ROS; DNA topoisomerase; c-Jun; COX-2
Berberine chloride hydrate targets DNA topoisomerase, an enzyme essential for DNA replication and transcription. By inhibiting topoisomerase, the compound disrupts DNA processes, leading to cell death. It also induces reactive oxygen species (ROS) generation, which can cause oxidative stress and apoptosis in cells. The compound's antibiotic activity is mediated through its effects on bacterial DNA replication and cellular metabolism. Its antineoplastic properties are attributed to its ability to induce ROS and inhibit topoisomerase, leading to cancer cell death. The compound's multi-targeted mechanism of action makes it a valuable tool for studying various cellular processes.

Berberine hydrate (Natural yellow 18 chloride hydrate; 1.25-160 μM; 72 hours) has a possible inhibitory effect on the growth of four colorectal cancer cell lines, LoVo, HCT116, SW480 and HT-29 [1]. Berberine chloride hydrate (1.25-160 μM; 24-72 hours) promotes time- and dose-dependent suppression of LoVo cell proliferation [1]. LoVo cells were treated to berberine chloride hydrate (10-80 μM) for 24 h. Cell cycle study of LoVo cells treated with 40 μM berberine by flow cytometry showed that cells accumulated in the G2/M phase [1]. Berberine chloride hydrate (10-80 μM) suppresses cyclin B1, cdc2 and cdc25c protein expression after 24 hours, especially at the 80.0 μM dose [1].
In vitro studies have demonstrated that berberine chloride hydrate is an effective inhibitor of DNA topoisomerase and induces ROS generation in various cell types. It exhibits antibiotic activity against a range of bacterial pathogens. The compound's antineoplastic properties have been demonstrated in various cancer cell lines, where it induces apoptosis through ROS generation and topoisomerase inhibition. Its ability to induce oxidative stress makes it a valuable tool for studying the role of ROS in cellular signaling and disease. The compound's activity profile suggests potential therapeutic applications in both infectious diseases and cancer.

In vivo growth of human colorectal adenocarcinoma is inhibited by berberine chloride hydrate (natural yellow 18 chloride hydrate; 10, 30, or 50 mg/kg/day; gastrointestinal gavage; for 10 consecutive days). In nude mice, berberine hydrate inhibited human colorectal cancer xenografts at 30 and 50 mg/kg/day by 33.1% and 45.3%, respectively [1].
In vivo, berberine chloride hydrate has been used in traditional Chinese medicine as an antibiotic for treating various infections. Its antineoplastic properties have been studied in animal models of cancer, where it has shown efficacy in inhibiting tumor growth. The compound's ability to induce ROS generation and inhibit topoisomerase contributes to its in vivo antitumor activity. However, comprehensive in vivo efficacy and safety studies are needed to fully evaluate its therapeutic potential. The compound's traditional use and documented biological activities make it a promising candidate for further drug development.

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.
In vitro non-cell enzyme assays for berberine chloride hydrate typically involve measuring the inhibition of DNA topoisomerase activity using purified topoisomerase enzymes and a DNA substrate. The compound is incubated with the enzyme and DNA, and the extent of DNA relaxation or supercoiling is measured by gel electrophoresis. IC₅₀ values are calculated from dose-response curves. The compound's ability to generate ROS can be measured using cell-free systems with fluorescent probes such as DCFH-DA. These assays provide quantitative data on the compound's direct effects on its molecular targets.

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 cyclin B1, cdc2 and cdc25c protein expression.
In vitro cell-based assays for berberine chloride hydrate use various cancer cell lines and bacterial strains. Cancer cells are treated with varying concentrations of the compound, and parameters such as cell viability (MTT or CCK-8 assays), apoptosis (Annexin V/PI staining), and ROS generation (DCFH-DA staining) are assessed. DNA topoisomerase inhibition is confirmed by analyzing DNA damage markers such as γ-H2AX. For antibiotic studies, bacterial cultures are used to determine minimum inhibitory concentrations (MIC) using broth microdilution or agar diffusion methods. The compound's effects on bacterial growth and viability are assessed by colony counting and optical density measurements.

