Testing Status of Agents at NTP
Goldenseal
Summary: In humans, goldenseal may cause convulsions and irritation of the mouth, throat, and stomach when taken orally in toxic doses (doses not specified). Paresthesia, paralysis, respiratory failure, and death may follow. Chronic use may inhibit vitamin B absorption, and sublethal doses (doses not specified) may induce labor if taken during pregnancy. Berberine, on the other hand, was found to markedly improve cardiac performance in patients with heart conditions when taken at 0.02 mg/kg/min (59 nmol/kg/min) for 30 minutes, followed by 0.2 mg/kg/min (0.59 mol/kg/min) for an additional 30 minutes. However, further elucidation of effects from berberine administration to heart failure patients is necessary due to the finding of ventricular tachycardia in some subjects. Berberine had an anesthetic effect when injected s.c (dose not specified). Both berberine and hydrastine produced parasympatholytic and anesthetic effects when applied to the eyes. Hydrastine induced labor when taken orally by pregnant women (0.5 g; 1.30 mmol).
No chemical disposition, metabolism, or toxicokinetics data were found for goldenseal or hydrastine. In humans, berberine sulfate is absorbed through the skin. Following oral administration (species not provided), berberine was absorbed slowly, taking 4 hours to reach peak concentrations in plasma and another 4 hours to clear. In rats orally administered tritiated berberine chloride, blood levels of the compound leveled off at 4 - 24 hours; peak levels in liver and muscles occurred at 12 hours, while urinary excretion peaked at 12-24 hours. At 48 hours, the majority of the administered dose had been excreted in feces. Following i.v. administration to rats, the highest concentrations of berberine were found in the kidneys, with lower concentrations in the liver, lung, and brain. In rabbits, 24 hours after administration by gavage, small amounts of berberine were found in the heart, liver, and kidneys. Uptake into cells and across epithelia may involve a cation exchange mechanism.
Berberine sulfate injection reduced urine volume and urinary concentrations of sodium and chloride in dogs and rats. Additionally, dogs had lower urinary creatinine concentrations and rats had lower urinary potassium levels.
No acute exposure data for goldenseal were located. In mice, the oral LD50 dose for berberine is 329 mg/kg (0.98 mmol/kg), the s.c. LD50 dose is 18 mg/kg (0.054 mmol/kg), and the i.p. LD50 for berberine sulfate is 24.3 mg/kg (0.056 mmol/kg). In rats, the i.p. LD dose for berberine is greater than 500 mg/kg (>1.49 mmol/kg); while the LD50 doses for berberine sulfate are 14.5 mg/kg (0.033 mmol/kg) intramuscularly, 88.5 or 205 mg/kg (0.20 or 0.47 mmol/kg) i.p., and greater than 1000 mg/kg (>2.31 mmol/kg) orally. In rabbits, the s.c. LDLo dose is 100 mg/kg (0.30 mmol/kg). In rats, the i.p. LD50 dose for hydrastine is 104 mg/kg (0.271 mmol/kg).
When tested as protection from amphetamine toxicity, berberine chloride (5 mg/kg; 0.013 mmol/kg) and berberine sulfate (15 mg/kg; 0.035 mmol/kg) injected i.p. had no significant effect in mice. Berberine sulfate reduced rectal temperature in albino rats injected i.p. with 50 mg/kg (0.12 mmol/kg) and a single intraintestinal injection (10 mg/kg; 0.023 mmol/kg) into the duodenum of rats had no effect on the volume or acidity of gastric fluid, nor did it effect the severity of gastric ulcers. When injected i.v. at 6 mg/kg (0.014 mmol/kg), it significantly increased the number of apomorphine-induced vomits in dogs. Berberine sulfate also reduced the blood pressure in rats, dogs, and cats following i.v. administration of 0.1-6.0 mg/kg (0.00023-0.014 mmol/kg).
Following i.p. administration at 10 and 15 mg/kg (0.023 and 0.035 mmol/kg), berberine sulfate induced lachrymation, pilomotor erection, and ptosis in mice, with maximal depression occurring two hours after drug administration. Cats administered 100 g/0.2 mL or 1 mg/0.2 mL (0.0013 or 0.013 mM) berberine chloride i.v. were sedated, inactive, showed no interest in surroundings, and did not consume food; these responses were more pronounced at the higher dose. In contrast, oral administration of berberine chloride to mice (dose not provided) did not depress spontaneous movement or coordinative motor activity and i.p. injection of 5 mg/kg (0.013 mmol/kg) berberine chloride also had no tranquilizing or anticonvulsant properties. Ocular application of berberine (0.1% solution) did not have an anesthetic effect on rabbits or dogs.
Conflicting information regarding hydrastine toxicity was found. In one study, hydrastine hydrochloride (29.8 mg/kg; 71.0 mmol/kg) caused convulsions within one minute in 50% of the mice administered one i.v. injection, while in another study, hydrastine did not cause convulsions in mice, even when administered at lethal doses (dose range not provided).
No subchronic information was found for goldenseal or hydrastine. Subchronic exposure of rats given berberine chloride by gavage, 100 or 500 mg/kg (0.27 or 1.34 mmol/kg) for 7 or 14 days, significantly reduced scopolamine-induced amnesia effects but 100 mg/kg (0.27 mmol/kg) administered for 14 days did not significantly alter motor activity. Subchronic exposure of rats to 500 mg/kg/day (1.15 mmol/kg/day) berberine sulfate orally for 6 weeks did not induce histopathological changes in tissues or organs.
No chronic exposure data or teratogenicity or embryotoxicity studies were found for any of the compounds.
No data were found on carcinogenicity, although some studies tested the potential anticarcinogenic effect of berberine. In two experiments using mice, berberine did not inhibit the growth of mouse sarcoma S-180 tumor cells in ascites form. Berberine injected i.p. into mice was effective in preventing the growth of P388 leukemia cells (administered i.v. or intracerebrally). Berberine sulfate also significantly inhibited the tumor yield and incidence of mice bearing tumors which were initiated with DMBA and promoted with teleocidin. In rats implanted in the brain with 9L-2 rat gliosarcoma cells, berberine administration effectively killed 81% of the gliosarcoma cells.
No genotoxicity data were found for goldenseal or hydrastine. Berberine binds to DNA by intercalation with an AT base pair preference. As a DNA intercalating agent, berberine caused a general reduction in cellular RNA and protein synthesis and overproduction of the beta and beta1 subunits of RNA polymerase in E. coli. In sonicated and superhelical calf thymus DNA, berberine chloride induced DNA unwinding. Berberine chloride induced his gene mutations in S. typhimurium strain TA98 in the absence but not in the presence of metabolic activation. It was not mutagenic in strain TA100, with or without metabolic activation. When S. cerevisiae cells were cultured in growth medium in the absence of metabolic activation, berberine chloride induced hom3-10 frameshift mutations and cytoplasmic 'petite' mutations in a dose dependent manner; it also induced cyh crossing over. However, berberine chloride did not induce mutations at the hom3-10 or 'petite' mutations when added to cells incubated in saline, nor did it induce leu gene conversions or lys1-1 and his1-7 point mutations when cells were cultured either in growth medium or saline. In a DNA damage assay, berberine chloride did not induce beta-galactosidase activity in E. coli strain PQ37 in the presence or absence of S9. Treatment with berberine induced a dose dependent increase in sister chromatid exchanges (SCE) in 9L rat intracerebral gliosarcoma cells.
No immunotoxicity data were found for goldenseal or hydrastine. Berberine inhibited DNA synthesis in mitogen-stimulated human lymphocytes, but did not inhibit polymorphonuclear leukocyte activation, nor was it cytotoxic to target cells in a C3H/He mouse MM2 tumor cell assay.
Berberine inhibited the growth of cultured mouse sarcoma-180 tumor cells, an effect that was ameliorated by cotreatment with glucose. It also inhibited the growth of cultured human brain tumor cells and HepG2 human hepatoma cells, and was cytotoxic to Molt-4 and L1210 human leukemia cells and P-388 murine leukemia cells. However, berberine was not cytotoxic towards C38 murine colon adenocarcinoma cells or L1210 mouse leukemia cells. Berberine and berberine chloride induced apoptosis in Balb/c 3T3 fibroblast cells and human HL-60 leukemia cells, respectively. In cultured 9L rat gliosarcoma cells, berberine induced lysis, encystation, and degeneration. Berberine chloride inhibited the growth of cultured HeLa cells, and was toxic to T2/D1 human teratocarcinoma cells and F9 murine teratocarcinoma cells. Berberine sulfate inhibited the incorporation of 32Pi into phospholipids induced by TPA and teleocidin. It also inhibited TPA-enhanced transport of 3H-3-O-methyl-D-glucose into mouse fibroblast 3T3 cells. Berberine reversed the resistance of BEL-7402 human liver cancer cells to the cytotoxic effects of vincristine, but not of MCF-7/Adr human breast cancer cells to adriamycin. Down-regulation of the Ki-ras2 protooncogene was induced by berberine chloride in treated T2/D1 human teratocarcinoma cells.
