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Greater Celandine's Ups and Downs−21 Centuries of Medicinal Uses of Chelidonium majus From the Viewpoint of Today's Pharmacology - Part 2
Antiviral
The glycosaminoglycan present in the latex inhibits intracellular human immunodeficiency virus HIV viral migration and blocks reverse transcriptase (Gerenčer et al., 2006). Moreover, individually tested five C. majus alkaloids: chelidonine [1], chelerythrine [9], sanguinarine [12], coptisine [31], and berberine [28] were able to inhibit the development of HIV-1. The first two decreased the activity of the virus reverse transcriptase at the concentrations 150-200 μg/ml, while sanguinarine [12], berberine [28], and coptisine [31] were already active at concentrations of 50-150 μg/ml (Tan et al., 1991).
The chloroform extract in the concentration of 35 μg/ml decreased the number of adenoviruses responsible for inducing acute fevelitis of the upper respiratory tract and conjunctiva in humans (Kéry et al., 1987). The experiments with animals showed that ethanol extract of C. majus inhibited encephalomyocarditis virus in 45% of experimental mice, whereas berberine [28] tested in the concentration range between 20 and 125 μg/ml inhibited influenza virus type A and B in chicken embryos with 33-99.97% efficiency (data previously reviewed by Kedzia et al., 2003). These results were presented only once, and from that time have never been confirmed or repeated.
Antiprotozoal
Herb and root water extracts, as well as sanguinarine [12] (2-4 μg/ml) were highly effective in the treatment of trichomoniasis caused by Trichomonas vaginalis. After 8-10 days of treatment there was no protozoa detected in genitals of nearly 64% young girl patients. Sanguinarine [12] was also found to inhibit the development of Entamoeba histolytica, responsible for the hepatic abscess (Kozicka and Radomanski, 1963; Vychkanova et al., 1969).
Liver and Biliary Tract
One of the most widespread and repeatedly mentioned indications of C. majus, both in European/Mediterranean and East Asian (TCM) tradition, was for various liver complaints. It may date back to "signatura rerum" rule from coloration of the latex and flowers but obviously must have been verified by observations. Nowadays, even though this indication has been supported by just a few in vitro and in vivo studies, caution is necessary for alleged hepatotoxicity. Also, clinical evidence is not sufficient to ultimately recommend this herb and galenic preparations thereof (European Medicines Agency, 2011).
Some of the hepatoprotective and choleretic/cholagogue activity could be more aptly attributed to the presence of hydroxycinnamic (caffeic) acids esters which have been quite frequently overlooked in alkaloid-focused studies (Weiskirchen, 2016).
The question whether the supposed stimulation of bile flow is caused only by cholagogue activity or also by increasing bile production or excretion was first addressed by Rentz (1947). Comparison of guinea pigs and rats (that do not have gall bladder and did not respond to the treatment) reaction to C. majus tincture suggested only cholecystokinetic mechanism of action, attributed by the author to the stimulation of smooth musculature by berberine [28]. However, the tincture composition was unknown.
Vahlensieck et al. (1995) indicated using isolated rat livers that beside the earlier reports on cholecystokinetic action, also increase of bile production contributes to the final outcome. The activity was not very high, reaching 20% increase by perfusion with C. majus extract. The activity of alkaloid and polyphenol fractions separately were only about half of that. It suggests an additive action of the complex mixture of all active constituents.
The antispasmodic activity of C. majus extract was tested in trials based on acetylcholine (ACh)-induced contraction in isolated rat ileal smooth muscle (Boegge et al., 1996). The extract was found to be moderate antagonist (12.7%; 2.0 × 10−4 g/ml organ bath) against (ACh)-induced contraction compared to caffeoylmalic acid [48] (6.9%; 2.5 × 10−5 g/ml) and coptisine [31] (16.5%; 1.0 × 10−5 g/ml). Also, individual alkaloids, i.e., chelidonine [1], stylopine [33], and coptisine [31] have been tested for relaxant activity on ileum smooth muscles (Hiller et al., 1998). Among them, chelidonine [1] and stylopine [33] showed papaverine-like musculotropic action, whereas coptisine [31] was ineffective in BaCl2 stimulation model. In carbachol and electric field induced contractions, all three alkaloids and ethanolic extracts were effective.
