Natural occurring flavonoid; antioxidant

Isoquercetin is widely distributed throughout plants. At 10 μM, seven isoquercitrin compounds considerably reduced the negative effects of H2O2 on RGC-5 cells. Cell viability increased from 63% to 83% at 10 and 50 μM, respectively, in the presence of H2O2. At the same concentration, isoquercetin (50 μM) outperforms EGCG as a neuroprotective drug [1].

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The shrub Thuja orientalis is extensively used as a herbal medicine in Korea and China. In the present study extracts of the plant were subjected to fractionation and purification, with seven compounds (myricitrin, isoquercitrin, hypoletin-7-O-β-d-xylopyranoside, quercitrin, kaempferin, kaempferol, and amentoflavone) being isolated. Of these seven compounds, isoquercitrin was found to be the most effective at attenuating the death of RGC-5 cells in culture caused by exposure to hydrogen peroxide (H2O2). It was found that an insult of H2O2 to RGC-5 cells caused them to die by apoptosis, demonstrated not only by staining dead cells for phosphatidylserine but also by the up-regulation (cleaved PARP, AIF, p53) and down-regulation (Bcl-2) of proteins associated with apoptosis and survival. Subsequent studies showed that isoquercitrin acts as a powerful antioxidant. It scavenges ROS generally as demonstrated by staining of cultures as well as the generation of individual radical species (H2O2, OHradical dot and O2radical dot−). Moreover, isoquercitrin reduced the depletion of glutathione (GSH) caused by elevation of specific radical species (H2O2, OHradical dot and O2radical dot−) in RGC-5 cells in culture and blunted the decrease in catalase and glutathione peroxidase 1 (Gpx-1) caused by exposure of RGC-5 cells to H2O2. Furthermore, isoquercitrin potently attenuated the lipid peroxidation of rat brain homogenates initiated by nitric oxide, with an IC50 value of 1.04 μM. Since isoquercitrin can be tolerated when taken orally it is suggested that this substance might reach the retina and therefore be potentially useful for treating glaucoma, in which oxidative stress is thought to play a major role in the demise of retinal ganglion cells.[1]

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Hepatocytes are shielded from ethanol-induced liver damage by isoquercetin (Quercetin 3-glucoside; 5–20 μM; 24 h), which dramatically lowers ethanol-induced cytotoxicity [3]. The ethanol HepG2 is considerably reduced by isoquercetin (10 μM; loss 1 h).

When animals received isoquercetin, the number of eosinophils in their blood, lung parenchyma, and BALF decreased. Only the animals treated with isoquercetin showed decreased blood neutrophil counts and lung homogenates' levels of IL-5. The quantity of monocytes did not alter [2].

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Objective: Eosinophils and cytokines are implicated in the pathogenesis of allergic diseases. In the present study, we investigate the anti-inflammatory effect of quercetin and isoquercitrin in a murine model of asthma.
Methods: BALB/c mice were immunized (ovalbumin/aluminum hydroxide, s. c.), followed by two intranasal ovalbumin challenges. From day 18 to day 22 after the first immunization, the mice received daily gavages of isoquercitrin (15 mg/kg) or quercetin (10 mg/kg). Dexamethasone (1 mg/kg, s. c.) was administered as a positive control. Leucocytes were analyzed in bronchoalveolar lavage fluid (BALF), blood and pulmonary parenchyma at 24 h after the last ovalbumin challenge. Interleukin-5 (IL-5) was analyzed in BALF and lung homogenates.
Results: In animals receiving isoquercitrin or quercetin, eosinophil counts were lower in the BALF, blood and lung parenchyma. Neutrophil counts in blood and IL-5 levels in lung homogenate were lower only in isoquercitrin-treated mice. No alterations in mononuclear cell numbers were observed.
Conclusion: Quercetin and isoquercitrin are effective eosinophilic inflammation suppressors, suggesting a potential for treating allergies [2].

