolic acid A and tanshinone IIA reduced mucin production and inflammation in the lungs. Administration of salvianolic acid A and tanshinone IIA reduced the expression and secretion of Th2 cytokines (IL-4 and IL-13) in the bronchoalveolar lavage fluid and lung tissues of mice with ovalbumin-induced allergic asthma. These findings provide evidence that salvianolic acid A and tanshinone IIA may be potential anti-allergic therapeutics.     

声光可调-近红外光谱法快速测定丹参药材中隐丹参酮的含量

目的:建立快速测定丹参药材中隐丹参酮含量的方法。方法:采用高效液相色谱法测定药材样品中隐丹参酮含量(作为参考值)。采用声光可调-近红外光谱法结合偏最小二乘法建立药材样品中隐丹参酮含量的定量模型:根据药材样品中隐丹参酮含量测定结果,采集35份药材样品,以一阶导数法联合平滑滤波系数法预处理光谱,药材样品中隐丹参酮含量测定最佳波段范围为1 250~2 150 nm。结果:药材样品中隐丹参酮含量测定方法学经验证符合要求。隐丹参酮定量模型的校正均方根偏差为0.014 6,预测均方根偏差为0.022 3,相关系数为0.976 6。内部验证偏差为2.41%,外部验证偏差为4.06%。结论:该方法快速准确,简便无污染,可用于丹参药材中隐丹参酮含量的快速测定。     

不同干燥方法对丹参毛状根有效成分含量及其抗氧化活性的影响

目的：探讨不同干燥方法对丹参毛状根中主要化学成分含量及其抗氧化活性的影响,尤其关注采后干燥对丹参毛状根酚酸类成分是否也有类似对植物根一样的胁迫诱导作用。方法丹参毛状根经冷冻干燥、真空干燥、阴干和晒干四种不同干燥方式处理后,采用高效液相色谱法测定毛状根中包括9种酚酸类和4种酮类成分的含量;利用1,1-二苯基-2-三硝基苯肼（ DPPH）自由基清除试验、超氧阴离子清除试验、脂质过氧化抑制试验测定毛状根提取物的抗氧化活性。结果与冷冻干燥相比,真空干燥和阴干对酚酸类成分的影响不大,但酮类成分下降约50％;晒干处理后二类成分均严重损失达90％左右;晒干样品提取物抗氧化活性也显著下降。结论冷冻干燥是最适合丹参毛状根的干燥方法;与丹参根不同,采后干燥对毛状根的酚酸类成分无诱导作用。     

Role of P-glycoprotein in restricting the brain penetration of tanshinone IIA, a major active constituent from the root of Salvia miltiorrhiza Bunge, across the blood-brain barrier

Tanshinone IIA (TSA) is a major constituent of Salvia miltiorrhiza Bunge widely used in the treatment of stroke. This current study aimed to investigate the nature of brain penetration of TSA using several in vitro and in vivo models. The uptake and efflux of TSA in primary rat brain microvascular endothelial cells (RBMVECs) were altered in the presence of a PgP inhibitor or multidrug-resistance-associated protein (Mrp1/2) inhibitor. A polarized transport of TSA was found in RBMVEC monolayers with facilitated efflux from the abluminal to the luminal side. The polarized transport of TSA was attenuated by PgP or Mrp1/2 inhibitors. In an in situ rat brain perfusion model, TSA crossed the blood-brain barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a PgP or Mrp1/2 inhibitor. The brain levels of TSA were only about 31% of that in the plasma and it increased to 74-77% of plasma levels when verapamil or quinidine was coadministered in rats. The entry of TSA to the central nervous system (CNS) significantly increased in rats subjected to middle cerebral artery occlusion or treatment with quinolinic acid. The normalized brain penetration of TSA in mdr1a((-/-)) mice was much higher than the wild-type mice. Taken collectively, these findings provide evidence that TSA has limited brain penetration through the blood-brain barrier owing to the contribution of PgP and possibly Mrp1/2.     

UPLC法同时测定丹参中11种成分的含量

目的:建立同时测定丹参中丹参素、原儿茶醛、咖啡酸、迷迭香酸、紫草酸、丹酚酸B、丹酚酸A、二氢丹参酮、隐丹参酮、丹参酮Ⅰ和丹参酮ⅡA11种化学成分含量的方法。方法:采用超高效液相色谱(UPLC)法。色谱柱为BEH C18(50 mm×2.1 mm,1.7μm),流动相为0.5%甲酸溶液-乙腈(梯度洗脱),流速为0.4 ml/min,检测波长为265 nm和280 nm。结果:该方法可在14 min内完成一次色谱分析,11种成分色谱峰之间有良好的分离度,各成分的质量浓度与各自峰面积积分值之间呈良好的线性关系,精密度、重复性、稳定性及加样回收率试验的RSD<2.0%。结论:该方法快捷、准确、重复性好,能同时测定丹参药材与饮片中11种成分的含量,可用于更全面地控制丹参的质量和丹参的相关研究。     

