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 to treat cerebral ischemia. This study aimed to investigate the metabolism, pharmacokinetics, and excretion of NBP in humans, and to identify the enzymes that generate the major metabolites. Following oral administration of 200 mg NBP, extensive metabolism occurred, with 23 metabolites identified in human plasma and urine. The main metabolic pathways included hydroxylation of the alkyl side chain, particularly at the 3-, ω-1-, and ω-carbons, as well as further oxidation and conjugation reactions. Approximately 81.6% of the administered dose was recovered in urine, primarily as NBP-11-acid (M5-2) and its glucuronide conjugates and monohydroxylated products. 10-Keto-NBP (M2), 3-Hydroxy-NBP (M3-1), 10-Hydroxy-NBP (M3-2), and M5-2 are the major circulating metabolites, with areas under the curve (AUCs) 1.6, 2.9, 10.3, and 4.1 times that of NBP, respectively. Reference standards for these four metabolites were obtained through microbial biotransformation using Cunninghamella blakesleana. In vivo morphological analysis revealed that multiple cytochrome P450 (P450) isoenzymes, particularly CYP3A4, 2E1, and 1A2, are involved in the formation of M3-1, M3-2, and 11-hydroxy-NBP. Using M3-2 and 11-hydroxy-NBP as substrates, subcellular fractionation assays showed that P450, alcohol dehydrogenases, and aldehyde dehydrogenases catalyzed the formation of M2 and M5-2. M5-2 is generated much faster than M2, and M5-2 can be β-oxidized in rat liver homogenate to phthaloacetic acid. In summary, our study shows that NBP can be fully absorbed and extensively metabolized into a variety of metabolites by various enzymes before being excreted in urine. [1]

The oral LD50 in rats was 2450 mg/kg. (Food and Cosmetic 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 belongs to the benzofuran class of compounds. Butylphthalide has been used in research trials for the prevention of restenosis. Butylphthalide has been reported to be found in Angelica sinensis, Ligusticum striatum, and other organisms with relevant data. See also: Celery seed (partial); Angelica sinensis root oil (partial). Background: Dl-3-n-butylphthalide (NBP) was initially isolated from celery seed and showed efficacy in animal stroke models. This study was a clinical trial designed to evaluate the efficacy and safety of continuous NBP administration in patients with acute ischemic stroke. Methods: A randomized, double-blind, double-dummy trial in China enrolled 573 patients with ischemic stroke within 48 hours of onset. Patients were randomly assigned to receive one of three treatment regimens: 14 days of NBP intravenous infusion followed by NBP capsules; 14 days of NBP intravenous infusion followed by aspirin; or 14 days of ozagrel intravenous infusion followed by aspirin. The efficacy endpoints were Barthel Index score and modified Rankin Scale (mRS) score on day 90. The differences in mRS scores among the three groups were compared using the χ² test (two-sided α = 0.05), and logistic regression analysis was performed to include baseline National Institutes of Health Stroke Scale (NIHSS) scores. Results: Among the 535 subjects included in the efficacy analysis, the mRS score in the 90-day NBP treatment group was significantly better than that in the 14-day ozagrel treatment group (P < 0.001). There was no significant difference in Barthel Index among the three groups on day 90. The incidence of adverse events was also similar among the three groups. Conclusion: 90 days of NBP treatment improves the prognosis at the third month post-stroke. NBP treatment (intravenous and oral) is safe (ChiCTR-TRC-09000483). [1]

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This study designed, synthesized, and bioactively evaluated a series of hydrogen sulfide (H2S)-releasing derivatives of 3-n-butylphthalide (NBP) (8a–g and 9a–f). Among them, compound 8e significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (AA) in vitro, with better effects than NBP, ticlopidine hydrochloride, and aspirin. In addition, 8e can slowly generate moderate concentrations of H2S in vitro, which may be beneficial for improving cardiovascular and cerebrovascular circulation. Most importantly, compound 8e can protect mice from collagen and adrenaline-induced thrombosis and showed stronger antithrombotic activity than NBP and aspirin in rats. In summary, compound 8e may be worthy of further investigation for the treatment of thrombosis-related ischemic stroke. [4]