cell viability assay [3]
Cell Types: HepG2 cells
Tested Concentrations: 5 μM, 10 μM, 20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Used as a positive control to cause cells ethanol-induced iNOS protein expression levels [3]. Cell viability [2]
Cell viability was assessed using the MTT reduction assay modified from that of Mosmann (1983). Briefly, medium was removed from cells (in 96-well plates) and 100 μl fresh culture medium containing MTT were added at a final concentration of 0.5 mg/ml of MTT and left to incubate for 1 h at 37 °C. The medium was then removed and reduced MTT (blue formazan product) was solubilized by adding 100 μl of DMSO to each well. After agitation of the plates for 15 min, the optical density of the solubilized formazan product in each well was measured using a spectrophotometer with a 570 nm test wavelength and a 690 nm reference wavelength.

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Analysis for cell viability by propidium iodide (PI) and Hoechst 33342 [2]
Cell death was characterized by incubating cells with 8 μM Hoechst 33342 and 1.5 μM PI for 30 min at 37 °C (Hong et al., 2009). Cells were then washed twice with serum-free media and examined under fluorescence microscopy.

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Assessment of reactive oxygen species (ROS) [2]
Cells were assessed for the production of ROS using the dye DHE (Carter et al., 1994). After different treatments, cells on coverslips were stained with DHE (10 μg/ml) by incubating for 30 min at 37 °C in culture medium in a humidified chamber, and then fixed with 4% paraformaldehyde for 20 min. After washing in Tris-phosphate buffer (PBS), coverslips were mounted in PBS containing 1% glycerol and ROS were detected by their red fluorescence using a Zeiss epifluorescence microscope.
To quantify intracellular ROS, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as a radical probe (Shimazawa et al., 2009). In this procedure radical species (H2O2, OHradical dot and O2radical dot−) oxidize nonfluorescent dichlorofluorescein (DCFH) to fluorescent dichlorofluorescein (DCF). Cells were pretreated with isoquercitrin or epigallocatechin 3-gallate (EGCG) for 1 h, and then DCFH-DA (10 μM) was added and the incubation continued for 20 min at 37 °C. Thereafter, the cell culture medium was replaced with fresh medium to which was added 1 mM H2O2 (H2O2 radical), 1 mM H2O2 plus 100 μM iron(II) perchlorate hexahydrate (hydroxyl radical) or KO2 at 1 mM (O2radical dot−). After an incubation period of 30 min at 37 °C, fluorescence was measured using excitation/emission wavelengths of 485/535 nm.

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Lipid peroxidation assay [2]
The procedure used was as described in previous studies (Zhang and Osborne, 2006). Following decapitation of a Wistar rat, the cerebral cortex was immediately homogenized in 10 volumes of ice-cold 0.9% saline (pH 7.0), centrifuged at 1000 × g for 10 min at 4 °C and the supernatant used for the lipid peroxidation assay by determining the amount of thiobarbituric acid reactive species (TBARS) formed. Briefly, aliquots of supernatant (0.5 ml) were preincubated at 37 °C for 5 min with 0.3 ml of 0.9% saline (pH 7.0) containing various concentrations of isoquercitrin or vehicle. Lipid peroxidation was initiated by the addition of 0.2 ml of 20 μM SNP or buffer. After 30 min of incubation at 37 °C, the test tubes were placed on ice to stop the lipid peroxidation reaction. TBARS were then determined as described by Ohkawa et al. (1979), by measuring coloured products resulting from the reaction of thiobarbituric acid, thought to be mainly malondialdehyde (a compound formed during lipid peroxidation) (Ohkawa et al., 1979). Briefly, the colour reaction was developed by the sequential addition of 0.2 ml 8.1% w/v sodium dodecyl sulphate, 1.5 ml 20% v/v acetic acid (pH 3.5) and 1.5 ml 0.8% w/v thiobarbituric acid. This mixture was incubated for 30 min in a boiling water bath. After allowing to cool, 2 ml of n-butanol:pyridine (15:1 v/v) was added and the reaction mixture was then centrifuged at 4000 × g for 10 min. Absorbance of the organic layer (at the top of the tube) was measured at 532 nm wavelength and the amount of TBARS determined using a standard curve of the malondialdehyde derivative 1,1,3,3-tetraethoxypropane (1–30 nmol). The protein concentration in whole-brain homogenate supernatant was determined using a bicinchoninic acid protein assay kit with bovine serum albumin as the standard.

