Cerebral ischemia

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 [3].

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DL-3-n-butylphthalide (NBP) is commonly used to treat ischemic strokes due to its antioxidative and anti-inflammatory effects. The present study aimed to examine the protective effects of NBP on myocardial ischemia-reperfusion injury (MIRI) by establishing a MIRI model in H9c2 cells. Cell viability assay using Cell Counting Kit-8, lactate dehydrogenase (LDH) cytotoxicity and lipid peroxidation malondialdehyde (MDA) content were assessed to detect cell activity, degree of cell injury and oxidative stress reaction. Reverse transcription-quantitative PCR was used to quantify the expression of inflammatory factors in H9c2 cells. Western blotting and immunofluorescence staining were used to detect the protein expression of PI3K/AKT and heat shock protein 70 (HSP70). The present results indicated that NBP significantly increased cell viability during ischemia-reperfusion. Moreover, NBP inhibited the release of LDH and the production of MDA. NBP treatment also significantly decreased the expression of inflammatory factors at the mRNA level. Additionally, NBP activated the PI3K/AKT pathway and upregulated the expression of HSP70 compared with cells in the MIRI model. LY294002, a PI3K inhibitor, reversed the protective effects of NBP and suppressed the expression of HSP70. The present study demonstrated that NBP protected H9c2 cells from MIRI by regulating HSP70 expression via PI3K/AKT pathway activation.[7]

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Effect of different concentrations of 3-n-butylphthalide/NBP on the viability of H9c2 cells [7]
The viability of H9c2 cells treated with different NBP doses was examined via the CCK-8 colorimetric assay. Compared with the MIRI group, 100 µM NBP increased cell viability (P<0.05; Fig. 1A). However, NBP concentrations ≥200 µM slightly decreased cell viability (Fig. 1A). The present results indicated that 100 µM NBP was the optimal concentration for protecting cells from MIRI.

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NBP/3-n-butylphthalide inhibits the oxidative stress response during MIRI [7]
When ischemia-reperfusion injury occurs in cardiomyocytes, oxidative stress also plays an important role. An MDA kit was used to explore the effect of NBP on the oxidative stress index after MIRI and the role of PI3K in this process. The oxidation level in the MIRI group was significantly higher compared with the CON group (P<0.05). However, NBP pretreatment significantly decreased oxidative stress (P<0.05). In contrast, LY294002 eliminated the antioxidant effect of NBP (P<0.05; Fig. 1B). Namely, NBP significantly decreased the oxidative stress response of H9c2 cells during MIRI, but this antioxidant protective effect was eliminated by blocking PI3K.

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NBP/3-n-butylphthalide protects H9c2 cells from MIRI [7]
In vitro, the destruction of the cell membrane structure caused by apoptosis or necrosis leads to LDH release into the culture medium. Therefore, LDH activity can indirectly indicate the degree of cell damage. To explore the effect of NBP on MIRI and the involvement of PI3K in MIRI, H9c2 cell viability was determined via CCK-8 colorimetric assay, while an LDH kit was used to determine the LDH content in the culture medium. Compared with the CON group, the cell viability of the MIRI group was significantly lower (P<0.05). However, the cell viability of the MIRI + NBP group was higher than that of the MIRI group (P<0.05). By contrast, addition of the PI3K inhibitor LY294002 decreased cell viability (P<0.05; Fig. 1C). Furthermore, the LDH content of the MIRI group was significantly higher than that of the CON group (P<0.05). Pretreatment with NBP decreased the LDH release (P<0.05), but addition of LY294002 reversed this effect (P<0.05; Fig. 1D). Thus, NBP significantly increased the viability of H9c2 cells after ischemia-reperfusion injury and reduced H9c2 cell injury. However, the PI3K inhibitor LY294002 reversed the protective effect of NBP on H9c2 cells.

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NBP/3-n-butylphthalide inhibits inflammation by decreasing the TNF-α and IL-1β mRNA expression [7]
Activation of inflammatory cytokines during MIRI aggravates the injury of cardiomyocytes. Therefore, to determine the association between the protective effect of NBP during MIRI and inflammatory factors, the mRNA expression of TNF-α and IL-1β was examined in H9c2 cells via RT-qPCR. Compared with the CON group, the expression of TNF-α and IL-1β in the MIRI group significantly increased (P<0.05). However, the expression of these genes decreased in the MIRI + NBP compared with the MIRI group (P<0.05). By contrast, addition of LY294002 increased the expression of TNF-α and IL-1β compared with the MIRI + NBP group (P<0.05; Fig. 1E and F). Therefore, it was revealed that NBP effectively reduced the expression of inflammatory factors during MIRI, while the application of the PI3K inhibitor LY294002 reversed this anti-inflammatory effect.

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NBP/3-n-butylphthalide activates the HSP70 and PI3K/AKT signaling pathway [7]
During MIRI, the expression of the stress-protective proteins HSP70 and PI3K/AKT was upregulated. Western blotting and immunofluorescence staining were used to determine the protein expression of HSP70 and PI3K/AKT after NBP treatment. The expression levels of p-AKT and PI3K in the MIRI group were significantly increased compared with the CON group (P<0.05). In the MIRI + NBP group, the expression of p-AKT and PI3K was increased compared with the MIRI group (P<0.05). By contrast, in the MIRI + NBP + LY294002 group, the expression of p-AKT and PI3K decreased compared with the MIRI + NBP group (P<0.05; Fig. 2A-D). This tendency was also observed in the immunofluorescence assay (Fig. 2E and F). Additionally, the expression level of HSP70 in the MIRI group increased compared with the CON group (P<0.05). NBP pretreatment further increased the expression of HSP70 (P<0.05), whereas LY294002 decreased it compared with the MIRI + NBP group (P<0.05; Fig. 3A and B). This tendency was also observed in the immunofluorescence assay (Fig. 3C). The present results indicated that NBP pretreatment could increase the expression level of PI3K, p-AKT and HSP70 following MIRI, and that the inhibition of the PI3K pathway also altered HSP70 expression.

