The comparative influence of coptisine bisulfate and berberine bisulfate on the evolution of acute toxic hepatitis: experimental study

RESEARCH ARTICLE

The comparative influence of coptisine bisulfate and berberine bisulfate on the evolution of acute toxic hepatitis: experimental study

Rodica Peredelcu^1*

¹Chair of pharmacology and clinical pharmacy, Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova

Corresponding author:

Rodica Peredelcu, assistant professor

Chair of pharmacology and clinical pharmacy

Nicolae Testemitanu State University of Medicine and Pharmacy

165, Stefan cel Mare si Sfant ave., Chisinau, Republic of Moldova, MD-2004

e-mail: rodica.peredelcu@usmf.md

Short title: Influence of coptisine and berberine in acute toxic hepatitis

What is not known yet about the topic

So far, the comparative influence of coptisine bisulfate and a reference substance, berberine bisulfate, on the development of acute toxic hepatitis has not been investigated.

Research hypothesis

The description of the obtained preclinical results would facilitate the study of the pharmacological properties of coptisine bisulfate in further clinical research as well as the adaptation of associated disease treatment.

Article's added novelty on this scientific topic

The article elucidates liver indicators obtained with coptisine bisulfate administered in doses of 1 mg/kg and 5 mg/kg for 7 and 14 days compared to berberine bisulfate in similar doses and terms. For the first time, the efficacy of coptisine bisulfate on acute toxic hepatitis was demonstrated compared to berberine bisulfate, which practically did not influence the evolution of experimental toxic hepatitis at investigated doses, being inferior to coptisine bisulfate.

ABSTRACT

Introduction. There are frequent reports of the pharmacological and toxicological effects of the celandine extracts, but very few works about the action of pure alkaloids, mainly berberine bisulfate. For these reasons, the current research goal was to evaluate the efficacy of coptisine bisulfate on hepatic parameters in experimental toxic hepatitis compared to berberine bisulfate.

Material and methods. It is a prospective, comparative, experimental study. The Agreement of the Ethics Committee of Research is obtained. There were 110 experimental animals included, healthy from a medical point of view and not enrolled in other scientific studies until the pre-clinical research. They were treated with carbon tetrachloride (CCl4) to induce toxic hepatitis and coptisine bisulfate (coptisine) and berberine bisulfate (berberine) were used as a remedy. The witness group included 5 intact animals, which received sterile olive oil subcutaneously. After euthanasia, the blood was collected for the study of biochemical parameters in the Central Laboratory of Scientific Research of the Nicolae Testemitanu State University of Medicine and Pharmacy. The statistical relevance between different groups was calculated based on the mean values, the deviation and the standard error, the t-Student significance test.

Results. In the modeling of acute toxic hepatitis by CCl4 there was a significant increase in ALT activity with a less important increase in AST activity. Administration of coptisine at a dose of 1 mg/kg for 7 days after CCl4 prevented the increase of transaminase activity, keeping them within the witness group ranges, which was confirmed by maintaining the De Ritis Ratio and at a dose of 5 mg/kg practically did not prevent the increase of the ALT activity with a decrease in the AST. Under these conditions, the De Ritis Ratio was 1.42 in the group with coptisine versus 1.18 in the CCl4 one. The use of berberine at the dose of 1 mg/kg for 7 days on the background of liver disease modeling revealed that the activity of AST was within the limits of the animals in the witness group, while that of ALT increased essentially from the witness group and even the CCl4 group; in the 5 mg/kg dose, transaminase activity increased both from the witness group and experimental hepatitis group, confirmed by the De Ritis Ratio values of animals with hepatic injury.

Conclusion. According to the results, we can deduce that in animals with experimental hepatitis, coptisine exhibits a protective effect at the dose of 1 mg/kg for 7 days, preventing the development of liver disease, while in the dose of 5 mg/kg practically does not prevent the toxic lesion of carbon tetrachloride, after 14 days even causing an increase in transaminase activity. Thus, we can report a dose-dependent effect, obtaining scientific data that extracts from Chelidonium majus may exhibit hepatotoxicity.