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Western blot analysis [2]
The RGC-5 cells (2.0 × 104) were seeded in 60 mm2 Petri-dishes. After incubation for 24 h, the dishes were rinsed with serum-free medium, and exposed to 300 μM H2O2 in the presence or absence of 10 μM isoquercitrin for 24 h. Cells were then scraped off, suspended in lysis buffer (1 M Tris pH 7.4, 2 M NaCl, 1 M EDTA, 10% NP40, and protease inhibitor cocktail), briefly sonicated and centrifuged at 12,000 × g for 30 min at 4 °C. The protein content was then determined using a Bio-Rad Protein Assay kit.

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Western Blot Analysis[3]
Cell Types: HepG2 Cell
Tested Concentrations: 10 μM
Incubation Duration: 1 hour
Experimental Results: Ethanol-induced reduction of iNOS protein expression.

Treatment with quercetin and isoquercitrin [2]
In order to determine the therapeutic effects of quercetin and isoquercitrin in a murine model of asthma, the following protocol was adopted. Mice immunized (on days 0 and 7) and challenged (on day 14 and 21) with ovalbumin as described above were randomly divided into 5 groups, designated Ova-only (untreated mice), Ova+vehicle (mice receiving oral propylene glycol:water, 1:1), Ova+dexamethasone (mice receiving subcutaneous injections of dexamethasone), Ova+isoquercitrin (mice receiving oral isoquercitrin) and Ova+quercetin (mice receiving oral quercetin). Equimolar amounts of quercetin (10 mg/kg) or isoquercitrin (15 mg/kg), or vehicle (propylene glycol:water, 1:1) were administered daily by gavage (0.3 mL) to each group of mice (n = 6) from days 18 to 22 after the fi rst immunization. One group treated with subcutaneous injections of dexamethasone (1 mg/kg, 0.25 mL) was used as a positive control of anti-infl ammatory activity.

mouse LD50 intraperitoneal >5 gm/kg Pharmaceutical Chemistry Journal, 19(326), 1985

[1]. Isoquercitrin is the most effective antioxidant in the plant Thuja orientalis and able to counteract oxidative-induced damage to a transformed cell line (RGC-5 cells). Neurochem Int. 2010 Dec;57(7):713-21.

[2]. Anti-inflammatory activity of quercetin and isoquercitrin in experimental murine allergic asthma. Inflamm Res. 2007 Oct;56(10):402-8.

[3]. Relative protective activities of quercetin, quercetin-3-glucoside, and rutin in alcohol-induced liver injury. J Food Biochem. 2019 Aug 5:e13002.

[4]. Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice. Am J Obstet Gynecol. 2018 Aug;219(2):197.e1-197.e8.

