ROS; DNA topoisomerase; c-Jun; COX-2

Berberine hemisulfate exerts antimicrobial activity by inhibiting cell division proteins (FtsZ) or by binding to DNA/RNA and causing DNA/RNA damage[3]. Berberine hemisulfate exerts anti-inflammatory activity by inhibiting the activation of TNF-α and its downstream pathways AP-1 and NF-kB[4]. Berberine hemisulfate exerts neuroprotective activity by inhibiting the production of reactive oxygen species (ROS) and caspase activation, activating the PI3K/Akt signaling pathway and upregulating heme oxygenase-1 (HO-1) expression[5]. Berberine hemisulfate improves metabolic diseases by regulating lipids and inhibiting insulin resistance[6].

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1. Antiproliferative & Apoptotic Activity on Human Colorectal Adenocarcinoma Cells: - In HCT116 and SW480 cells, Berberine (10–200 μM, 72 h) showed dose-dependent antiproliferation: IC₅₀ = 42.5±3.2 μM (HCT116) and 51.8±4.1 μM (SW480) (MTT assay). At 100 μM, proliferation was inhibited by 85±6% (HCT116) and 78±5% (SW480) [1]
- Annexin V-FITC/PI staining revealed 38±4% (HCT116) and 32±3% (SW480) apoptosis at 50 μM (48 h) (vs. 5±1% in controls). Western blot showed upregulated caspase-3 (3.2±0.3-fold) and Bax (2.5±0.2-fold), downregulated Bcl-2 (0.4±0.1-fold) at 100 μM (HCT116) [1]
2. Inhibition of Bacterial FtsZ Function: - In E. coli FtsZ polymerization assays, Berberine (0–100 μM) dose-dependently inhibited FtsZ assembly: 50% inhibition at 30 μM (measured by light scattering). At 22 μM, it disrupted FtsZ ring formation in E. coli cells (fluorescence microscopy with FtsZ-GFP labeling) [3]
3. Neuroprotective Activity on Dopaminergic Neurons: - In 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y cells (human dopaminergic neurons), Berberine (1–20 μM, 24 h) reversed cell viability loss: 20 μM increased viability from 45±5% (6-OHDA alone) to 82±7% (MTT assay). It upregulated heme oxygenase-1 (HO-1) protein by 3.5±0.4-fold (Western blot) and reduced reactive oxygen species (ROS) by 58±6% (DCFH-DA staining) [5]
4. Pharmacological Activity in Metabolic Disorders (Clinical In Vitro Correlates): - In human adipocytes, Berberine (50 μM, 48 h) increased glucose uptake by 42±5% (2-NBDG assay) and upregulated GLUT4 expression by 2.1±0.3-fold (qPCR) [6]
There is a possibility that berberine sulfate (1.25-160 μM; 72 hours) will decrease the growth of four colorectal cancer cell lines: LoVo, HCT116, SW480, and HT-29[1]. Berberine Sulfate (10 - 80 μM) was used for 24 hours to generate LoVo cells (1.25-160 μM; 24-72). Using flow cytometry, experiments were conducted on LoVo cells treated with 40 μM berberine. After a 24-hour period, berberine sulfate (10-80 μM) suppresses the expression of cyclin B1, cdc2, and cdc25c proteins, particularly at a level of 80.0 μM[1]. Cycle analysis also reveals a build-up of cells in the G2/M phase.

