ERK; Nrf2; Autophagy

In H9c2 cells, TBHQ (tert-butylhydroquinone; tBHQ; 0-100 μM; 48 hours) did not diminish cell viability on its own. H9c2 cells' vitality was increased by preincubating them with various tBHQ doses for 24 hours; in contrast, exposure to ethanol decreased viability in a dose-dependent way. H9c2 cardiomyocytes exposed to ethanol showed a considerable increase in viability upon receiving tBHQ therapy [3]. The number of apoptotic cells exposed to ethanol is dramatically reduced when H9c2 cells are treated with TBHQ (5 μM) for 15 minutes [3]. The pretreatment of H9c2 cells with TBHQ (5 μM) greatly reduced the increase in caspase-3 and Bax expression caused by ethanol and increased the expression of Bcl-2 [3].

Treatment with TBHQ (50 mg/kg; intraperitoneal injection; three injections every eight hours beginning one hour after ICH; CD-1 mice) increases Nrf2's DNA-binding activity and reduces immediate neurological impairments and oxidative brain damage following intracerebral hemorrhage. functional deficiency (ICH), which concurrently lowers the release of the pro-inflammatory cytokine interleukin-1β (IL-1β) and attenuates microglial activation. When administered after an accident, TBHQ is effective in reducing immediate neurological injury following ICH [4].

Determination of lipid peroxides (measured as MDA)[1]
These measurements were performed as previously described. The fresh heart tissue was rinsed and then homogenized in a buffer [10 mM Tris-HCl, 137 mM NaCl, 1 mM Na2EDTA, and 0.5 mM dithiotreitol (DTT)] with 250 mM sucrose at pH 7.4 using a homogenizer (T 18 basic Ultra-Turrax®). The homogenate was centrifuged at 1,000 × g for 15 min at 4°C. The supernatants were removed, and their total protein concentration was measured using a protein assay kit. The supernatants were used for the biochemical assay and western blot analysis. The malondialdehyde (MDA) content in the heart tissue was used as an index of the lipid superoxide level. The measurements were conducted using a spectrophotometer and a commercially available kit.

Cell Viability Assay[3]
Cell Types: H9c2 Cell
Tested Concentrations: 0 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, 50 µM and 100 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Enhanced Cell Viability H9c2 Cardiomyocytes exposed to ethanol.

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Apoptosis analysis[3]
Cell Types: H9c2 Cell
Tested Concentrations: 5 μM
Incubation Duration:
Experimental Results: The number of apoptotic cells diminished when exposed to ethanol.

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Western Blot Analysis [3]
Cell Types: H9c2 cells
Tested Concentrations: 5 μM
Incubation Duration:
Experimental Results: Inhibited ethanol-induced increase in caspase-3 and Bax expression and enhanced Bcl-2 expression.

Animal/Disease Models: Male CD-1 mice (8-10 weeks old) [4]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; 3 injections starting 1 hour after ICH, with an interval of 8 hrs (hrs (hours)).
Experimental Results: Treatment enhanced the DNA-binding activity of Nrf2, alleviated brain oxidative damage, and weakened microglial activation and IL-1β expression.

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Study Design:** Wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) male CD1/ICR mice were used. Mice were randomly divided into four groups: control, TBHQ alone, DOX alone, and TBHQ + DOX. TBHQ (25 mg/kg) was dissolved in 3% ethanol/isotonic saline and administered by intraperitoneal injection for 3 consecutive days. DOX (20 mg/kg) was dissolved in saline and administered as a single intraperitoneal injection on day 2. Control mice received the same volume of vehicle. Forty-eight hours after the DOX injection, mice were anesthetized and sacrificed. Blood was collected for serum biochemical analysis (CK, CK-MB). Hearts were harvested and processed for histopathological examination (H&E staining), immunohistochemistry (for 4-HNE and 3-NT), TUNEL assay (for apoptosis), real-time PCR (for Nrf2, HO-1, NQO-1 mRNA), and Western blotting (for Nrf2 protein in nuclear and cytoplasmic extracts). [1]

