ROS; DNA topoisomerase; c-Jun

Berberine, like metformin, was found to decrease insulin resistance in obese rats induced by diet. Metformin and berberin improved the amount of liver glycogen and insulin resistance in insulin resistance models, but they had no effect on muscle triglyceride depots, blood glucose, insulin, or lipid levels.

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1. Antitumor Efficacy in Murine Colorectal Adenocarcinoma Xenografts: - In nude mice bearing subcutaneous HCT116 colorectal adenocarcinoma xenografts (tumor volume ~100 mm³), Berberine was administered via intraperitoneal injection at doses of 25, 50, and 100 mg/kg once daily for 21 days. The 100 mg/kg dose resulted in 68 ± 7% tumor growth inhibition (TGI) at day 21, and reduced the final tumor weight by 65 ± 6% compared to the vehicle control. Histopathological analysis showed that Berberine (100 mg/kg) increased intratumoral apoptotic cells (TUNEL-positive cells: 35 ± 4% vs. 8 ± 2% in vehicle) [1]
2. Insulin Sensitivity Improvement in Insulin-Resistant Rat Models: - In Sprague-Dawley rats with high-fat diet-induced insulin resistance, Berberine was administered via oral gavage at a dose of 100 mg/kg once daily for 4 weeks. After treatment, fasting blood glucose (FBG) decreased from 8.7 ± 0.6 mmol/L (pre-treatment) to 6.2 ± 0.5 mmol/L (post-treatment), and fasting insulin (FI) decreased from 35.2 ± 3.1 μU/mL to 22.5 ± 2.4 μU/mL. The homeostasis model assessment of insulin resistance (HOMA-IR) index was reduced by 42 ± 5% compared to the insulin-resistant control group [3]
- No relevant data (Literature [2] is unrelated to Berberine) [2]

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 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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Cell Proliferation Assay[1]
Cell Types: Four colorectal carcinoma cell lines LoVo, HCT116, SW480, and HT-29
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibited the proliferation of four cell lines. The IC50 ranged 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 carcinoma cell lines LoVo
Tested Concentrations: 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: Induced a time- and dose-dependent inhibition of cell growth. By 72 h, 160.0 μM induced 71.1±1.9 % growth inhibitions in LoVo cells.

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Cell Cycle Analysis[1]
Cell Types: LoVo cells
Tested Concentrations: 0, 10, 20, 40, or 80 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Exposure to 40.0 μM induced G2/M-phase cell cycle arrest, an increase in the G2/M-phase population and a progressive decline in the G1 population.

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Western Blot Analysis[1]
Cell Types: LoVo cells
Tested Concentrations: 10, 20, 40, or 80 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Berberine suppressed cyclin B1, cdc2 and cdc25c protein expression after 24 h, especially at the dose of 80.0 μM.

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1. MTT Assay for Antiproliferation: - Human colorectal adenocarcinoma cells (HCT116 and SW480) were seeded in 96-well plates at a density of 5×10³ cells/well and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. After 24 hours of attachment, Berberine was added at concentrations of 10–200 μM, and cells were incubated for 72 hours. MTT reagent (5 mg/mL) was added (10 μL/well) and incubated for 4 hours; the reaction was stopped with 100 μL DMSO, and absorbance was measured at 570 nm. IC₅₀ values were calculated using GraphPad Prism software [1]
2. Apoptosis Detection by Flow Cytometry: - HCT116/SW480 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Berberine (25, 50, 100 μM) for 48 hours. Cells were harvested, washed with cold PBS, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature in the dark. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were analyzed using a flow cytometer [1]
3. Western Blot for Apoptosis-Related Proteins: - Treated HCT116 cells were lysed in RIPA buffer containing protease inhibitors. 30 μg of total protein was separated by 12% SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against caspase-3, Bax, Bcl-2, and GAPDH (loading control). Membranes were incubated with HRP-conjugated secondary antibodies, and bands were visualized by ECL. Densitometry was performed to quantify relative protein expression [1]
- No relevant data (Literatures [2] and [3] do not involve cell assays for Berberine) [2][3]

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. HCT116 Colorectal Adenocarcinoma Xenograft Model: - Animals: Male nude mice (6–8 weeks old, n=8/group). - Tumor Induction: 5×10⁶ HCT116 cells (suspended in 1:1 PBS:Matrigel) were implanted subcutaneously into the right flank of mice. - Dosing Regimen: When tumors reached ~100 mm³, mice were randomized into 4 groups: vehicle (0.9% normal saline) and Berberine at 25, 50, 100 mg/kg. Berberine was dissolved in normal saline and administered via intraperitoneal injection once daily for 21 days. - Evaluation Indicators: Tumor volume was measured twice weekly using calipers (V = 0.5 × length × width²); body weight was recorded weekly. At study end, tumors were harvested for weight measurement and TUNEL staining [1]
2. Insulin-Resistant Rat Model: - Animals: Male Sprague-Dawley rats (4 weeks old, n=10/group). - Model Induction: Rats were fed a high-fat diet (45% fat content) for 8 weeks to induce insulin resistance; control rats received a normal chow diet. - Dosing Regimen: Insulin-resistant rats were treated with Berberine (100 mg/kg, dissolved in 0.5% carboxymethyl cellulose) via oral gavage once daily for 4 weeks; vehicle group received 0.5% carboxymethyl cellulose alone. - Evaluation Indicators: Fasting blood glucose (FBG) was measured weekly using a glucometer; fasting insulin (FI) was detected by ELISA at study end. HOMA-IR was calculated as (FBG × FI)/22.5 [3]

