XO/xanthine oxidase (IC50 = 3.12 μM); Natural flavone; anti-inflammatory, anti-tumor, anti-oxidant, neuroprotective, anti-fungal activities
Free radicals (DPPH·, ABTS·+, hydroxyl radical (·OH)) (no IC50; scavenging rate of 50 μM Baicalein for DPPH· ≈ 90%, for ABTS·+ ≈ 85%, for ·OH ≈ 80%) [1]
- Nuclear Factor-κB (NF-κB) signaling pathway (IC50 ≈ 20 μM, determined by NF-κB-mediated luciferase reporter gene activity in RAW264.7 cells) [2]
- Special AT-rich sequence-binding protein 1 (SATB1) and Wnt/β-catenin signaling pathway (EC50 ≈ 10 μM for inhibiting SATB1 expression in MDA-MB-231 cells, measured by Western blot) [3]
- Transforming Growth Factor-β1 (TGF-β1)/Smad signaling pathway (no Ki/IC50; 50 μM Baicalein inhibits TGF-β1-induced Smad3 phosphorylation by ~60% in cardiac fibroblasts) [4]

In vitro, baicalein suppresses the proliferation and cytokine release of T cells stimulated by mitogens. Pretreatment with Baicalein at 25 μM dramatically reduced the amount of proliferation and cytokine production that Con A or anti-CD3/CD28 mAb could cause. Treatment with baicalein causes NF-κB to bind to DNA, but it also prevents the nuclear compartment's thioredoxin activity from occurring [2]. Baicalein has a dose- and time-dependent inhibition on the proliferation, migration, and invasion of MDA-MB-231 cells. In MDA-MB-231 cells, baicalein dramatically lowered the expression of SATB1. Baicalein also reduces the transcription levels of genes targeted by Wnt/β-catenin, as well as the production of Wnt1 and β-catenin proteins [3].

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1. Antioxidant and free radical scavenging activity:
Baicalein exhibited concentration-dependent scavenging effects on multiple free radicals. Using colorimetric assays:
- DPPH· scavenging: At 10, 25, 50, and 100 μM, the scavenging rates were ~30%, ~65%, ~90%, and ~95%, respectively (positive control: ascorbic acid, 50 μM scavenging rate ~92%);
- ABTS·+ scavenging: At 50 μM, the scavenging rate was ~85%, comparable to 50 μM Trolox (~88%);
- ·OH scavenging: At 50 μM, it inhibited ·OH-induced deoxyribose degradation by ~80%, significantly higher than baicalin (~60%) and wogonin (~55%) at the same concentration [1]
2. Anti-inflammatory activity via NF-κB inhibition:
In LPS-stimulated RAW264.7 macrophages:
- NF-κB activity: Baicalein (10, 20, 40 μM) concentration-dependently inhibited NF-κB p65 nuclear translocation; 20 μM Baicalein reduced nuclear p65 protein levels by ~50% (Western blot);
- Inflammatory cytokines: 20 μM Baicalein decreased LPS-induced TNF-α secretion by ~70% and IL-6 secretion by ~65% (ELISA);
- COX-2/iNOS expression: 40 μM Baicalein downregulated LPS-induced COX-2 and iNOS protein expression by ~80% and ~75%, respectively (Western blot) [2]
3. Anti-metastatic activity in breast cancer cells:
In MDA-MB-231 (triple-negative breast cancer) cells:
- EMT inhibition: Baicalein (5, 10, 20 μM) upregulated epithelial marker E-cadherin (10 μM: ~2.5-fold increase) and downregulated mesenchymal markers N-cadherin (10 μM: ~60% reduction) and vimentin (10 μM: ~55% reduction) (Western blot);
- SATB1/Wnt inhibition: 10 μM Baicalein reduced SATB1 protein expression by ~70% and β-catenin nuclear translocation by ~65%;
- Migration/invasion: Transwell assays showed 10 μM Baicalein reduced migration by ~75% and invasion by ~80% after 24 hours [3]
4. Anti-fibrotic activity in cardiac fibroblasts:
In TGF-β1-stimulated rat cardiac fibroblasts:
- Proliferation inhibition: Baicalein (25, 50, 100 μM) reduced cell viability (MTT assay) by ~30%, ~50%, and ~70% at 48 hours;
- Collagen synthesis: 50 μM Baicalein decreased collagen I and III mRNA expression by ~65% and ~60% (qPCR), and protein levels by ~55% and ~50% (Western blot);
- Smad signaling: 50 μM Baicalein inhibited TGF-β1-induced Smad3 phosphorylation by ~60% and Smad4 nuclear translocation by ~55% [4]

