Metabolic Activation and Covalent Protein Binding of Berberrubine: Insight into the Underlying Mechanism Related to Its Hepatotoxicity.

INTRODUCTION: Berberrubine (BRB), an isoquinoline alkaloid, is a major constituent of medicinal plants Coptis chinensis Franch or Phellodendron chinense Schneid. BRB exhibits various pharmacological activities, whereas exposure to BRB may cause toxicity in experimental animals. METHODS: In this study, we thoroughly investigated the liver injury induced by BRB in mice and rats. To explore the underlying mechanism, a study of the metabolic activation of BRB was conducted. Furthermore, covalent modifications of cysteine residues of proteins were observed in liver homogenate samples of animals after exposure to BRB, by application of an exhaustive proteolytic digestion method. RESULTS: It was demonstrated that BRB-induced hepatotoxicities in a time- and dose-dependent manner, based on the biochemical parameters ALT and AST. H&E stained histopathological examination showed the occurrence of obvious edema in liver of mice after intraperitoneal (i.p.) administration of BRB at a single dose of 100 mg/kg. Slight hepatotoxicity was also observed in rats given the same doses of BRB after six weeks of gavage. As a result, four GSH adducts derived from reactive metabolites of BRB were detected in microsomal incubations with BRB fortified with GSH as a trapping agent. Moreover, four cys-based adducts derived from reaction of electrophilic metabolites of BBR with proteins were found in livers. CONCLUSION: These results suggested that the formation of protein adducts originating from metabolic activation of BRB could be a crucial factor of the mechanism of BRB-induced toxicities.

Berberine associated photodynamic therapy promotes autophagy and apoptosis via ROS generation in renal carcinoma cells.

Renal cell carcinoma (RCC) consists of the most lethal common urological cancer and the clinical practice has shown that resistant RCC to commons therapies is extremely high. Berberine is an isoquinoline alkaloid, presents in different kinds of plants and it has long been used in Chinese medicine. It has several properties, such as antioxidant, anti-inflammatory, anti-diabetic, anti-microbial and anti-cancer. Moreover, berberine has photosensitive characteristics and its association with photodynamic therapy (PDT) is effective against tumor cells. This study aimed to evaluate the effects of berberine associated with PDT in renal carcinoma cell lines. The cellular viability assay showed increased cytotoxicity in concentration and time-dependent manner. Berberine presented efficient internalization in all cell lines analyzed. In addition, after treatment with berberine associated with PDT, it was observed a high phototoxicity effect with less than 20 % of viable cells. In this study we observed that the increase of reactive oxygen species (ROS) levels was accompanied by an increase of autophagy levels and apoptosis by caspase 3 activity, suggesting cell death by both mechanisms. Additionally, three target genes of anti-cancer drugs were differentially expressed in 786-O cells, being that Vascular Endothelial Growth Factor-D (FIGF) and Human Telomerase Reverse Transcriptase (TERT) gene presented low expression and Polo Like Kinase 3 (PLK3) presented overexpression after treatment with berberine associated with PDT. In this study, the proposed treatment triggered metabolites changes related to cell proliferation, tumorigenesis and angiogenesis. Thus, it was possible to suggest that berberine has promising potential as a photosensitizing agent in a photodynamic therapy, because it induced significant anticancer effects on renal carcinoma cells.

Intracellular Accumulation as an Indicator of Cytotoxicity to Screen Hepatotoxic Components of Chelidonium majus L. by LC-MS/MS.

A novel strategy was developed to identify hepatotoxic compounds in traditional Chinese medicines (TCMs). It is based on the exposure of HL-7702 cells to a TCM extract, followed by the identification and further determination of potential hepatotoxic compounds accumulated in the cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a case study, potential hepatotoxic components in Chelidonium majus L. were screened out. Five alkaloids (sanguinarine, coptisine, chelerythrine, protopine, and chelidonine) were identified by LC-MS/MS within 10 min, and their intracellular concentrations were first simultaneously measured by LC-MS/MS with a run time of 4 min. A cell viability assay was performed to assess the cytotoxicity of each alkaloid. With their higher intracellular concentrations, sanguinarine, coptisine, and chelerythrine were identified as the main hepatotoxic constituents in Ch. majus. The study provides a powerful tool for the fast prediction of cytotoxic components in complex natural mixtures on a high-throughput basis.

