Natural product; anti-inflammatory; NF-κB

前胡素A和前胡素C互为总状花序，是传统上用作中草药的前胡属植物的主要生物活性成分。在本研究中，我们研究了前喷发素A和C激活组成型雄甾烷受体并诱导HepG2细胞中人多药耐药相关蛋白2表达的能力。分别通过定量实时PCR、Western blot和CDF摄取试验测定多药耐药相关蛋白2的mRNA水平、蛋白表达和转运活性的变化。通过瞬时转染特定的组成型雄甾烷受体siRNA，我们还测定了组成型雄甾醇受体敲除对多药耐药相关蛋白2 mRNA和蛋白表达的影响。结果表明，前胡芦甲素和前胡芦乙素C显著诱导了多药耐药相关蛋白2的mRNA和蛋白的表达，并增强了多药耐药性相关蛋白2的转运活性。进一步的研究表明，通过瞬时转染特定的组成型雄甾烷受体siRNA，mRNA和蛋白质的上调被减弱，这表明多药耐药相关蛋白2的上调是由组成型雄甾醇受体介导的。综上所述，我们的研究结果表明，前胡芦甲素和前胡芦乙素C可以在体外通过组成型雄甾烷受体介导的途径显著上调多药耐药相关蛋白2的表达，这应该被视为一种草药药物相互作用[3]。

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（-）-Praeruptorin A/前胡素A已在日本前胡和泰国前胡中报道。（±）-Praeruptorin A能有效放松回肠和气管平滑肌。（+）-前胡素A可以通过减少平滑肌细胞（SMCs）的面积、胶原含量和SMCs中的[Ca2+]i来改善血管肥大[1]。

白花前胡甲素是一种天然存在于中药白花前胡根部的香豆素类化合物，该药材常用于治疗某些呼吸系统疾病和高血压。虽然先前研究表明(±)-白花前胡甲素对气管和动脉标本具有舒张作用，但关于其对映体的功能特性知之甚少。本研究通过制备型Daicel Chiralpak AD-H色谱柱成功分离并鉴定出两种对映体，观察比较了它们对主动脉环的舒张作用。(+)-白花前胡甲素在抑制KCl和苯肾上腺素诱导的完整内皮大鼠离体主动脉环收缩方面，显示出比(-)-白花前胡甲素更强的舒张活性。去除内皮显著减弱了(+)-白花前胡甲素的舒张作用，但对(-)-对映体影响不大。用N(ω)-硝基-L-精氨酸甲酯（L-NAME，一氧化氮合酶抑制剂）或亚甲蓝（MB，可溶性鸟苷酸环化酶抑制剂）预处理主动脉环，会导致两种对映体的舒张作用发生与内皮去除相似的变化。分子对接研究也表明(+)-白花前胡甲素比(-)-对映体更符合一氧化氮合酶药效团特征。另一方面，两种白花前胡甲素对映体都能轻微减弱由肌浆网(SR)内Ca2+释放诱导的大鼠主动脉环收缩。这些发现表明(+)-和(-)-白花前胡甲素对大鼠离体主动脉环具有不同的舒张作用，这可能主要归因于内皮一氧化氮合酶催化的一氧化氮合成。[1]

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(±)-白花前胡甲素对映体对KCl或PE诱导的大鼠主动脉环收缩的影响[1]
分别用高浓度KCl（60 mM）或PE（1 μM）预收缩主动脉环。达到平台期后，分别累积加入(+)-白花前胡甲素和(−)-白花前胡甲素（KCl诱导收缩时浓度为1-30 μM，PE诱导收缩时为3-100 μM），获得浓度-舒张反应曲线。 在内皮完整的主动脉环中，(±)-白花前胡甲素对映体在1-100 μM浓度范围内对基础张力无显著影响（数据未显示），但两者均对KCl（60 mM）或PE（1 μM）诱导的收缩表现出浓度依赖性舒张作用（图5A和C）。对于KCl诱发的收缩，(+)-和(−)-白花前胡甲素的IC50值分别为12.1±1.3 μM和20.9±0.8 μM，Emax值分别为90.7±1.4%和68.6±5.2%。对于PE诱发的收缩，(+)-和(−)-对映体的IC50值分别为35.4±3.6 μM和45.8±2.5 μM，Emax值分别为86.3±2.6%和79.8±2.4%。结果表明，在内皮完整的大鼠主动脉环中，(+)-白花前胡甲素对KCl或PE诱导收缩的抑制作用强于(−)-对映体。