Quercetin 3-O-beta-D-glucopyranoside is a quercetin O-glucoside that is quercetin with a beta-D-glucosyl residue attached at position 3. Isolated from Lepisorus contortus, it exhibits antineoplastic activityand has been found to decrease the rate of polymerization and sickling of red blood cells It has a role as an antineoplastic agent, a plant metabolite, a bone density conservation agent, an osteogenesis regulator, an antioxidant, a histamine antagonist, an antipruritic drug and a geroprotector. It is a quercetin O-glucoside, a tetrahydroxyflavone, a beta-D-glucoside and a monosaccharide derivative. It is functionally related to a beta-D-glucose. It is a conjugate acid of a quercetin 3-O-beta-D-glucopyranoside(1-).
Isoquercetin has been used in trials studying the treatment of Kidney Cancer, Renal cell carcinoma, Advanced Renal Cell Carcinoma, Thromboembolism of Vein in Pancreatic Cancer, and Thromboembolism of Vein VTE in Colorectal Cancer, among others.
Isoquercitrin is under investigation in clinical trial NCT04622865 (Masitinib Combined With Isoquercetin and Best Supportive Care in Hospitalized Patients With Moderate and Severe COVID-19).
Isoquercitrin has been reported in Camellia sinensis, Geranium carolinianum, and other organisms with data available.
Isoquercetin is an orally bioavailable, glucoside derivative of the flavonoid quercetin and protein disulfide isomerase (PDI) inhibitor, with antioxidant and potential antithrombotic activity. As an antioxidant, isoquercetin scavenges free radicals and inhibits oxidative damage to cells. As a PDI inhibitor, this agent blocks PDI-mediated platelet activation, and fibrin generation, which prevents thrombus formation after vascular injury. In addition, isoquercetin is an alpha-glucosidase inhibitor. PDI, an oxidoreductase secreted by activated endothelial cells and platelets, plays a key role in the initiation of the coagulation cascade. Cancer, in addition to other thrombotic disorders, increases the risk of thrombus formation.
See also: Ginkgo (part of); Calendula Officinalis Flower (part of); Moringa oleifera leaf (part of) ... View More ...

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Quercetin 3-O-beta-D-glucofuranoside is a quercetin O-glucoside in which a glucofuranosyl residue is attached at position 3 of quercetin via a beta-glycosidic linkage. It has a role as a metabolite. It is a beta-D-glucoside, a quercetin O-glucoside, a monosaccharide derivative and a tetrahydroxyflavone.
Isoquercitrin is under investigation in clinical trial NCT04622865 (Masitinib Combined With Isoquercetin and Best Supportive Care in Hospitalized Patients With Moderate and Severe COVID-19).
3-(((2S,3R,4R,5R)-5-((R)-1,2-Dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one has been reported in Camellia sinensis, Caragana spinosa, and other organisms with data available.
Quercetin 3-O-glucoside is a metabolite found in or produced by Saccharomyces cerevisiae.

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In conclusion, it was demonstrated that the major compound associated with T. orientalis that blunts the negative effect of H2O2 on RGC-5 cells in culture is isoquercitrin. The neuroprotective effect of isoquercitrin seems to be related primarily to the antioxidant characteristics of the substance. Many other flavonoids (e.g. EGCG, baicalin) also have powerful antioxidant properties and attempts have been made to establish whether a relationship exists between the structure and antioxidative activity of different flavonoids (Dugas et al., 2000, Lien et al., 1999, Rice-Evans et al., 1996). One possible reason why isoquercitrin is such a powerful antioxidant might be due to it having a catechol group in its B ring, which would allow for better electron donating and thus efficient metal chelation and electron delocalization (Yang et al., 2001). However, it should be noted that flavonoids have a variety of functions, with some having no apparent antioxidant activity (e.g. genistein). The unique herbal properties of T. orientalis that allow the successful treatment of conditions such as gout, rheumatism, diarrhoea and chronic tracheitis (Zhu et al., 2004) are undoubtedly partly related to the constituent isoquercitrin. Since isoquercitrin can be taken orally and possibly crosses the blood–brain barrier to reach the retina (Paulke et al., 2008, Paulke et al., 2006), its use for treating glaucoma to attenuate ganglion cell death is certainly worthy of consideration.[1]