Natural Berberine-Based Chinese Herb Medicine Assembled Nanostructures with Modified Antibacterial Application.

The abuse of traditional antibiotics has caused a series of health problems including antimicrobial resistance, which threatens human health. Therefore, searching for broad sources of antimicrobial agents and developing multidimensional strategies to combat bacterial infections are urgent. Here, we reported two natural self-assembling modes between berberine (BBR) and flavonoid glycosides: nanoparticles (NPs) and nanofibers (NFs), which were both mainly governed by electrostatic and hydrophobic interactions. These two nanostructures exhibited different antibacterial properties from BBR. NPs showed significantly enhanced bacteriostatic activity, whereas NFs displayed a much weaker effect than BBR. The distinguishing properties can be attributed to the different spatial configurations and self-assembly processes of NPs and NFs. Flavonoid glycosides and BBR first formed a one-dimensional complex unit and subsequently self-assembled into three-dimensional nanostructures. With the hydrophilic glucuronic acid toward the outside, NPs exhibited stronger affinity to bacteria, thereby inducing the collapse of the bacteria population and the decrease in biofilm. In addition, in vitro hemolysis tests, cytotoxicity tests, and in vivo zebrafish toxicity evaluation showed that the obtained self-assemblies had good biocompatibility. This supramolecular self-assembly strategy can be applied to construct other nanoscale antibacterial drugs and thus provides weapons for the development of self-delivering drugs in bacterial infection treatment.

Characterization of the cytotoxicity of selected Chelidonium alkaloids in rat hepatocytes.

Phytomedicinal preparations containing extracts of the plant Chelidonium majus (Greater Celandine) have been used in the therapy of upper abdominal disorders. C. majus alkaloids (CAL) were suspected to be responsible for reported cases of liver symptoms including cases of acute liver failure in patients upon treatment with certain C. majus preparations. Based on these reports, a safe oral daily dose limit of not more than 2.5 mg CAL was established in the EU. However, C. majus extracts and individual CAL were not able to elicit similar adverse effects when given orally to pigs or rats. We found that CAL differ considerably in their cytotoxicity in rat hepatocytes in culture. The cationic congeners chelerythrine, coptisine and sanguinarine were the most toxic ones (EC20 values ≤2 µM) while the neutral congeners chelidonine, dihydrosanguinarine and protopine were less toxic, with a rank order of toxicity of coptisine > chelerythrine > sanguinarine > chelidonine > protopine > dihydrosanguinarine. Calculation of octanol-water partition coefficients revealed that the most cytotoxic CAL in hepatocytes were the cationic polar ones. At cytotoxic concentrations sanguinarine led to a marked decrease in reduced and oxidized intracellular glutathione while the much less cytotoxic dihydrosanguinarine did not. After glutathione depletion with menadione, CAL toxicity was only slightly enhanced. Comparison of the cytotoxic concentrations to reported liver levels in experimental animals suggests that the latter were too low to cause hepatotoxicity, probably due to an extremely low oral availability of certain CAL.

Berberine impairs embryonic development in vitro and in vivo through oxidative stress-mediated apoptotic processes.

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H2 O2 -induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine-triggering or preventing apoptosis-has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5-10 µM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine-induced injury of mouse blastocysts appeared to be attributable to oxidative stress-triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.

Pre-clinical toxicity of a combination of berberine and 5-aminosalicylic acid in mice.