Berberine did not induce phototoxicity in E. coli strains RT7h, RT8h, RT9h, or RT10h.
Berberine acted as an anticholinergic in isolated guinea pig ileum, isolated tracheal muscles of dogs, and isolated rectus muscle of frogs. Berberine sulfate exhibited an antiadrenergic response in isolated rabbit aortic strips, guinea pig seminal vesicle, and rat aortas. At low concentrations, it elicited a spasmogenic response in isolated guinea pig ileum, while at higher concentrations, a spasmolytic effect was observed. Berberine sulfate had no effect on tracheal muscle preparations from dogs or guinea pigs, isolated rabbit aortic strips, depolarized guinea pig ileum, or guinea pig seminal vesicle, but showed an antihistaminic response in isolated guinea pig ileum. It also potentiated calcium chloride-induced contractions and PGE1-, PGF1-, and PGF2-induced contractions in isolated guinea pig ileum. Berberine sulfate was reported to both inhibit and potentiate PGF2-induced contractions in isolated guinea pig ileum. It had a negative inotropic effect on isolated heart preparations and rabbit intestine; it had a positive inotropic and positive chronotropic effect on spontaneously beating atria of rats, guinea pigs, and rabbits. In isolated guinea pig ventricular papillary muscle, berberine inhibited the hypoxic-condition- or cromakalim-induced shortening of action potential and effective refractory period.
Hydrastine had no effect on adrenaline-induced contractions in rabbit aortic strips, nor on unstimulated guinea pig ileum or mouse vas deferens preparations, but it had a negative inotropic but positive chronotropic effect on spontaneously beating rat atrium, and inhibited electrically evoked contractions in guinea pig ileum. Hydrastine induced a positive inotropic effect on electrically evoked contractions of isolated mouse vas deferens.
Berberine sulfate reduced bull sperm motility in a dose dependent manner.
Berberine and protoberberine alkaloids have varying DNA binding affinities, but generally binding is very weak; substitution in bulky groups of protoberberine alkaloids inhibited DNA binding. Another study found that only quaternary salts bind with DNA. Berberine inhibited topoisomerase II, but not topoisomerase I. The increased planarity of berberine is thought to account for its enhanced activity in topoisomerase II inhibition. Cleavage of both methylenedioxy and methoxyl groups from berberine forms a compound which is a potent topoisomerase I poison; minor variations in the protoberberines may substantially alter their pharmacological properties.
With regard to induction of convulsion
following i.v. injection into mice, (-)-hydrastine was much less
potent than (+)-hydrastine. Further, (-)-beta-hydrastine and (±)-beta-hydrastine
were less potent than (+)-alpha-hydrastine and (±)-alpha-hydrastine
in inducing binding to the GABAA receptor in rat brain synaptic
membranes.
9.1.1 Human Data
Goldenseal can cause convulsions, and irritation of the mouth, throat, and stomach when taken orally in toxic doses (doses not specified) (Hamon, 1990). Paresthesia, paralysis, respiratory failure, and death may follow. Chronic use of goldenseal may inhibit absorption of vitamin B. If taken in sublethal doses (doses not specified) during pregnancy, goldenseal may induce labor.
In a group of 12 patients with heart conditions refractory to conventional medical treatment, berberine markedly improved cardiac performance (Marin-Neto et al., 1988). Berberine was administered for 30 minutes by intravenous (i.v.) infusion at a rate of 0.02 mg/kg/min (0.000059 mmol/kg/min) followed by infusion at a rate of 0.2 mg/kg/min (0.00059 mmol/kg/min) for an additional 30 minutes. However, 4 of the 12 patients experienced "torsades de pointes" morphology (ventricular tachycardia characterized by a marked QT prolongation on the electrocardiogram) within 20 hours after berberine infusion, suggesting further studies need to be undertaken before berberine administration is extended to other heart failure patients.
Berberine had an anesthetic effect when injected (amount not specified) s.c. in a volunteer (Seery and Raymond, 1940). Additionally, berberine (0.5%) had parasympatholytic and anesthetic activity when applied to the eyes of one human volunteer (Medow and Greco, 1975). In another study conducted by Medow and Greco (1976), dilation of the pupils, temporary paralysis of pupil ciliary muscles, and corneal anesthesia were observed in a human volunteer treated ocularly with 3 drops of 0.1% and 0.5% berberine in methyl cellulose. Eye treatment with hydrastine (0.5%) exhibited the same parasympatholytic and anesthetic effects as berberine in a study of one human volunteer (Medow and Greco, 1975).
As occurs with goldenseal, oral administration of 0.5 g (1.3 mmol)
hydrastine to pregnant women may induce labor (Grismondi et al.,
1979).
9.1.2 Chemical Disposition, Metabolism, and Toxicokinetics
Chemical disposition, metabolism, and toxicokinetics data were not found for goldenseal or hydrastine. The following summarizes information found for berberine and berberine salts.
In humans, berberine sulfate is absorbed through the skin (HSDB, 1997). In another study, berberine was slowly absorbed following oral administration (dose and species not provided) (Rennick, 1981; cited by Baird et al., 1997). It took 4 hours to reach a peak concentration in plasma and another 4 hours for berberine to clear. As an organic cation, berberine uptake into cells and across epithelia may involve a cation exchanger mechanism.
When 35 mg/kg (0.10 mmol/kg) berberine was administered i.v. to Sprague-Dawley rats, the highest concentrations were found in the kidney, with lower concentrations found in the liver, lung, and brain, respectively (time since administration not provided) (Bodor and Brewster, 1983). Berberine was rapidly lost from the tissues, except for the brain.
In rats (strain not provided) orally administered tritiated berberine chloride (dose not provided), blood levels of the compound leveled off at 4-24 hours (Sakurai et al., 1976). Peak levels in the liver and muscles occurred at 12 hours, while urinary excretion peaked at 12-24 hours. At 48 hours, 2.7% of the administered dose had been excreted in the urine and 86% of the dose had been excreted in feces.
In another study (Yamahara et al., 1972), 72 hours after oral administration of 10 mg (0.027 mmol) berberine chloride to rats (strain not provided), 60-91% of the dose was recovered in feces.
Twenty-four hours after administration of berberine (dose not provided) by gavage to rabbits, only small amounts (not specified) of the compound were detected in the heart, liver, and kidneys (Wang et al., 1995).
Berberine sulfate (10 or 30 mg/kg; 0.023 or 0.069 mmol/kg) administered i.p. to adult male albino rats preloaded orally with normal saline caused a reduction in urinary volume over the following 24-hour period accompanied by a decline in urinary sodium and an increase in urinary potassium and chloride concentrations (Sabir et al., 1978). There was no change in urinary pH.
In dogs (breed not provided) i.v. administered sodium chloride solution, subsequent treatment with berberine sulfate (0.1-10 mg/kg; 0.00023-0.023 mmol/kg) by i.v. injection consistently reduced the urine volume and urinary concentrations of sodium, chloride, and creatinine (Sabir et al., 1978). Urinary potassium concentration and urinary pH remained unchanged. The duration of the observation period was not specified.
9.1.3 Acute Exposure
Acute toxicity values for berberine, berberine sulfate, and hydrastine
are presented in Tables 1, 2, and 3, respectively;
acute toxicity values for goldenseal were not available. Other
in vivo acute exposure data presented in this section are
outlined in Table 4.
9.1.3.1 Oral Administration
No data were found for hydrastine.
There was no depression of spontaneous
movement or coordinative motor activity in mice (strain not provided)
treated orally with berberine chloride (dose and duration of exposure
not provided) (Yamahara, 1976). There was also no inhibition of
chemical- and electro-shock-induced convulsion, morphine-induced
Straub's tail reaction, apomorphine-induced masticating motion,
or aggressive behavior induced by electrical stimulation. Berberine
chloride also did not potentiate a loss of righting reflex induced
by hypnotics (compounds not specified).
9.1.3.2 Ocular Administration
No data were found for hydrastine.
Ocular application of a 0.1% solution
of berberine (solvent not specified) to rabbits and dogs had no
anesthetic effect (Seery and Raymond, 1940).
In two studies, berberine sulfate at 0.1-6.0 mg/kg (0.00023-0.014 mmol/kg) (Sabir and Bhide, 1971) and 0.15-0.30 mg/kg (0.00035-0.00069 mmol/kg) (Sabir et al., 1978) reduced the blood pressure of rats (strain not provided). There was a reduction in blood pressure, left ventricular systolic pressure, and left ventricular end diastolic pressure in rats (strain not provided) injected i.v. with berberine (dose and duration of exposure not provided) (Fang et al., 1987). Heart rate was initially increased, but then gradually decreased, and cardiac contractility values were unchanged to increased.