Key words: coptisine, berberine, carbon tetrachloride, hepatotoxicity, transaminases.

INTRODUCTION

Celandine, Chelidonium majus L., is a medicinal plant of the Papaveraceae family, also called “holy weed”, “nipplewort”, and “swallowwort”. It is considered resistant both to torrid and dry summer and winter’s cold weather, when it can be found under the snow. Celandine has been known since antiquity; the works of Paracelsus, Dioscordies, showed the miraculous and beneficial properties of the plant. Today it can be found in Europe, North America, Asia, and in the Republic of Moldova as well.

The increased interest in the pharmacological effects of the plant has been preserved to this day, with scientific researchers increasingly trying to investigate the pharmacological activity of the alkaloids present in Chelidonium majus.

According to Zielinska S. et al. (2018) and Ye X. et al. (2009), one of the most widespread indications of the celandine is the liver pathology. It is considered that the hepatoprotective properties are attributed to the isoquinoline alkaloids - berberine, coptisine, sanguinarine, chelerythrine etc., which have a beneficial effect on the liver functions and exhibit a range of pharmacological effects such as antioxidant, choleretic, anti-inflammatory and immunomodulatory (coptisine, protopine, berberine, chelerythrine, sanguinarine, chelidonic acid) [11, 12].

Gîlca M. et al. (2010), Maji A. et al. (2015), have shown in their experimental studies that celandine extract does not alter liver function, and the extract and alkaloids can prevent CCl4-induced and paracetamol-induced liver damage by decreasing enzyme activity (ALT, AST, ALP) and bilirubin levels [3, 6]. An important role is represented by the antioxidant effect with the negative effect of the oxidative stress on the hepatocytes, being more pronounced in the extract from the celandine which contains a number of alkaloids. This effect is caused, in the view of Ye X. et al. (2009) and Zielinska S. et al. (2018), by the capture of free radicals (hydroxyl, peroxyl, hypochlorite) and anions (superoxide, singlet) by increasing the activity of antioxidant enzymes (catalase, superoxide reductase, superoxide dismutase MnSOD) and improving markers of cell destruction and inflammation: decreased level of TNF-α, IL-6, NFκB, p65, cas-3, iNOS [11, 12].

In the WHO Monograph (2010), it is noted that the oral administration of the extract from the celandine (doses of 12.5, 62.5 and 125 mg/kg twice a week for 3 weeks) resulted in a true reduction in activity of ALT, AST, alkaline phosphatase, LDH and bilirubin levels with the cholesterol level stabilizing, modified at the administration of carbon tetrachloride. Histological examination showed a decrease in the number of necrotizing hepatocytes and a double increase in the elimination of the bile [13].

Mitra et al. (1996), Gîlca M. et al. (2010), demonstrated, in experimental studies, the hepatoprotective effect of berberine and coptisine in laboratory animals with carbon tetrachloride induced toxic hepatitis. They have reduced cell necrosis, lack of fibrosis and reduced lipid levels. Mechanisms of hepatoprotective action have supposedly ameliorated oxidative stress by restoring lipid peroxidation, intracellular reactive oxygen species, reduced glutathione (GSH) and normalization of apoptosis decreased by nucleus fragmentation, cytochrome C release, caspase 3 activation [3, 7]. According to the data obtained by Chai F. et al. (2018), coptisine showed a protective effect on hepatic injuries for mice during a 7 day course by reducing the level of lipopolysaccharides and D-galactosamine (LPS/D-GalN). Hepatotoxicity caused by the toxin was also decreased in vivo in mice and ex vivo in hepatocyte cultures [2]. Biochemical parameters (ALT, AST and alkaline phosphatase activities) were reduced to intermediate levels in control animals and those treated with carbon tetrachloride according to data obtained by Ye X. et al. (2009) and Hu Y. et al. (2017) [5, 11].