In a different contraction model - isolated and perfused porcine uterus, the commercial alcoholic extract exhibited two-phase response, initially stimulating very strong contractions followed by a longer relaxation period (Kuenzel et al., 2013). These properties were suggested as potentially useful in supporting artificial insemination or facilitating fertilization by acceleration of sperm movement toward fallopian tubes. However, this indication was rather unknown in traditional usage and would be a novel application of C. majus.
Cytoprotective
The macerated ethanol extract from juice expressed from pulped fresh plant material (according to the homeopathic recipes) was able to counteract carbon tetrachloride induced hepatotoxicity (Mitra et al., 1996). The effects of extract administration included reduced cell necrosis, absence of fibrosis, and lower lipid accumulation. Here again, no reliable data on the composition of the tested extract was available. C. majus primary tincture (German Homeopathic Pharmacopeia), diluted 100x and 1,000x was significantly effective against cadmium-induced hepatoxicity in HepG2 and primary rat hepatocyte models (Gebhardt, 2009). The activity was stronger than the proprietary compound preparation and similar to the recognized hepatoprotective herb - Silybum marianum. The putative mechanisms of cytoprotective activity was associated to oxidative stress relieve as demonstrated by improvement of several parameters such as lipid peroxidation, intracellular Reactive Oxygen Species, reduced glutathione (GSH) level, as well as diminished apoptosis symptoms (nuclei fragmentation, cytochrome C release, caspase 3 activation). Hepatotoxicity caused by cadmium was also ameliorated in vivo in mice and ex vivo in hepatocyte cultures. In mice, administration of 50 or 75 mg/kg body weight chelidonine [1], also in form of nanocapsules, improved histopathological picture of livers damaged by Cd treatment. Also, biochemical parameters such as ALT, AST, and ALP activities were lowered to the levels intermediate between control animals and Cd treated ones. Moreover, the expression of cell death related genes Bax and Bcl-2 was modulated to the levels closer to the Cd untreated animals. In all tested hepatotoxicity parameters, nanoencapsulated chelidonine [1] was more efficient. It was corroborated with the ca. 1/3 higher distribution of chelidonine [1] from nanoformulation into the liver tissues. The putative mechanisms relate to alleviation of oxidative stress as revealed by improvement of antioxidant status (lower lipid peroxidation, higher GSH level, and SOD and CAT activities) and various cell death and inflammation markers (decreased protein level of TNF-α, IL-6, NFκB, p65, cas-3, iNOS) (Paul et al., 2013).
Notwithstanding, the literature evidence supporting beneficial properties in hepatobiliary disorders from both in vitro, in vivo, or ex vivo studies is still less abundant than case reports on liver toxicity. This, quite surprising disparity, should motivate pharmacologists and clinical researchers to do further and more insightful studies to explain the mechanisms of action and pinpoint the most active constituents or their combinations.
Antiproliferative, Pro-apoptotic, and Cytotoxicity to Cancer Cell Lines
As in case of liver and biliary tract disorders, the antitumor properties have been indicated since antiquity. Today, this kind of activity belongs to the most intensely investigated. Unlike the hepatoprotective properties, this kind of properties has been quite well-documented in a high number of studies. Mostly, some of the major alkaloids are expected to be able to cause cell death or stop proliferation of cancerous cells. This is based on the ability of berberine [28], chelerythrine [9], sanguinarine [12], and to some extent also other alkaloids to intercalate DNA that should interfere with replication and cell division (Philchenkov et al., 2008; Basu et al., 2013; Noureini et al., 2017). However, other mechanisms have been also discovered, albeit most studies used in vitro experiments on cell lines. Chelidonine [1] appeared to exert its cytostatic activity through interactions with microtubules and thereby causing cell cycle arrest (Panzer et al., 2001; Havelek et al., 2016a).