Berberine sulfate reduced the blood pressure in dogs and cats (0.1-6.0 mg/kg [0.00023-0.014 mmol/kg]) (strains not provided) (Sabir and Bhide, 1971).
There was a significant increase in tone and amplitude of intestinal movements in dogs administered 1 or 5 mg/kg (0.0023 or 0.012 mmol/kg) berberine sulfate i.v. (Kulkarni et al., 1972).
| Table 1. Acute Toxicity Values for Berberine | ||||||
| oral | mouse (sex and strain n.p.) | 329 mg/kg
(0.98 mmol/kg) | Journal of Pharmacy 82:726 (1962; cited by RTECS, 1996) | |||
| s.c. | mouse (sex and strain n.p.) | 18 mg/kg
(0.054 mmol/kg) | Russian Pharmacology and Toxicology (English translation of Farmakologiya I Toksikologiva 31:129 (1968; cited by RTECS, 1996) | |||
| - | - | - | ||||
| i.p. | rat (sex and strain n.p.) | >500 mg/kg
(>1.49 mmol/kg) | Natl. Acad. Sci.5:17 (1953; cited by RTECS, 1996) | |||
| s.c. | rabbit (sex and strain n.p.) | 100 mg/kg
(0.30 mmol/kg) | Abdernalden's Handbuch der Biologischen Arbeitsmethoden 4:1289 (1935; cited by RTECS, 1996) | |||
Abbreviations: i.p. = intraperitoneal; n.p. = not
provided; s.c. = subcutaneous
| Table 2. Acute Toxicity Values for Berberine sulfate | ||||||
| i.m. | rat (sex and strain n.p.) |
(0.033 mmol/kg) | Kowalewski et al. (1975) | |||
| i.p. | rat (albino, sex n.p.) | 205 mg/kg
(0.47 mmol/kg) | Kulkarni et al. (1972) | |||
| - | rat (sex and strain n.p.) |
(0.20 mmol/kg) | Kowalewski et al. (1975) | |||
| - | mouse (male and female albino) |
(0.056 mmol/kg) | Sabir and Bhide (1971) | |||
| oral | rat (sex and strain n.p.) | >1000 mg/kg
(>2.31 mmol/kg) | Kowalewski et al. (1975) | |||
Abbreviations: i.m. = intramuscular; i.p. = intraperitoneal;
n.p. = not provided
| Table 3. Acute Toxicity Values for Hydrastine | ||||||
| i.p. | rat (sex and strain n.p.) | 104 mg/kg
(0.248 mmol/kg) | MacDougal (year n.p.; cited by RTECS, 1996) | |||
Abbreviations: i.p. = intraperitoneal; n.p. = not provided
| Table 4. Acute Exposure to Berberine and Hydrastine | ||||||
| 9.1.3.1 Oral Administration | ||||||
| mouse (strain and age n.p.) | n.p. | berberine hydrochloride, purity n.p. | n.p. | n.p. | There was no depression of spontaneous movement or coordinative motor activity. There was also no inhibition of chemical- and electro-shock-induced convulsion, morphine-induced Straub's tail reaction, apomorphine-induced masticating motion, or aggressive behavior induced by electrical stimulation. Berberine hydrochloride did not potentiate a loss of righting reflex induced by hypnotics (compounds not specified). | Yamahara (1976) |
| 9.1.3.2 Ocular Administration | ||||||
| rabbit (strain and age n.p.) | n.p. | berberine, purity n.p. | 0.1% solution (solvent n.p.) | n.p. | There was no anesthetic effect. No other experimental details were provided. | Seery and Raymond (1940) |
| dog (strain and age n.p.) | n.p. | berberine, purity n.p. | 0.1% solution (solvent n.p.) | n.p. | There was no anesthetic effect. No other experimental details were provided. | Seery and Raymond (1940) |
| 9.1.3.3 Intravenous Injection | ||||||
| rat (strain and age n.p.) | n.p. | berberine sulfate, purity n.p. | 0.15-0.30 mg/kg (0.00035-0.00069 mmol/kg) or 0.1-6.0 mg/kg (0.00023-0.014 mmol/kg) | n.p. | There was a reduction in blood pressure. Rats were anesthetized with urethane. | Sabir et al. (1978); Sabir and Bhide (1971) |
| rat (strain and age n.p.) | n.p. | berberine, purity n.p. | n.p. | n.p. | There was a reduction in blood pressure, left ventricular systolic pressure, and left ventricular end diastolic pressure. Heart rate was initially increased, but then gradually decreased. Cardiac contractility values were unchanged to increased. The rats were conscious during testing. | Fang et al. (1987) |
| dog (strain and age n.p.) | n.p. | berberine sulfate, purity n.p. | 0.1-6.0 mg/kg (0.00023-0.014 mmol/kg) | n.p. | There was a reduction in blood pressure. Dogs were anesthetized with sodium pentobarbitone (35 mg/kg i.p.). | Sabir and Bhide (1971) |
| cat (strain and age n.p.) | - | - | - | - | There was a reduction in blood pressure. Cats were anesthetized with sodium pentobarbitone (35 mg/kg i.p.). | - |
| Table 4. Acute Exposure to Berberine and Hydrastine (continued) | ||||||
| dog (strain and age n.p.) | n.p. | berberine sulfate, purity n.p. | 1 or 5 mg/kg (0.23 or 0.012 mmol/kg) | n.p. | There was a significant increase in tone and amplitude of intestinal movements. The dogs were anesthetized. No other experimental details were given. | Kulkarni et al. (1972) |
| dog (strain and age n.p.) | exposed: 6
controls: 6 male and female, not broken down by sex | berberine sulfate, purity n.p. | 1 or 6 mg/kg (0.23 or 0.014 mmol/kg) | single dose; observation period n.p. | Apomorphine hydrochloride (0.1 mg/kg) was administered intramuscularly 1 h after injection of berberine sulfate. Berberine sulfate at 6 mg/kg (0.014 mmol/kg) significantly increased the number of vomits in dogs, as compared to dogs injected only with apomorphine hydrochloride. | Sabir et al. (1978) |
| cat (strain and age n.p.) | 3, sex n.p. | berberine hydrochloride | 100 g/ 0.2 mL (0.0013 mM) in 1 cat and 1 mg/ 0.2 mL (0.013 mM) in 2 cats | single dose; observed continuously for 3 h an then unspecified intervals until the cats returned to normal | Cats were sedated, inactive, disinterested, and did not consume food. Effects were dose-dependent. The cat administered 100 g/0.2 mL (0.0013 mM) recovered after 20 h. At the 1 mg/0.2mL (0.013 mM) dosage, the recovery time was 36-40 h. | Shanbhag et al. (1970) |
| mouse (strain and age n.p.) | n.p. | hydrastine hydrochloride, purity n.p. | n.p. | single injection; 1- min observation period | The CD50 (dose producing convulsions in 50% of animals within 1 minute of injection) for (+)-hydrastine hydrochloride was 0.16mg/kg (0.00038 mmol/kg) and for (-)-hydrastine hydrochloride was 29.8 mg/kg (71.0 mmol/kg). | Huang and Johnston (1990) |
| mice (strain and age n.p.) | n.p. | hydrastine, purity n.p. | n.p. | single dose; observed for the 1st 30 m and then 12 and 24 h later | -Hydrastine did not induce convulsions, even at lethal doses. No other experimental details were given. | Bartolini et al. (1990) |
| 9.1.3.4 Intraperitoneal Injection | ||||||
| albino mice (age n.p.) | M, number n.p. | berberine hydrochloride (purity n.p.) | 5 mg/kg (0.013 mmol/kg) after 4 mg/kg amphetamine | single dose; observation period n.p. | Amphetamine administration markedly increased motor activity. Berberine administration at peak activity of amphetamine sedated the mice in 5 min., reducing motor activity below the control (no treatment). | Shanbhag et al. (1970) |
| - | 40 (10 at each dose level), sex n.p. | - | 5 mg/kg (0.013 mmol/kg) berberine followed in _ h by 25, 30, 35, or 40 mg/kg pentobarbitone | - | Berberine significantly increased sleeping time at the 35 and 40 mg/kg pentobarbitone dose levels. | - |
| albino mice (age n.p.) | 10M | berberine hydrochloride (purity n.p.) | 5 mg/kg (0.013 mmol/kg) berberine followed in 0.5 h by 15 mg/ kg amphetamine | single dose; observed for 20 h | No significant modification in aggregate or segregate amphetamine toxicity. | Shanbhag et al. (1970) (cont.) |
| - | 30 (10 at each dose level), sex n.p. | berberine hydrochloride (purity n.p.) | 5 mg/kg (0.013 mmol/kg) berberine followed in 0.5 h by 20, 50, or 75 mg/ kg leptazol | single dose; observation period n.p. | Berberine had no significant action on convulsions caused by leptazol. | - |
| - | 20M (10 treatment and 10 control) | - | 5 mg/kg (0.013 mmol/kg) berberine followed a shock of 120 mA for 0.2 sec. | - | Berberine did not protect mice from supramaximal electroshock convulsions. | - |
| freshly weaned albino mice | 32 (16 controls, and 8 at each dose level), sex n.p. | berberine sulfate (purity n.p.) | 10 and 15 mg/kg (0.023 and 0.035 mmol/kg) | single dose; observed after 5, 15, and 30 min. and after 1, 2, and 4 h and then daily for 4 days. | Maximal overall depression by berberine exposure observed after 2 h. Stimulation of respiration observed more with the higher dose group. Complete recovery noted after 24 h. | Mardikar et al. (1973) |