Zielinska S. et al. (2018), have found, in clinical trials with products containing Chelidonium majus, that significant improvements in clinical, instrumental and laboratory parameters (bilirubin, transaminases, blood test) have been reported in patients with hepatobiliary disorders (cholelithiasis, colangitis, cholecystitis, postcolecistectomic syndrome, alcoholic hepatitis). Preclinical researches performed on hamsters and rats (which have had heir gallbladder removed and have not responded to traditional treatment) have demonstrated cholecystokinetic action by stimulating the gallbladder musculature with berberine [12].

Based on the above-mentioned scientific literature, the hepatoprotective effect of coptisine and berberine, protoberberine alkaloids, with a content rich in Chelidonium majus, was found. Taking into consideration that the Republic of Moldova is considered an endemic area of hepato-biliary diseases, it is necessary to study the new hepatoprotective treatment methods with increased yield and efficiency. The pharmaco-economic benefit of celandine is that it is not demanding to specific grow conditions, thus can be found practically in all seasons and in all areas of the country. Another positive side is the new, less costly, method of obtaining coptisine bisulfate salt, allowing a deeper investigation of some pharmacological actions of coptisine, discovered by lecturers I. Casian and A. Casian, from the Laboratory of Analysis, Standardization and Drug Control (Scientific Drug Center of the Nicolae Testemitanu SUMPh). Since berberine has been examined in larger studies, where the hepatoprotective effect has been established, but its toxic effect has been confirmed as well, the reason for its necessity was to carry out preclinical comparative research of berberine and coptisine. Preclinical evidence of the toxicity and hepatoprotective effect of coptisine was insufficient, which contributed to the investigation of its hepatoprotective properties.

MATERIAL AND METHODS

Our research includes an experimental, comparative and prospective study conducted at the Department of Pharmacology and Clinical Pharmacy, which was performed according to the Ethics Committee's requirements for the control and surveillance of animal experiments. The research protocol was submitted for approval to the Research Ethics Committee and a favorable notice was obtained on 24 March 2012.

Coptisine bisulfate was obtained from the Chelidonium majus hydroalcoholic extract by preliminary purification of the plant extract, treatment with sulfuric acid at a pH of 3.0-3.5 and crystallization of coptisine bisulfate at a pH of 1.0-1.2. The procedure developed by Casian I. and Casian A. (2011) is simple, economical and allows for a deeper investigation of some pharmacological actions of coptisine, as well as the implementation of this substance in the pharmaceutical industry [1].

In the preclinical study, 110 white rats of both genders, who were medically healthy and who were not included in other preclinical researches until the beginning of the research, were raised in the vivarium of the Nicolae Testemitanu State University of Medicine and Pharmacy. The body weight of the rats was 170-250 grams, the age – 12 weeks. The experimental animals were divided into 2 groups of 5 animals, which consisted of the control groups and 10 groups of 10 animals (control and coptisine + berberine treated groups) under stable humidity conditions of 40-60% and a constant temperature of 24±200C in a separate room, being placed in polypropylene cages with wood sawdust as bedding. All animals had free access to food and water 40 minutes after the oral administration of the investigated substance. In view of the fact that the experimental research had a duration of 7 days and 14 days, the animals were repeatedly weighed every 3 days, the administered coptisine and berberine doses being adjusted to the body mass oscillations, thus ensuring the correct doses of administration.

Acute Toxic Hepatitis (ATH) in rats was modeled by parenteral injection of sterile carbon tetrachloride solution with a 50% concentration in olive oil, resulting from the calculation of 0.4 ml/100 g for 4 days.

In order to determine the action of coptisine and berberine on acute modeled toxic hepatitis, comparative studies of the influence of coptisine at doses of 1 and 5 mg/kg and berberine at doses of 1 and 5 mg/kg within 7 and 14 days on the evolution of experimental hepatitis were developed, with the analysis of the obtained biochemical parameters: alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), bilirubin and its fractions when using the above-mentioned substances.