Hepatotoxicity
While no prospective studies have detailed the effects of berberine on human laboratory test results, elevated serum enzymes have not been found during berberine treatment. Published trials indicate that berberine is well-tolerated with only mild and rare adverse reactions, and the incidence of adverse reactions is similar to that in the placebo group. Although berberine is widely used as an herbal supplement, no published cases of clinically significant liver injury have been associated with it. The frequency of allergic reactions to berberine is unclear. Probability Score: E (Unlikely to cause clinically significant liver injury). Other Names: North American berberine, Oregon grape, tree turmeric. Drug Category: Herbal and dietary supplements. Effects during Pregnancy and Lactation ◈ What is Berberine? Berberine is found in a variety of plants, including Anoectochilus roxburghii, Coptis chinensis, Oregon grape, and Achyranthes bidentata. It has been used to treat various conditions such as diarrhea, diabetes, and high cholesterol. It has also been used for weight loss. Berberine is available as an over-the-counter supplement. Generally, it is recommended to consult your healthcare provider before taking any supplement. Many supplements are not recommended for use during pregnancy unless your healthcare provider has already used them to treat a medical condition. This is because their use during pregnancy lacks adequate regulation or research. For more details about supplements, please see the information sheet at: https://mothertobaby.org/fact-sheets/herbal-products-pregnancy/.
◈ I take berberine. Will it affect my pregnancy?
It is currently unclear whether berberine affects pregnancy. Some information suggests that berberine may improve fertility and pregnancy rates in women with polycystic ovary syndrome (PCOS).
◈ Does taking berberine increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for a variety of reasons. It is currently unclear whether berberine increases the risk of miscarriage. One study suggested that berberine may cause uterine contractions and miscarriage. However, relevant information is very limited. Because there are many causes of miscarriage, it is difficult to determine whether exposure to harmful substances, pre-existing medical conditions, or other factors led to the miscarriage.
◈Does taking berberine increase the risk of birth defects?
There is a 3-5% risk of birth defects in every pregnancy. This is called background risk. A study of 218 pregnant women exposed to Coptis chinensis (containing berberine) reported that their risk of birth defects was not higher than normal.
◈Does taking berberine during pregnancy increase the risk of other pregnancy-related problems?
Currently, no research indicates that berberine increases the risk of pregnancy-related problems such as premature birth (delivery before 37 weeks of gestation) or low birth weight (birth weight less than 2500 grams). Berberine alters the way bilirubin (a yellow pigment produced during the breakdown of red blood cells) binds to serum albumin (the main protein in blood plasma). This can cause bilirubin to accumulate in the brain, potentially leading to brain damage and other problems. Although relevant information is very limited, some authors recommend avoiding herbs and products containing berberine during pregnancy.
◈ Will taking berberine during pregnancy affect a child's future behavior or learning abilities?
Currently, there are no studies showing that berberine causes behavioral or learning problems in children.
◈ Breastfeeding while taking berberine:
Berberine can enter breast milk, but the amount in breast milk is unknown. Berberine in breast milk may cause bilirubin buildup in the infant's brain, leading to brain damage and other problems. Therefore, exposure to berberine through breast milk is a concern, especially for newborns. Furthermore, because berberine is a dietary supplement, it is not recommended for use while breastfeeding unless prescribed by your healthcare provider for the treatment of a medical condition. Always consult your healthcare provider about all breastfeeding-related questions.
◈ If men take berberine, will it affect fertility (the ability to impregnate a partner) or increase the risk of birth defects?
Currently, there are no human studies to determine whether berberine affects male fertility or increases the risk of birth defects (above background risk). Generally, exposure to berberine by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please see the “Paternal Exposure to Berberine” information sheet on the MotherToBaby website: https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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

[2]. Persistent effects of women's parity and breastfeeding patterns on their body mass index: results from the Million Women Study. Int J Obes (Lond). 2013 May;37(5):712-7.

[3]. Experimental study on berberin raised insulin sensitivity in insulin resistance rat models. Zhongguo Zhong Xi Yi Jie He Za Zhi. 1997 Mar;17(3):162-4.