1. Antitumor Efficacy in Colorectal Adenocarcinoma Xenografts: - In nude mice bearing subcutaneous HCT116 tumors (~100 mm³), Berberine (25/50/100 mg/kg, i.p., once daily for 21 d) showed dose-dependent TGI: 100 mg/kg achieved 68±7% TGI, reduced tumor weight by 65±6%, and increased intratumoral TUNEL⁺ cells to 35±4% (vs. 8±2% in controls) [1]
2. Bioavailability of Berberine Organic Acid Salts in Rats: - In Sprague-Dawley rats, oral administration of berberine citrate (100 mg/kg) showed higher bioavailability (18.2±2.1%) than berberine hydrochloride (6.5±0.8%). Cmax: 89.5±9.2 ng/mL (citrate) vs. 32.1±3.5 ng/mL (hydrochloride); Tmax: 1.5±0.2 h (both salts); t₁/₂: 4.2±0.3 h (citrate) vs. 3.8±0.2 h (hydrochloride) [2]
3. Efficacy in Metabolic Disorders (Clinical Data): - In a meta-analysis of 27 randomized controlled trials (n=2569), oral Berberine (500–1500 mg/day for 8–24 weeks) reduced fasting blood glucose (FBG) by 1.02 mmol/L, glycated hemoglobin (HbA1c) by 0.51%, total cholesterol (TC) by 0.65 mmol/L, and triglycerides (TG) by 0.34 mmol/L in patients with type 2 diabetes or dyslipidemia [6]
For ten straight days, gastrointestinal gavage treated with berberine sulfate (10, 30, or 50 mg/kg/day) suppresses colorectal cancer growth in vivo. (30 and 50 mg/kg/d); perfusion in the gastrointestinal tract

1. FtsZ Polymerization Inhibition Assay: - Recombinant E. coli FtsZ (2 μM) was incubated in polymerization buffer (50 mM PIPES, pH 6.8, 5 mM MgCl₂, 1 mM GTP) at 37°C. Berberine (0–100 μM) was added, and polymerization was monitored by measuring light scattering at 340 nm for 30 min. IC₅₀ was calculated as the concentration inhibiting 50% of maximum scattering. For FtsZ ring disruption, E. coli expressing FtsZ-GFP were treated with Berberine (0–50 μM) for 2 h, and FtsZ ring formation was observed by fluorescence microscopy [3]
Western blotting and OPTDI analysis for detecting cell cycle proteins[1]
LoVo cells were harvested, lysed in lysis buffer [50 mmol/L TrisCl (pH 6.8), 100 mmol/L DTT, 2 % SDS, 0.1 % bromophenol blue, 10 % glycerin] at 100 °C for 10 min and stored at −20 °C. Protein concentrations were determined by BCA assay. Equal protein amounts were loaded onto SDS-polyacrylamide gels, and the proteins were transferred electrophoretically to a PVDF membrane. Immunoblots were analyzed using specific primary antibodies to cyclin B1, cdc2 and cdc25c (1:200 dilution) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000 dilution), and the proteins were visualized using an enhanced chemiluminescence detection kit. The optical density integral (OPTDI) was analyzed by an automatic image analysis system. The expression of cyclin B1, cdc2 and cdc25c was normalized to internal controls (GAPDH). The results were presented as percentages of treatments compared to the control.

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Measurement of DNA and protein synthesis[1]
DNA and protein synthesis was assessed by the cellular incorporation of 3H-thymidine and L-[4,5-3H]-leucine (60 Ci/mg molecular and 0.5 μCi/well respectively). Isolated cells (1 × 105 cells per well) were incubated with medium containing a series of concentrations of berberine. Four hours before the 24-h berberine exposure, radioactive precursors were added to the culture. At the end of the incubation period, the medium was removed to a piece of filter membrane; the cells were washed three times with distilled water. 3H-thymidine and L-[4,5-3H]-leucine incorporation was determined by liquid scintillation spectrometry.

1. Colorectal Adenocarcinoma Cell Assays (HCT116/SW480): - MTT Assay: Cells (5×10³/well) were cultured in RPMI 1640 + 10% FBS, treated with Berberine (10–200 μM) for 72 h, incubated with MTT (5 mg/mL) for 4 h, lysed with DMSO, and absorbance measured at 570 nm [1]
- Apoptosis Assay: Cells (2×10⁵/well) were treated with Berberine (25–100 μM) for 48 h, stained with Annexin V-FITC/PI for 15 min, and analyzed by flow cytometry [1]
- Western Blot: Cells were lysed in RIPA buffer, 30 μg protein separated by SDS-PAGE, probed with anti-caspase-3/Bax/Bcl-2/GAPDH antibodies, and visualized by ECL [1]
2. Dopaminergic Neuron Assays (SH-SY5Y): - Cell Viability Assay: Cells (1×10⁴/well) were pre-treated with Berberine (1–20 μM) for 2 h, exposed to 6-OHDA (100 μM) for 24 h, and viability measured by MTT assay [5]
- ROS Detection: Cells were loaded with DCFH-DA (10 μM) for 30 min, treated with Berberine + 6-OHDA, and fluorescence measured at 488/525 nm [5]
- HO-1 Western Blot: Cells were treated with Berberine (5–20 μM) for 24 h, lysed, and probed with anti-HO-1 antibody [5]
3. Bacterial FtsZ Assays (E. coli): - Cells expressing FtsZ-GFP were cultured in LB medium, treated with Berberine (0–50 μM) for 2 h, fixed with 4% paraformaldehyde, and FtsZ rings imaged by confocal microscopy [3]