Sodium tanshinone IIA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin II in cultured neonatal rat cardiac cells

Recent studies support the view that in addition to its effect on both phase I and phase II xenobiotic metabolizing enzymes, the synthetic chemopreventive agent oltipraz also increases the nucleotide excision repair (NER) which represents the major pathway of elimination of chemical carcinogen DNA adducts. Since most carcinogens are activated in the liver, we investigated the influence of oltipraz on NER activity of this target tissue by using two different approaches. First, we employed an assay based on the measurement of DNA repair in cisplatin-damaged plasmid DNA incubated in the presence of cell-free extracts prepared from either rat liver or human hepatoma HepG2 cells treated by oltipraz. Secondly, we analyzed the removal of aflatoxin B(1)-derived DNA adducts formed in primary human hepatocytes exposed to oltipraz after treatment with this mycotoxin. Whatever the strategy used, NER activity was not altered in liver cells. These data demonstrated that liver cells actively repair bulky DNA adducts by NER and that oltipraz does not influence their NER activity neither in vivo nor in vitro, consequently strongly suggesting that the chemopreventive agent oltipraz is acting before the initiation step of cancer development.     

Effect of Salvia miltiorrhiza Bunge injection on anticardiolipin antibody production induced by beta2 glycoprotein.

AIM: To explore the therapeutic effect and the mechanism of Chinese herbs on antiphospholipid syndrome (APS) by observing the effect of Salvia miltiorrhiza Bunge injectio (SmBI) on anticardiolipin antibody (aCL) induced by beta2 glycoprotein I (beta2-GP I). METHODS: Sixty female mice randomly fell into 6 groups: group A, B, C, D was injected through abdominal cavity with different dosage of SmBI daily; after 14 d, group A, B, C, E was immunized with 150 microg of purified human beta2-GP I in complete Freund's adjuvant subcutaneously; group F as control. The titre of aCL were detected by enzyme linked immunosorbent assay; subsets of T cell were grouped by streptavidin-biotin complex technique; and the activity of IL-2 was measured by MTT chromatometry. RESULTS: (1) Compared with group E, the absorbance (A) of aCL in group A, B, and C was decreased (P < 0.05 or P < 0.01). By linear correlation, the dosage is negatively correlated with the A values of aCL in 1, 2, and 3 weeks (P < 0.01). (2) Compared with group E, TH/TS ratio was reduced in group A, B, and C (P < 0.05 or P < 0.01); there is no significant differences between group D and F (P>0.05). By linear correlation, the dosage is negatively correlated with TH/TS ratio (P < 0.01). (3) Compared with E, the activity of IL-2 in group B and C decreased significantly (P < 0.01). By linear correlation, there is negative correlation between dosage and IL-2 activity (P < 0.01). There is no significant difference between D and F (P > 0.05). (4) There is positive correlation between TH/TS ratio and IL-2 activity in different dilutions (P<0.01). CONCLUSION: The mechanism of suppressive effect of SmBI on aCL induced by beta2-GP I may be realized by resuming the elevated TH/TS ratio and IL-2 activity. The state that SmBI have no effect on normal mice indicates that SmBI has selective immunoregulative functive.     

Simultaneous determination of cryptotanshinone, tanshinone I and tanshinone IIA in traditional Chinese medicinal preparations containing Radix salvia miltiorrhiza by HPLC

A reversed-phase high performance liquid chromatographic method was established for the simultaneous determination of tanshinones in five kinds of traditional Chinese medicinal preparations (TCMPs) containing Radix salvia miltiorrhiza (Chinese herbal name: Danshen). Tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA were successfully separated on a Diamonsil C18 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of methanol, tetrahydrofuran, water and glacial acetic acid (20:35:44:1, v/v/v/v), employing isocratic elution at a flow rate of 1.0 mL/min. Detection was accomplished at 254 nm. The compounds were identified by comparing their retention times and UV spectra in the 200-400 nm range with authentic standards. Regression equations revealed good linear relationship between the peak areas of the constituents and their concentrations (correlation coefficients: 0.9998 for cryptotanshinone, 0.9999 for tanshinone I and 1.0000 for tanshinone IIA). The relative standard deviations (n=6) of retention time and peak area were less than 0.25% and 1.00%, respectively. The recoveries were between 96.2% and 102.5%. The proposed method has been successfully applied to the simultaneous determination of the tanshinones in five kinds of Chinese herbal preparations containing Danshen within 20 min.