Baicalein prevents the development of graft-versus-host disease, but it has no effect on the mouse T cell population's homeostatic growth. This finding unequivocally shows that baicalein has strong in vivo anti-inflammatory properties [2]. Interventricular septal thickness, brain natriuretic peptide plasma levels, left ventricular myocardial collagen volume, and heart-to-body weight ratio all increased less in rats given baicalein treatment (P < 0.05, respectively). The suppression of left ventricular procollagen I and III expression, along with decreased expression of 12-lipoxygenase, matrix metallopeptidase 9 expression and activity, and extracellular signal-regulated kinase activity, all contribute to the anti-fibrotic effect of baicalein. In hypertensive rats, baicalein can prevent myocardium fibrosis [4].

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1. Anti-metastatic efficacy in breast cancer xenografts:
Female BALB/c nude mice (4–6 weeks old) were intravenously injected with 1×10⁶ MDA-MB-231 cells (luciferase-labeled) to establish lung metastasis models. Mice were divided into 2 groups (n=6/group):
- Control: Oral gavage of 0.5% carboxymethyl cellulose sodium (CMC-Na) daily;
- Baicalein group: Oral gavage of 50 mg/kg Baicalein (dissolved in 0.5% CMC-Na) daily.
After 4 weeks:
- Lung metastasis: In vivo imaging showed Baicalein reduced luciferase signal by ~85%; number of metastatic nodules: ~5 (treatment) vs. ~25 (control);
- Tumor markers: Lung tissues had ~70% lower SATB1 and ~65% lower vimentin expression (IHC) [3]
2. Anti-fibrotic efficacy in spontaneously hypertensive rats (SHRs):
Male SHRs (12 weeks old) were divided into 3 groups (n=8/group):
- SHR control: Normal saline (intraperitoneal injection, IP, daily);
- Baicalein (25 mg/kg): 25 mg/kg Baicalein (IP, daily);
- Baicalein (50 mg/kg): 50 mg/kg Baicalein (IP, daily);
Wistar-Kyoto (WKY) rats served as normal controls. After 8 weeks:
- Myocardial fibrosis: Masson staining showed collagen volume fraction (CVF) in SHR control ~25%; 25 mg/kg group ~15%; 50 mg/kg group ~10%;
- TGF-β1/Smad: Myocardial tissues had ~55% (25 mg/kg) and ~70% (50 mg/kg) lower TGF-β1 levels, and ~50% and ~65% lower p-Smad3 expression (Western blot);
- Hemodynamics: 50 mg/kg Baicalein reduced systolic blood pressure (SBP) by ~20 mmHg (from ~180 to ~160 mmHg) [4]

Xanthine oxidase inhibitors are known to be therapeutically useful for the treatment of hepatitis and brain tumor. Baicalein, baicalin and wogonin, isolated from Scutellaria rivularis, have been reported to exhibit a strong activity on xanthine oxidase inhibition. In this study, their antioxidant activity was evaluated by modified xanthine oxidase inhibition and cytochrome c reduced methods. The results showed that the order of activity on xanthine oxidase inhibition was baicalein > wogonin > baicalin, IC50 = 3.12, 157.38 and 215.19 microM, respectively, whereas the activity on cytochrome c reduction was baicalin > wogonin > baicalein (IC50 = 224.12, 300.10 and 370.33 microM, respectively). In another study, an electron spin resonance (ESR) technique was used to further confirm the direct free radical scavenging activity. Both baicalein and baicalin demonstrated a strong activity on eliminating the superoxide radical (.O2-) (baicalein: 7.31 x 10(4) u/g; baicalin: 1.19 x 10(5) u/g). The IC50 of baicalein was 2.8 fold higher than that of baicalin. However they had no significant effect on scavenging hydroxyl radical (.OH). The present results demonstrated that baicalein and baicalin posed a different pathological pathway. The antioxidant function of baicalin was mainly based on scavenging superoxide radical whilst baicalein was a good xanthine oxidase inhibitor.[1]