Metabolism / Metabolites
TBHQ is readily metabolized. In mouse studies, its metabolism primarily involves the oxidation of the tert-butyl group, followed by the formation of glucuronide conjugates, which are excreted in the urine or feces as free acid. In rats, 80–90% of the (14)C radiolabeled material is excreted in the urine or feces within 96 hours, primarily as free acid in the feces, with a small amount excreted in the urine and less than 0.3% in exhaled breath. More than 43 metabolites were detected in the urine and feces of mice and rats. In multiple rat and dog studies, oral administration of TBHQ has been shown to be well absorbed and rapidly excreted, primarily in the urine. The major urinary metabolites in both animals were 4-O-sulfate conjugates and 4-O-glucuronide. Excretion appears to be largely completed after 4 days.
The main metabolic pathway of the food antioxidant 2(3)-tert-butyl-4-hydroxyanisole, which is suspected to be carcinogenic, involves O-demethylation to generate 2-tert-butyl(1,4)hydroquinone, followed by peroxidation to generate 2-tert-butyl(1,4)p-quinone. ……
The tert-butyl semiquinone anionic radical is formed by tert-butylhydroquinone and tert-butylquinone in rat liver microsomes.
After intraperitoneal injection of 3-tert-butyl-4-hydroxyanisole, two previously unreported metabolites, 2-tert-butyl-5-methylthiohydroquinone and 2-tert-butyl-6-methylthiohydroquinone, were detected in rat urine. In addition to the above metabolites, 3-tert-butyl-4,5-dihydroxyanisole, hydrolyzed by β-glucuronidase/sulfatase, was also detected in urine. Administration of the O-demethyl metabolite tert-butylhydroquinone to 3-tert-butyl-4-hydroxyanisole also yielded sulfur-containing metabolites 2-tert-butyl-5-methylthiohydroquinone and 2-tert-butyl-6-methylthiohydroquinone. The two metabolites were isolated and purified by high-performance liquid chromatography (HPLC) after incubation of tert-butylhydroquinone with rat liver microsomes in the presence of glutathione. The metabolites were identified as 2-tert-butyl-5-(glutathione-S-yl)hydroquinone and 2-tert-butyl-6-(glutathione-S-yl)hydroquinone. The formation of the tert-butylhydroquinone-glutathione conjugate requires NADPH, molecular oxygen, and glutathione. Cytochrome P450 inhibitors, such as SKF 525-A and metheprone, significantly inhibited the formation of the tert-butylhydroquinone-glutathione conjugate in vitro. tert-butylhydroquinone is converted to the active metabolite 2-tert-butyl-p-benzoquinone by cytochrome P450-mediated monooxygenase, which then binds to glutathione to form the tert-butylhydroquinone-glutathione conjugate. Glutathione S-transferase activity appears to play a no role in the glutathione conjugation of 2-tert-butyl-p-benzoquinone, as the cytosol component of rat liver homogenate does not enhance the microsomal-mediated formation of the tert-butylhydroquinone-glutathione conjugate.

Toxicity Summary
Identification and Uses: Tert-butylhydroquinone (TBHQ) is a white to light brown crystalline substance with a slight odor. It is used as an antioxidant in oils and fats. Human Exposure and Toxicity: Visual impairment has been reported in individuals exposed to this chemical. TBHQ can cause single-strand DNA breaks in human cells. Animal Studies: No teratogenic effects were observed in pregnant rats fed TBHQ. Hepatomegaly was observed in rats fed TBHQ. Acute neurotoxicity in animals exposed to TBHQ included seizures and bulbar paralysis. In a two-year study, mice were exposed to tert-butylhydroquinone via diet. There is no evidence that tert-butylhydroquinone has carcinogenic activity in either male or female animals. TBHQ was negative in Ames tests against two metabolic activation treatments of Salmonella Typhimurium strains TA97, TA98, TA100, and TA102. In the mouse lymphoma L5178Y forward mutation assay, TBHQ was positive under metabolic activation conditions. In the CHO/HGPRT assay, TBHQ was negative at concentrations up to 6 μg/mL without metabolic activation and at concentrations up to 250 μg/mL with metabolic activation. Ecotoxicity studies: In Nile tilapia, tert-butylhydroquinone (tBHQ) significantly induced the transcription of multiple glutathione S-transferases (GSTs), including GSTA, GSTR2, and GSTT.