Our previous study demonstrated that a combination of alternative medicine berberine and conventional 5-aminosalicylic acid (5-ASA) showed promise to be a novel therapeutic strategy for ulcerative colitis (UC). This present study aims to sketch the pre-clinical toxicity profile of this combination (1:10 dose ratio) on mice. In acute toxicity test, the determined median lethal dose (LD50) was 278.7 mg/kg berberine plus 2787 mg/kg 5-ASA. The results from subacute toxicity test demonstrated that no toxic signs of clinical symptoms, no significant changes in hematological or biochemical parameters were detected in mice treated with 14 + 140, 28 + 280 or 56 + 560 mg/kg of berberine plus 5-ASA treatment. Histological examinations revealed that accompanied with an increase in spleen weight, frequently recorded enlargement and white pulp hyperplasia of spleen were detected in mice when exposed to three doses of combination treatments. Further in vitro assessment suggested that the spleen toxicity was originated from berberine by its inhibition in cell viability and cell proliferation of lymphocytes. The results of this study indicate that the combination of berberine and 5-ASA shows a slight toxic effect on spleen, suggesting that this combination should be used with caution for patients.

Naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption.

Pharmacological activities of some natural products diminish and even disappear after purification. In this study, we explored the mechanisms underlying the decrease of acute oral toxicity of Coptidis Rhizoma extract after purification. The water solubility, in vitro absorption, and plasma exposure of berberine (the major active compound) in the Coptidis Rhizoma extract were much better than those of pure berberine. Scanning electron microscopy, laser scanning confocal microscopy (LSCM), and dynamic light scattering experiments confirmed that nanoparticles attached to very fine precipitates existed in the aqueous extract solution. The LSCM experiment showed that the precipitates were absorbed with the particles by the mouse intestine. High-speed centrifugation of the extract could not remove the nanoparticles and did not influence plasma exposure or acute oral toxicity. However, after extract dilution, the attached precipitates vanished, although the nanoparticles were preserved, and there were no differences in the acute oral toxicity and plasma exposure between the extract and pure berberine. The nanoparticles were then purified and identified as proteinaceous. Furthermore, they could absorb co-dissolved berberine. Our results indicate that naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption. These findings should inspire related studies in other natural products.

Mitochondria and NMDA receptor-dependent toxicity of berberine sensitizes neurons to glutamate and rotenone injury.

The global incidence of metabolic and age-related diseases, including type 2 diabetes and Alzheimer's disease, is on the rise. In addition to traditional pharmacotherapy, drug candidates from complementary and alternative medicine are actively being pursued for further drug development. Berberine, a nutraceutical traditionally used as an antibiotic, has recently been proposed to act as a multi-target protective agent against type 2 diabetes, dyslipidemias, ischemic brain injury and neurodegenerative diseases, such as Parkinson's and Alzheimer's disease. However, the safety profile of berberine remains controversial, as isolated reports suggest risks with acute toxicity, bradycardia and exacerbation of neurodegeneration. We report that low micromolar berberine causes rapid mitochondria-dependent toxicity in primary neurons characterized by mitochondrial swelling, increased oxidative stress, decreased mitochondrial membrane potential and depletion of ATP content. Berberine does not induce caspase-3 activation and the resulting neurotoxicity remains unaffected by pan-caspase inhibitor treatment. Interestingly, inhibition of NMDA receptors by memantine and MK-801 completely blocked berberine-induced neurotoxicity. Additionally, subtoxic nanomolar concentrations of berberine were sufficient to sensitize neurons to glutamate excitotoxicity and rotenone injury. Our study highlights the need for further safety assessment of berberine, especially due to its tendency to accumulate in the CNS and the risk of potential neurotoxicity as a consequence of increasing bioavailability of berberine.

Berberine induces apoptosis and DNA damage in MG­63 human osteosarcoma cells.

Berberine, an isoquinoline alkaloid extracted from the dry root of Coptidis Rhizoma, has been found to exhibit marked anticancer effects on a panel of established cancer cells. Among the human osteosarcoma lines treated, MG­63 cells were found to be the most sensitive. The present study investigated the potential genotoxic effect of berberine on MG­63 human osteosarcoma cells. The effect of berberine on cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5­diphenyltetrazolium bromide assay and cell apoptosis was analyzed by flow cytometry and a DNA ladder assay. γH2AX focus formation was used to detect DNA damage in MG-63 cells. Berberine induced a significant increase in apoptosis in MG-63 cells in a concentration- and time-dependent manner, as determined by DNA fragmentation analysis and flow cytometry. Furthermore, berberine induced significant concentration- and time-dependent increases in DNA damage compared with that in the negative control. In conclusion, these observations indicated that berberine induced apoptosis and DNA damage in MG­63 cells.