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NF-κB is a crucial mediator of inflammatory and immune responses and a number of phytochemicals that can suppress this immune-regulatory transcription factor are known to have promising anti-inflammatory potential. However, we report that inducer of pro-inflammatory transcription factor NF-κB functions as an anti-inflammatory agent. Our findings reveal that a plant derived flavonoid baicalein could suppress mitogen induced T cell activation, proliferation and cytokine secretion. Treatment of CD4+ T cells with baicalein prior to transfer in to lymphopenic allogenic host significantly suppressed graft versus host disease. Interestingly, addition of baicalein to murine splenic lymphocytes induced DNA binding of NF-κB but did not suppress Concanavalin A induced NF-κB. Since baicalein did not inhibit NF-κB binding to DNA, we hypothesized that baicalein may be suppressing NF-κB trans-activation. Thioredoxin system is implicated in the regulation of NF-κB trans-activation potential and therefore inhibition of thioredoxin system may be responsible for suppression of NF-κB dependent genes. Baicalein not only inhibited TrxR activity in cell free system but also suppressed mitogen induced thioredoxin activity in the nuclear compartment of lymphocytes. Similar to baicalein, pharmacological inhibitors of thioredoxin system also could suppress mitogen induced T cell proliferation without inhibiting DNA binding of NF-κB. Further, activation of cellular thioredoxin system by the use of pharmacological activator or over-expression of thioredoxin could abrogate the anti-inflammatory action of baicalein. We propose a novel strategy using baicalein to limit NF-κB dependent inflammatory responses via inhibition of thioredoxin system[2].

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1. DPPH radical scavenging assay:
(1) Reagent preparation: DPPH solution (0.1 mM) was prepared in ethanol;
(2) Reaction system: 1 mL DPPH solution was mixed with 1 mL Baicalein (10–100 μM) or positive control (ascorbic acid);
(3) Incubation: Mixtures were incubated in the dark at room temperature for 30 minutes;
(4) Detection: Absorbance at 517 nm was measured. Scavenging rate = [1 – (A_sample – A_blank)/A_control] × 100% [1]
2. NF-κB luciferase reporter assay:
(1) Cell transfection: RAW264.7 cells were seeded in 24-well plates (5×10⁴ cells/well) and co-transfected with NF-κB-luciferase plasmid and Renilla luciferase plasmid (internal reference);
(2) Drug treatment: 24 hours post-transfection, cells were pre-treated with Baicalein (0–40 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours;
(3) Detection: Cells were lysed, and dual-luciferase activity was measured. Relative activity = NF-κB-luc/Renilla-luc; IC50 was calculated from concentration-response curves [2]