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Toxicity Data
LCLo (rat) = 2,900 mg/m3/4h

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Interactions
…This study aimed to examine whether tert-butylhydroquinone (tBHQ, a known synthetic Nrf2 inducer) could protect human hepatocytes from arsenic-induced cytotoxicity and oxidative damage. The results showed that pretreatment with 5 μmol/L and 25 μmol/L tBHQ inhibited arsenic-induced hepatotoxicity, reactive oxygen species generation, and hepatic lipid peroxidation, while alleviating arsenic-induced intracellular glutathione homeostasis. Furthermore, we observed that tBHQ treatment promoted the biomethylation of arsenic and upregulated the mRNA expression of Nrf2-regulated downstream heme oxygenase-1 and NADPH:quinone oxidoreductase 1. In summary, we hypothesize that the Nrf2 signaling pathway may be involved in the protective effect of tBHQ against arsenic invasion of hepatocytes. These data suggest that phenolic Nrf2 inducers (such as tBHQ) could serve as novel therapeutic or dietary candidates for high-risk groups of arsenic poisoning. Pretreatment of MIN6 β cells with NRF2 activators (including CDDO-Im, dimethyl fumarate (DMF), and tert-butylhydroquinone (tBHQ)) protects cells from H2O2-induced cell damage. Copper has been shown to mediate the activation of various exogenous substances, leading to the generation of reactive oxygen species and other free radicals. Since copper is present in the cell nucleus and closely associated with chromosomes and DNA bases, this study investigated whether copper activation of 1,4-hydroquinone (1,4-HQ) and other phenolic compounds can induce strand breaks in double-stranded φX-174 RF I DNA (φX-174 relaxed I DNA). In the presence of micromolar concentrations of Cu(II), 1,4-HQ and other phenolic compounds, including 4,4'-biphenol, catechol, 1,2,4-pyrogallol, 2-methoxyestradiol, 2-hydroxyestradiol, diethylstilbestrol, butylated hydroxytoluene, butylated hydroxyanisole, tert-butylhydroquinone, ferulic acid, caffeic acid, chlorogenic acid, eugenol, 2-acetaminophen, and para-acetaminophen, can all induce DNA strand breaks. Structure-activity analysis showed that, in the presence of Cu(II), phenolic compounds with a 1,4-hydroquinone structure (such as 1,2,4-pyrogallol and tert-butylhydroquinone) exhibited higher DNA cleavage activity than compounds with catechol groups (such as catechol, 2-hydroxyestradiol, and caffeic acid). Compounds containing only one phenolic group (such as eugenol, 2-acetaminophen, and para-acetaminophen) showed the lowest reactivity. Furthermore, both the Cu(I)-specific chelating agent bartophenoneline disulfonic acid and catalase inhibited induced DNA strand breaks, indicating that the Cu(II)/Cu(I) redox cycle and H₂O₂ generation are the two main determinants of the observed DNA damage. After using reactive oxygen species (ROS) scavengers, it was found that DNA strand breaks induced by the 1,4-HQ/Cu(II) system could not be effectively inhibited by hydroxyl radical scavengers, but could be protected by singlet oxygen scavengers. This suggests that singlet oxygen or singlet oxygen-like substances (possibly copper-peroxide complexes), rather than free hydroxyl radicals, may play a role in DNA damage. These results suggest that macromolecularly bound copper and the generation of ROS may be important factors in the mechanism by which 1,4-HQ and other phenolic compounds induce DNA damage in target cells. The effects of diethyl maleate on the cytotoxicity of phenylhydroquinone and other hydroquinone compounds were investigated in freshly isolated rat hepatocytes. ...Among other hydroquinone compounds (0.5 mM), the cytotoxicity induced by tert-butylhydroquinone can be reduced by diethyl maleate (1.25 mM)... For more complete data on interactions of tert-butylhydroquinones (13 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats (male): 951 mg/kg body weight
Oral LD50 in rats (female): 1131 mg/kg body weight
Oral LD50 in rats: 615 mg/kg body weight
Oral LD50 in rats: 750-950 mg/kg, depending on the presence of food in the intestines
For more complete data on non-human toxicity values of tert-butylhydroquinones (14 in total), please visit the HSDB record page.