The animals were divided into 12 experimental groups. Rats from groups 1 and 7 received sterile olive oil solution subcutaneously once per day at a dose of 0.4 ml/100 g for 4 days, serving as reference batches; the rats of groups 2 and 8 received a 50% carbon tetrachloride solution in sterile olive oil at a dose of 0.4 ml/100 g for 4 days, being considered as controls. Rats from groups 3 and 9, besides receiving CCl4, were given enteric coptisine bisulfate from the first day, at a dose of 1 mg/kg for 7 and 14 days respectively, and those in batches 4 and 10 were given coptisine bisulfate at the dose of 5 mg/kg for 7 and 14 days, respectively. Berberine bisulfate, at a dose of 1 mg/kg for 7 and 14 days, was administered to the animals in groups 5 and 11, and at a dose of 5 mg/kg – to the animals in groups 6 and 12, being associated with 50% CCl4 solution.

Rats from lots 1-6 were euthanized with ether on the 8th day, while those in groups 7-12 – on the 15th day of pathology production, with blood and biochemical parameters characteristic of liver disease, documented in the labortory of biochemistry of the Central Laboratory of Scientific Research, Nicolae Testemitanu SUMPh.

The statistical processing of the obtained data was calculated based on the mean values, the deviation and the standard error, the t-Student significance test using the software package “Statistics for Windows, Release 6.0 StatSoft, Inc.”

RESULTS

It was found that ATH increased the activity of ALT, which denotes the alteration of hepatocytes by releasing 80% of the cytoplasmic fraction of ALT from the total activity of the enzyme in the liver. At the same time, the serum AST activity increased by 40%, the cytoplasmic fraction of which is only 20% of the total enzyme in the liver. Based on this, the de Ritis ratio decreased twice. When coptisine was administered at a dose of 1 mg/kg for 7 days (rats with experimental toxic hepatitis), transaminase activity was restored to the values of the animals in the witness group, while maintaining the de Ritis ratio. Coptisine, at the dose of 5 mg/kg, practically did not prevent the increase of ALT activity, reducing the AST activity, so the de Ritis ratio constituted 1.42 in the coptisine group versus 1.18 in ATH. When berberine was used at the dose of 1 mg/kg for 7 days under the same conditions, the significant increase in ALT activity values was recorded, compared to the witness and ATH groups, and the de Ritis ratio declined significantly. When administering berberine at the dose of 5 mg/kg for 7 days, the transaminase activity increased, as compared to the witness group and the carbon tetrachloride group, confirmed by the de Ritis ratio (the values being as the ones of the animals with hepatic injury) (Figures 1, 2 and 3).

Fourteen days after the experimental hepatitis modeling, the ALT activity values were restored nearly at the witness group level, the AST being below its values; the de Ritis ratio was 1.6, compared to 2.3 in the witness group. When coptisine was administered at a dose of 1 mg/kg, the values of AST activity remained within the limits of the witness group, while ALT values insignificantly increased, with the de Ritis ratio increasing to 1.8 versus 1.6 in the CCl4 group. It should be noted that at the dose of 5 mg/kg for 2 weeks, coptisine increased the ALT activity, while the AST decreased below the witness group values. At doses of 1 mg/kg and 5 mg/kg, berberine used in acute toxic hepatitis increased the ALT activity, compared to the animals who remained intact and the animals from the carbon tetrachloride group. The de Ritis ratio was below the control group values (Figures 1, 2, 3).

The activity of alkaline phosphatase within 7 days, after modeling experimental toxic hepatitis, increased non-essentially. In both doses, for 7 days, coptisine prevented the increase in ALP activity. Berberine, at a dose of 1 mg/kg for 7 days, increased ALP activity, and in the 5 mg/kg dose, the activity of the enzyme significantly decreased in both the experimental toxic hepatitis and the witness group. After 14 days of modeling liver toxicity, the alkaline phosphatase activity increased, resulting in the cholestatic syndrome. After 2 weeks of coptisine and berberine administration in both doses, a significant reduction in alkaline phosphatase activity was confirmed (Figure 4).

