Cerebral ischemia

在本研究中，设计、合成了一系列3-正丁基邻苯二甲酸（NBP）释放硫化氢（H2S）的衍生物（8a–g和9a–f），并对其进行了生物评价。最有前景的化合物8e在体外显著抑制二磷酸腺苷（ADP）和花生四烯酸（AA）诱导的血小板聚集，优于NBP、盐酸噻氯匹定和阿司匹林。此外，8e在体内可缓慢产生中等水平的H2S，有利于改善心血管和脑循环[3]。

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DL-3-正丁基苯酞（NBP）因其抗氧化和抗炎作用而常用于治疗缺血性中风。本研究旨在通过在H9c2细胞中建立心肌缺血再灌注损伤（MIRI）模型，研究NBP对MIRI的保护作用。使用细胞计数试剂盒-8进行细胞活力测定，评估乳酸脱氢酶（LDH）细胞毒性和脂质过氧化丙二醛（MDA）含量，以检测细胞活性、细胞损伤程度和氧化应激反应。逆转录定量PCR用于定量H9c2细胞中炎性因子的表达。采用Western blot和免疫荧光染色检测PI3K/AKT和热休克蛋白70（HSP70）的蛋白表达。目前的结果表明，NBP在缺血再灌注期间显著提高了细胞存活率。此外，NBP抑制LDH的释放和MDA的产生。NBP治疗还显著降低了炎症因子在mRNA水平上的表达。此外，与MIRI模型中的细胞相比，NBP激活了PI3K/AKT通路并上调了HSP70的表达。LY294002是一种PI3K抑制剂，可逆转NBP的保护作用并抑制HSP70的表达。本研究表明，NBP通过PI3K/AKT通路激活调节HSP70表达，保护H9c2细胞免受MIRI的侵袭。[7]

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不同浓度的3-正丁基苯酞/NBP对H9c2细胞存活率的影响[7]
通过CCK-8比色法检测用不同NBP剂量处理的H9c2细胞的存活率。与MIRI组相比，100µM NBP提高了细胞存活率（P<0.05；图1A）。然而，NBP浓度≥200µM会略微降低细胞存活率（图1A）。目前的结果表明，100µM NBP是保护细胞免受MIRI的最佳浓度。

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NBP/3-正丁基苯酞抑制MIRI期间的氧化应激反应[7]
当心肌细胞发生缺血再灌注损伤时，氧化应激也起着重要作用。使用MDA试剂盒探讨NBP对MIRI后氧化应激指数的影响以及PI3K在这一过程中的作用。MIRI组的氧化水平明显高于CON组（P<0.05）。然而，NBP预处理显著降低了氧化应激（P<0.05）。相比之下，LY294002消除了NBP的抗氧化作用（P<0.05；图1B）。也就是说，NBP在MIRI期间显著降低了H9c2细胞的氧化应激反应，但这种抗氧化保护作用被阻断PI3K所消除。

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NBP/3-正丁基苯酞保护H9c2细胞免受MIRI[7]
在体外，细胞凋亡或坏死引起的细胞膜结构破坏导致LDH释放到培养基中。因此，LDH活性可以间接指示细胞损伤的程度。为了探索NBP对MIRI的影响以及PI3K在MIRI中的参与，通过CCK-8比色法测定H9c2细胞的存活率，同时使用LDH试剂盒测定培养基中的LDH含量。与CON组相比，MIRI组的细胞存活率显著降低（P<0.05）。然而，MIRI+NBP组的细胞存活率高于MIRI组（P<0.05）。相比之下，添加PI3K抑制剂LY294002会降低细胞存活率（P<0.05；图1C）。此外，MIRI组的LDH含量显著高于CON组（P<0.05）。NBP预处理降低了LDH的释放（P<0.05），但添加LY294002逆转了这一作用（P<0.05；图1D）。因此，NBP显著提高了缺血再灌注损伤后H9c2细胞的存活率，并减少了H9c2细胞损伤。然而，PI3K抑制剂LY294002逆转了NBP对H9c2细胞的保护作用。