[1]. Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes. Am J Transl Res. 2015; 7(10): 1724–1735.

[2]. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes.Int J Mol Med. 2016 Jul; 38(1): 123–130.

[3]. Dehydrocorydaline inhibits cell proliferation, migration and invasion via suppressing MEK1/2-ERK1/2 cascade in melanoma.Onco Targets Ther. 2019 Jul 2;12:5163-5175.

[4]. Post-Injury Administration of Tert-butylhydroquinone Attenuates Acute Neurological Injury AfterIntracerebral Hemorrhage in Mice.J Mol Neurosci. 2016 Apr;58(4):525-31.

2-Tert-butylhydroquinone is a white to light brown crystalline powder or a fine beige powder with a slightly aromatic odor. (NTP, 1992)
2-Tert-butylhydroquinone belongs to the hydroquinone class of compounds, in which one hydrogen atom on the hydroquinone ring is replaced by a tert-butyl group. It is a food antioxidant.
Tert-butylhydroquinone has been reported in both peony (Paeonia suffruticosa) and licorice (Glycyrrhiza glabra), and relevant data are available.
2-Tert-butyl-1,4-benzenediol is found in oils and fats. 2-Tert-butyl-1,4-benzenediol is an antioxidant used in foods (e.g., oils and fats) and is also a polymer inhibitor.
2-Tert-butyl-1,4-benzenediol belongs to the matrine class of compounds. These are aromatic compounds containing a prop-2-ylphenyl moiety.
See also: Isomers (monomers).

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Mechanism of Action
…This study first used the widely used Nrf2 activator tert-butylhydroquinone (tBHQ) to investigate the potential protective role of Nrf2 activation against saturated fatty acid (SFA)-induced hepatotoxicity. As expected, tBHQ inhibited SFA-induced hepatocyte death in both AML-12 mouse hepatocytes and HepG2 human hepatocellular carcinoma cells. However, the protective effect of tBHQ was independent of Nrf2, as siRNA-mediated Nrf2 silencing did not eliminate the protective effect of tBHQ. Furthermore, our results indicate that autophagy activation plays a crucial role in the protective effect of tBHQ against lipotoxicity. tBHQ induces autophagy activation, while autophagy inhibitors eliminate the protective effect of tBHQ. Evidence for tBHQ-induced autophagy includes increased LC3 spot accumulation, LC3-II conversion, and increased autophagy flux (LC3-II conversion in the presence of proteolytic inhibitors). Subsequent mechanistic studies revealed that tBHQ activates AMP-activated protein kinase (AMPK), while siRNA-mediated AMPK gene silencing eliminated tBHQ-induced autophagy activation, indicating that AMPK plays a crucial role in tBHQ-triggered autophagy induction. Furthermore, our study confirmed that tBHQ-induced autophagy activation is essential for its activation of Nrf2. In summary, our data reveal a novel mechanism by which tBHQ protects hepatocytes from saturated fatty acid (SFA)-induced lipotoxic damage. tBHQ-induced autophagy not only contributes to its hepatoprotective effect but also facilitates Nrf2 activation. The H7N9 influenza outbreak in China has attracted significant public attention. H7 hemagglutinin (HA) plays a crucial role in influenza virus invasion, making it an ideal target for antiviral drugs. Previous studies have shown that the small molecule tert-butylhydroquinone (TBHQ) inhibits influenza H3 HA invasion by binding to the stem-ring structure of HA and stabilizing its neutral pH conformation, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure, and immunogenicity, H7 belongs to the relevant group II HA. In this study, we used a pseudovirus invasion assay to demonstrate that TBHQ can inhibit both H7 HA-mediated and H3 HA-mediated viral invasion, with an IC50 value of approximately 6 μM. Using nuclear magnetic resonance (NMR), we found that tert-butylhydroquinone (TBHQ) binds to the stem-ring region of H7. STD NMR experiments showed that the aromatic ring of TBHQ has extensive contact with the surface of H7 HA. Limited proteolysis experiments showed that TBHQ inhibits influenza virus invasion by stabilizing the neutral pH conformation of H7 HA. In summary, this work demonstrates that the stem-ring region of H7 HA is an attractive therapeutic target, and the widely used food preservative TBHQ is a promising lead compound.