The selected examples of cell line-based studies on cytotoxic properties of C. majus and its major alkaloids are summarized in Table 2.
In more detail, quite many different, but mostly human cell lines were used as model systems (for references see Table 2), representing leukemias (Jurkat with several modifications to study certain cell death mechanisms), Raji, MT-4, MOLT-4, HL-60, U-937, HEL-92.1.7, CCRF/CEM, CCRF/ADR5000), colon carcinomas (Caco-2, HT-29, HCT116, SW480), breast cancer (MCF-7, MDA-MB231), pancreatic cancer (human PANC-1, murine PANC02), lung cancer ((A549, H460), prostate cancer (DU-145), cervical cancer (HeLa), ovarian carcinoma (A2780), liver cancer (HepG2), gastric cancer (SGC-7901), vulvar squamous cell carcinoma (A431), oesophageal squamous carcinoma (WHCO5), and mouse melanoma (B16F10). Non-cancerous lines, such as lung fibroblasts (MRC-5, WI-38), skin fibroblasts (Hs27) immortalized cells from mice (3T3), green monkey (Vero), humans (293N3S, HS-27, HaCaT), or SV-40 transformed bronchial epithelium (BEAS-2B) were also used.
From most of the published mechanistic studies a clear distinction can be established between mechanisms of action of chelidonine [1] and sanguinarine [12]/berberine [28]/chelerythrine [9].
Sanguinarine [12], chelerythrine [9], and berberine [28] possess strong affinity to binding G-quadruplex in telomeres which leads to blocking telomerase activity in fast proliferating cells (Noureini et al., 2017).
Unlike the quarternary alkaloids, chelidonine [1] is only a weak DNA intercalating agent and does not induce lethal mutations or DNA damage. Its mechanism of action is suggested to rely on interactions with spindle microtubules leading to cell cyle arrest and mitotic catastrophe, inhibition of ABC transporters thus abolishing multidrug resistance and finally modulation of gene transcription (telomerase, cell death-related, cell cycle-controlling). These properties combined with the stronger-acting intercalating alkaloids can make the whole alkaloid fraction a unique multifaceted agent targeting cancer cells.
For example, El-Readi et al. (2013) demonstrated complex interaction of chelidonine [1] and alkaloid-rich extract with several signaling pathways, including those responsible for cell cycle, cell death, and proliferation. Using microarrays confirmed by RT-PCR data, it was shown that a set of genes associated with multidrug resistance (e.g., ABC transporters and CYP) were significantly downregulated (from 50% in case of CYP3A4 to 99% in ABCG2). On the other hand, caspase-3 and 8 genes were upregulated (up to 27-fold). These results explain the strong augmenting of doxorubicin toxicity to resistant Caco-2 and CCRF/ADR5000 cells. Upon low-dose treatment with chelidonine [1] (20 μM), the LC50 decreased from 3.67 μM (Caco-2) and 32.16 μM (CCRF/ADR5000) to 0.42 and 7.4 μM, respectively. Similar results were obtained with an adequate concentration of extract (5 μg/ml) containing (in decreasing order) chelidonine [1], coptisine [31], stylopine [33], and protopine [37] as four major compounds and smaller amounts of other alkaloids. In the more resistant CCRF/ADR5000 cells the effect of extract was even stronger than pure chelidonine [1], suggesting that some of the other alkaloids are more efficient P-gp inhibitors. Induction of apoptosis was the major mechanism of cytotoxicity, also supported by increase of caspase activity and annexin staining.
These results support the potential of chelidonine [1] (and at least some other C. majus alkaloids) as ideal agents to overcome MDR as targeting both transport proteins, metabolic enzymes and pro-apoptotic pathways.