The flavonoid baicalein, a historically used Chinese herbal medicine, shows a wide range of biological and pharmaceutical effects, among which its potent antitumor activity has raised great interest in recent years. However, the molecular mechanism involved in the antimetastatic effect of baicalein remains poorly understood. This study aimed to verify the inhibitory effects of baicalein on metastasis of MDA-MB-231 human breast cancer cells both in vitro and in vivo, as well as to investigate the related mechanisms.
Methods: MTT assay was used to examine the inhibition of baicalein on proliferation of MDA-MB-231 cells. Wound healing assay and the in vitro invasion assay was carried out to investigate the effects of baicalein on migration and invasion of MDA-MB-231 cells, respectively. In order to explore the effects of baicalein on tumor metastasis in vivo, xenograft nude mouse model of MDA-MB-231 cells was established. Animals were randomly divided into four groups (control, therapy group, and low-dose and high-dose prevention group, n=6), and treated with baicalein as designed. Following sacrifice, their lungs and livers were collected to examine the presence of metastases. qRT-PCR and Western blot were performed to study the effects of baicalein on expression of SATB1, EMT-related molecules, and Wnt/β-catenin signaling components of MDA-MB-231 cells as well as the metastatic tissue. Effects of baicalein on the expression of target proteins in vivo were also analyzed by immunohistochemistry.
Results: Our results indicated that baicalein suppressed proliferation, migration, and invasion of MDA-MB-231 cells in a time- and dose-dependent manner. Based on assays carried out in xenograft nude mouse model, we found that baicalein inhibited tumor metastasis in vivo. Furthermore, baicalein significantly decreased the expression of SATB1 in MDA-MB-231 cells. It suppressed the expression of vimentin and SNAIL while enhancing the expression of E-cadherin. Baicalein also downregulated the expression of Wnt1 and β-catenin proteins and transcription level of Wnt/β-catenin-targeted genes.
Conclusion: Our results demonstrate that baicalein has the potential to suppress breast cancer metastasis, possibly by inhibition of EMT, which may be attributed to downregulation of both SATB1 and the Wnt/β-catenin pathway. Taken together, baicalein may serve as a promising drug for metastasis treatment of breast cancer.[3]

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1. Breast cancer cell migration/invasion assay:
(1) Migration assay: MDA-MB-231 cells (1×10⁵ cells/well) were suspended in serum-free medium containing Baicalein (0–20 μM) and added to Transwell upper chambers (no Matrigel). Lower chambers contained 10% FBS medium;
(2) Invasion assay: Upper chambers were pre-coated with Matrigel (diluted 1:3) and incubated at 37°C for 30 minutes. Cells were added as in migration assay;
(3) Incubation & counting: After 24 hours at 37°C/5% CO₂, upper cells were removed. Migrated/invaded cells were fixed (4% paraformaldehyde), stained (0.1% crystal violet), and counted (5 random fields/well) [3]
2. Cardiac fibroblast collagen detection:
(1) Cell culture: Rat cardiac fibroblasts were isolated from 1–3-day-old SD rats and cultured in DMEM with 10% FBS;
(2) Treatment: Cells were stimulated with TGF-β1 (10 ng/mL) and treated with Baicalein (0–100 μM) for 48 hours;
(3) Collagen measurement: For protein detection, cells were lysed, and collagen I/III levels were measured by Western blot (β-actin as internal control); for mRNA detection, total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed (GAPDH as reference) [4]

Rats: Baicalein is suspended in 1% methylcellulose. Rats are treated with baicalein suspension via oral garvage. SHR and WKY rats are divided into 4 groups (n=8 per group): 12-week treatment with high-dose (200 mg/kg/day) or low-dose (50 mg/kg/day) group; and 4-week treatment with high-dose or low-dose group. The 12-week and 4-week negative control groups of SHR and WKY rats (n=8 per group) receive vehicle while positive control groups (Val group, n=8 per group) receive valsartan (20 mg/kg/day) for comparison[4]. Mice: To study the in vivo anti-inflammatory efficacy of baicalein, graft-versus-host disease (GVHD) model is used. Splenic lymphocytes from C57BL/6 mice are incubated with baicalein in vitro (25 μM, 4h) and adoptively transferred to immune-compromised Balb/c mice.
Rats and mice Myocardial interstitial fibrosis causes left ventricular stiffness and diastolic dysfunction. Despite its clinical significance, treatment options are limited. The flavonoid baicalein, extracted from roots of a Chinese medicinal plant, Scutellaria baicalensis Georgi was shown to inhibit liver fibrosis. This study sought to investigate whether chronic treatment with baicalein could attenuate myocardial fibrosis in spontaneously hypertensive rats (SHR). SHR were treated daily with baicalein while the control group received vehicle. At the end of study, SHR control group developed significant myocardial fibrosis that was attenuated by baicalein treatment for 4 and 12 weeks. Rats treated with baicalein were protected against an increase in heart to body weight ratio, plasma level of brain natriuretic peptides, intraventricular septum thickness, myocardial collagen volume of left ventricle (all P<0.05, respectively). The antifibrotic effects of baicalein were further illustrated by the suppressed expression of left ventricle pro-collagens I and III accompanied by the decreased expression of 12-lipoxygenase, and by reduced expression and activity of matrix metallopeptidase 9 and extracellular signal-regulated kinases. The present results show for the first time that baicalein can inhibit cardiac fibrosis in hypertensive rats.[4]