Animal/Disease Models: 5weeks old BALB/c nu/nu (nude) mice with human colorectal adenocarcinoma LoVo xenografts [1]
Doses: 10, 30, or 50 mg/kg/day
Route of Administration: Gastrointestinal gavage; continuous 10-day
Experimental Results: At doses of 30 and 50 mg/kg/day, 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).
In vivo animal studies for berberine chloride hydrate employ models of cancer and bacterial infection. For anticancer studies, xenograft models using various cancer cell lines are used. The compound is administered orally or intraperitoneally, and tumor growth is monitored. Parameters such as tumor volume, tumor weight, and survival are recorded. Tumor tissues are analyzed for markers of apoptosis, ROS generation, and topoisomerase inhibition. For antibiotic studies, animal models of bacterial infection are used, and parameters such as bacterial clearance from blood and tissues, survival rates, and reduction of infection symptoms are assessed. Pharmacokinetic studies in these models provide information about the compound's absorption, distribution, metabolism, and excretion.

Berberine chloride hydrate has a molecular weight of 389.83 g/mol and a molecular formula of C₂₀H₂₀ClNO₅. It is a naturally derived isoquinoline alkaloid salt. The compound is soluble in water and organic solvents. It should be stored under appropriate conditions as recommended by the manufacturer. Detailed pharmacokinetic parameters such as absorption, distribution, metabolism, and excretion have been characterized in preclinical studies. As a small molecule alkaloid, berberine chloride hydrate is expected to have moderate oral bioavailability. The compound's ability to induce ROS generation and inhibit topoisomerase contributes to its biological activity.

The toxicity profile of berberine chloride hydrate has been evaluated in preclinical studies. The compound is generally well-tolerated at therapeutic doses, though it can cause gastrointestinal irritation at high doses. Its ability to induce ROS generation suggests that it may cause oxidative stress at high concentrations. The compound's antineoplastic properties indicate that it may have significant biological effects that require careful evaluation. As a research compound, berberine chloride hydrate is not intended for human therapeutic use without appropriate regulatory approval. Standard laboratory safety precautions should be followed when handling the compound, including the use of appropriate personal protective equipment and working in a well-ventilated area.

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

Berberine chloride hydrate is an alkaloid commonly extracted from plants like Berberis species and isolated from the Chinese herbal medicine Huanglian (Coptis chinensis). It is also known as Natural Yellow 18 chloride hydrate. The compound acts as an antibiotic and induces reactive oxygen species (ROS) generation. It inhibits DNA topoisomerase and exhibits antineoplastic properties. Berberine chloride hydrate is commonly used as an antibiotic. Its ability to induce ROS generation and inhibit topoisomerase makes it a valuable tool for studying cancer biology and infectious diseases. The compound's antineoplastic properties suggest potential applications in cancer therapy. Not approved for clinical use; intended for research purposes only.

C20H20CLNO5

389.8295

389.103

68030-18-2

Berberine chloride;633-65-8;Berberine;2086-83-1

155074

Light yellow to yellow solid

Huanglian

3.834

1

6

2

27

488

0

[Cl-].O1C([H])([H])OC2=C1C([H])=C1C(=C2[H])C2C([H])=C3C([H])=C([H])C(=C(C3=C([H])[N+]=2C([H])([H])C1([H])[H])OC([H])([H])[H])OC([H])([H])[H].O([H])[H]

BPNJXFPOPCFZOC-UHFFFAOYSA-M

InChI=1S/C20H18NO4.ClH.H2O/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;;/h3-4,7-10H,5-6,11H2,1-2H3;1H;1H2/q+1;;/p-1

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;chloride;hydrate

Natural Yellow 18 chloride hydrate

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)

DMSO : ≥ 3.9 mg/mL (~10.00 mM)
H2O : ~1 mg/mL (~2.57 mM)

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.)