The interference of chelidonine [1] with the cell proliferation apparatus was also demonstrated using planarian stem cell model (Isolani et al., 2012) in which the gene expression of mcm2 (essential DNA replication factor) or inx-11 (a gap-junction protein essential for regeneration) was decreased by 20 μM of the alkaloid. The stem cells were affected but not differentiated populations such as neuronal or intestinal.
Quite a particular story pertains to the mentioned earlier patented product referred to as a semisynthetic derivative of alkaloids - a trimeric structure with thiophosphoric acid moiety connecting alkaloid molecules. It has been mentioned in the literature as NSC-631570 or Ukrain®. However, one of the major doubts regarding this product is its chemical identity (Panzer et al., 2000; Habermehl et al., 2006; Jesionek et al., 2016). Despite the earlier manufacturer claims, it couldn't be positively confirmed that it is indeed a thiophosphoric acid complex and at least in some batches, it appeared as a rich mixture of native alkaloids, that have been identified using reliable chromatographic and spectroscopic methods.
Several papers additionally report cytotoxic activity of Ukrain® on other cell lines such as prostate cancer (LNCaP, PC-3), glioblastoma (T60, T63, primary cancer line), pancreatic ductal adenocarcinoma (HPAF-II, HPAC, PL45, renal clear cell or papillary cell-derived lines (Caki-1, Caki-2, ACHN), melanoma (B16F10) and murine (TUBO, 4T1) or human (SKBR-3) breast cancer (see Table 2 for respective references).
However, due to the conflicting data about the chemical identity of this product, the previous results would have to be repeated or reinterpreted. If indeed Ukrain® is just a version of alkaloid mix, all the research done using it as an active substance would extend the scope of anticancer properties of C. majus native alkaloids. One of the early interesting findings is radiosensitizing influence of Ukrain® (Cordes et al., 2002, 2003) and it should be verified using non-modified alkaloid-rich extract and individual compounds to find out which of them or a combination would be responsible for this property. Also, other studies reporting Ukrain® as an active principle need re-evaluation with native alkaloids as probably constituting major component in the patented preparation. Moreover, Ukrain® remains the only preparation from C. majus that has a vast literature documenting its anti-cancer and also other properties in clinical (see the respective section in the present paper), pre-clinical and in vivo studies.
Anti-inflammatory and Immunomodulating
Some of the versatile traditional uses of C. majus can be explained, as in many other herbs, by anti-inflammatory potential targeting various pathways in the organism as well as modulation of immune response. Both have been confirmed in many studies using in vitro cellular models, as well as in vivo.
The ability to inhibit inflammation or, in some cases, to stimulate immune response and mitigate excessive reactiveness can contribute to the postulated anticancer properties and improve symptoms of gastric disorders as well.
Schneider et al. (2016) used human colon cell line (NCM460) to check anti-inflammatory action of composite preparations containing C. majus marketed and popular in Europe and any of the individual components. C. majus extract was among the most potent in a couple of checked parameters, such as inhibition of IL-8, MCP, and I-TAC secretion, thus contributing significantly to the overall and apparently synergistic combination of active principles. This activity was likely to influence the clinical outcome mentioned below (Abdel-Aziz et al., 2017).
Mostly, the total extracts or isolated alkaloids were tested, but in our opinion, other components such as hydroxycinnamic derivatives, flavonoids and chelidonic acid are likely to contribute significantly. Chelidonic acid [45] was efficient in mouse models of ovoalbumin-elicited allergic rhinitis (Oh et al., 2011) and ulcerative colitis (Kim et al., 2012). This compound also attenuated inflammatory responses by reducing levels and gene expression of several mediators and enzymes in colon tissues (COX-2, HIF1α, PGE2) and in allergic mice (IL-4, IL-1β, COX-2, caspase-1, and increase of IFN-γ). In human mast cell line HMC-1 stimulated for inflammatory response by the phorbol ester (TPA) and calcium ionophore A23187, chelidonic acid [45] inhibited IL-6 expression by blocking NFκB (Shin et al., 2011).