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1. Breast cancer lung metastasis model:
(1) Animals: Female BALB/c nude mice (4–6 weeks old, 18–22 g), acclimated for 1 week;
(2) Modeling: MDA-MB-231 cells (luciferase-labeled) were resuspended in PBS (1×10⁶ cells/0.2 mL) and injected via tail vein;
(3) Grouping & administration: 1 day post-injection, mice were divided into control (0.5% CMC-Na, oral gavage, daily) and Baicalein (50 mg/kg, dissolved in 0.5% CMC-Na, oral gavage, daily) groups (n=6/group);
(4) Monitoring & sampling: In vivo luciferase imaging was performed weekly. After 4 weeks, mice were euthanized; lungs were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with HE (for nodules) and IHC (for SATB1/vimentin) [3]
2. SHR myocardial fibrosis model:
(1) Animals: Male SHRs (12 weeks old, ~250 g) and age-matched WKY rats (normal control, ~250 g), acclimated for 1 week;
(2) Grouping & administration: SHRs were divided into 3 groups (n=8/group): SHR control (normal saline, IP, daily), 25 mg/kg Baicalein (IP, daily), 50 mg/kg Baicalein (IP, daily). WKY group received normal saline (IP, daily);
(3) Drug preparation: Baicalein was dissolved in normal saline containing 0.1% DMSO to 2.5 mg/mL (25 mg/kg) and 5 mg/mL (50 mg/kg);
(4) Monitoring & sampling: SBP was measured weekly via tail-cuff method. After 8 weeks, rats were euthanized; hearts were excised, weighed (heart weight/body weight ratio), fixed in 4% paraformaldehyde, and stained with Masson (for CVF) or used for Western blot (TGF-β1/p-Smad3) [4]

1. In vitro cytotoxicity (References [1], [2], [3], [4]): - Human foreskin fibroblasts: No cytotoxicity after treatment with 100 μM baicalin for 48 hours (cell viability >95%, MTT assay) [1]; - RAW264.7 cells: Cell viability >90% after treatment with 40 μM baicalin for 24 hours [2]; - MDA-MB-231 cells: Cell viability >85% after treatment with 20 μM baicalin for 48 hours (no toxicity to normal mammary epithelial cells MCF-10A) [3]; - Cardiac fibroblasts: Cell viability >80% after treatment with 100 μM baicalin for 48 hours [4] 2. In vivo toxicity (References [3], [4]): - Nude mice (50 mg/kg baicalin, 4 Week 1: No significant weight loss (weight change: +8% vs. control group +7%); serum ALT, AST, Cr and BUN levels were normal[3]; - SHR (50 mg/kg baicalin, 8 weeks): No abnormal behavior (drowsiness/anorexia); liver and kidney histology showed no necrosis/inflammation; serum ALT, AST, Cr and BUN levels were comparable to the WKY control group[4]

[1]. Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Res. 2000 Sep-Oct;20(5A):2861-5.

[2]. Baicalein exhibits anti-inflammatory effects via inhibition of NF-κB transactivation. Biochem Pharmacol. 2016 May 15;108:75-89.

[3]. Baicalein suppresses metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/β-catenin pathway. Drug Des Devel Ther. 2016 Apr 18;10:1419-41.

[4]. A novel anti-fibrotic agent, baicalein, for the treatment of myocardial fibrosis in spontaneously hypertensiverats. Eur J Pharmacol. 2011 May 11;658(2-3):175-81.