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NBP/3-正丁基苯酞通过降低TNF-α和IL-1βmRNA表达来抑制炎症[7]
MIRI期间炎性细胞因子的激活会加重心肌细胞的损伤。因此，为了确定NBP在MIRI期间的保护作用与炎症因子之间的关联，通过RT-qPCR检测了H9c2细胞中TNF-α和IL-1β的mRNA表达。与CON组相比，MIRI组TNF-α和IL-1β的表达显著增加（P<0.05）。然而，与MIRI组相比，MIRI+NBP中这些基因的表达降低（P<0.05）。相比之下，与MIRI+NBP组相比，添加LY294002增加了TNF-α和IL-1β的表达（P<0.05；图1E和F）。因此，研究表明，NBP有效地降低了MIRI期间炎症因子的表达，而PI3K抑制剂LY294002的应用逆转了这种抗炎作用。

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NBP/3-正丁基苯酞激活HSP70和PI3K/AKT信号通路[7]
在MIRI期间，应激保护蛋白HSP70和PI3K/AKT的表达上调。Western blot和免疫荧光染色用于测定NBP处理后HSP70和PI3K/AKT的蛋白表达。与CON组相比，MIRI组p-AKT和PI3K的表达水平显著升高（p<0.05）。与MIRI组相比，MIRI+NBP组p-AKT和PI3K的表达增加（p<0.05）。相比之下，在MIRI+NBP+LY294002组中，与MIRI+NBP组相比，p-AKT和PI3K的表达降低（p<0.05；图2A-D）。在免疫荧光分析中也观察到了这种趋势（图2E和F）。此外，MIRI组HSP70的表达水平较CON组升高（P<0.05）。与MIRI+NBP组相比，NBP预处理进一步增加了HSP70的表达（P<0.05），而LY294002则降低了其表达（P<0.05；图3A和B）。在免疫荧光分析中也观察到了这种趋势（图3C）。本研究结果表明，NBP预处理可以增加MIRI后PI3K、p-AKT和HSP70的表达水平，抑制PI3K通路也会改变HSP70的表达。

在 SAMP8 小鼠中，丁基苯酞（160 mg/kg，IR）可以缓解记忆和学习缺陷[3]。

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3- n -丁苯酞是治疗急性缺血性脑卒中的有效药物。然而，其对慢性脑缺血引起的神经元损伤的影响仍然知之甚少。因此，本研究结扎15月龄大鼠双侧颈动脉，模拟老年人慢性脑缺血。然后给老龄大鼠灌胃3-正丁苯酞。3- n -丁苯酞改善慢性脑缺血大鼠大脑皮层和海马神经元形态，提高胆碱乙酰转移酶活性，降低丙二醛和β淀粉样蛋白水平，显著改善认知功能。上述结果提示，3-正丁苯酞可减轻慢性脑缺血引起的氧化应激，改善胆碱能功能，抑制β淀粉样蛋白积累，从而改善脑神经元损伤和认知缺陷。[2]

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中药提取物3-N-butylphthalide (NBP)在中国用于临床治疗缺血性患者。它已被证明在体内和体外都有多种神经保护作用。本研究探讨了NBP对衰老加速小鼠易感性-8 （SAMP8）动物模型学习记忆衰退的影响。4月龄SAMP8小鼠灌胃NBP 2个月后，空间学习记忆能力显著提高。SAMP8小鼠中隔核和对角带纵肢胆碱乙酰转移酶（choline acetyltransferase, ChAT）阳性神经元的损失减慢，海马、大脑皮层和前脑ChAT蛋白和mRNA表达下降。这些结果表明，在4个月大时开始的NBP治疗可以保护SAMP8小鼠的学习/记忆缺陷，并且这种效果可能是通过防止中枢胆碱能系统的衰退来介导的。[3]

关于线粒体功能的保护，早期动物研究表明，dl-NBP可以提高线粒体中Na/K-ATP酶和Ca-ATP酶的活性。众所周知，Na/K-ATP酶和Ca-ATP酶是维持细胞膜电位并参与物质运输以调节细胞体积的重要酶。NBP可以维持线粒体膜的流动性和线粒体膜的稳定性，防止线粒体肿胀[5]。