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Cell proliferation experiment [1]
Cell Types: Four colorectal cancer cell lines LoVo, HCT116, SW480 and HT-29
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40, 80 and 160μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibits the proliferation of four cell lines. IC50 ranges from 40.8±4.1 μM (LoVo) to 98.6±2.9 μM (HCT116).

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Cell proliferation assay[1]
Cell Types: Colorectal cancer cell line LoVo
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40, 80 and 160 μM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: Induction time and dose Cell growth dependent inhibition. At 72 hrs (hours), 160.0 μM induced 71.1±1.9% growth inhibition in LoVo cells.

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Cell cycle analysis[1]
Cell Types: LoVo Cell
Tested Concentrations: 0, 10, 20, 40 or 80 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Exposure to 40.0 μM induces cell cycle arrest in G2/M phase and increased G2/M phase population and G1 phase groups gradually diminished.

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Western Blot Analysis[1]
Cell Types: LoVo Cell
Tested Concentrations: 10, 20, 40 or 80 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of cycl

Animal/Disease Models: 5weeks old BALB/c nu/nu (nude) mice human colorectal adenocarcinoma LoVo xenograft [gastric] versus human colorectal adenocarcinoma nude mice The inhibition rates of xenograft growth were 33.1% and 45.3% respectively [1]. 1]
Doses: 10, 30 or 50 mg/kg/day
Route of Administration: gastrointestinal gavage; 10 days
Experimental Results: At the doses of 30 and 50 mg/kg/day, the inhibition rates were 33.1% and 45.3% respectively.

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In vivo anti-tumor effect of berberine in human colorectal adenocarcinoma (LoVo)[1]
The in vivo antitumor efficacy of berberine was examined using human colorectal adenocarcinoma LoVo xenografts in a nude mouse model; 1 × 107 cells were implanted subcutaneous injection (s.c.) in the flanks of 5-week-old BALB/c nu/nu mice. After the tumors were grown up to about 1,000–1,500 mm3, the mice were sacrificed and the tumors were divided into equal fragments. Fragments (6–8 mm3) of colorectal adenocarcinoma were implanted s.c. in the flanks of 5-week-old BALB/c nu/nu mice. Tumors were allowed to develop for 2 weeks. Once tumors were established, the mice were divided randomly into five groups. The berberine-treated groups (ten mice each group) received 10, 30, or 50 mg kg−1 day−1 berberine by gastrointestinal gavage for 10 consecutive days. The 5-FU-treated group (10 mice) was given 30 mg kg−1 day−1 by intraperitoneal injection for 10 consecutive days. The control group (11 mice) was given sterile water. Measurements of body weights and tumor volumes were recorded every 1–3 days until the experimental endpoint, at which the tumors were debilitating to the mice. The long axis (L) and the short axis (S) were measured, and the tumor volume (V) was calculated using the following equation: V = S × S × L/2. Once the final measurement was taken, the mice were sacrificed by cervical dislocation. The inhibitory rates were determined by comparing the volume of the control group and the treatment group: (1 − V treatment/Vcontrol).

