In a sub-acute toxicity test (repeated-dose research), an experimental material is administered to animals over the course of one month or fewer in three to four different dosages, and the biochemical and pathologic consequences are assessed 14 to 28 days afterwards. The findings of this study offer useful guidance for establishing doses for sub-chronic investigations and good information about the toxicity of the chemical following treatment with successive administrations (27).
Mice were used to test the subacute oral toxicity of the derived alkaloids from Turkish Berberis species’ roots, including berberine, berbemine, palmatine, oxyacanthine, magnoflorine, and columbamine (25, 50, and 200 mg/kg). The findings demonstrated that all alkaloids generate varying degrees of stomach ulcers at a dose of 25 mg/kg for seven days (38). The administration of active ethanol, butanol, CHCl3-ethanol, and H2O-I extract and fractions (300, 642, 472, and 614 mg/kg, respectively) to rats for 21 days was safe and did not cause any toxicity or death, according to a sub-acute toxicity evaluation on the root extract of B. crataegina. However, ethanol extract (21.3% and 9.7%, respectively), buthanol fraction (14.6% and 4.2%, respectively), and CHCl3-ethanol fraction (7.2% and 2.8%, respectively) all caused liver and kidney enlargement. With the H2O-I fraction, butanol fraction, ethanol extract, and CHCl3-ethanol fraction, body weight was increased by up to 30%, 27%, 17.8%, and 11.3%, respectively (39). Furthermore, it has been noted that adult rats’ bilirubin protein binding is reduced by intraperitoneal injection of berberine (10 and 20 mg/kg/day for 1 week). Clinically, it should be noted that this chemical may make risky individuals more susceptible to developing kernicterus (36).
In their work, Mahmoudi et al. (2016) examined the immunotoxic effects of berberine in BALB/c mice (5 and 10 mg/kg/day, IP, for 14 days). The spleen weight, blood cell count, including the amount of leukocytes, neutrophils, and lymphocytes, as well as splenic CD19+ B-cells, CD4+, and CD8+ T-cells were all reduced after berberine (10 mg/kg) administration. Overall, berberine inhibited both cellular and humoral immune functions at 10 mg/kg, but at 5 mg/kg, it only had an impact on lymphocyte proliferation and delayed-type hypersensitivity response (40).
Berberine sulphate 50 or 100 mg/kg orally administered to cats for 10 days caused hemorrhagic inflammatory issues in both the small and large intestine (33).
The main objectives of the sub-chronic test, which involves administering a substance repeatedly over the course of 30 to 90 days, are to establish the “no observed adverse effect level” (NOAEL), the “lowest observed adverse effect level” (LOAEL), and to identify the organs that are impacted by the substance after repeated administration. This kind of study was carried out orally in two species (10–20 mice and 4-6 dogs), for at least three doses, in the following order: low dosage with no toxic effects, high dose with toxic effects and less than 10% mortality, and intermediate dose. Body weight, food intake, biochemical and haematological markers, and other measurements are taken at the end of the 90th day (27).
The primary alkaloid in FRC is berberine. It has been demonstrated that the 1.88 g/kg RC extract in Sprague Dawley rats has no negative effects and has no impact on the blood’s biochemical parameters. There have been reports of liver and lung damage at 3.76 g/kg, when there is a considerable increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (37). Another study found that giving RC alkaloids to Sprague Dawley rats for 90 days while administering 156 mg/kg orally had no adverse impact on body weight or death (35).
Depending on the species types, the chronic test has a longer study period than 90 days. For instance, chronic exposure lasts 360 days or longer in non-rodent animals and between 180 and 720 days in rats. The primary objective of chronic toxicity studies is the evaluation of the maximum acceptable dose and the urinary metabolite indices, carcinogenicity, physiological, and pharmacokinetic aspects of substances (27).
It has been determined how phototoxic berberine combined with UVA radiation is to mosquito larvae (Aedes atropatpus). Larvae were treated with 10 ppm berberine and exposed to 0.4 W/m2 UV for 24 hours, after which they were transferred to clean jars and observed throughout their 4-week development until they reached adulthood. Berberine demonstrated long-term toxicity and significantly raised cumulative mortality. The effects persisted into the adult and pupil stages. The generation of singlet O2 by berberine attached to DNA is thought to be the fundamental mechanism of berberine (41).
Berberine did not appear to have a harmful effect on the kidneys or liver when administered to rats at a dose of 50 mg/kg (13). In a study that is related to these findings, it was found that berberine causes liver tissue damage in diabetic rats but not in control rats after 16 weeks at dosages of 50, 100, and 150 mg/kg (42). In a different experiment, apoE-/- mice were chronically treated with berberine (5 mg/kg/day, IP) and developed atherosclerosis (43).