在内皮去除的主动脉环中，(+)-白花前胡甲素和(−)-对映体也显示出对KCl或PE诱导收缩的浓度依赖性舒张作用（图5B和D）。对于KCl诱发的收缩，(+)-和(−)-对映体的IC50值分别为22.7±1.5 μM和22.8±2.9 μM，Emax值分别为65.6±3.5%和65.3±8.1%。对于PE诱发的收缩，IC50值分别为42.9±4.1 μM和44.0±1.0 μM，Emax值分别为70.2±3.9%和69.1±3.0%。这些发现表明内皮去除显著减弱了(+)-白花前胡甲素的舒张作用，但对(−)-对映体影响不大，且(+)-白花前胡甲素的作用涉及内皮依赖性和非依赖性舒张机制。

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NO/环磷酸鸟苷(cGMP)通路在(±)-白花前胡甲素对映体舒张KCl诱导的大鼠主动脉环收缩中的作用[1]
为确定NO/cGMP通路是否参与(±)-白花前胡甲素对映体的舒张作用，分别在内皮完整的大鼠主动脉环中，于KCl给药前30分钟加入L-NAME（100 μM，NO合酶抑制剂）和MB（10 μM，催化cGMP形成的可溶性鸟苷酸环化酶(sGC)抑制剂）。
L-NAME或MB在测试浓度下对大鼠主动脉环的基础张力无影响（数据未显示）。在L-NAME预处理的主动脉环中，(+)-白花前胡甲素和(−)-对映体的IC50值分别为21.0±4.1 μM和22.4±1.6 μM，Emax值分别为70.3±8.5%和64.2±7.6%（图6A）。在MB预处理的主动脉环中，(+)-和(−)-白花前胡甲素的IC50值分别为20.2±0.5 μM和22.1±1.1 μM，Emax值分别为80.0±1.3%和70.2±4.2%（图6B）。与内皮去除类似，L-NAME或MB预处理均显著减弱了(+)-白花前胡甲素对KCl诱导收缩的舒张作用，但对(−)-对映体影响不大，且两种对映体的舒张效果趋于相同。这些发现表明NO/cGMP信号通路主要参与(+)-而非(−)-白花前胡甲素的舒张作用。
为阐明(+)-白花前胡甲素舒张效能对NO阻断的敏感性是否源于NO（或cGMP）与其协同作用，我们观察了(±)-白花前胡甲素对乙酰胆碱诱发舒张（NO依赖性舒张）的影响。结果显示：(+)-和(−)-白花前胡甲素均不影响乙酰胆碱对完整内皮大鼠离体主动脉环的舒张作用。单独乙酰胆碱、乙酰胆碱加(+)-白花前胡甲素（30 μM）、乙酰胆碱加(−)-对映体（30 μM）的IC50值分别为4.0±0.7 μM、4.3±0.6 μM和4.2±0.4 μM。该结果证明(+)-白花前胡甲素并非通过与NO协同发挥舒张效应1。

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(±)-白花前胡甲素对映体对细胞内Ca2+释放诱导的大鼠主动脉环收缩的影响[1]
在无Ca2+的K-H液中，PE（1 μM）可通过释放细胞内Ca2+诱发内皮去除大鼠主动脉环的短暂收缩。如图7所示，(±)-白花前胡甲素对映体仅轻微减弱PE诱导的收缩且效能相近。对照组与(+)-白花前胡甲素（3 μM、10 μM、30 μM）处理组的收缩比值（T2/T1）分别为93.3±2.1%、92.2±2.5%、88.7±3.2%和81.9±4.3%；(−)-对映体处理组的T2/T1值分别为94.6±3.7%、91.8±1.9%、85.8±3.5%和80.1±5.1%。选择性IP3R抑制剂肝素（100 μg/ml）则显著抑制PE诱导的收缩（对照组与肝素组的T2/T1值分别为98.1±7.5%和6.5±1.3%）。这表明(±)-白花前胡甲素对映体可轻微减弱IP3R（肌醇-1,4,5-三磷酸受体）介导的细胞内Ca2+释放诱导的主动脉环收缩1。