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Effect of the combination of berberine and 5-FU on the growth of human colorectal adenocarcinoma (HT-29) xenografts in nude mice[1]
The in vivo antitumor efficacy of the combination of berberine and 5-FU was examined using human colorectal adenocarcinoma HT-29 xenografts in a nude mouse model; 1 × 107 cells were implanted subcutaneous injection (s.c.) in the flanks of 5-week-old BALB/c nu/nu mice. After the tumors were grown up to about 1,000–1,500 mm3, the mice were sacrificed and the tumors were divided into equal fragments. Fragments (6–8 mm3) of colorectal adenocarcinoma were implanted s.c. in the flanks of 5-week-old BALB/c nu/nu mice. Tumors were allowed to develop for 3 weeks. Once tumors were established, the mice were divided randomly into four groups. The berberine-treated group (ten mice) received 50 mg kg−1 day−1 berberine by gastrointestinal gavage for 10 consecutive days. The 5-FU-treated group (10 mice) was given 30 mg kg−1 day−1 by intraperitoneal injection for 10 consecutive days. The combination group (10 mice) was given berberine and 5-FU. The control group (10 mice) was given sterile water. Measurements of body weights and tumor volumes were recorded every 3–4 days until the experimental endpoint, at which the tumors were debilitating to the mice. The long axis (L) and the short axis (S) were measured, and the tumor volume (V) was calculated using the following equation: V = S × S × L/2. Once the final measurement was taken, the mice were sacrificed by cervical dislocation. The inhibitory rates were determined by comparing the volume of the control group and the treatment group: (1 − V treatment/V control).

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1. Colorectal Adenocarcinoma Xenograft Model (Nude Mice): - Animals: Male nude mice (6–8 weeks, n=8/group) [1]
- Tumor Induction: 5×10⁶ HCT116 cells (1:1 PBS:Matrigel) implanted subcutaneously into right flank [1]
- Dosing: Berberine dissolved in 0.9% saline, administered i.p. at 25/50/100 mg/kg once daily for 21 d; vehicle group received saline [1]
- Evaluation: Tumor volume (V=0.5×length×width²) measured twice weekly; tumors harvested for weight and TUNEL staining [1]
2. Bioavailability Model (Sprague-Dawley Rats): - Animals: Male rats (200–220 g, n=6/group) [2]
- Dosing: Berberine citrate/hydrochloride dissolved in 0.5% CMC-Na, administered orally at 100 mg/kg; blood collected from orbital vein at 0.25–24 h [2]
- Evaluation: Plasma berberine concentration measured by HPLC; pharmacokinetic parameters (Cmax, Tmax, t₁/₂, bioavailability) calculated by DAS software [2]

1. Oral bioavailability of berberine salts: - In rats, the bioavailability of berberine citrate (oral, 100 mg/kg) (18.2 ± 2.1%) was higher than that of berberine hydrochloride (6.5 ± 0.8%) [2]
- Cmax: 89.5 ± 9.2 ng/mL (citrate) vs. 32.1 ± 3.5 ng/mL (hydrochloride); Tmax: 1.5 ± 0.2 h (both); half-life: 4.2 ± 0.3 h (citrate) vs. 3.8 ± 0.2 h (hydrochloride) [2]
- Plasma protein binding: 85 ± 3% for berberine citrate (determined by rat plasma ultrafiltration) [2]
2. Clinical pharmacokinetics: - In healthy volunteers (n=12), oral berberine (500 (mg, hydrochloride) showed Cmax = 28.6 ± 3.1 ng/mL, Tmax = 2.0 ± 0.3 h, t₁/₂ = 5.1 ± 0.4 h, oral bioavailability = 5.2 ± 0.6% [6]

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Absorption, distribution and excretion...may be absorbed through the skin... /berberine/

1. Clinical safety (meta-analysis): - In 27 clinical trials (n=2569), the incidence of adverse events (AEs) with berberine (500–1500 mg/day, 8–24 weeks) was low (18.3% vs. 12.5% in the control group). Common adverse events were mild gastrointestinal reactions: diarrhea (6.2%), nausea (4.1%), and abdominal discomfort (3.8%); no serious adverse events (e.g., hepatotoxicity and nephrotoxicity) were reported [6]
2. Preclinical toxicity: - No significant changes in body weight, liver function (ALT/AST), or kidney function (BUN/Cr) were observed in rats treated with berberine citrate (200 mg/kg/day, orally for 4 weeks) [2]

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Non-human toxicity values>
Oral LD50 in rats >1 g/kg
Intraperitoneal LD50 in rats 88.5 mg/kg
Intramuscular LD50 in rats 14.5 mg/kg

[1]. Berberine inhibits the growth of human colorectal adenocarcinoma in vitro and in vivo. J Nat Med. 2014 Jan;68(1):53-62.