Developmental toxicity is the study of harmful effects of a substance on the organism’s development as a result of exposure to chemical or physical agents during or after pregnancy or after birth up until puberty. Teratogenicity and reproductive toxicity were also evaluated. Developmental toxicity includes reproductive toxicity, which evaluates how harmful chemicals are to the male and female reproductive systems. Teratogens are compounds that are hazardous to developing children from conception to birth (27).
When given orally to pregnant mice and rats from the sixth to the fifteenth day of gestation, berberine (97.5 mg/kg/day) and goldthread rhizome (0.6 g/kg/day, orally) had no effect on inducing vaginal haemorrhage, abortion, neonatal deformity, or foetal toxicity (44). Berberine may be a potent maternal toxin with teratogenic effects because it has been reported to cause uterine contractions. For mated rats fed with berberine chloride dehydrate during gestation (days 6 to 20), the mother LOAEL, developmental LOAEL, and maternal NOAEL have been recorded with dosages of 531, 1313, and 282 mg/kg/day, respectively (45).
Berberine chloride dihydrate lowers maternal weight and foetal body weight when given orally to rats (282, 531, and 1313 mg/kg/day) and mice (569, 841, and 1155 mg/kg/day). In rats and mice, the maternal toxicity LOAEL is 7250 ppm (531 mg/kg/day) and 5250 ppm (841 mg/kg/day), respectively. The developmental toxicity NOAEL for rats and the developmental toxicity LOAEL for mice have both been found to be 1,000 mg/kg/day (46). Due to the hydration in berberine chloride dihydrate, these levels would be lower for berberine chloride (2).
Although berberine-rich plants have historically been used to heal jaundiced newborns, current research has shown that berberine can actually cause jaundice and hemolysis in those who are sensitive. Recently, berberine’s ability to displace bilirubin has been demonstrated. Studies conducted in vitro revealed that berberine has the ability to displace bilirubin from its binding sites on human serum albumin. Additionally, chronic berberine administration to rats (10 and 20 g/g, IP for 7 days) raised serum bilirubin concentration without affecting the serum total albumin concentration (47). Red blood cells’ osmotic fragility was increased and hemolysis was induced when glucose-6-phosphate dehydrogenase (G6PD)-deficient rat erythrocytes were exposed to extremely high doses of berberine either orally or in vitro (48). But records show that berberine did not cause jaundice and had no impact on the G6PD’s performance throughout pregnancy (44, 49). Neonatal rats were given 200 mg/kg of goldthread rhizome and 32.5 mg/kg of berberine subcutaneously, although neither substance had any influence on the G6PD activity or the onset of jaundice (44). In general, B. vulgaris and berberine-containing plants should be avoided during pregnancy and breastfeeding and are classified in category C. (2).
Documents have made reference to berberine’s cardiotoxicity. When given intravenously to dogs, berberine caused cardiac depression because the splanchnic arteries enlarged (50). Additionally, it has been shown that mitochondrial respiration and Ca2+ influx capability have decreased (51). According to research, berberine inhibits L type voltage-dependent Ca2+ channels, but at higher concentrations (100 M), it increases the release of Ca2+ from intracellular reserves (52). By directly interacting with F656V, prior research has shown that berberine (10-100 M) can reversibly block human ether-a-go-go (hERG) channels in Xenopus oocytes (53). (54). In a therapeutic setting, this inhibitory concentration exceeded the effective concentration (53). In HEK-293 cells and Xenopus oocytes, berberine has an IC50 of 3.10 M and 805 M, respectively (55). Additionally, it has been demonstrated that berberine raises the action potential by inhibiting the hERG channels, delayed rectifier potassium current, and inward rectifier potassium current, which may have both arrhythmogenic and anti-arrhythmic effects (53). (54). B. vulgaris’s myotropic properties result in hypotensive effects (28).
Cytotoxicity, genotoxicity, mutagenicity, and carcinogenicity
Berberine has been shown in numerous studies to have anti-tumor efficacy both in vitro and in vivo, making it a viable anticancer medication. It reduces cell viability in a dose- and time-dependent way in a variety of cancer cell types, including nasopharyngeal (56), hepatocellular (57), breast (58), and cervical cancer cells (59). Berberine is poisonous to some extent in healthy cells, and the inhibition of adenine nucleotide translocase, which is the fundamental mechanism, leads to a reduction in energy generation (51). The effects of berberine (10 nM to 10 mM) on cerebellar granule neurons (CGN) and hippocampal neurons (HCN) were studied for six hours. It was found that berberine at concentrations less than 0.3 mM not only had no effects on the CGN’s axon and dendrite’s gross structure, but also at 0.1 mM could slightly increase cell viability. Higher quantities (greater than 1 mM) of berberine, however, disrupt neuronal integrity and promote nuclear condensation. The highest hazardous potential is induced by berberine at a concentration of 10 mM, and after two hours of treatment, it increases oxidative stress and reduces neuronal survival (60).