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K+通道阻断剂对(±)-白花前胡甲素对映体舒张PE诱导大鼠主动脉环收缩的影响[1]
为探究K+通道开放是否参与(±)-白花前胡甲素对映体的舒张作用，在标准K-H液中去内皮主动脉环中，于PE（1 μM）给药前30分钟加入TEA（5 mM，假定K+通道阻断剂）。
图8A显示两种对映体以相近效能减弱PE诱导的收缩，(+)-白花前胡甲素和(−)-对映体的IC50值分别为42.9±4.1 μM和44.0±1.0 μM。TEA预处理未改变对映体的舒张作用，其IC50值分别为43.1±3.8 μM和44.1±6.4 μM（图8B）。该结果提示K+通道开放不参与(±)-白花前胡甲素对映体的舒张机制1。

Praeruptorin A/PA和CKL在肠道细菌中的孵化[2]
按照参考文献中提出的方案制备脑心输注（BHI）培养基和肠道微生物群溶液。大鼠肠道细菌对PA或CKL的生物转化是在0.5 mL的培养系统中进行的，该培养系统含有50μL的肠道微生物菌群溶液、5μL的PA或CKL-DMSO储备溶液（任何一种化合物的终浓度为25μmol/L）和445μL BHI培养基。在GasPak EZ Anaerobe袋系统中，于37°C下厌氧培养4小时，并加入500μL冰冷的甲醇淬灭。0分钟温育、无菌群溶液反应和无PA/CKL温育作为对照。每个反应进行三次。然后通过离心和过滤连续处理样品。随后，对滤液进行LC-UV-MS/MS分析。

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RLMs和HLMs中Praeruptorin A/PA的I期代谢[2]
在37°C下，在总共200μL的含有PA（终浓度：25μmol/L）、RLM或HLM（1 mg/mL）、NADPH再生系统（4 mmol/L MgCl2、1 mmol/Lβ-NADP+、1 mmol/L G-6-P和1 U/mL G-6-PD）和100 mmol/L磷酸钾缓冲液（pH 7.4）的孵育溶液中，在肝微粒体蛋白质中实现PA的代谢。分别在0、5、10、15、20、30、60、90、120分钟时通过倒入200μL冰冷的甲醇并涡旋以充分混合来终止反应。随后，将每个培养物在4°C下以15000×g离心10分钟，然后将所得上清液通过0.45μm尼龙膜过滤器过滤，然后进行HPLC-UV分析。分别孵育0分钟、60分钟不含β-NADP+、60分钟无PA和60分钟含变性肝微粒体蛋白的样品作为对照。每个反应进行三次。为了鉴定PA代谢物，选择从20分钟反应中获得的培养物，并将其加载到LC-UV-MS/MS系统中。 为了鉴定在没有NADPH再生系统的情况下参与PA水解的酶，将HLMs或RLMs与羧酸酯酶抑制剂BNPP（500μmol/L）或PMSF（500μmol/L）预孵育5分钟，然后加入PA以启动另一个20分钟的孵育。使用0分钟孵育和20分钟无化学抑制剂的反应作为对照。每个实验进行三次。