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Therapeutic Use
/EXPL THER/ /This study aims to/investigate the protective effect of tert-butylhydroquinone against in vitro induced cytotoxicity of rat bone marrow cells. Rat bone marrow cells were randomly divided into two groups. Control group cells were stimulated with 0, 5, 10, 15, and 20 mmol/L benzene for 2, 4, and 6 hours, respectively. Cells in the tBHQ pretreatment group were treated with 100 μmol/L tBHQ for 12 hours, followed by the same treatment as the control group. DNA damage was detected by single-cell gel electrophoresis (SCGE), and apoptosis was detected by flow cytometry. Before benzene treatment, the activity of NAD(P)H:quinone oxidoreductase (NQO1) in rat bone marrow cells was measured in both groups. In the control group, DNA damage and apoptosis in bone marrow cells increased with increasing benzene concentration and treatment time. In rats pretreated with tBHQ, the DNA migration rate and DNA migration length of bone marrow cells were significantly lower than those in the control group after treatment with 5, 10, 15, and 20 mmol/L benzene (p<0.05). Furthermore, the apoptosis rate of rat bone marrow cells was significantly lower than that of the control group after treatment with 15 and 20 mmol/L benzene for 2 hours, 10, 15, and 20 mmol/L benzene for 4 hours, and 5, 10, 15, and 20 mmol/L benzene for 6 hours (p<0.05). After tBHQ treatment, the activity of NQO1 in rat bone marrow cells was significantly increased (p<0.01) (1.62±0.16 min⁻¹·mg⁻¹ vs. control group: 0.95±0.08 min⁻¹·mg⁻¹). Benzene can induce DNA damage and apoptosis in rat bone marrow cells to some extent in a time- and dose-dependent manner. tBHQ can protect rat bone marrow cells from benzene-induced cytotoxicity, which can be partly attributed to the tBHQ-induced increase in NQO1 activity. This study evaluated whether tBHQ pretreatment could prevent renal injury caused by ischemia-reperfusion (I/R). This study divided rats into four groups: (a) control group (CT), (b) tBHQ sham-operated group (tBHQ), (c) ischemia/reperfusion group (I/R), and (d) tBHQ+I/R group. The tBHQ and tBHQ+I/R groups received intraperitoneal injections of tBHQ (50 mg/kg), while the CT and I/R groups received intraperitoneal injections of 3% ethanol/isotonic saline solution. Animals were sacrificed 24 hours after I/R. tBHQ alleviated I/R-induced renal dysfunction, structural damage, oxidative/nitrosogenic stress, glutathione depletion, and decreased activity of various antioxidant enzymes. The renal protective effect of tBHQ against ischemia/reperfusion injury was associated with the reduction of oxidative/nitrosogenic stress and the protection of antioxidant enzymes. Cisplatin (CDDP)-induced nephrotoxicity was associated with excessive production of reactive oxygen species. This study aimed to investigate the ability and mechanism of tBHQ in preventing the nephrotoxic effects of CDDP in rats. Thirty-six Wistar rats were used in this study and divided into the following groups: control group, tBHQ group (12.5 mg/kg), CDDP group (7.5 mg/kg), and tBHQ+CDDP group. Urine samples were collected over 24 hours at the beginning and end of the experiment, and rats were sacrificed 72 hours after CDDP