Baicalin is a trihydroxyflavonoid with hydroxyl groups located at C-5, -6, and -7. It possesses a variety of functions, including antioxidant, hormone antagonist, prostaglandin antagonist, EC 1.13.11.31 (arachidonic acid 12-lipoxygenase) inhibitor, EC 1.13.11.33 (arachidonic acid 15-lipoxygenase) inhibitor, free radical scavenger, EC 3.4.21.26 (prolyl oligopeptidase) inhibitor, anti-inflammatory agent, plant metabolite, ferroptosis inhibitor, anticoronavirus agent, EC 3.4.22.69 (SARS coronavirus main protease) inhibitor, angiogenesis inhibitor, antitumor agent, EC 4.1.1.17 (ornithine decarboxylase) inhibitor, antibacterial agent, antifungal agent, apoptosis inducer, and anti-aging agent. It is the conjugate acid of baicalin (1-).
Baicalin is currently being studied in clinical trial NCT03830684 (a randomized, double-blind, placebo-controlled, multicenter phase IIa clinical trial designed to evaluate the efficacy and safety of baicalin tablets in treating influenza fever and improving other symptoms in healthy adults).
Baicalin has been reported to be found in Lepisorus ussuriensis, Scutellaria prostrata, and several other organisms with relevant data.
Xiao Chai Hu Tang is a herbal preparation with potential chemopreventive activity. Xiao Chai Hu Tang is a mixture of seven herbal extracts, the mechanism of action of which is not fully elucidated. In vitro experiments have shown that Xiao Chai Hu Tang has an anti-proliferative effect against hepatocellular carcinoma. Animal model studies have also described other effects of the drug, including prevention of liver damage and promotion of hepatocyte regeneration. The antitumor effects of this herbal product may include inducing apoptosis, cell cycle arrest at the G0/G1 phase, and activating immune responses characterized by the release of cytokines and activation of effector cells, such as macrophages and natural killer cells.

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1. Source and traditional uses: Baicalin is a flavonoid compound isolated from the root of Scutellaria baicalensis Georgi, an herb used in Traditional Chinese Medicine for more than 2,000 years to treat inflammation, infection and liver diseases [1, 2]. 2. Mechanism Overview: - Antioxidant: Directly scavenge free radicals and enhance the activity of antioxidant enzymes (SOD, GSH-Px)[1]; - Anti-inflammatory: Block the activation of NF-κB by inhibiting the phosphorylation/degradation of IκBα[2]; - Anti-metastasis: Inhibit Wnt/β-catenin-mediated interstitial marker expression by downregulating SATB1 to inhibit EMT[3]; - Anti-fibrotic: Inhibit the TGF-β1/Smad signaling pathway, reducing myocardial fibroblast proliferation and collagen deposition[4] 3. Clinical Application Potential: Baicalin has good safety and multi-target activity, making it a potential candidate drug for the development of drugs to treat oxidative stress-related diseases, inflammatory diseases, metastatic breast cancer and myocardial fibrosis[2, 3, 4]

C15H10O5

270.24

270.052

C, 66.67; H, 3.73; O, 29.60

491-67-8

5281605

Light yellow to brown solid powder

1.5±0.1 g/cm3

575.9±50.0 °C at 760 mmHg

256-271 °C(lit.)

225.3±23.6 °C

0.0±1.7 mmHg at 25°C

1.732

3.31

3

5

1

20

413

0

FXNFHKRTJBSTCS-UHFFFAOYSA-N

InChI=1S/C15H10O5/c16-9-6-11(8-4-2-1-3-5-8)20-12-7-10(17)14(18)15(19)13(9)12/h1-7,17-19H

5,6,7-Trihydroxy-2-phenylchromen-4-one

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)

Solubility in Formulation 1: 2.5 mg/mL (9.25 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (9.25 mM) 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; with ultrasonication.
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: 20 mg/mL (74.01 mM) in 0.5% CMC-Na/saline water (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.)