Stylopine [33] added to the cell culture of in lipopolysaccharide-stimulated RAW264.7 macrophages was inhibiting production of several proinflammatory molecules such as nitric oxide, PGE2, TNF-α, IL-1β, and IL-6 (Jang et al., 2004). Also, iNOS and COX-2 protein levels were lowered. However, the cyclooxygenase activity inhibition was not selective. Chelidonine [1] and 8-hydroxydihydrosanguinarine [14a] in the same model inhibited NO production and iNOS and COX-2 gene transcription (Park et al., 2011). These results suggest the underlying inhibition of NFκB as potential mechanism but it wasn't investigated in this study. However, in HCT-1 colon cancer cell line treated with chelidonine [1], the NFκB activation was blocked by inhibition of IκBα degradation and nuclear translocation of p65 (an NFκB subunit) as well as mitogen-activated protein kinase pathway activation by blocking c-Jun N-terminal kinase and p38 phosphorylation (Zhang et al., 2018).
Alkaloid fraction and sanguinarine [12] were efficient against carrageenan-induced rat paw edema but chelerythrine [9] showed lower activity (Lanfeld et al., 1981). However, in the later study by Mikołajczak et al. (2015), various fractions of water extract at relatively high doses of 200 mg/kg body weight failed to alleviate the inflammation in the similar model. The crude water extract treatment actually aggravated the paw inflammation. Conversely, the extracts containing mainly coptisine [31] and chelidonine [1] were effective in hot plate test for antinociceptive properties that suggests a supramedullary way of action.
Chelerythrine [9] inhibited inflammatory and pain reaction in several in vivo and cell models employed by Niu et al. (2011). In vivo, i.p administration of the alkaloid (1-5 mg/kg) alleviated mouse ear edema, rat paw edema, and abdominal constriction (pain reaction). Also, the isolated peritoneal macrophages upon treatment with 0.0001-1 μg/ml chelerythrine [9] had dose dependently reduced PGE2 and COX-2 expression.
In NC/Nga mice model for atopic dermatitis induced by DNCB (1-chloro-2,4-dinitrobenzene), the hydroethanolic extract from aerial parts alleviated several measures of dermatitis such as itching behavior and skin severity (Yang et al., 2011). Interestingly, the oral administration at doses of 200 mg and 400 mg/kg were even more efficient than topical application as 1 and 2% smear. The IgE levels were put back to control values upon the higher dose oral administration and reduced by about 50% after topical treatment. Also, IL-4 and TNF-α serum levels were significantly reduced but remained higher than in control animals.
In an animal model of ovoalbumine-provoked asthma, chelidonine [1] suppressed eosinophile-mediated inflammation. The activity at the doses of 1 and 5 mg/kg body weight was similar to 0.5 mg/kg body weight dexamethasone. Among the several monitored parameters such as different pro-inflammatory cell population counts in bronchoalveolar lovage fluid and lungs, IgE, and cytokine protein and transcripts levels, some were inhibited even stronger than by dexamethasone (total BALF cells, Gr-1+/CD11b+ cells, IL-4), whereas others were inhibited either similarly or weaker than the standard drug. It suggests a specific mechanism involving STAT6 and Foxp3 transcription pathways (Kim et al., 2015). Yet another inflammation-based condition absent from list of traditional indications, in which C. majus was found to be efficient is arthritis (Lee et al., 2007). In the mice model of collagen-induced arthritis, aqueous-methanol extract at the oral doses of 40 and 400 mg/kg body weight, suppressed progress of joint damage as well as a set of studied inflammation-related cellular and biochemical parameters. In the higher dose regime, the incidence of arthritis decreased from 100 to < 40% during 4 weeks. Cell invasion into lymph nodes, spleen, thymus and synovial fluid was inhibited in all organs, but most marked effect was in lymph nodes and joints. Among the tested T cells populations, there was an insignificant decrease of CD4+, CD8+, and CD3e+ cells, but a quite remarkable decrease of CD19+ B cells, almost to the level of non-arthritic animals. On the other hand, the number of regulatory CD4+CD25+ T cells increased significantly that suggests mobilizing adaptive response of the immune system to counteract and balance the excessive inflammatory processes. Suppression of inflammation mediators such as IL-6, TNFα, IFNγ was observed as well as lowered level of IgG and IgM but the latter only upon the higher dose treatment. However, the extract was not standardized an no particular compound or phytochemical class could be pinpointed as determining this potent anti-arthritic action.