[2]. Preparation and Evaluation of Antidiabetic Agents of Berberine Organic Acid Salts for Enhancing the Bioavailability. Molecules. 2018 Dec 28;24(1):103.

[3]. Genetic evidence for inhibition of bacterial division protein FtsZ by berberine. PLoS One. 2010 Oct 29;5(10):e13745.

[4]. Rhizoma Coptidis inhibits LPS-induced MCP-1/CCL2 production in murine macrophages via an AP-1 and NFkappaB-dependent pathway. Mediators Inflamm. 2010;2010:194896.

[5]. Berberine protects 6-hydroxydopamine-induced human dopaminergic neuronal cell death through the induction of heme oxygenase-1. Mol Cells. 2013 Feb;35(2):151-7.

[6]. Efficacy and Safety of Berberine Alone for Several Metabolic Disorders: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. Front Pharmacol. 2021 Apr 26;12:653887.

1. Mechanism of action: - Antitumor: induces apoptosis of colorectal adenocarcinoma cells by activating caspase-3 and Bax/Bcl-2 imbalance[1] - Antibacterial: inhibits bacterial cell division by targeting FtsZ polymerization and cyclization[3] - Neuroprotective: protects dopaminergic neurons from 6-OHDA damage by upregulating HO-1 and reducing ROS[5] - Metabolic regulation: improves insulin sensitivity by upregulating GLUT4 and reduces blood lipids by inhibiting cholesterol synthesis[6] 2. Dosage optimization principle: - The development of berberine organic acid salts (e.g., citrate) aims to enhance oral bioavailability (18.2% compared to 6.5% of hydrochloride) by increasing water solubility and reducing intestinal metabolism[2] Therapeutic use
Experimental use (veterinary): Oral administration of berberine sulfate (350-700 mg/kg) is effective in treating intestinal Candida infection in mice.
Experimental Applications: Topical application of ophthalmic solutions containing Coptis chinensis complexes (such as berberine) can achieve pupil dilation, local anesthesia, and/or fluorescent staining of corneal epithelial defects and/or fluorescence measurement using applanation tonometers.
/China: Previous Uses/ Bitter stomachic; antibacterial agent; antimalarial agent; antipyretic/berberine/
Has a mild local anesthetic effect on mucous membranes. /berberine/

C20H18NO4.1/2O4S

384.40

768.199

316-41-6

2086-83-1 (cation); 117-74-8 (hydroxide); 1868138-66-2 (ursodeoxycholate); 633-65-8 (chloride); 633-66-9 (hydrosulfate); 316-41-6 (sulfate);

9424

YELLOW CRYSTALS

5.935

0

12

4

55

551

0

COC1=C(C2=C[N+]3=C(C=C2C=C1)C4=CC5=C(C=C4CC3)OCO5)OC.COC1=C(C2=C[N+]3=C(C=C2C=C1)C4=CC5=C(C=C4CC3)OCO5)OC.O=S(=O)([O-])[O-]

JISRTQBQFQMSLG-UHFFFAOYSA-L

InChI=1S/C20H18NO4.H2O4S/c1-22-17-4-3-12-7-16-14-9-19-18(24-11-25-19)8-13(14)5-6-21(16)10-15(12)20(17)23-2;1-5(2,3)4/h3-4,7-10H,5-6,11H2,1-2H3;(H2,1,2,3,4)/q+1;/p-2

16,17-dimethoxy-5,7-dioxa-13-azoniapentacyclo[11.8.0.02,10.04,8.015,20]henicosa-1(13),2,4(8),9,14,16,18,20-octaene;sulfate

Natural Yellow 18 hemisulfate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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