NBP的主要药理作用包括重建微循环、保护线粒体功能、抑制氧化应激、抑制炎症反应和抑制神经元凋亡。它还被发现具有抗血小板聚集、抗血栓形成和抗动脉粥样硬化等作用[5]。

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细胞培养[7]
将H9c2细胞（1x106细胞/ml）接种在含有高葡萄糖DMEM（10%FBS；1%青霉素/链霉素）的25-cm2细胞培养瓶中。细胞在37˚C、95%空气和5%二氧化碳的细胞培养箱中培养。H9c2细胞培养24小时后，随机分为四组：i）对照组（CON）；ii）MIRI组；iii）3-正丁基苯酞/NBP预处理组（MIRI+NBP）；iv）PI3K抑制剂组（MIRI+NBP+LY294002）。CON细胞在37˚C和95%空气中培养。MIRI细胞在37˚C下与5%CO2、93%N2和2%O2一起孵育6小时，然后复氧4小时。MIRI+NBP细胞用100µM NBP预处理2小时，然后缺氧6小时，再复氧4 h。MIRI+NBP+LY294002细胞用10µM LY294002（28）预处理1小时，然后用NBP处理2小时。

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H9c2 MIRI模型的构建[7]
为了模拟缺血再灌注损伤的体内模型，将细胞培养基替换为不含FBS的低葡萄糖DMEM，并在37˚C、5%CO2、93%N2和2%O2的条件下孵育细胞。在3-正丁基苯酞/NBP处理前1小时，共加入10µM的LY294002。NBP预处理在缺氧前持续2小时。MIRI后，用高糖DMEM（10%FBS；1%青霉素/链霉素）代替培养基，将细胞在培养箱（37˚C；5%CO2；95%空气）中培养4小时。

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确定最佳3-正丁基苯酞/NBP浓度和细胞存活率[7]
将H9c2细胞（1x104个细胞/孔）接种在96孔板中24-48小时，然后用不同剂量的NBP（1、50、100、200、300和500µM）预处理。再灌注后，丢弃原始培养基，向每个孔中加入100µl含有10mg/ml细胞计数试剂盒-8的培养基。在无光培养1小时后测量450nm处的吸光度。每组的最终细胞存活率用以下公式计算：细胞存活率（%）=（实验组空白）/（仅MIRI组空白）x100。

Animal/Disease Models: Aged male SAMP8 mice and SAMR1 mice [3]
Doses: 40 mg/kg, 80 mg/kg, 160 mg/kg
Route of Administration: Orally administered daily for 60 days
Experimental Results: The escape latency on day 7 was shortened. Improved learning deficits in SAMP8 mice.

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Ten rats were selected in the sham-operated group. Forty-five days after modeling, 27 rats survived, and those with equal cognitive levels were selected and randomly divided into three groups (n = 9 per group): (1) the model or (2) 30 or (3) 120 mg/kg 3-N-butylphthalide/NBP. [2]

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The chronic cerebral ischemia rat model and drug treatment [2]
Rats (280 mg/kg) were anesthetized by intraperitoneal (i.p.) injection of chloral hydrate. A neck operation was conducted to separate the bilateral common carotid arteries, which were then ligated permanently with 5-0 operation silk suture (Zhao et al., 2013). We injected 200,000 units of penicillin (i.p.) per day after the operation for 3 successive days. The bilateral common carotid arteries were separated without ligation in the sham-operated group. Chronic cerebral ischemia rat models were established in all three groups. Forty-five days after the operation, rats in the sham-operated and model groups were intragastrically administered peanut oil (2 mL/kg), and rats in the 30 or 120 mg/kg 3-N-butylphthalide/NBP groups were intragastrically administered 30 or 120 mg/kg 3-N-butylphthalide/NBP, respectively, dissolved in peanut oil for 45 successive days.