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使用混合的大鼠血浆、人血浆、hCES1和hCES2水解Praeruptorin A/PA[2]
按照文献中提出的程序，使用各自的探针底物咪达普利和伊立替康（CPT-11）验证了hCES1和hCES2的活性。从五只无毒雄性SD大鼠中新鲜制备混合大鼠血浆，并从澳门红十字会获得混合人血浆作为礼物。PA（25μmol/L）与大鼠血浆（最终含量：1 mg/mL）、人血浆（最终内容：1 mg/mL）、hCES1（最终含量为1单位）或hCES2（最终含量）在磷酸钾缓冲溶液（pH 7.4，100 mmol/L）中于37°C下孵育60分钟，最终孵育体积为200μL。有两种类型的对照样品，包括与变性血浆蛋白一起孵育和在没有PA的情况下孵育。每种反应都进行了三次处理。通过加入等体积的冰冷甲醇终止培养。通过离心去除沉淀的蛋白质。过滤上清液，将滤液等分（10μL）注入LC-UV-MS/MS系统。

(+)-Praeruptorin A and (−)-Praeruptorin A were dissolved in PEG400. [1]
To investigate the effects of compounds on the contraction induced by intracellular Ca2+ release, the aortic rings were exposed to Ca2+-free solution, and PE (1 μM) were used to induce the first transient contraction (T1). Thereafter, the rings were washed twice with standard Krebs–Henseleit solution (at least 40 min of incubation period for refilling the intracellular Ca2+ stores) and then twice with Ca2+-free K–H solution (15 min of incubation period). Then PE was used to induce the second transient contraction (T2) in the absence or presence of (+)-Praeruptorin A and (−)-Praeruptorin A, added 30 min before PE application. Heparin (100 μg/ml) was used as a positive control. The ratio of the second contraction to the first contraction (T2/T1) was calculated [1].

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Preparation of urine and fecal samples [2]
Male SD rats (220 ± 15 g) were acclimated in laboratory for one week prior to the experiments, housed in separate cages at a temperature of 23 ± 1 °C with a 12 h light/dark cycle and 50% relative humidity, free access to standard diet and water. All the rats were fasted over night before the experiments yet free access to water.
Three rats as PA-group were orally administered of Praeruptorin A/PA in a 50% aqueous 1,2-propylene glycol solution (v/v) while the control group (3 rats) was treated with the solvent (50% aqueous 1,2-propylene glycol solution, v/v). The urine and fecal samples were collected over 0–48 h after treatment and pooled within group. Pooled urine from PA-group and control group were diluted with 3 folds of methanol, vortexed and centrifuged to remove precipitate and then introduced to the LC-UV–MS/MS system, respectively. At the meanwhile, pooled fecal samples from PA-group or control group were extracted with acetonitrile at 10 mL per gram fecal sample for 30 min using ultrasonic water bath and then filtered through 0.45 μm membrane before analysis.

(±)-Praeruptorin A (PA) is the major bioactive component in Peucedani Radix (Chinese name: Qian-hu), and exhibits dramatically anti-hypertensive effect typically through acting as a calcium channel blocker. The current study aims on the characterization of the metabolic profiles of PA in vitro and in vivo using high performance liquid chromatography (HPLC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). A total of 12 phase I metabolites (M1-12) in rat liver microsomes (RLMs), 9 phase I metabolites (M1-3, M5-6 and M9-12) in human liver microsomes (HLMs), 2 hydrolyzed products in rat plasma (M11 and M12), none metabolite in human plasma, none metabolite in rat intestinal bacteria, 7 metabolites (M1, M4-7, M13 and M15) in PA-treated rat urine and 6 metabolites (M1, M4-7 and M15) in PA-treated feces were detected and tentatively identified using predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (predictive MRM-IDA-EPI) mode in combination with enhanced mass spectrum-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode in the mass spectrometer domain, respectively, while TOF-MS was adopted to confirm the identification. Further, 2 glucuronidated metabolites (M13-14) in RLMs and none metabolite in HLMs of cis-khellactone (CKL), which was the main actual form of PA in vivo, were generated, while its sulfated product was not observed in either rat liver S9 fractions (RS9) or human liver S9 fractions (HS9). Oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation were demonstrated to be the predominant metabolic types for PA in vitro and in vivo. Judging from the decrement of peak areas, PA was metabolized quickly in both RLMs and HLMs, indicating extensively hepatic first-pass elimination. Taken together, the metabolic fates of (±)-praeruptorin A in vitro and in vivo were elucidated in current study, and Q-trap-MS coupled with LightSight™ software can be adopted as a useful tool for quick detection and identification of metabolites in complex biological matrices. [2]