| - | n.p. | - | 15 mg/kg (0.035 mmol/kg) | single dose; observation period n.p. | The rotating rod test was used with previously-trained mice. Berberine-treated animals lost balance quickly and fell down in less than 300 sec. | - |
| - | 10 for each test, sex n.p. | - | - | single dose; 2 and 4 h., respectively for the two tests. | Previously screened mice were used in both the chimney and the traction tests. Mice which could reach the 20 cm mark within 30 sec. without treatment (chimney test) were not able to perform the test for up to 4 h after berberine treatment. In the traction test, the animal must touch a wire within 5 sec. to be sucessul. 50% of the animals could not perform the test in the 2 h immediately after drug administration. | - |
| freshly weaned albino mice | 20, sex n.p. | berberine sulfate (purity n.p.) | 15 mg/kg (0.035 mmol/kg) | single dose; 15 minute observation period | In the evasion test, mice were placed in a small open box within a large trough to test residual curiosity. Two inclined planes allowed escape from the small box. Berberine treatment significantly lowered residual curiosity levels. | Mardikar et al. (1973) (cont.) |
| - | 20 (10 treated and 10 control), sex n.p. | - | - | single dose; half hourly for 4 _ h | Berberine treatment lowered rectal temperature, with a peak drop after 2 h. | - |
| - | 20 (10 treated and 10 control) | - | treatment:15 mg/kg (0.035 mmol/kg) berberine followed in 2 h by 40 mg/kg pentobarbitone
control: 40 mg/kg pentobarbitone | n.p. | Berberine pretreatment dramatically increased pentobarbitol-induced sleeping time. | - |
| - | 40 (20 treated and 20 control) | - | treatment: 15 mg/kg (0.035 mmol/kg) berberine followed by 14 mg/kg amphetamine
control: 14 mg/kg amphetamine | 24 h | Amphetamine-induced mortality rates were not changed by berberine pretreatment. However, immediately after amphetamine administration, animals pretreated with berberine did not show hyperactivity as compared to controls. | - |
| rats (strain and age n.p.) | 30 (10 in each treatment group), sex n.p. | berberine hydrochloride (purity n.p.) | 5 mg/kg (0.013 mmol/kg) berberine in untreated rats, rats treated with 5 mg/kg morphine, or 15 mg/kg pentobarbitone
(berberine was injected _ h prior to treatment) | single dose; observation period n.p. | Hot wire test was used to test pain threshold. Berberine did not produce any effect. | Shanbhag et al. (1970) |
| rat (albino adult) | M, number n.p. | berberine sulfate, purity n.p. | 10, 30, or 50 mg/kg (0.023, 0.069, or 0.12 mmol/kg) | single dose; 5-h observation period | The highest dose significantly lowered rectal temperature; hypothermia persisted over the entire observation period. The 2 lower doses had no effect on rectal temperature. Rectal temperature was the only parameter evaluated. | Sabir et al. (1978) |
| rat (albino, age n.p.) | 10 M in each group (exposed and controls) | berberine sulfate, purity n.p. | 10 or 30 mg/kg (0.023 or 0.069 mmol/kg) | single dose; 5-h observation period | All rats were previously injected with Brewer's yeast suspension s.c. Only rats that exhibited an increase in temperature of at least 1.5C after 18 h were included in this study. Berberine sulfate (10 or 30 mg/kg; 0.023 or 0.069 mmol/kg) significantly lowered the rectal temperature of pyretic rats. | Sabir et al. (1978) (cont.) |
| cat (strain and age n.p.) | 9 (3 at each dose level), sex n.p. | berberine hydrochloride (purity n.p.) | 5, 20, or 40 mg/kg (0.013, 0.054, or 0.11 mmol/kg) in a volume of 5 mL | single dose; observed continuously for 3 h and then hourly for the next 12 h | The following effects were noted: sedation, retching, urination, and defecation with straining in 8 cats (effects lasted less than 2 h); rage reaction in 1 cat (returned to normal after 1 h). | Shanbhag et al. (1970) |
| 9.1.3.5 Intraintestinal Injection | ||||||
| rat (Shay, age n.p.) | 12 in both exposed and control groups, sex, n.p. | berberine sulfate, purity n.p. | 10 mg/kg (0.023 mmol/kg) | single dose; 18-h observation | Dose was injected into the duodenum. Controls injected with distilled water. Berberine sulfate had no effect on the volume or acidity of gastric fluid, or on the severity of gastric ulcers. | Sabir et al. (1978) |
Berberine sulfate significantly increased the number of apomorphine-induced vomits in dogs (strain not provided) injected i.v. with 6 mg/kg (0.014 mmol/kg) (Sabir et al., 1978). A lower dose of berberine sulfate (1 mg/kg; 0.0023 mmol/kg) had no effect on the number of vomits.
Cats showed a dose-dependant response to berberine chloride when given in doses of 100 g/0.2 mL (0.0013 mM) and 1 mg/0.2 mL (0.013 mM), respectively (Shanbhag et al., 1970). With berberine chloride administration, cats were sedated, inactive, showed no interest in surroundings, and did not consume food.
Hydrastine hydrochloride caused convulsions
in male mice (strain not provided) when treated once by i.v. injection
(Huang and Johnston, 1990). The CD50 (dose producing
convulsions in 50% of animals within 1 minute of injection) was
29.8 mg/kg (71.0 mmol/kg). However, in another study, hydrastine
did not induce convulsions even when administered at lethal doses
(dose range not provided) to mice (strain not provided) (Bartolini
et al., 1990).
9.1.3.4 Intraperitoneal Injection
No data were found for hydrastine.
Berberine administered i.p. appears to affect the central nervous system, producing sedation in mice (Shanbhag et al., 1970). However, it was found to have no tranquilizing or anticonvulsant properties in white mice administered a 5 mg/kg (0.013 mmol/kg) i.p. dose of berberine chloride. Berberine also had no analgesic properties when administered i.p. (5 mg/kg, 0.013mmol/kg) as treatment for aggregate or segregate amphetamine toxicity in mice.
Berberine sulfate doses of 10 and 15 mg/kg (0.023 and 0.035 mmol/kg) administered i.p. to male and female albino mice produced maximal depression of the autonomic system two hours after drug administration (Mardikar et al., 1973). It caused lachrymation, pilomotor erection, and ptosis. Treatment with 15 mg/kg (0.035 mmol/kg) lowered rectal temperature significantly, with a peak drop occurring after two hours. No significant effect was identified when berberine sulfate (15 mg/kg; 0.035 mmol/kg i.p.) was tested as protection against amphetamine toxicity.
Berberine had no analgesic properties when administered i.p. (5 mg/kg, 0.013 mmol/kg) as a pain threshold reducer in rats (Shanbhag et al., 1970). Berberine sulfate (50 mg/kg; 0.12 mmol/kg) significantly lowered the rectal temperature in adult male albino rats injected once i.p. and observed for 5 hours (Sabir et al., 1978). Administration of lower doses (10 or 30 mg/kg; 0.023 or 0.069 mmol/kg) had no effect on the normal rectal temperatures. However, when the lower doses were administered to male albino rats that had been previously injected s.c. with Brewer's yeast suspension, rectal temperature was significantly decreased.
Berberine produced sedation in cats
(Shanbhag et al., 1970). Nine cats were dosed with 5, 20, or 40
mg/kg (0.013, 0.054, 0.11 mmol/kg) berberine hydrochloride (3
at each dose level). Eight cats experienced sedation, retching,
urination, and defecation with straining, with effects lasting
less than 2 hours; one cat experienced a rage reaction, but returned
to normal after 1 hour.
9.1.3.5 Intraintestinal Injection
No data were found for hydrastine.
A single injection of berberine sulfate
(10 mg/kg; 0.023 mmol/kg) into the duodenum of Shay rats had no
effect on the volume or acidity of gastric fluid, or on the severity
of gastric ulcers (Sabir et al., 1978).
9.1.4 Short-Term and Subchronic Exposure
No information relating to short-term and subchronic exposure was found for goldenseal or hydrastine. The studies described in this section are presented in Table 5.
Berberine chloride (150 mg/kg; 0.00040 mmol/kg) did not stimulate liver regeneration in partially hepatectomized Charles River and Holtzman male rats when injected s.c. daily for 7 days (Gershbein and Pedroso, 1985). The rats were killed 3 days after the last injection.