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Objective: 3-n-butylphthalide (NBP) is one of the chemical components of celery oil. It has a series of pharmacological mechanisms, including rebuilding microcirculation, protecting mitochondrial function, inhibiting oxidative stress, and inhibiting neuronal apoptosis. Due to the complex multi-target pharmacological mechanism of NBP, its clinical applications are increasing, and more and more clinical studies and animal experiments are focusing on NBP. This review aims to comprehensively and systematically summarize the application of NBP in neurological diseases and briefly outline its application in non-neurological diseases. In addition, this article summarizes the research progress of NBP in animal experimental models in recent years. Data Sources: PubMed and Wanfang databases were searched up to November 2018. Search terms included “3-n-butylphthalide,” “microcirculation,” “mitochondria,” “ischemic stroke,” “Alzheimer’s disease,” “vascular dementia,” “Parkinson’s disease,” “cerebral edema,” “carbon monoxide poisoning,” “traumatic central nervous system injury,” “autoimmune diseases,” “amyotrophic lateral sclerosis,” “epilepsy,” “diabetes,” “diabetic cataract,” and “atherosclerosis.” Literature Selection: The main literature sources were English articles or articles with available English abstracts, with some sources from Chinese articles. There were no restrictions on article type. References were also drawn from the reference lists of the searched articles and the authors' bibliographic databases. Results: NBP has become an important adjunctive treatment for ischemic stroke. In vascular dementia, the clinical application of NBP in treating severe cognitive impairment syndromes caused by insufficient cerebral perfusion during cerebrovascular disease is also increasing. There is evidence that NBP also has therapeutic effects on neurodegenerative diseases. Many animal studies have found that it can also improve symptoms of other neurological diseases, such as epilepsy, cerebral edema, and cognitive decline caused by severe acute carbon monoxide poisoning. Furthermore, NBP also has therapeutic effects on non-neurological diseases such as diabetes, diabetic cataracts, and atherosclerosis. Mechanistically, NBP primarily improves microcirculation and protects mitochondria. Its broad pharmacological effects also include inhibition of oxidative stress, neuronal apoptosis, inflammatory responses, and antiplatelet and antithrombotic effects. Conclusion: The multiple pharmacological mechanisms of NBP involve many complex molecular mechanisms; however, many of its unknown pharmacological effects require further investigation. [5] 3-n-Butylphthalide (NBP) [(±)-3-Butyl-1(3H)-isobenzofuranone] is a potent and widely used drug for the clinical treatment of ischemic stroke. Racemic NBP was approved for marketing by the State Food and Drug Administration (SFDA) of China in 2004, in the form of soft capsules and infusion. The recommended dose of NBP is 200 mg three times a day. Previous pharmacological studies have shown that NBP can exert neuroprotective effects by increasing local cerebral blood flow in ischemic areas and inhibiting the release of glutamate and serotonin (Yan and Feng, 1998; Yan et al., 1998; Chong and Feng, 1999; Xu and Feng, 2001). Recent studies have shown that NBP can alleviate amyloid-induced cell death in neuronal cultures, improve cognitive impairment in animal models of Alzheimer's disease, and prevent neuronal cell death following 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 have been extensively studied, its absorption, distribution, metabolism, and excretion mechanisms remain unclear; only a few studies have explored its metabolism in rats (Peng and Zhou, 1996; Wang et al., 1997). Based on fragmentation analysis of speculative metabolites and their tetramethylsilane derivatives, four hydroxylated metabolites were detected in the urine of rats after oral administration. Another in vivo study of radiolabeled 3H-NBP showed that NBP is rapidly absorbed and extensively metabolized. The metabolites are mainly excreted in the urine. One of the urinary metabolites was identified as 10-hydroxy-NBP, while another metabolite was presumed to be 3-hydroxy-NBP, but its structure has not yet been reliably identified. To date, there is little research on the biotransformation of NBP in the human body. Therefore, a deeper understanding of the metabolic process of NBP in the human body and the identification of the enzymes involved in its biotransformation will provide a reliable basis for the safety evaluation of NBP, the avoidance of potential drug interactions, and the further discovery of new anti-stroke drugs (Li et al., 2011). In view of this, this study aims to: (1) study the metabolism of NBP in the human body after oral administration of 200 mg NBP soft capsules using ultra-high performance liquid chromatography-ultraviolet/quadrupole time-of-flight mass spectrometry (UPLC-UV/Q-TOF MS); (2) characterize the pharmacokinetics and elimination characteristics of NBP in the human body; (3) evaluate the role of cytochrome P450 (P450), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and β-oxidation in the biotransformation of NBP. [6]

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This study has some major limitations. First, animal models are needed to further study the current regulatory pathways. Second, the protective effect of NBP against myocardial ischemia-reperfusion injury (MIRI) has not yet been demonstrated in clinical treatment, and further research is needed on the route of administration and dosage. Third, H9c2 cells are rat myoblasts rather than cardiomyocytes; therefore, they differ from adult cardiomyocytes in terms of characteristics and protein expression. Future research will verify the current hypothesis in primary cardiomyocytes. In summary, NBP upregulates HSP70 through the PI3K/AKT pathway, reducing the inflammatory response, oxidative stress and damage of H9c2 cells, thereby alleviating MIRI. These findings may provide new therapeutic targets 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.)