A couple of reports indicate a potential of C. majus against Alzheimer's progression due to significant inhibition of acetylcholinesterase (AChE) without influence on butyrylcholinesterase (BuChE), which is a desired profile for potential drug-likeliness. The extract inhibited AChE in vitro by 98% at a concentration of 200 μg/ml and BuChE by only 13%. The isolated alkaloid 8-hydroxydihydrochelerythrine [10a] was the most active and AChE-selective (IC50 0.61 μM and selectivity index 56.7) (Cho et al., 2006). In another study, chelidonine [1] and other active alkaloids were completely unselective with similar IC50 values against AChE and BuChE (Cahlíková et al., 2010). Interestingly, the specific AChE inhibiting activity by coptisine [31]-rich extract was discovered in vivo in a herbivore insect Lymantria dispar in which this activity contributes to killing the pest (Zou et al., 2017). By this property the plant is protecting itself from herbivore attack and it would be a feasible biological explanation of the therapeutic potential existence in the wild growing species.
The analgetic properties of alkaloids, mentioned before (Mikołajczak et al., 2015) can be explained by the observations of interaction with glycine transporters (Shin et al., 2003; Jursky and Baliova, 2011). The water extract inhibited the glycine-activated and enhanced glutamate-activated ion current in isolated rat periaqueductal gray (PAG) neurons studied by patch-clamp technique (Shin et al., 2003). Chelerythrine [9] and sanguinarine [12] selectively inhibited GlyT1 (but not GlyT2) in the micromolar concentrations (5-10 μM) while berberine [28] showed no inhibition in transfected HEK293T cells. GlyT1 inhibition was time-dependent, noncompetitive and increased with glycine concentration. Interestingly, chelerythrine [9] effect was reversible while sanguinarine [12] persisted through washing out (Jursky and Baliova, 2011).
Clinical Studies
Quite typically for many traditionally used medicinal plants, the clinical evidence of efficacy remains scarce and C. majus is no exception. Therefore, the relatively numerous pharmacological studies need rigorous verification by properly designed and supervised clinical studies. At present, most of the allegedly curative properties toward some important complaints remain unconfirmed, even if there is strong pre-clinical evidence. The millennia long tradition of use becomes confirmed in a significant part even if some mythical indications turned out to be invalid or perhaps have been misunderstood or distorted when passed through generations of practitioners and authors.
Several clinical studies exist on the compound preparation STW-5 containing 10% of C. majus herb extract (Drug-to-Extract Ratio = 1:3) as one of nine ingredients. The gastric disorders are the main indications and was evidenced both clinically and pharmacologically (Von Arnim et al., 2007; Abdel-Aziz et al., 2017). Certainly, there is no proof that C. majus was the essential ingredient in this preparation but the functional dyspepsia/postprandial distress syndrome it seems to be one of the most active (Abdel-Aziz et al., 2017) influencing acid regulation and antrum contraction as well as moderate mitigation of inflammatory reactions. The mechanisms behind these effects were also studied in vitro using cell lines showing anti-inflammatory activity (Schneider et al., 2016). IFN-γ dependent stat1 phosphorylation was postulated as a putative mechanism of action in which C. majus extract was among the averagely active, but apparently the whole composition had superior properties suggesting synergistic rather than additive effect.