Administration by gavage of berberine chloride (100 or 500 mg/kg; 0.27 or 1.34 mmol/kg in 0.9% saline) for 7 or 14 days significantly reduced scopolamine-induced amnesia effects in male Sprague-Dawley rats (Peng et al., 1997) when amnesia was measured using performance of a passive avoidance response task. The antiamnesic effect of berberine chloride was significantly increased by administration of physostigmine or neostigmine, and was completely reversed by administration of scopolamine N-methyl bromide. There was not a significant change in motor activity in male Sprague-Dawley rats treated by gavage with berberine chloride (100 mg/kg; 0.27 mmol/kg in 0.9% saline) for 14 days (Peng et al., 1997). Combined administration of scopolamine and berberine chloride (500 mg/kg; 1.34 mmol/kg) also did not affect motor activity of rats, as compared to the motor activity of rats treated only with scopolamine.
There were no histopathological changes
in tissues or organs of rats (strain not given) treated orally
with berberine sulfate (500 mg/kg/day; 1.15 mmol/kg/day) for 6
weeks (Kowalewski et al., 1975). It was not specified which tissues
and organs were examined.
No data were found.
| Table 5. Short-Term and Subchronic Exposure to Berberine | ||||||
| Species, Strain, Age | ||||||
| Charles River and Holtzman rats (age n.p.) | 13-18 M | berberine chloride | 150 mg/kg (0.00040 mmol/kg), s.c. | berberine chloride administered for 7 d | Rats were partially hepatectomized before berberine hydrochloride administration.
Berberine chloride did not stimulate liver regeneration. | Gershbein and Pedroso (1985) |
| rat (Sprague- Dawley, age n.p.) | exposed: 12-18 M per group
controls: 12-18 M | berberine hydro- chloride, purity n.p. | 100 or 500 mg/kg (0.27 or 1.34 mmol/kg) by gavage in 0.9% saline | 7 or 14 days; training trial carried out 1 h after administration of last dose | Rats were previously treated i.p. with 1.0 mg/kg scopolamine and tested for compound-induced amnesia using performance of a passive avoidance response task as an indicator. Administration of berberine hydrochloride (100 or 500 mg/kg; 0.27 or 1.34 mmol/kg) for 7 or 14 days significantly improved scopolamine-induced amnesia. The antiamnesic effect of berberine hydrochloride was significantly increased by administration of physostigmine (0.02 mg/kg i.p.) or neostigmine (0.02 mg/kg i.p.), and was completely reversed by administration of scopolamine N-methylbromide (0.05 mg/kg i.p.). | Peng et al. (1997) |
| - | exposed: 6 M per group
controls: 6 M | - | - | 14 days; motor activity measurement carried out 1 h after administration of last dose | There was no change in motor activity of berberine hydrochloride-treated rats, as compared to vehicle-treated controls. Combined administration of scopolamine (1.0 mg/kg i.p.) and berberine hydrochloride (500 mg/kg; 1.34 mmol/kg) also did not affect motor activity of rats, as compared to the motor activity of rats treated only with scopolamine. | - |
| rat (strain and age n.p.) | n.p. | berberine sulfate, purity n.p. | 500 mg/kg/d orally (1.15 mmol/kg/d) | 6 wk; observation period n.p. | There were no histopathological changes in tissues or organs (not specified). | Kowalewski et al. (1975) |
9.2 Teratogenicity and Embryotoxicity
No data were found.
No data on carcinogenicity were found,
although some studies were conducted to test the anticarcinogenicity
of berberine. These studies are described in Section 9.4.
The studies described in this section are presented in Table 6; no information was available for goldenseal or hydrastine.
Berberine injected i.p. into dd mice inoculated with mouse sarcoma-180 ascites cells did not exhibit antitumor activity (measured as the total packed cell volume [TPCV] of sarcoma-180 ascites cells) (Hoshi et al., 1976). Mice were administered 3, 10, or 30 mg/kg (0.0089, 0.030, or 0.089 mmol/kg) berberine i.p. daily for 5 days, starting 24 hours after i.p. transplant of the ascites cells.
Creasey (1979) performed a comparative study of the biochemical interactions of berberine on sarcoma-180 tumor cells in the ascites form using in vivo and in vitro analyses.
The first in vivo analysis investigated the effect of berberine on the lifespan of Swiss white mice inoculated i.p. with sarcoma-180 ascites cells and then administered 2.5, 5, 10, 15, or 20 mg/kg/day berberine chloride (0.0067, 0.013, 0.027, 0.040, or 0.054 mmol/kg/day) i.p. for 5 days. The berberine chloride doses were initially given 24 hours after transplantation of tumor cells. Survival of tumor-inoculated mice was not increased by treatment with berberine chloride; in fact a dose-dependent decrease in life span compared to mice inoculated with tumor cells was noted. The second in vivo analysis assessed Swiss white mice administered 10 mg/kg (0.027 mmol/kg) berberine chloride i.p. 30 minutes before injection of [1-14C]glycine or [methyl-3H]thymidine. Berberine chloride had only minimal effects on the incorporation of the precursors, inhibiting incorporation of glycine into protein by 17% and thymidine into DNA by 14%. In a third analysis, cells were harvested 30 minutes after treating Swiss white mice in vivo with 10 mg/kg (0.027 mmol/kg) berberine chloride i.p. The harvested cells were washed and then incubated with labeled glycine in vitro. Treatment with berberine chloride resulted in an 85% inhibition of glycine into protein. In a fourth analysis using an in vitro approach, 5 g/mL (0.013 M) berberine chloride and glucose (varying doses) were incubated in medium with the sarcoma-180 cell suspension for 30 minutes before the addition of [14C]glycine or [3H]thymidine. This treatment partially or completely blocked the berberine chloride-induced inhibition of glycine and
| Table 6. Anticarcinogenic Activity of Berberine | ||||||
| sarcoma-180 ascites transplanted i.p. into dd mice | total packed cell volume (TPCV) | berberine, purity n.p. | 3, 10, or 30 mg/kg/d (0.0089, 0.030, or 0.089 mmol/kg/d) i.p. for 5 d, starting 24 h after transplant of tumor cells | Berberine had no antitumor activity. | TPCV was measured 7 days after transplant of ascites. | Hoshi et al. (1976) |
| sarcoma-180 ascites transplanted i.p. into Swiss white mice | survival | berberine hydrochloride, purity n.p. | 2.5, 5, 10, 15, or 20 mg/kg/d (0.0067, 0.013, 0.027, 0.040, or 0.054 mmol/kg/d) i.p. for 5 d, starting 24 h after transplant of tumor cells | There was no prolongation of survival. Life span decreased with increasing berberine hydrochloride dose. | Only 3 injections of the 15 and 20 mg/kg doses were tolerated by the mice. | Creasey (1979) |
| - | incorporation of thymidine into DNA and glycine into protein | - | 10 mg/kg (0.027 mmol/kg) i.p. 30 min before injection of [1-14C]glycine or [methyl-3H]thymidine | Glycine and thymidine uptake were inhibited by 17% and 14%, respectively, in vivo. | To test effects of glucose, berberine hydrochloride and glucose were incubated in vitro 30 min before the addition of [1-14C]glycine or [methyl-3H]thymidine. Addition of glucose to the incubation medium partially or completely blocked the berberine hydrochloride-induced inhibition of thymidine into DNA and glycine into protein, respectively. The author noted that the failure of berberine to inhibit growth in vivo may be related to the effects of glucose. | - |
| - | incorporation of glycine into protein | - | 10 mg/kg (0.027 mmol/kg) i.p. 30 min. before cells were harvested, washed, and then incubated with [1-14C]glycine in vitro. | Incorporation of glycine into protein was inhibited by 85%. | - | - |
| BDF mice (male, age n.p.) | 24 (6 at each treatment level and 6 in control group) | berberine, purity n.p. | P388 lymphocytic leukemia cells administered i.p. or intracerebrally. Berberine administered i.p. 3 times a day on day 2, 6, and 10 at treatment levels of 5, 10, or 20 mg/kg (0.015, 0.030, or 0.059 mmol/kg). | Prevented growth of lymphocytic leukemia cells. | - | Bodor and Brewster (1983) |
| Table 6. Anticarcinogenic Activity of Berberine (continued) | ||||||
| mouse (8-wk-old female ICR) | tumor induction | berberine sulfate, purity n.p. | initiation: Single application of 100 g DMBA onto dorsal skin
promotion: Teleocidin (2.5 g) applied dermally twice/wk for 18 wk starting 1 wk after initiation. Berberine sulfate (0.5 mg; 0.0012 mmol) in ethanol/dimethyl sulfoxide applied dermally 40 min before each teleocidin application | Tumor yield and incidence of tumor-bearing mice was significantly inhibited by berberine sulfate. At wk 18, approximately 85% of controls had tumors versus approximately 12% of berberine-sulfate-treated mice. The location of the tumors was not specified. | Controls received DMBA and teleocidin only. Berberine sulfate was not administered alone to any animals. | Nishino et al. (1986) |
| 9L or BCNU-resistant 9L-2 rat gliosarcoma cells implanted in brain of adult male Fischer 344 rats | in vitro colony forming efficiency of implanted cells | berberine, purity n.p. | 9L cells: berberine alone (10 mg/kg; 0.030 mmol/kg i.p.); BCNU alone (4.43 mg/kg i.p.); or berberine (10 mg/kg; 0.030 mmol/kg i.p.) and BCNU (4.43 mg/kg i.p.)