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This is the first report on the metabolic characterization of Praeruptorin A/PA in vivo and CKL in vitro. The metabolites were detected and tentatively identified using high performance liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry and LightSight™ software using predictive MRM-IDA-EPI in combination with EMS-IDA-EPI mode. Metabolic pathways including hydrolysis, oxidation, intra-molecular transacylation and glucuronidation were observed in vitro to generated 14 metabolites (M1–14). On the other hand, six (M1, 5–7 and 13) and five (M1 and 5–7) metabolites in M1–13 were detected in PA-treated urine and fecal samples, respectively, along with an additional oxidated product (M15) forming by a step-wise oxidation at C-4′ position. The crucial hydrolysis in plasma and CYP450-catalyzed transformation, rather than gut flora, were revealed to be responsible for the low bioavailability in rats, while the oral bioavailability of PA in human beings is expected to be higher than that in rats. In addition, the present study also suggested that predictive MRM-IDA-EPI coupled with EMS-IDA-EPI mode, which were developed using LightSight™ software, can be utilized as a useful tool for quick detection and identification of metabolite in complex biological matrices. [2]

[1]. (+/-)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis. Chem Biol Interact. 2010 Jul 30;186(2):239-46.

[2]. Metabolic characterization of (±)-praeruptorin A in vitro and in vivo by high performance liquidchromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry. J Pharm Biomed Ana. 2014 Mar:90:98-110.

[3]. Effects of praeruptorin A and praeruptorin C, a racemate isolated from Peucedanum praeruptorum, on MRP2 through the CAR pathway. Planta Med. 2013 Nov;79(17):1641-7.

Praeruptorin A is a member of coumarins.
(+)-Praeruptorin A has been reported in Peucedanum japonicum, Prionosciadium thapsoides, and Ligusticum lucidum with data available.

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(-)-Praeruptorin A has been reported in Peucedanum japonicum and Prionosciadium thapsoides with data available.

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(±)-Praeruptorin A has been previously demonstrated to be able to relax vascular smooth muscles as the main bioactive constituent of P. praeruptorum roots. However, the action characteristics and underlying mechanisms of the enantiomers remain unclear. In the present study, we found that both (+)-praeruptorin A and (−)-praeruptorin A showed a concentration-dependent relaxation of isolated rat aortic rings with functional endothelium contracted by high K+, and (+)-praeruptorin A was more potent than (−)-praeruptorin A. Of note, endothelium removal and pretreatment with l-NAME or MB significantly attenuated the relaxant effect of (+)-praeruptorin A but not (−)-praeruptorin A, and resulted in the relaxant potencies of the two enantiomers tending to be same. These findings strongly suggested that (+)-Praeruptorin A exerted both endothelium-dependent and -independent relaxation of vascular smooth muscles, and (−)-praeruptorin A only exerted endothelium-independent one.

Furthermore, K+ channels also participate in the regulation of muscle contractility and vascular tone. Direct activation of K+ channels on arterial smooth muscle cells should hyperpolarize the cell membrane, and inhibit Ca2+ influx and smooth muscle contraction. In the present study, TEA (a nonselective K+ channel blocker) pretreatment did not alter the relaxant effects of (±)-Praeruptorin A enantiomers, suggesting that K+ channel opening might be not involved in the relaxation of the enantiomers.