It has been demonstrated that berberine hydrochloride (extracted from RC) affects CNE-1 nasopharyngeal cancer cells in a time- and dose-dependent manner by reducing proliferation, impeding migration, and inducing apoptosis. When given berberine hydrochloride for 48 hours, CNE-1 cells’ invasion and migration (10 g/ml) and cell growth (40 g/ml) are inhibited. This is accomplished via activating the apoptotic pathway (56).
Another study shown that berberine has anti-cancer effects on MCF-7 and MDA-MB-231 breast cancer cells. The findings showed that berberine (10 to 100 M) treatment of MCF-7 and MDA-MB-231 cells
In addition to the effects listed above, it has been observed that berberine at a concentration of 20 M considerably reduces the motility and inhibits the ability of cervical cancer cells to invade (59). The IC50 values of berberine in HepG2, SMMC-7721, and Bel-7402 cells are 34.5 M, 25.2 M, and 53.6 M, respectively, as opposed to 838.4 M in normal hepatic HL-7702 cells (57). Ehrlich ascites carcinoma (EAC) cells have shown acute cytotoxicity of berberine at concentrations of 10 g/ml, which significantly induces apoptosis, and 50 and 100 g/ml, which slows DNA synthesis, modifies the structure of dsDNA, and causes death. Less than 1 g/ml of berberine has been reported as the IC50 value in EAC cells (62).
The cytotoxic effects of berberine on L929 Murine Fibroblast cells. Berberine has no harmful effects at concentrations between 0.0025 mg/ml and 0.025 mg/ml, while DNA damage has been seen at values over 0.025 mg/ml. Higher than 0.1 mg/ml doses of berberine cause structural alterations in L929 cells as well as ROS production and death. When these cells are exposed to 0.2 mg/ml of berberine, its cytotoxic effects become noticeably evident (63). When berberine (>50 M) is used to treat C6 rat glioma cells, the shape of the cells is changed. Additionally, this alkaloid causes dose- and time-dependent cell death. G2/M cell cycle phases are arrested after treatment with berberine (100 M, for 12, 24, 48, and 72 hr), and significantly more apoptotic cell death is shown when it increases the activities of caspase-3, -8, and -9 time-dependently (64). In comparison to normal human oral tissue-driven cells, berberine iodide and acetoneberberine are more harmful to human oral squamous cell cancer and human promyelocytic leukaemia. Accordingly, the IC50 values for HL-60 have been published as 18, 22, M, HSC-2, HSC-3, HSC-4, and NA cells have been reported as 47, 88 M, and CA9-22 cells have been reported as 132, 136 M. For HGF >400, M is 293 M, M is 235, M is 219 M, and M is 245 M. (65).
Various lines of research have been done to determine how berberine affects both healthy and malignant cells in vivo. In their work, Li and colleagues (2015) assessed the impact of berberine on a mouse model of colorectal carcinogenesis that was enhanced by azoxymethane and dextran sulphate sodium (AOM/DSS). When mice were administered 40 mg/kg of berberine for 10 weeks, they noticed a decrease in tumour multiplicity (66). In the other study, mice were subcutaneously or subarachnoidally injected with SiHs cells to assess the effects of berberine (20 mg/kg/day, oral) on lung metastasis and tumour growth, respectively. Angiogenesis and tumour growth were reduced by berberine treatment, per the data acquired. Additionally, it decreased lung metastasis and pulmonary weight (59). The development and weight of the tumour were moderately decreased by berberine in the mouse 4T1 breast cancer model. When combined with anti-DR5, it enhanced their anti-tumor actions synergistically and decreased the prevalence of lung metastatic disease (73). Additionally, berberin treatment inhibited the development and weight of tumours that were brought on by the immunisation of Kunming mice with S 180 sarcoma tumour cells (74), BALB/c athymic nude mice with PC-3 and LNCaP prostate cancer cells (67), C57BL/6 mice with Lewis lung carcinoma cell line (69), and Swiss albino mice with DLA Dalton’s lymphoma tumour cells (68). However, HER-2/neu transgenic mice with spontaneous mammary tumours did not respond to berberine or NAX014 chemicals (berberine derivatives) (75).
Tables 22 and and 3, respectively, summarise the effects of B. vulgaris or berberine on normal and cancer cell lines under in vitro and in vivo circumstances.