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A variety of pharmacological agents, such as PAF receptor antagonists and 5-lipoxygenase inhibitors, have been shown to have inhibitory effects on in vivo eosinophil accumulation. In addition, it has been demonstrated that quercetin and isoquercitrin, respectively, reduce PAF-induced and leukotriene-induced bronchoconstriction in a murine model of asthma. Therefore, it is possible that quercetin and isoquercitrin inhibit PAF and leukotriene production in cells. The results of the present study show that quercetin and isoquercitrin both attenuate infl ammation in a murine model of asthma. The effects described herein, as well as those observed by others investigators, together with the broad spectrum of the biological effects of these substances, strongly suggest that quercetin and isoquercitrin have therapeutic potential for the treatment of asthma and other allergic diseases. Therefore, the study of these two substances could lead to the development of an effective anti-asthma therapy or of a means of identifying novel anti-asthma targets.[2]

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Alcoholic liver diseases has been known to be one of the major health risks worldwide. The purpose of this study was aimed to demonstrate the relative protective effect of quercetin, quercetin-3-glucoside, and rutin on alcohol-induced damage in hepatocytes. The hepatotoxicity, antioxidant enzymatic defense mechanisms, and pro-inflammatory mediators were examined for evaluating the hepatoprotective effects of quercetins in hepG2 cells. The results revealed that quercetin and its glucoside derivatives significantly prevented ethanol-induced hepatotoxicity by decreasing hepatic aminotransferase activities and inflammatory response in HepG2 cells. Moreover, the quercetins significantly induced detoxifying enzymes via the nuclear accumulation of the NF-E2-related factor 2 (Nrf2) and induction of antioxidant response element (ARE) gene. These hepatoprotective activities were observed to be more effective with quercetin aglycone than quercetin glucosides. From the above findings, the present study imply that quercetin aglycone may have a vital function in the therapeutic and preventive strategies of alcoholic liver diseases. PRACTICAL APPLICATIONS: Quercetin is commonly present in fruits and vegetables as aglycone and glucoside-derived forms. In the present study, quercetin and its glycosides was shown to alleviate oxidative stress, glutathione depletion, and pro-inflammatory cytokines in alcohol-induced HepG2 cells via the Nrf2/ARE antioxidant pathway. Moreover, quercetin aglycone had better protective effects against alcohol-induced liver damage in vitro, compared to its glycosylated form. The present study proposed that quercetin aglycone may be a more efficient hepatoprotective agent than its glucoside derivatives such as rutin in the amelioration of alcohol-induced liver diseases.[3]

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Background: Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. Objective: This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. Study design: Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. Results: Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P < .05). Conclusion: Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.[4]

C21H20O12

464.3763

464.095

C, 54.32; H, 4.34; O, 41.34

482-35-9

482-35-9 (EMIQ); 482-35-9 (Isoquercetin); 17-39-5 (quercetin); 21637-25-2 (Isoquercitrin)

5280804

Light yellow to yellow solid

1.9±0.1 g/cm3

872.6±65.0 °C at 760 mmHg

238 - 242 °C

307.5±27.8 °C

0.0±0.3 mmHg at 25°C

1.803

1.75

8

12

4

33

758

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1C(C2=C(C([H])=C(C([H])=C2OC=1C1C([H])=C([H])C(=C(C=1[H])O[H])O[H])O[H])O[H])=O

OVSQVDMCBVZWGM-QSOFNFLRSA-N

InChI=1S/C21H20O12/c22-6-13-15(27)17(29)18(30)21(32-13)33-20-16(28)14-11(26)4-8(23)5-12(14)31-19(20)7-1-2-9(24)10(25)3-7/h1-5,13,15,17-18,21-27,29-30H,6H2/t13-,15-,17+,18-,21+/m1/s1

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one

Trifoliin A; Isoquercitrin; Isoquercitroside; Trifoliin; Isohyperoside; Isotrifolin; Isotrifoliin; Isoquercitrin; iso-quercetin; Isoquercitin; Quercetin-3-O-glucoside; Quercetin-3-glucoside

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~19.23 mg/mL (~41.41 mM)

Solubility in Formulation 1: ≥ 1.92 mg/mL (4.13 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 19.2 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: ≥ 1.92 mg/mL (4.13 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 19.2 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: ≥ 1.92 mg/mL (4.13 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 19.2 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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