In conclusion, both (+)-Praeruptorin A and (−)-praeruptorin A can produce a concentration-dependent relaxation of isolated rat aortic rings contracted by KCl. The action of (+)-praeruptorin A is more potent than (−)-praeruptorin A. The most important reason for the difference is probably that (+)-praeruptorin A but not (−)-praeruptorin A can well agree to the pharmacophores of eNOS, and activates NO/cGMP signaling pathway. [1]

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Due to the powerful capability of CYP450s and plasma for the metabolism of Praeruptorin A/PA, the observation of CKL as the major product and the slight glucuronidation of CKL in RLMs (the decrement of CKL was less than 10% calculating by the peak areas), the mainly actual form of PA in rats were tentatively characterized as CKL, which is consistent with our previous report. On the other hand, the potent actual form of PA in human would include the proto type and CKL. In sight of the lower activity observed for CKL, the therapeutic effect of PA could last for a longer time in human beings than rats. However, M15, a step-wisely oxidated product of PA, was observed as one of the main metabolites in PA-treated urine, indicating that this metabolites might offer a significant contribution for the PA in vivo process and should be taken in account in the further studies. The active component is likely to become traceful in the body owing to absorption/distribution barrier and/or biotransformation hurdle, and the interference from endogenous substances always makes it hard to detect and identify the drug-related components in biological samples. Hence, high sensitive and selective techniques are imperiously demanded to meet the needs of metabolic characterization. In current study, the limit of detections of PA and its metabolites were less than 2 nmol/L using the developed predictive MRM-IDA-EPI method. Moreover, due to the employment of precursor-product ion transitions, this method is expected to possess high selectivity. However, the key point for the characterization of metabolic profile using the proposed method lies on the prediction prior to the LC–MS/MS measurement. In current study, the presumption was performed on the basis of knowledge obtained for angular-type pyranocoumarin metabolism and the common metabolic pathways that might occur for the xenobiotics in vivo. In fact, some softwares, PALLAS MetabolExpert (CompuDrug) and METEOR MetabolicExpert (Lhasa) for instance, are available for the prediction of metabolites in silico, which could promote the speculation for in vivo metabolism. On the other side, EMS-IDA-EPI mode, the LODs of which were less than 100 nmol/L for either PA or its metabolites, was also introduced to avoid the miss of potential metabolites during prediction, in spite that none additional product was afforded. Therefore, the proposed method provides a preferable analytical choice for the characterization of metabolites in biological matrices, while EMS-IDA-EPI could adopted as the complementary tool at the meanwhile. [2]

C21H22O7

386.3952

386.136

C, 65.28; H, 5.74; O, 28.98

73069-27-9

21499-23-0; 14017-71-1; 73069-25-7; 73069-27-9

38347607

White to off-white solid

1.3±0.1 g/cm3

486.8±45.0 °C at 760 mmHg

211.5±28.8 °C

0.0±1.2 mmHg at 25°C

1.574

4.18

92.04

0

7

5

28

720

2

O1C2C([H])=C([H])C3C([H])=C([H])C(=O)OC=3C=2C([H])(C([H])(C1(C([H])([H])[H])C([H])([H])[H])OC(C(=C([H])C([H])([H])[H])C([H])([H])[H])=O)OC(C([H])([H])[H])=O

XGPBRZDOJDLKOT-NXIDYTHLSA-N

InChI=1S/C21H22O7/c1-6-11(2)20(24)27-19-18(25-12(3)22)16-14(28-21(19,4)5)9-7-13-8-10-15(23)26-17(13)16/h6-10,18-19H,1-5H3/b11-6-/t18-,19-/m0/s1

[(9S,10S)-10-acetyloxy-8,8-dimethyl-2-oxo-9,10-dihydropyrano[2,3-f]chromen-9-yl] (Z)-2-methylbut-2-enoate

(+)-Praeruptorin A; Praeruptorin A; 73069-27-9; 73069-25-7; (+-)-Praeruptorin A; 2-Butenoic acid, 2-methyl-,(9S,10S)-10-(acetyloxy)-9,10-dihydro-8,8-dimethyl-2-oxo-2H,8H-benzo[1,2-b:3,4-b']dipyran-9-yl ester, (2Z)-; CCRIS 7247; ( inverted exclamation markA)-Praeruptorin A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~33.3 mg/mL (~86.3 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.47 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (6.47 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.47 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。