In SAMP8 mice, butylphthalide (160 mg/kg, ir) can alleviate memory and learning deficiencies [3].

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3-N-butylphthalide is an effective drug for acute ischemic stroke. However, its effects on chronic cerebral ischemia-induced neuronal injury remain poorly understood. Therefore, this study ligated bilateral carotid arteries in 15-month-old rats to simulate chronic cerebral ischemia in aged humans. Aged rats were then intragastrically administered 3-n-butylphthalide. 3-N-butylphthalide administration improved the neuronal morphology in the cerebral cortex and hippocampus of rats with chronic cerebral ischemia, increased choline acetyltransferase activity, and decreased malondialdehyde and amyloid beta levels, and greatly improved cognitive function. These findings suggest that 3-n-butylphthalide alleviates oxidative stress caused by chronic cerebral ischemia, improves cholinergic function, and inhibits amyloid beta accumulation, thereby improving cerebral neuronal injury and cognitive deficits. [2]

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The herbal extract 3-N-butylphthalide (NBP) is used in clinical practice for ischemic patients in China. It has been shown to have various neuroprotective effects both in vitro and in vivo. In the present study, the effects of NBP on learning and memory decline in the senescence-accelerated mouse prone-8 (SAMP8) animal model were investigated. Intragastric NBP administration to 4-month-old SAMP8 mice for 2 months significantly improved spatial learning and memory ability. Moreover, the loss of choline acetyltransferase (ChAT)-positive neurons in the medial septal nucleus and the vertical limb of the diagonal band in SAMP8 mice was slowed down, as was the decline in the protein and mRNA expression of ChAT in the hippocampus, cerebral cortex, and forebrain. These results demonstrated that NBP treatment starting at the age of 4 months protected from the learning/memory deficits with aging of SAMP8 mice, and that this effect might be mediated by preventing the decline of the central cholinergic system. [3]

Regarding the protection of mitochondrial function, early animal studies showed that dl-NBP improves the activities of Na/K-ATPase and Ca-ATPase in the mitochondrion. As is known, Na/K-ATPase and Ca-ATPase are important enzymes that maintain cell membrane potential and participate in material transportation to regulate cell volume. NBP can maintain mitochondrial membrane fluidity and the stability of the mitochondrial membrane, preventing mitochondrial swelling [5].

The main pharmacologic effects of NBP include reconstructing microcirculation, protecting mitochondrial function, inhibiting oxidative stress, inhibiting inflammatory responses, and inhibiting neuronal apoptosis. It has also been found to have effects including anti-platelet aggregation, anti-thrombosis, and anti-atherosclerosis [5].

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Cell culture [7]
H9c2 cells (1x106 cells/ml) were seeded in 25-cm2 cell-culture flasks containing high-glucose DMEM (10% FBS; 1% penicillin/streptomycin). The cells were cultured in a cell incubator with 95% air and 5% CO2 at 37˚C. After H9c2 cells were cultured for 24 h, they were randomly assigned to four groups: i) Control group (CON); ii) MIRI group; iii) 3-n-butylphthalide/NBP pretreatment group (MIRI + NBP); and iv) PI3K inhibitor group (MIRI + NBP + LY294002). CON cells were cultured at 37˚C with 95% air. MIRI cells were incubated at 37˚C with 5% CO2, 93% N2 and 2% O2 for 6 h, then reoxygenated for 4 h. MIRI + NBP cells were pretreated with 100 µM NBP for 2 h before being subjected to hypoxia for 6 h, followed by reoxygenation for 4 h. MIRI + NBP + LY294002 cells were pretreated with 10 µM LY294002(28) for 1 h before treatment with NBP for 2 h, followed by hypoxia for 6 h and reoxygenation for 4 h.

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Construction of the H9c2 MIRI model [7]
To simulate the in vivo model of ischemia-reperfusion injury, the cell culture medium was replaced with low-glucose DMEM without FBS, and the cells were incubated at 37˚C with 5% CO2, 93% N2 and 2% O2. A total of 10 µM of LY294002 was added 1 h before 3-n-butylphthalide/NBP treatment. NBP pretreatment lasted for 2 h before hypoxia. After MIRI, the medium was replaced with high-glucose DMEM (10% FBS; 1% penicillin/streptomycin) and the cells were cultured in an incubator (37˚C; 5% CO2; 95% air) for 4 h.

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Determination of optimal 3-n-butylphthalide/NBP concentration and cell viability [7]
H9c2 cells (1x104 cells/well) were seeded in 96-well plates for 24-48 h, and then pretreated with different doses of NBP (1, 50, 100, 200, 300 and 500 µM). After reperfusion, the original medium was discarded and 100 µl medium containing 10 mg/ml Cell Counting Kit-8 were added to each well. The absorbance at 450 nm was measured after culture for 1 h without light. The final cell viability of each group was calculated with the following formula: Cell viability (%)=(experimental group-blank)/(MIRI only group-blank) 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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (10.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (10.93 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (10.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 13.33 mg/mL (70.07 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)