3-n-Butylphthalide (NBP) is a cardiovascular drug currently used for the treatment of cerebral ischemia. The present study aims to investigate the metabolism, pharmacokinetics, and excretion of NBP in humans and identify the enzymes responsible for the formation of major metabolites. NBP underwent extensive metabolism after an oral administration of 200 mg NBP and 23 metabolites were identified in human plasma and urine. Principal metabolic pathways included hydroxylation on alkyl side chain, particularly at 3-, ω-1-, and ω-carbons, and further oxidation and conjugation. Approximately 81.6% of the dose was recovered in urine, mainly as NBP-11-oic acid (M5-2) and glucuronide conjugates of M5-2 and mono-hydroxylated products. 10-Keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-hydroxy-NBP (M3-2), and M5-2 were the major circulating metabolites, wherein the areas under the curve values were 1.6-, 2.9-, 10.3-, and 4.1-fold higher than that of NBP. Reference standards of these four metabolites were obtained through microbial biotransformation by Cunninghamella blakesleana. In vitro phenotyping studies demonstrated that multiple cytochrome P450 (P450) isoforms, especially CYP3A4, 2E1, and 1A2, were involved in the formation of M3-1, M3-2, and 11-hydroxy-NBP. Using M3-2 and 11-hydroxy-NBP as substrates, human subcellular fractions experiments revealed that P450, alcohol dehydrogenase, and aldehyde dehydrogenase catalyzed the generation of M2 and M5-2. Formation of M5-2 was much faster than that of M2, and M5-2 can undergo β-oxidation to yield phthalide-3-acetic acid in rat liver homogenate. Overall, our study demonstrated that NBP was well absorbed and extensively metabolized by multiple enzymes to various metabolites prior to urinary excretion. [1]

rat LD50 oral 2450 mg/kg Food and Cosmetics Toxicology., 17(251), 1979

[1]. Ninety-day administration of dl-3-n-butylphthalide for acute ischemic stroke: a randomized, double-blind trial. Chin Med J (Engl). 2013;126(18):3405-10.

[2]. 3-N-butylphthalide improves neuronal morphology after chronic cerebral ischemia. Neural Regen Res. 2014 Apr 1;9(7):719-26.

[3]. Improvement of cognitive deficits in SAMP8 mice by 3-n-butylphthalide. Neurol Res. 2014 Mar;36(3):224-33.

[4]. Design, synthesis and biological evaluation of hydrogen sulfide releasing derivatives of 3-n-butylphthalide as potential antiplatelet and antithrombotic agents. Org Biomol Chem. 2014 Aug 21;12(31):5995-6004.

[5]. Application and prospects of butylphthalide for the treatment of neurologic diseases. Chin Med J (Engl). 2019;132(12):1467-1477.

[6]. Metabolism and pharmacokinetics of 3-n-butylphthalide (NBP) in humans: the role of cytochrome P450s and alcohol dehydrogenase in biotransformation. Drug Metab Dispos. 2013 Feb;41(2):430-44.

[7]. DL-3-n-butylphthalide protects H9c2 cardiomyoblasts from ischemia/reperfusion injury by regulating HSP70 expression via PI3K/AKT pathway activation. Exp Ther Med. 2021 Jul 15;22(3):1008.

Butylphthalide is a member of benzofurans.
Butylphthalide has been used in trials studying the prevention of Restenosis.
Butylphthalide has been reported in Angelica gigas, Ligusticum striatum, and other organisms with data available.
See also: Celery Seed (part of); Angelica sinensis root oil (part of).

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Background: Dl-3-n-butylphthalide (NBP), first isolated from the seeds of celery, showed efficacy in animal models of stroke. This study was a clinical trial to assess the efficacy and safety of NBP with a continuous dose regimen among patients with acute ischemic stroke. Methods: A randomized, double-blind, double-dummy trial enrolled 573 patients within 48 hours of onset of ischemic stroke in China. Patients were randomly assigned to receive a 14-day infusion of NBP followed by an NBP capsule, a 14-day infusion of NBP followed by aspirin, or a 14-day infusion of ozagrel followed by aspirin. The efficacy measures were Barthel index score and the modified Rankin scale (mRS) at day 90. Differences among the three groups on mRS were compared using χ(2) test of proportions (with two-sided α = 0.05) and Logistic regression analysis was conducted to take the baseline National Institutes of Health Stroke Scale (NIHSS) score into consideration. Results: Among the 535 subjects included in the efficacy analysis, 90-day treatment with NBP was associated with a significantly favorable outcome than 14-day treatment with ozagrel as measured by mRS (P < 0.001). No significant difference was found among the three groups on Barthel index at day 90. The rate of adverse events was similar among the three groups. Conclusions: The 90-day treatment with NBP could improve outcomes at the third month after stroke. The NBP treatment (both intravenous and oral) is safe (ChiCTR-TRC-09000483). [1]