A series of older clinical trials were performed on a product containing C. majus extract to test the efficacy in patients with bile tract and gall bladder complaints and gall stones. These studies, reviewed in detail in the EMA report, suggest significant improvement of many clinical parameters. These included: subjective complaints (pain attacks, feeling of fullness), physician's examination (sonography of gall bladder and liver, liver palpation, meteorism, jaundice) and laboratory tests (bilirubin, transaminases, blood sedimentation). The patient conditions in which the preparation containing C. majus alkaloids (in daily doses of ca. 0.2 mg alkaloid sum as chelidonine [1]) was administered with positive outcome were for example: cholelithiasis, cholangitis/cholecystitis, post-cholecystectomy, and alcohol toxic liver damage. In the entire area of gastrointestinal/hepatic complaints, one can estimate the number of human subjects involved in to date published clinical literature as exceeding 1,500.
An impressive number of studies suggest antitumor properties of the apparently semisynthetic product - Ukrain® that showed efficiency in several in vitro studies on an assortment of neoplastic and non-transformed cell lines (Capistrano et al., 2015) but it by large failed to demonstrate clinically relevant activity in humans. Several case reports have been published that suggest its beneficial action in a range of malignancies, such as melanoma, metastatic breast cancer, various carcinomas.
The randomized clinical trials (RCTs) using Ukrain® were reviewed over a decade ago (Ernst and Schmidt, 2005) with a conclusion that despite the intensive publishing activity of several groups testing Ukrain® against miscellaneous malignacies with very promising outcome, most of these data are full of shortcomings that prevent unequivocal credibility. Since then, only a few more studies have been realized but it is still far from definite resolving of this issue, especially in the situation that the chemical analyses, some of which were even co-authored by the inventor (Jesionek et al., 2016) proved that it is not the compound which was initially claimed.
Most of the RTCs published were full of inconsistencies or even appeared unreliable due to the insufficient information regarding the methodology. Despite the spectacular results in such malignancies as pancreatic or terminal colorectal cancer, serious doubts remain about the evidence provided in some of these trials. Some of the studies are so poorly documented that it would undermine the validity of the results. For example, missing or suspect randomization method, missing methods for tumor dimensions measuring, unclear protocols, subjective outcome evaluation, lack of proper statistics. Also, a great majority of the clinical trials and case reports were published in one journal. Nonetheless, these few reports on Ukrain® remain the only available clinical data focusing specifically on C. majus and strongly suggesting extraordinary antitumor potential.
Since the potential conflicts of interest, reliability of the clinical and chemical data of this product have been disputed repeatedly (Farrugia and Slevin, 2000, 2001; Nowicky, 2001; Ernst and Schmidt, 2005), more studies are indeed mandatory also in this aspect of C. majus as a source of highly potent substances with multidirectional mechanisms of action. Nonetheless, it should be borne in mind that approval of an extract/mixture based drug for clinical practice in such a sensitive field as oncology is highly improbable at the moment, and most likely it would remain so in the near future. Thus, further studies are mandatory that would focus on using individual alkaloids as lead structures for drugs that would target multiple anticancer mechanisms. On the other hand, clinical data on efficacy of various C. majus products used in complementary or adjuvant therapy may facilitate the tedious process of drug development without changing the existing paradigm of chemotherapy based on combination of chemically defined substances. Quite differently from the relatively safe (with limitations described below) gastrointestinal and cutaneous indications, the cautious policies of EMA and other agencies demonstrated in case of declared anticancer actions are reasonable and should be continued.
Toxicology and Safety Issues
Repeatedly, studies and case reports occur that suggest hepatic injury/hepatotoxicity. It is especially important because one of the main indications of C. majus relates to liver and biliary tract disorders due to its cholagogue and hepatoprotective activities. The incidence of hepatotoxic cases and the possible clinical importance and safety issues have been reviewed recently (Pantano et al., 2017). Specifically, the results from animal studies are ambiguous and suggest a rather complex mixture of various mechanisms, such as inducing or alleviating oxidative stress or modulation of hepatic enzymes such as MAO and SOD, or slowing down mitochondrial respiration. The mitochondrial toxicity was related to DNA intercalating properties of sanguinarine [12] and chelerythrine [9].