Berberine induces double-strand DNA breaks in Rev3 deficient cells.

Berberine is a natural isoquinoline alkaloid, the majority of which is extracted from Huang Lian and other medicinal herbs. Numerous studies have revealed that berberine exhibits anticancer activity, however the mechanisms underlying this effect remain elusive. To examine these mechanisms, we analyzed the effects of berberine on a panel of DNA repair deficient chicken B lymphocyte (DT40) clones. Our results revealed that DT40 cells deficient in Rev3 (Rev3-/-), a translesion DNA synthesis (TLS) gene, were hypersensitive to berberine. Following berberine treatment, cell cycle analysis identified that G2/M arrest was increased in Rev3-/- cells. Furthermore, compared with wild-type cells (WT), berberine also induced a significant increase in double-strand breaks (DSBs) in Rev3-/- cells, as revealed by chromosomal aberration (CA) analysis. These results suggest that berberine is able to induce DNA damage, and that the Rev3 associated DNA repair pathway participates in the processes that aid its repair.

Mechanism study of goldenseal-associated DNA damage.

Goldenseal has been used for the treatment of a wide variety of ailments including gastrointestinal disturbances, urinary tract disorders, and inflammation. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice; however, the mechanism of goldenseal-associated liver carcinogenicity remains unknown. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity.

Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes.

The recurrence of breast cancer is associated with drug-resistance of cancer stem cells (CSCs), while overexpression of cell membrane ATP-binding cassette (ABC) transporters and resistance of mitochondrial apoptosis-related proteins are responsible for the drug-resistance of CSCs. The targeting berberine liposomes were developed to modulate the resistant membrane and mitochondrial proteins of breast CSCs for the treatment and prevention of breast cancer relapse. Evaluations were performed on human breast CSCs and CSC xenografts in nude mice. The targeting berberine liposomes were shown to cross the CSC membrane, inhibit ABC transporters (ABCC1, ABCC2, ABCC3, ABCG2) and selectively accumulate in the mitochondria. Furthermore, the pro-apoptotic protein Bax was activated while the anti-apoptotic protein Bcl-2 was inhibited resulting in opening of the mitochondrial permeability transition pores, release of cytochrome c, and activation of caspase-9/caspase-3 enzymes. Significant efficacy of the administrations in mice was observed, indicating that the targeting berberine liposomes are a potential therapy for the treatment and prevention of breast cancer relapse arising from CSCs.

Safety evaluation of main alkaloids from Rhizoma Coptidis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma Coptidis (RC), a widely used traditional Chinese medicine, has been used for the treatment of heat-clearing and detoxifying, but there is very little information on its safety. AIM OF THE STUDY: To provide information on the safety of RC, we evaluated the toxicity of the crude RC and RC alkaloids (berberine, coptisine, palmatine and epiberberine) including cytotoxicity, acute toxicity in mice and sub-chronic toxicity in rats. MATERIALS AND METHODS: The cytotoxicity of RC alkaloids was tested in HepG2 and 3T3-L1 cells by the MTT assay. The acute toxicity of RC alkaloids was tested in mice and the mortality was calculated at the end of experiment. For sub-chronic toxicity study, the rats were treated with the RC alkaloids at a dose of 156 mg/kg/day and RC at a dose of 521 mg/kg/day for 90 days. Mortality, clinical signs, body weight changes, organ weights, urinalysis and hematological parameters, gross necropsy and histopathology were monitored during the study period. RESULTS: The cell assay indicates that the IC(50) values of berberine, coptisine, palmatine and epiberberine in HepG2 cells were 48.17, 64.81, 112.80 and 120.58 µg/mL, which in 3T3-L1 cells were 41.76, 56.48, 84.32 and 104.18 µg/mL, respectively. In the acute toxicity assay, the LD(50) values of four alkaloids were 713.57, 852.12, 1533.68 and 1360 mg/kg, respectively. However, in the sub-chronic toxicity study, no mortality and morbidity were observed which could be related to RC alkaloids and RC treatment. Besides, there was no abnormality in clinical signs, body weights, organ weights, urinalysis, hematological parameters, gross necropsy and histopathology in any of the animals after the oral administration of RC alkaloids and RC. CONCLUSIONS: Taking these results together, we came to the conclusion that the toxicity of berberine is the maximum and palmatine is the minimal in four RC alkaloids. The currently recommended doses of RC alkaloids and RC consumed are relatively safe.