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In the present study, a series of hydrogen sulfide (H2S) releasing derivatives (8a–g and 9a–f) of 3-n-butylphthalide (NBP) were designed, synthesized and biologically evaluated. The most promising compound 8e significantly inhibited the adenosine diphosphate (ADP) and arachidonic acid (AA)-induced platelet aggregation in vitro, superior to NBP, ticlopidine hydrochloride and aspirin. Furthermore, 8e could slowly produce moderate levels of H2S in vitro, which could be beneficial for improving cardiovascular and cerebral circulation. Most importantly, 8e protected against the collagen and adrenaline induced thrombosis in mice, and exhibited greater antithrombotic activity than NBP and aspirin in rats. Overall, 8e could warrant further investigation for the treatment of thrombosis-related ischemic stroke.[4]

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Objective: The 3-N-butylphthalide (NBP) comprises one of the chemical constituents of celery oil. It has a series of pharmacologic mechanisms including reconstructing microcirculation, protecting mitochondrial function, inhibiting oxidative stress, inhibiting neuronal apoptosis, etc. Based on the complex multi-targets of pharmacologic mechanisms of NBP, the clinical application of NBP is increasing and more clinical researches and animal experiments are also focused on NBP. The aim of this review was to comprehensively and systematically summarize the application of NBP on neurologic diseases and briefly summarize its application to non-neurologic diseases. Moreover, recent progress in experimental models of NBP on animals was summarized. Data sources: Literature was collected from PubMed and Wangfang database until November 2018, using the search terms including "3-N-butylphthalide," "microcirculation," "mitochondria," "ischemic stroke," "Alzheimer disease," "vascular dementia," "Parkinson disease," "brain edema," "CO poisoning," "traumatic central nervous system injury," "autoimmune disease," "amyotrophic lateral sclerosis," "seizures," "diabetes," "diabetic cataract," and "atherosclerosis." Study selection: Literature was mainly derived from English articles or articles that could be obtained with English abstracts and partly derived from Chinese articles. Article type was not limited. References were also identified from the bibliographies of identified articles and the authors' files. Results: NBP has become an important adjunct for ischemic stroke. In vascular dementia, the clinical application of NBP to treat severe cognitive dysfunction syndrome caused by the hypoperfusion of brain tissue during cerebrovascular disease is also increasing. Evidence also suggests that NBP has a therapeutic effect for neurodegenerative diseases. Many animal experiments have found that it can also improve symptoms in other neurologic diseases such as epilepsy, cerebral edema, and decreased cognitive function caused by severe acute carbon monoxide poisoning. Moreover, NBP has therapeutic effects for diabetes, diabetes-induced cataracts, and non-neurologic diseases such as atherosclerosis. Mechanistically, NBP mainly improves microcirculation and protects mitochondria. Its broad pharmacologic effects also include inhibiting oxidative stress, nerve cell apoptosis, inflammatory responses, and anti-platelet and anti-thrombotic effects. Conclusions: The varied pharmacologic mechanisms of NBP involve many complex molecular mechanisms; however, there many unknown pharmacologic effects await further study.[5]