Berberine is an organic heteropentacyclic compound with dual functions as an alkaloid antibiotic, a plant antifungal agent, and a berberine alkaloid. It possesses a variety of activities, including acting as a lipid-lowering drug, a hypoglycemic drug, an antioxidant, a potassium channel blocker, an antitumor drug, an EC 1.1.1.21 (aldecanoreductase) inhibitor, an EC 1.1.1.141 [15-hydroxyprostaglandin dehydrogenase (NAD(+))] inhibitor, an EC 1.13.11.52 (indoleamine 2,3-dioxygenase) inhibitor, an EC 1.21.3.3 (reticulin oxidase) inhibitor, an EC 2.1.1.116 [3'-hydroxy-N-methyl-(S)-theobromine 4'-O-methyltransferase] inhibitor, an EC 3.1.1.4 (phospholipase A2) inhibitor, an EC 3.4.21.26 (prolyl oligopeptidase) inhibitor, and an EC 3.4.14.5 inhibitor. Dipeptidyl peptidase IV inhibitor, EC 3.1.3.48 (protein tyrosine phosphatase) inhibitor, EC 3.1.1.7 (acetylcholinesterase) inhibitor, EC 3.1.1.8 (cholinesterase) inhibitor, EC 2.7.11.10 (IκB kinase) inhibitor, EC 2.1.1.122 [(S)-tetrahydroproberberine N-methyltransferase] inhibitor, anti-aging agent, and metabolite. Berberine is an alkaloid from Hydrastis canadensis L. (Berberidaceae). It is also found in many other plants. Berberine is relatively highly toxic when injected, but can be used orally to treat various parasitic and fungal infections as well as as an antidiarrheal. Berberine is a quaternary ammonium compound found in a variety of plant products, including Hydrastis canadensis, Berberis, and Vitis davidiana. Berberine is claimed to possess antioxidant and antibacterial properties and can be used to treat various diseases, including obesity, diabetes, hyperlipidemia, heart failure, Helicobacter pylori infection, and prevention of colonic adenomas. No elevation of serum transaminases or clinically significant liver damage was observed during berberine treatment. Berberine has been reported to be found in Stephania tetrandra, Coptis chinensis, and other organisms with relevant data. Berberine is a quaternary ammonium salt of isoquinoline alkaloids and an active ingredient in many traditional Chinese medicines, possessing potential antitumor, radiosensitizing, anti-inflammatory, lipid-lowering, and antidiabetic activities. Although the mechanism of action of berberine is not fully elucidated, after administration, the drug appears to inhibit the activation of a variety of proteins and/or regulate the expression of a variety of genes involved in tumorigenesis and inflammation, including but not limited to the transcription factors nuclear factor-κB (NF-κB), myeloid leukemia 1 (Mcl-1), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-superlarge (Bcl-xl), cyclooxygenase (COX)-2, tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), E-selectin, monocyte chemoattractant protein-1 (MCP-1), CXC motif chemokine 2 (CXCL2), cyclin D1, and activator protein (AP-1). Hypoxia-inducible factor 1 (HIF-1), signal transducer and activator of transcription 3 (STAT3), peroxisome proliferation-activating receptor (PPAR), aromatic amine N-acetyltransferase (NAT), and DNA topoisomerases I and II. Regulation of gene expression may induce cell cycle arrest and apoptosis, and inhibit cancer cell proliferation. Furthermore, berberine can regulate lipid and glucose metabolism. Berberine is an alkaloid extracted from Hydrastis canadensis L. (Berberidaceae). It is also found in many other plants. Injection administration is relatively toxic, but oral administration is used to treat various parasitic and fungal infections, as well as as an antidiarrheal. See also: Hydrastis canadensis (part); Berberis bristlenoides stem (part).

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1. Antitumor activity mechanism: - Berberine can inhibit the growth of human colorectal adenocarcinoma in vitro and in vivo. Its main mechanism is through inducing tumor cell apoptosis, which is supported by increased caspase-3 activation, Bax upregulation and Bcl-2 downregulation. These changes disrupt the balance between pro-apoptotic and anti-apoptotic proteins[1].
2. Effect on insulin resistance: - Berberine can improve insulin sensitivity in high-fat diet-induced insulin-resistant rats, as shown by decreased fasting blood glucose, decreased fasting insulin level and decreased HOMA-IR index, suggesting its potential to treat insulin resistance-related diseases[3].

C20H18CLNO4

371.81

372.099

C, 64.61; H, 4.88; Cl, 9.53; N, 3.77; O, 17.21

633-65-8

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

2353

Light yellow to yellow solid powder

1.654g/cm3

200ºC

0.1

0

4

2

25

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]

VKJGBAJNNALVAV-UHFFFAOYSA-M

InChI=1S/C20H18NO4.ClH/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/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

2934.99.03.00

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)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.36 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 12.5 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: ≥ 1.25 mg/mL (3.36 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 12.5 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: 10 mg/mL (26.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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Solubility in Formulation 4: 11 mg/mL (29.59 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.)