Lipopolysaccharide increased the acute toxicity of the Rhizoma coptidis extract in mice by increasing the systemic exposure to Rhizoma coptidis alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma coptidis is used as an antidysenteric in clinics in China. However, patients suffering from dysentery are susceptible to the acute toxicity of Rhizoma coptidis. The current study investigates the effects of lipopolysaccharide (LPS), which are a key pathogenic factor in dysentery, on the acute toxicity of a Rhizoma coptidis extract in mice; possible mechanisms are proposed. MATERIALS AND METHODS: Acute toxicity and pharmacokinetic experiments in mice were conducted. The plasma concentration of Rhizoma coptidis alkaloids in mice was determined using liquid chromatography/tandem mass spectrometry. The activity of acetylcholinesterase (AChE) in the tissue homogenate was determined using an AChE determination kit. RESULTS: Pretreatment with LPS for 16 h increased the acute toxicity of the oral Rhizoma coptidis extract. Systemic exposure to Rhizoma coptidis alkaloids was also increased by LPS pretreatment. Neostigmine significantly increased whereas pyraloxime methylchloride reduced the acute toxicity of the Rhizoma coptidis extract. LPS pretreatment alone showed no significant effect on the activity of thoracoabdominal diaphragm AChE. However, it enhanced the inhibitory effect of the Rhizoma coptidis extract. LPS pretreatment did not affect the acute toxicity of various dosages of tail vein-injected berberine. CONCLUSIONS: LPS increased the acute toxicity of the oral Rhizoma coptidis extract in mice by increasing the systemic exposure to the Rhizoma coptidis alkaloids.

Investigating the potential for toxicity from long-term use of the herbal products, goldenseal and milk thistle.

Two-year toxicity studies were conducted on the widely used herbal products, goldenseal and milk thistle, in male and female F344/N rats and B6C3F1 mice. With goldenseal root powder, the primary finding was an increase in liver tumors in rats and mice, and with milk thistle extract, a decrease in spontaneous background tumors including mammary gland tumors in female rats and liver tumors in male mice. Increased tumorigenicity in rodents exposed to goldenseal root powder may be due in part to the topoisomerase inhibition properties of berberine, a major alkaloid constituent in goldenseal, or its metabolite, berberrubine. In the clinic, use of topoisomerase-inhibiting agents has been associated with secondary tumor formation and inhibition in DNA repair processes. In contrast, the radical-scavenging and antioxidant properties of silibinin and other flavonolignans in milk thistle extract may have contributed to the decrease in background tumors in rodents in the present studies. The fate of the active constituents of goldenseal and milk thistle is similar in humans and rodents; therefore, the modes of action may translate across species. Further studies are needed to extrapolate the findings to humans.

Anti-herpes simplex virus effects of berberine from Coptidis rhizoma, a major component of a Chinese herbal medicine, Ching-Wei-San.

Berberine is an alkaloid extracted from Coptidis rhizome. Among the individual herbal components of a Chinese herb medicine, Ching-Wei-San, Coptidis Rhizoma has the most potent antimicrobial activity. By high-pressure liquid chromatography, the quantitative analysis of berberine from 6.25-mg/mL (w/v) Coptidis rhizome extract or 50.00-mg/mL (w/v) Ching-Wei-San was determined to be 0.26 mg/mL. To explore the potential use of Ching-Wei-San against herpes simplex virus (HSV) infection, the cytotoxicity, anti-HSV-1 and anti-HSV-2 activity in Vero cells were assayed. The selectivity index of berberine was about 1.2-1.5 times higher than that of Coptidis rhizome extract and Ching-Wei-San. Moreover, the antiviral activities correspond to the content of berberine in the aqueous solution. Berberine may interfere with the viral replication cycle after virus penetration and no later than the viral DNA synthesis step, and its activities were not affected by the preparation processes. Berberine, the natural plants that contain this component, including Coptidis rhizome, and Ching-Wei-San have all shown anti-HSV effects.