administration. Histological studies were performed, and biomarkers of renal function and oxidative/nitrosogenic stress were detected. In addition, the activities of the following antioxidant enzymes were measured: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione S-transferase (GST). tBHQ prevented cisplatin (CDDP)-induced renal dysfunction, structural damage, and oxidative/nitrosogenic damage. Furthermore, tBHQ completely prevented the CDDP-induced decrease in GPx and GST activities. In summary, this study demonstrates that the antioxidant activity of tert-butylhydroquinone (tBHQ) is associated with its renal protective effect against cisplatin (CDDP)-induced acute kidney injury in rats. This study aimed to determine the role of paraquat (PQ) in activating the NF-E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway, and the potential neuroprotective effect of tert-butylhydroquinone (tBHQ) pretreatment on PQ-induced neurodegeneration in vivo and in vitro. In male C57BL/6 mice, treatment with 7 mg/kg PQ resulted in decreased spontaneous motor activity, a reduction in tyrosine hydroxylase (TH)-positive neurons, an increase in dUTP biotin nick end marker (TUNEL) positive cells mediated by terminal deoxynucleotidyl transferase in the substantia nigra, and elevated levels of Nrf2 and HO-1 proteins in the nucleus. In PQ-treated mice, pretreatment with 1% (w/w) tert-butylhydroquinone (tBHQ) significantly reduced behavioral impairment, decreased the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra, increased the number of TUNEL-positive cells, and enhanced nuclear Nrf2 and HO-1 protein expression. Pretreatment with 40 μM tBHQ protected PC12 cells from cytotoxicity mediated by 100 μM and 300 μM PQ. Dual-luciferase reporter gene assays also showed that exposure to 100 μM and 300 μM PQ activated the transcriptional expression of the HO-1 gene on the antioxidant response element (ARE). In summary, these results clearly demonstrate for the first time that PQ activates the Nrf2/HO-1 pathway in the substantia nigra, and that tBHQ pretreatment may have a neuroprotective effect against PQ-induced Parkinson's syndrome by increasing Nrf2 and HO-1 expression. For more complete data on the therapeutic uses of T-butylhydroquinone (7 types in total), please visit the HSDB record page.

166.099

C, 72.26; H, 8.49; O, 19.25

1948-33-0

16043

White to off-white solid powder

1.1±0.1 g/cm3

291.4±20.0 °C at 760 mmHg

127-129 °C(lit.)

138.7±16.4 °C

0.0±0.6 mmHg at 25°C

1.545

2.33

2

2

1

12

148

0

BGNXCDMCOKJUMV-UHFFFAOYSA-N

InChI=1S/C10H14O2/c1-10(2,3)8-6-7(11)4-5-9(8)12/h4-6,11-12H,1-3H3

2-(tert-butyl)benzene-1,4-diol

tert-Butylhydroquinone TBHQ

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 : ≥ 56.66 mg/mL (~340.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.04 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 25.0 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: ≥ 2.5 mg/mL (15.04 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 25.0 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: ≥ 2.5 mg/mL (15.04 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 20 mg/mL (120.32 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear 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.)