In the performed researches, the alkaline phosphatase/alanine aminotransferase (ALP/ALT) activity was determined in order to determine the evolution of hepatic lesions. In experimental hepatitis, on the 7th day, the ALP/ALT ratio was reduced, confirming the presence of the cytolysis syndrome. Coptisine, at a dose of 1 mg/kg, maintained the enzyme ratio within the witness group, while the dose of 5 mg/kg reduced the value of this ratio (reducing ALP activity, ALT being unchanged). Berberine, at the dose of 1 mg/kg, contributed to the increase in the ALP/ALT ratio, similar to the group of animals subjected to toxic hepatitis (increase in ALP and ALT activity). At a dose of 5 mg/kg, it caused an evident decrease in the ratio (reduction of ALP activity and increase of ALT activity) (Figure 5).

After 14 days, the ALP/ALT ratio increased, compared to the witness group, from 7.68 to 10.54, confirming the presence of the cholestasis phenomena. Coptisine showed a regulatory effect in both doses, helping to restore ALP activity with normalization of the ratio. It should be noted that ALT activity was not increased at the 5 mg/kg dose, thus significantly diminishing the ALP/ALT ratio. Berberine, in both doses, reduced the nominated ratio values by restoring ALP activity but increasing the ALT level (Figure 5).

The activity of gamma-glutamyltransferase decreased 7 days after the onset of toxic hepatitis, without being affected by it after 14 days. Both the investigated alkaloids, at doses of 1 mg/kg and 5 mg/kg for 7 days, contributed to the substantial reduction of the GGT activity in experimentally modeled animals. After 2 weeks, coptisine at the dose of 5 mg/kg and berberine at doses of 1 mg/kg and 5 mg/kg decreased GGT values (Figure 6).

In animals undergoing carbon tetrachloride influence, on day 8, the increase in total, direct and indirect bilirubin was confirmed. Coptisine and berberine, in both the doses, administered for 7 days, within the hepatotoxic background, caused the parallel increase of all bilirubin fractions (Figure 7).

The total direct and indirect bilirubin levels decreased insignificantly on the 15th day after acute toxic hepatitis modeling. At the 1 mg/kg dose, under the influence of coptisine for 2 weeks, the total, direct and indirect bilirubin decreased, whereas the bilirubin fractions increased in the 5 mg/kg dose, compared to the witness and hepatic lesion groups. An inversely proportional phenomenon was observed with the administration of berberine over the same period of time, with the increase in the total, direct and indirect bilirubin level, at the dose of 1 mg/kg, and the decrease in the bilirubin level at the 5 mg/kg dose, in the animals exposed to the hepatotoxin (Figure 8).

DISCUSSION

It is rightly believed that the Republic of Moldova is an endemic area of hepatitis, more often, of viral origin, but hepatitis caused by hepatotoxic factors such as intoxication, alcohol abuse, etc. are also frequent. In the acute phase of the disease, treatment is directed to detoxification and hepatoprotection.

According to scientific literature, an important role in the treatment of hepatoprotectors is attributed to alkaloids extracted from the celandine (coptisine, berberine, sanguinarine).

Chai F. et al. (2018), determined that in the case of 7-days administration, coptisine improved the liver condition in rats, induced by lipopolysaccharide and D-galactosamine (LPS/D-GalN) by reducing ALT and AST values, increasing glutathione and superoxide reductase levels, while maintaining the morpho-structural function of hepatocytes. According to in vitro research, the injury to HepG2 cells under the influence of coptisine appeared, which also denotes the protective effect on the hepatocytes [2]. The hepatoprotective effect was also established in berberine, which, at a 50 mg/kg dose for 8 weeks, prevented the body mass decrease, reduced ALT, AST, ALP and increased albumin levels in lead-induced toxic hepatitis, according to data obtained by Hasanein P. et al. (2017). Also, the alkaloid prevented the oxidative stress by increasing lipid peroxidation, diminishing thiol groups in the liver and activating the antioxidant system [4].