Acute toxicity of berberine and its correlation with the blood concentration in mice.

The aim of this study was to investigate the LD(50) (median lethal dosage) of berberine (BBR) through three different routes of injection in mice: intravenous (i.v.) injection, intraperitoneal (i.p.) injection, and intragastric (i.g.) oral administration. The concentration of BBR in blood from their i.g. doses (10.4, 20.8, 41.6, and 83.2 g/kg) and the content relationship of BBR among different injections were analyzed by high-performance liquid chromatography (HPLC). The LD(50) of BBR from i.v. and i.p. injections is 9.0386 and 57.6103 mg/kg, respectively; but no LD(50) was found in the i.g. group. A significant difference in bioavailability was observed between the different routes. Furthermore, the concentration of BBR in the blood from different i.g. doses was also significantly different. However, we discovered an interesting phenomenon indicating that the absorption of BBR by oral administration has a limit, therefore, explaining the difficulty in obtaining an LD(50) of BBR for i.g. injection. From the analysis of BBR content in blood after various administrations, we hypothesized that not only does the concentration of BBR in blood contribute to its acute toxicity, but also the routes of administration may be an important facet that affects this toxicity evaluation.

Berberine inhibits the expression of TNFalpha, MCP-1, and IL-6 in AcLDL-stimulated macrophages through PPARgamma pathway.

Macrophages are the main source of cytokines in atherosclerotic plaques. Modified low-density lipoproteins may stimulate macrophages to produce large quantities of proinflammatory cytokines that promote atherosclerosis. Berberine is the main component of the traditional Chinese medicine umbellatine, which has a widespread effect and was used to treat many diseases clinically. Our previous study found that berberine could increase adipophilin expression in macrophages, which is a target gene of PPARgamma. PPARgamma agonist could decrease proinflammatory cytokines in macrophage. In this study, we investigated the effects and the mechanism of action of berberine on the expression and secretion of TNFalpha, MCP-1, and IL-6 in vitro to identify new pharmacological actions of berberine. The results of RT-PCR and ELISA shows that berberine may inhibit the expression and secretion of the tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6) in macrophages stimulated by acetylated low-density lipoprotein (AcLDL), whereas the peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 could attenuate this effect of berberine. This study demonstrates that berberine may inhibit the expression and production of TNF-alpha, MCP-1, and IL-6 in AcLDL-stimulated macrophages. This effect might be partially mediated through PPARgamma activity.

Effect of Alstonia scholaris in enhancing the anticancer activity of berberine in the Ehrlich ascites carcinoma-bearing mice.

The chemomodulatory activity of Alstonia scholaris extract (ASE) was studied in combination with berberine hydrochloride (BCL), a topoisomerase inhibitor, in Ehrlich ascites carcinoma-bearing mice. The tumor-bearing animals were injected with various doses of ASE, and 8 mg/kg of BCL (one-fifth of the 50% lethal dose) was combined with different doses of ASE (60-240 mg/kg). The combination of 180 mg/kg of ASE with 8 mg/kg of BCL showed the greatest antitumor effect; the number of tumor-free survivors was more, and the median survival time and the average survival time increased up to 47 and 40.5 days, respectively, when compared with either treatment alone. Similarly, when 180 mg/kg of ASE was combined with different doses of BCL (2-12 mg/kg), a dose-dependent increase in the anticancer activity was observed up to 8 mg/kg of BCL. However, a further increase in the BCL dose to 10 and 12 mg/kg resulted in toxic side effects. The best effect was observed when 180 mg/kg of ASE was combined with 6 or 8 mg/kg of BCL, where an increase in the antineoplastic activity was reported. The efficacy of the combination of 180 mg/kg of ASE was also tested with 6 mg/kg body weight of BCL in various stages of tumorigenesis, and it was effective when given in the early stages, although the efficiency decreased with an increase in the tumor developmental stages.