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3-n-Butylphthalide (NBP) [(±)-3-butyl-1(3H)-isobenzofuranone] is a potent and widely used drug for the treatment of ischemic stroke in clinic. Racemic NBP was approved for marketing in 2004 by the State Food and Drug Administration (SFDA) of China in the form of soft capsule and infusion drip. The recommended dose of NBP is 200 mg, taken three times a day. Previous pharmacological studies have demonstrated that NBP exhibits neuroprotective effects by increasing the regional cerebral blood flow in the ischemic zone and inhibiting the release of glutamate and 5-hydroxytryptamine (Yan and Feng, 1998; Yan et al., 1998; Chong and Feng, 1999; Xu and Feng, 2001). Recent studies have revealed that NBP displays beneficial effects in attenuating amyloid-induced cell death in neuronal cultures, improving cognitive impairment in an animal model of Alzheimer’s disease and preventing neuronal cell death after focal cerebral ischemia in mice via the c-Jun N-terminal kinase pathway (Peng et al., 2008, 2010; Li et al., 2010). Although the pharmacological properties of NBP were intensively investigated, its absorption, distribution, metabolism, and excretion are not well understood; only a few studies have investigated its metabolism in rats (Peng and Zhou, 1996; Wang et al., 1997). On the basis of the fragmentation of tentative metabolites and their tetramethylsilane derivatives, four hydroxylated metabolites were observed in the urine after an oral administration in rat. In another in vivo study of radiolabeled 3H-NBP, NBP was absorbed rapidly and metabolized extensively. The metabolites were mainly excreted in urine. One of the urinary metabolites was confirmed as 10-hydroxy-NBP, whereas the other metabolite was proposed to be 3-hydroxy-NBP without robust structure elucidation. To date, few studies have investigated the biotransformation of NBP in humans. Thus, a clear understanding of the metabolism of NBP in humans and the identification of the enzymes involved in its biotransformation would provide solid evidence for the safety evaluation of NBP, avoidance of potential drug-drug interaction, and inspiration for further discovery of new anti-stroke drugs (Li et al., 2011). In light of these concerns, the present study aims to (1) investigate the metabolism of NBP in humans after an oral administration of 200 mg NBP soft capsules via ultraperformance liquid chromatography-UV/quadruple time-of-flight mass spectrometry (UPLC-UV/Q-TOF MS); (2) characterize the pharmacokinetic and elimination profiles of NBP in humans; and (3) evaluate the roles of cytochrome P450s (P450), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and β-oxidation in NBP biotransformation.[6]

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Certain main limitations can be taken into account in the present work. Firstly, the present regulation pathway needs to be further investigated in an animal model. Secondly, the protective effect of NBP on MIRI has not been reflected in clinical treatment, and further studies are required to explore the drug administration route and dosages. Thirdly, H9c2 cells are rat myoblasts and not cardiomyocytes; therefore, they present differences in their characteristics and protein expression compared with adult cardiomyocytes. Future studies will investigate the present hypotheses in primary cardiomyocytes. In summary, NBP upregulated HSP70 through the PI3K/AKT pathway and reduced the inflammatory response, oxidative stress and injury of H9c2 cells, thereby attenuating MIRI. These findings may provide a novel therapeutic target for the clinical treatment of MIRI.[7]

C12H14O2

190.242

190.099

C, 75.76; H, 7.42; O, 16.82

6066-49-5

Butylphthalide-d3

61361

Colorless to light yellow liquid

1.1±0.1 g/cm3

312.8±31.0 °C at 760 mmHg

128.3±22.2 °C

0.0±0.7 mmHg at 25°C

1.525

Celery species

3.08

26.3

0

2

3

14

212

0

O1C(C2=C([H])C([H])=C([H])C([H])=C2C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O

HJXMNVQARNZTEE-UHFFFAOYSA-N

InChI=1S/C12H14O2/c1-2-3-8-11-9-6-4-5-7-10(9)12(13)14-11/h4-7,11H,2-3,8H2,1H3

3-Butyl-2-benzofuran-1(3H)-one

N-butylphthalide; 3-n-Butylphthalide; 6066-49-5; Phthalide, 3-butyl-; 3-Butyl-1(3H)-isobenzofuranone; FEMA No. 3334; dl-3-n-butylphthalide; Butylphthalide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~250 mg/mL (~1314.13 mM)
H2O : ~100 mg/mL (~525.65 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (10.93 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (10.93 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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: ≥ 2.08 mg/mL (10.93 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 20.8 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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配方 4 中的溶解度: 13.33 mg/mL (70.07 mM) in 50% PEG300 50% 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。
*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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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。