9L-2 cells: berberine alone (10 mg/kg; 0.030 mmol/kg i.p.); BCNU alone (6.66 mg/kg i.p.); or berberine (10 mg/kg; 0.030 mmol/kg i.p.) and BCNU (6.66 mg/kg i.p.) (dose was administered 14-15 d after tumor cell implantation) | In 9-L tumor-bearing rats, berberine alone and BCNU alone achieved 81% and 76% cell death, respectively. Treatment with both compounds produced 95.2% cell death. In 9L-2 tumor-bearing rats, berberine alone was inactive. | Rats were killed 24 h after berberine and/or BCNU treatment. Brain tumors were removed and converted into single-cell suspensions which were incubated for 12-14 days. | Zhang et al. (1990) |
thymidine incorporation. The author concluded that the failure of berberine to inhibit growth in vivo may be related to the effects of glucose.
Berberine (purity not given) at 5, 10, and 20 mg/kg (0.015, 0.030, and 0.059 mmol/kg) i.p. was effective in preventing the growth of P388 leukemia cells administered i.v. or intracerebrally to male BDF mice (Bodor and Brewster, 1983). Berberine was administered on days 2, 6, and 10.
In a 2-stage mouse skin carcinogenicity study, berberine sulfate significantly inhibited the tumor yield and the incidence of tumor-bearing animals initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted with teleocidin (Nishino et al., 1986). Female ICR mice were initiated with a single application of DMBA onto dorsal skin. One week later, teleocidin was applied dermally twice/week for 18 weeks; berberine sulfate (0.5 mg; 0.0012 mmol) dissolved in ethanol/dimethyl sulfoxide was applied dermally 40 minutes before each teleocidin application. At week 18, approximately 85% of controls had tumors, whereas only approximately 12% of berberine-sulfate-treated mice did. Berberine sulfate was not administered alone to any animal.
Zhang et al. (1990) evaluated the
anticarcinogenic activity of berberine and 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU) in adult male Fischer 344 rats implanted in the brain with
9L or BCNU-resistant 9L-2 rat gliosarcoma cells. Rats bearing
9L tumor cells were treated i.p., 14-15 days after implantation,
with berberine alone (10 mg/kg; 0.030 mmol/kg), BCNU alone (4.43
mg/kg), or a combination of berberine (10 mg/kg; 0.030 mmol/kg)
and BCNU (4.43 mg/kg). Rats bearing 9L-2 tumor cells were similarly
treated, but received a higher dose of BCNU (6.66 mg/kg). Rats
were killed 24 hours after treatment. Brain tumors were removed
and converted into single-cell suspensions which were incubated
for 12-14 days. In 9-L tumor-bearing rats, berberine alone and
BCNU alone achieved 81% and 76% cell death, respectively. Treatment
with both compounds produced 95.2% cell death. In 9L-2 tumor-bearing
rats, berberine alone was inactive.
No information was found for goldenseal
or hydrastine. The studies on berberine that are described in
this section are also presented in Table 7.
Berberine chloride induced DNA unwinding in sonicated and superhelical calf thymus DNA (Davidson et al., 1977). It binds to DNA by intercalation (Rungsitiyakorn et al., 1981; Smekal and Kubova, 1982), with an AT base pair preference (Kumar et al., 1993).
| Table 7. Genotoxicity of Berberine | |||||||
| 9.5.1 Acellular Assays | |||||||
| sonicated and superhelical calf thymus DNA | induction of DNA unwinding | n.p. | berberine chloride | 0.002-0.1 mol | positive for both sonicated and superhelical DNA | - | Davidson et al. (1977) |
| sonicated calf thymus DNA | DNA binding | n.p. | berberine, purity n.p. | 0.000034 mol | positive (probably by intercalation) | - | Rungsitiyakorn et al. (1981) |
| calf thymus DNA | DNA binding | n.p. | berberine chloride, chemically pure | 1 and 48.44 M | positive (binds by intercalation) | Smekal and Kubova (1982) | |
| calf thymus DNA | DNA binding | n.p. | berberine chloride, purity n.p. | n.p. | positive (showing an AT base pair preference) | - | Kumar et al. (1993) |
| 9.5.2 Prokaryotic Systems | |||||||
| Salmonella typhimurium strains TA98 and TA100 | his gene mutations | +/- | berberine hydrochloride, purity n.p. | n.p. | positive (TA98 without S9)
negative (TA98 with S9; TA100 with or without S9) | Berberine hydrochloride was weakly mutagenic (2 revertants/g/plate) in TA98 in the absence of S9. | Nozaka et al. (1990) |
| Escherichia coli strain PQ37 | induction of -galactosidase activity | +/- | berberine chloride, purity n.p. | 1, 5, 10, 50, 100, 500, 1000, 5000, or 10000 ng/plate (0.0027, 0.013, 0.027, 0.13, 0.27, 1.34, 2.68, 13.4, or 26.8 nM) for 2 h | negative at all doses, with and without S9 | SOS chromotest was used. | Pasqual et al. (1993) |
| 9.5.3 Lower Eukaryotic Systems | |||||||
| Saccharomyces cerevisiae haploid strain XV-185-14c | hom3-10 frameshift mutation | - | berberine chloride, purity n.p. | in saline, 75, 150, 225, or 300 g/mL (0.20, 0.40, 0.60, or 0.80 M)
in growth medium, 10, 20, 30, 40, or 50 g/mL (26.8, 53.6, 80.5, 107, or 134 nM) | positive (20-50 g/mL; 53.6-134 nM in growth medium)
negative at 10 g/mL (26.8 nM) in growth medium and 75-300 g/mL (0.20-0.80 M) in saline | Cells were maintained either in saline or in growth medium. | Pasqual et al. (1993) |
| - | cytoplasmic 'petite' mutation | - | - | - | - | ||
| Table 7. Genotoxicity of Berberine (continued) | |||||||
| S. cerevisiae haploid strain XV-185-14c | cyh crossing-over | - | berberine chloride, purity n.p. | in saline, 75, 150, 225, or 300 g/mL (0.20, 0.40, 0.60, or 0.80 M)
in growth medium, 25, 50, 75, or 100 g/mL (67.1, 134, 201, or 268 M) | positive at 25-100 g/mL (67.1-268 M) in growth medium
negative at 75-300 g/mL (0.20-0.80 M) in saline | cells were maintained either in saline or in growth medium | Pasqual et al. (1993) (cont.) |
| - | leu gene conversion | - | - | - | negative | - | - |
| lys1-1 point mutation | - | - | in saline, 75, 150, 225, or 300 g/mL (0.20, 0.40, 0.60, or 0.80 M)
in growth medium, 10, 20, 30, 40, or 50 g/mL (26.8, 53.6, 80.5, 107, or 134 nM) | negative | - | - | |
| - | his1-7 point mutation | - | - | - | - | - | - |
| 9.5.4 In Vitro Mammalian Systems | |||||||
| 9L rat intracerebral gliosarcoma cells | sister chromatid exchange (SCE) | n.p. | berberine, purity n.p. | 25-150 g/mL (0.07-0.45 M) for 2 h | There was a dose-dependent increase in SCE, up to 2.7-fold at the highest dose, as compared to untreated cells. | Cells were washed after treatment and incubated for 2 replication cycles (approximately 30 h) | Zhang et al. (1990) |
Berberine chloride (dose not provided) induced his gene mutations in S. typhimurium strain TA98 in the absence but not the presence of metabolic activation, and was nonmutagenic in strain TA100 in the presence or absence of metabolic activation (Nozaka et al., 1990).
Using the SOS chromotest, berberine
chloride (1-10,000 ng, 0.0000027-0.027 M for 2 hours) did not
induce beta-galactosidase activity in E. coli strain PQ37
in either the presence or absence of S9 (Pasqual et al., 1993).