Mohammadzadeh N. et al. (2016) proved that in tetrachloride-induced toxic hepatitits, berberine eased the hepatic injuries by lowering the ALT, AST, ALP and lipid peroxidation processes, with the elevation of the superoxide dismutase activity and the reduction of glutathione reductase. Particular importance was attributed to the reduction of the inflammatory process in the liver under the action of berberine, by the reduction of the levels of TNF-α, TGF-β1, COX-2 și iNOS [8].

According to clinical studies conducted by Zielinska S. et al. (2018), Chelidonium majus extract produced an essential efficacy in clinical, instrumental and laboratory parameters (bilirubin, transaminases, blood test) in patients with hepatobiliary disorders [12].

Based on the results obtained in the animals subjected to the toxic action of CCl4, it was found that when using coptisine at the dose of 1 mg/kg, the protective effect was established, which prevented the development of experimental liver injury (Figures 1, 2 and 3) by restoring the activity of transaminases, in particular the AST activity, for 14 days with the inclusion of compensatory mechanisms of acceleration of the neutralization of affected hepatocytes. At the same time, after 7 days, the alkaloid at the 5 mg/kg dose poorly influenced the toxic lesion of CCl4, whereas after 14 days, the activity of transaminases increased, so we can state the dose-dependent effect of the coptisine (1 mg/kg), knowing the fact that celandine extracts themselves can cause hepatotoxicity [9, 10].

At both doses, for 7 days, coptisine prevented the increase in alkaline phosphatase activity, thus actively influencing the acute toxic hepatitis by reducing the cholestasis syndrome.

Compared to coptisine, studies have shown that berberine in both doses has virtually no hepatoprotective effect on transaminases and alkaline phosphatase, which were increased by carbon tetrachloride.

Administration of coptisine and berberine at doses of 1 mg/kg and 5 mg/kg for 7 days resulted in a more pronounced decrease in GGT activity in animals with hepatic disease, which plays an important role in the transport of aminoacids and catabolizes the metabolism of glutathione-S (substituent of various xenobiotics) conjugates. Thus, the named alkaloids can be considered as remedies which improve the protein synthesis function of the liver.

The dose-dependent effect was established on all bilirubin fractions, restored by coptisine at the 1 mg/kg dose and increased at the 5 mg/kg dose when used for 2 weeks. An opposite effect of coptisine was found in the administration of berberine, which, at the dose of 1 mg/kg, increased the total, direct and indirect bilirubin, and at the dose of 5 mg/kg – reduced their content.

Analyzing the obtained data, we can state that Chelidonium majus alkaloids possess a hepatoprotective effect through various mechanisms, such as: inhibiting lipid peroxidation by reducing the reactive species of oxygen, increasing the antioxidant activity, correcting metabolic disorders and reducing cholestasis phenomena.

CONCLUSIONS

Following the evaluation of comparative hepatoprotective action of coptisine and berberine in experimental HTA, it was found that:

1. The acute toxic hepatitis model in rats has been shown to have cytolysis syndrome by enhancing transaminase activity and cholestasis syndrome by increasing ALP and bilirubin fractions.
2. Coptisine showed a dose-dependent hepatoprotective effect by restoring ALT, AST, ALP, GGT and bilirubin fractions at low doses and early liver disease terms.
3. Berberine produced a less pronounced effect on the hepatic lesions, the activity of enzymes being even increased at the dose of 5 mg/kg, which confirms the literature data about the favorability of its toxicity in the composition of the celandine extract.

Declaration of conflicting interests

The author states the lack of conflicts of interest in the preparation of this article.

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