9.5.3 Lower Eukaryotic Systems
Berberine chloride was tested for induction of mutation and recombination in S. cerevisiae which were either cultured and maintained in growth medium or were incubated in saline (Pasqual et al., 1993). All experiments were conducted in the absence of metabolic activation. In cells maintained in growth medium, berberine chloride (10-50 g/mL; 0.027-0.13 M) induced hom3-10 frameshift mutations and cytoplasmic 'petite' mutations in a dose-dependent manner; at doses of 25-100 g/mL (0.07-0.27 M), it induced cyh crossing-over. However, berberine chloride (75-300 g/mL; 0.20-0.80 M) did not induce hom 3-10 or 'petite' mutations when added to cells incubated in saline, nor did it induce leu gene conversions when added to cells maintained in growth medium (25-100 g/mL;0.07-0.27 M) or in saline (75-300 g/mL; 0.20-0.80 M). Berberine chloride did not induce lys1-1 or his1-7 point mutations at any dose tested in either medium.
9.5.4 In Vitro Mammalian Systems
There was a dose-dependent increase
in sister chromatid exchange (SCE) in 9L rat intracerebral gliosarcoma
cells treated with 25-150 g/mL (0.07-0.45 M) berberine for 2 hours
(Zhang et al., 1990).
No data were found for goldenseal or hydrastine. The immunotoxicity studies described in this section are also presented in Table 8.
In an in vitro cellular proliferation study using mitogen-stimulated human peripheral blood lymphocytes, the authors concluded that the effects of berberine, particularly the anti-inflammatory
| Table 8. Immunotoxicity of Berberine | |||||||
| Test System | |||||||
| Human peripheral blood lymphocytes, in vitro | inhibition of [3H]thymidine incorporation | berberine, purity n.p. | 2.5-20 g/mL (7.43-59.4 nM) | 85% inhibition with 20 g/mL (59.4 nM) berberine for phytohaemagglutinin and concanavalin A-stimulated lymphocytes and with 10 g/mL (29.7 nM) for pokeweed mitogen-treated lymphocytes.
Berberine induced a dose-dependent inhibition of DNA synthesis. | Cells were stimulated to divide by the addition of phytohaemagglutinin, concanavalin A, or pokeweed mitogen. | Ckless et al. (1995) | |
| C3H/He mouse MM2 transplantable ascites tumor | inhibition of PMN activation and cytotoxicity | berberine, purity n.p. | n.p. | Berberine showed neither toxicity to the target tumor cells nor inhibition of PMN activation. | - | Kinoshita et al. (1992) | |
action, may occur from the inhibition of DNA-synthesis (Ckless et al., 1995). In lymphocyte cultures stimulated with phytohemagglutinin and concavalin A, berberine at 20 g/mL (0.059 M) inhibited DNA synthesis by 85%. Inhibition of 85% was achieved in lymphocytes stimulated with pokeweed at a dose of 10 g/mL.
Kinoshita et al. (1992) investigated
the effects of berberine on polymorphonuclear leukocyte (PMN)
activation using a PMN cytotoxicity assay system. Berberine (dose
not specified) showed neither toxicity to the target MM2 tumor
cells from a spontaneous mammary carcinoma in a C3H/He mouse nor
inhibition of PMN activation.
9.7.1 Cytotoxicity and Apoptosis In Vitro
Berberine chloride (1 or 5 g/mL; 0.0027 or 0.013 M) caused a marked, but temporary, inhibition of growth of cultured mouse sarcoma-180 ascites cells (Creasey, 1979). Berberine chloride (4 or 40 g/mL; 0.011 or 0.11 M) also interfered with the in vitro production of nucleic acids, residual proteins, and lipids, and the oxidation of glucose in mouse sarcoma-180 ascites cells. The synthesis of nucleic acids, residual proteins, and lipids was measured by incorporation of thymidine[methyl-3H], glycine[1-14C], and sodium acetate[2-14C], respectively.
In HeLa cells, berberine sulfate (25 or 50 M for 4 hours) inhibited the incorporation of 32Pi into phospholipids induced by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA; 50 nM) and teleocidin (23 nM) (Nishino et al., 1986). The cells were not incubated with berberine sulfate alone. Berberine sulfate (25, 50, or 100 M for 4 hours) inhibited TPA (50 nM)-enhanced transport of 3H-3-O-methyl-D-glucose into mouse fibroblast Swiss 3T3 cells.
Berberine (25-150 g/mL; 0.07-0.45 M) was marginally effective in inhibiting the in vitro growth of human brain tumor cell lines; dose-dependent colony inhibition occurred in only 2/6 cells lines tested (Zhang et al., 1990). The cells were treated for 2 hours, washed, and then incubated in berberine-free medium for 12-14 days. Incubation of the cells for 2 hours with berberine (25-150 g/mL; 0.07-0.45 M), followed by BCNU (23 M) had an additive effect; dose-dependent colony inhibition was observed in 6/6 cell lines treated with both compounds and then washed and incubated for 12-14 days.
In NT2/D1 human teratocarcinoma cells, berberine chloride at doses of 0.5 mg/mL (1.34 M) and higher (higher doses not specified) was toxic; cell degeneration and death occurred 1 day after treatment (Chang et al., 1990; Chang, 1991). Lower doses (0.01-0.2 mg/mL; 0.027-0.54 M) of berberine chloride were not toxic, but induced morphologic differentiation. In another portion of the experiment conducted by Chang et al. (1990; Chang, 1991), the latency period for induction of morphologic differentiation by berberine chloride in T2/D1 cells was investigated. Cells were incubated with 0.1 mg/mL (0.27 M) berberine chloride for 1, 2, 3, 4, or 5 days. Morphologic differentiation was observed even with only 1 day of treatment. Differentiation started to occur the following day and rapidly progressed during subsequent days.
In F9 murine teratocarcinoma cells, however, neither cytotoxicity nor morphologic differentiation was observed following treatment of the cells with 0.1-0.2 mg/mL (0.27-0.54 M) berberine chloride (Chang et al., 1990; Chang, 1991). Higher doses (not provided) were cytotoxic, but did not induce morphologic differentiation. The duration of the incubation period was not specified.
When the effect of berberine on the expression of glucocorticoid receptors (GR) and its relation to cell cycle progression of HepG2 human hepatoma cells was examined, berberine (1-50 M for 1-3 days) caused a dose-dependent inhibition of growth in HepG2 cells (Chi et al., 1994). However, the inhibition was not cytotoxic because cells continue to grow after removal of berberine from the culture media; viability of berberine-treated cells was greater than 90% in all treatment groups. Berberine also significantly reduced the S-phase fraction and increased GR levels. Berberine inhibited cell secretion of alpha-fetoprotein.
Berberine (dose not provided), either alone or in combination with argon laser treatment, induced lysis, encystation, and degeneration in 9L rat glioma cells (Chen et al., 1994).
In Molt-4 and L1210 human leukemia cells, berberine-induced cytotoxicity was enhanced by arabinose-cytidine monophosphate (ara-CMP) and isoguanosine monophosphate (IGMP) (Lee et al., 1995).
Berberine chloride treatment of human HL-60 leukemia cells induced a dose-dependent decrease in cell viability and an increase in apoptosis when cells were treated with 5 to 50 g/mL (0.013.4-0.134 M) berberine chloride and observed over a period of 48 hours (Kuo et al., 1995). When the cells were treated with 25 g/mL (0.037 M) and observed over the same time period, changes in the cell cycle distribution were observed after 6 hours; the number of S-phase cells and the amount of DNA content decreased, but no changes were observed in the total number of G1 and G2-M cells. The authors suggested that cells in S-phase may be the cell subpopulation which is undergoing rapid apoptosis following berberine chloride exposure.
Berberine (0.032-500 mg/mL; 0.10-1486 M) exhibited cytotoxicity against P-388 murine leukemia cells (Lee et al., 1995). Incubation of the cells with a mixture of berberine and arabinose cytidine monophosphate (0.0.32-500 mg/mL, 1:1 molar ratio) or of berberine and isoguanosine monophosphate (0.0.32-500 mg/mL, 1:1 molar ratio) enhanced the berberine-induced cytotoxicity by 2 to 20 times.
Berberine reversed the resistance of BEL-7402 human liver cancer cells to vincristine (VCR)-induced cytotoxicity by 8.3-fold (Pan and Tian, 1996). Cells were incubated with VCR (dose not provided) and 0 or 27.0 M berberine for 72 hours. The cells exhibited innate resistance to VCR. However, berberine did not reverse the resistance of MCF-7/Adr human breast cancer cells to adriamycin (ADR)-induced cytotoxicity. Cells were incubated with 10 M ADR and 0 or 10 M berberine for 3 hours. MCF-7/Adr cells were selected by stepwise exposure of MCF-7 parental cells to ADR.
Berberine at 200 g/mL (0.59 M) but not 100 g/mL (0.30 M) induced apoptosis in over 90% of Balb/c 3T3 fibroblast cells (Wen Yang et al., 1996).
Berberine (16 g/disk) was not cytotoxic
towards C38 murine colon adenocarcinoma cells or L1210 mouse leukemia
cells (Valeriote et al., 1996).
Treatment with berberine chloride
(0.1 to 0.02 mg/mL; 0.27-0.05 M, purity not specified) induced
down-regulation of the Ki-ras2 protooncogene when measured as
the reduction in the amount of poly (A)+ RNA (Chang
et al. 1990; Chang, 1991). Treated T2/D1 human teratocarcinoma
cells were compared to untreated T2/D1 cells and untreated MRC-5
embryonic human lung fibroblast cells. Actin-mRNA production was
not impaired even after berberine-induced differentiation. It
was not clear whether the down-regulation of Ki-ras2 expression
was causally related to neuronal differentiation.
9.7.3 Topoisomerase Inhibition
Berberine inhibited topoisomerase
II but not topoisomerase I in vitro (Makhey et al., 1995).
Berberine did not induce phototoxicity
in E. coli strains RT7h, RT8h, RT9h, or RT10h (Tuveson
et al., 1986). Approximately 105 cells/mL were placed
onto thin-layer chromatographic plates spotted with psoralen and
alpha-T. The cells were treated with berberine (dose not provided)
in agar for 2 minutes and were then exposed to 43.7 kJ of broad-spectrum
near UV.
9.7.5 Anticholinergic Activity
In isolated guinea pig ileum, pretreatment with 20 g/mL (0.046 M) berberine sulfate blocked the response to 0.1 g/mL acetylcholine (Kulkarni et al., 1972). Pretreatment of isolated tracheal muscle of dogs with 20-30 g/mL (0.046-0.069 M) berberine sulfate blocked the response to 0.1 g/mL acetylcholine by more than 80%, and 60 g/mL (0.14 M) berberine sulfate completely inhibited the response of rectus muscle of frogs to 0.1 g/mL acetylcholine.
In depolarized guinea pig ileum at doses of 0.001-0.01 mg/mL (0.0023-0.023 M), berberine sulfate inhibited carbachol-induced contractions by 27-60%; the inhibition was quickly reversible (Sabir et al., 1978).
In isolated rabbit aortic strips, berberine sulfate (0.003- 0.03 mg/mL; 0.0069-0.069 M) inhibited adrenaline and noradrenaline-induced contractions in a dose-dependent manner (Sabir et al, 1978). Berberine chloride (0.01-0.5 mg/mL; 0.023-1.15 M) inhibited adrenaline-induced contractions in isolated guinea pig seminal vesicle by approximately 10, 30, 50, and 70% at doses of 0.01, 0.03, 0.06, and 0.10 mg/mL (0.023, 0.069, 0.14, and 0.23 M), respectively. Noradrenaline-induced contractions were also inhibited by approximately 14, 17, 34, 44, and 80% by 0.01, 0.03, 0.06, 0.10, and 0.30 mg/mL (0.023, 0.069, 0.14, 0.23, and 0.69 M) berberine sulfate, respectively.
Palmery et al. (1993) found that berberine (4.33 mg/mL; 12.9 M) inhibited adrenaline-induced contractions in rabbit aortic strips, but did not produce vasoconstrictive effects.
In a study investigating the mediators of induced contractions, berberine relaxed potassium chloride- and phenylephrine-induced contractions in isolated aortas of Sprague-Dawley rats (Lee and Chang, 1996). Berberine exhibited a greater inhibition of the contractile response induced by phenylephrine than by potassium chloride, which implied that berberine exhibits alpha-adrenoceptor blocking activity. The aortas were incubated with 1.0-10 M berberine and phenylephrine for 10 minutes or with 1.0-10 M berberine and potassium chloride for 30 minutes.
Hydrastine (4.05 mg/mL; 10 M) had
no effect on adrenaline-induced contractions in rabbit aortic
strips and did not produce vasoconstrictive effects (Palmery et
al., 1993).
In isolated guinea pig ileum, 60
g/mL (0.14 M) berberine sulfate completely inhibited the response
to 0.1 g/mL histamine (Kulkarni et al., 1972).
9.7.8 Spasmolytic/Spasmogenic Effects
In isolated guinea pig ileum, berberine sulfate elicited a spasmogenic response at concentrations of 0.001-0.01 mg/mL (0.0023-0.023 M) but had a spasmolytic effect at concentrations of 0.020-0.10 mg/mL; 0.046-0.230 M (Kulkarni et al., 1972). It did not inhibit contractions in tracheal muscle preparations from dogs and guinea pigs (dose not provided).
Berberine sulfate had no effect on the uninduced contraction rate of isolated rabbit aortic strips, depolarized guinea pig ileum, or guinea pig seminal vesicle at concentrations of 0.003- 0.03 mg/mL (0.0069-0.069 M), 0.001-0.30 mg/mL (0.0023-0.69 M), and 0.01-0.5 mg/mL (0.023-1.15 M), respectively (Sabir et al., 1978).
Hydrastine was inactive on unstimulated
guinea pig ileum and unstimulated mouse vas deferens preparations
at doses of 30-500 M and 10-300 M, respectively (Bartolini et
al., 1990).
9.7.9 Inotropic/Chronotropic Effects
In isolated rabbit heart preparations, berberine sulfate (dose not provided) had a negative inotropic effect (decreased the amplitude of contraction) (Kulkarni et al., 1972). At concentrations of 0.010-0.050 mg/mL (0.023-0.115 M), berberine sulfate also had a negative inotropic effect and caused changes in tone (not described) in isolated rabbit intestine.
Berberine sulfate (0.003-0.10 mg/mL; 0.0069-0.23 M) had positive inotropic (increased amplitude of contraction) and positive chronotropic (increased rate of contraction) effects on spontaneously beating isolated atria of rats, guinea pigs, and rabbits (Sabir et al., 1978). On rabbit atria, the amplitude of contraction was increased by 8, 15, 30, and 70% and the rate of contraction was increased by 10, 14, 20, and 30% with 0.003, 0.01, 0.03, and 0.10 mg/mL (0.0069, 0.023, 0.069, and 0.23 M) berberine sulfate, respectively (Sabir et al., 1978). Percentages of increase were not given for rats or guinea pigs.
Hydrastine produced dose-related
positive inotropic but negative chronotropic effects in spontaneously
beating rat atrium (Bartolini et al., 1990). It also inhibited
electrically evoked contractions in isolated longitudinal muscle
of guinea pig ileum at high doses (ED50 = 620, CI = 386-993 M).
In the range of 10-500 M, hydrastine induced a dose-related positive
inotropic effect on electrically evoked contractions of isolated
mouse vas deferens (Bartolini et al., 1990).
9.7.10 Inhibitory Effects on Potassium Channels
In isolated guinea pig ventricular papillary muscle, incubation with 3 to 100 M berberine for 30 minutes partially inhibited (low dose) or completely abolished (high dose) the shortening of action potential duration and effective refractory period induced by hypoxic conditions or by cromakalim (Wang et al., 1996).
In guinea pig ventricular myocytes
under whole-cell voltage clamp conditions, berberine (3 to 100
M) partially or completely inhibited cromakalim-induced outward
K+ currents (Wang et al., 1996). In inside-out membrane
patches exposed to 0.1 mM ATP, berberine inhibited KATP
channel activity in a dose-dependent manner.
9.7.11 Effects on Calcium Chloride-Induced Contractions
In depolarized guinea pig ileum,
berberine sulfate (0.001-0.003 mg/mL; 0.0023-0.0069 M) potentiated
calcium chloride-induced contractions by 7-23% and did not produce
an inhibitory effect even when administered at doses as high as
0.30 mg/mL (0.69 M) (Sabir et al., 1978). Berberine sulfate reversed
the inhibitory effect of polysorbate 80 and papaverine on calcium
chloride-induced contractions by 50-60%.
9.7.12 Effects on Prostaglandin-Induced Contractions
In 6/9 experiments conducted by Sabir
et al. (1978) using isolated guinea pig ileum, berberine sulfate
(0.001-0.006 mg/mL; 0.0023-0.014 M) inhibited PGE2-induced contractions
by 10-64%; in 7 other experiments, berberine sulfate (0.0001-0.03
mg/mL; 0.00023-0.069 M) inhibited PGF2alpha-induced contractions
by 8-67%. At 0.001-0.01 mg/mL (0.0023-0.023 M), berberine sulfate
potentiated prostaglandin (PG)E1-induced contractions by 18-280%
and PGF1alpha-induced contractions by 20-560% in isolated guinea
pig ileum. In 3/9 experiments investigating PGE2-induced contractions,
berberine sulfate potentiated the induced contractions by 15-165%.
In 5 other experiments, berberine sulfate (0.001-0.01 mg/mL; 0.0023-0.023
M) increased PGF2alpha-induced contractions by 84-300%.
9.7.13 Inhibition of Sperm Motility
British Friesian bull sperm samples
treated in vitro with berberine sulfate became immotile
in 217 minutes at 3 mg/mL (6.92 M); in 153 minutes at 10 mg/mL
(23.1 M); in 20 minutes at 50 mg/mL (120 M); and immediately after
treatment with 100 mg/mL (231 M) berberine sulfate (Sabir et al.,
1978).
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