In humans, numerous reports have been recorded since the 1990's. The main symptoms included cholestasis and mild to severe liver impairments with quite well documented causality in a majority of cases. In total, over 50 such cases have been reported from Europe, mostly from Germany (Etxenagusia et al., 2000; Stickel et al., 2003).
However, no certain constituent has been directly linked to the toxicity of the herb. On the contrary, it has been suggested that drug interactions rather than intrinsic toxicity are responsible for reported cases. Also, individual hypersensitivity or allergy has to be considered. Latex contained in the fresh plant is also possibly more toxic or allergenic than the dried material (EMA report, 2011).
In an in vitro study on HepG2 cells treated with various solvent extracts (Orland et al., 2014), the biotransformation and toxicity-related gene expression was enhanced, but the dichloromethane extract richest in chelidonine [1] and total alkaloids was the weakest inducer and least cytotoxic, whereas ethanolic extracts containing more coptisine [31] and sanguinarine [12] were more cytotoxic. However, the in vivo relevance of these results is uncertain. In rats, the high doses (up to 3g/kg body weight; Mazzanti et al., 2009) did not elicit any symptoms of hepatic injury and did not alter liver function.
Despite proven interaction of sanguinarine [12] and other alkaloids with DNA, no genotoxicity was observed using well established methods such as Ames tests or in vivo DNA damage (EMA report, 2011).
Nonetheless, uncontrolled internal use of unstandardized preparations should be discouraged and appropriate pharmacovigilance measures should be implemented to prevent unnecessary complications following C. majus administration. The comprehensive pharmacotoxicological investigation is also needed and should encompass establishing toxic doses range of various forms and preparations as well as individual constituents.
The European Medicine Agency therefore published the following recommendation:
"Two possible therapeutic indications were proposed for the monograph:
Traditional herbal medicinal product:
1) For symptomatic relief of digestive disorders such as dyspepsia and flatulence (oral intake)
2) For treatment of warts, callus and corns (cutaneous use)" (EMA, 2011).
However, based on the reported undesirable effects, including cases of liver damage, special precautions are necessary, especially during pregnancy and lactation, people suffering from liver diseases or taking liver-damaging drugs.
Conclusions and Future Outlooks
Much effort has been put into the recognition of bioactive compounds contained in the extracts of C. majus and the mechanisms of their action. Numerous reports have been published about its effectiveness in treatment of different medical complaints, such as gastrointenstinal and hepatobiliary disorders or cutaneous ailments caused by a microbial or viral infection. Next to the huge number of scientific reports indicating the beneficial effect of C. majus, information about its potential toxic properties emerged. The use of herbal remedy carries the risk of adverse effects, which is why it is important to know the raw material. Official recommendations do not exclude the use of C. majus as traditional herbal medicinal product. Therefore, further research on the mechanisms of action during treatment should be carried out. It is also important to control the phytochemical composition of the raw material both in conventional growing conditions and in vitro cultures. The more so, because the potential risk of carcinogenesis and hepatotoxicity is still not well documented.
Finally, we conclude that the millenia long history of Chelidonium in folk, traditional, and official medicine is far from coming to the end. On the contrary, recent years witnessed a revival of advanced pharmacological and mechanistic approaches using both native complexes and individual components in discovery of the therapeutic potential of this herb. We are quite convinced that in the near future, at least some of the already known and evidence-based properties should and would find their place in officially recognized therapeutic procedures. Moreover, new discoveries should broaden the scope of traditional usage and deliver better preparations, especially combining different classes of active molecules such as proteins, alkaloids, chelidonic acid, and perhaps also polyphenols. To achieve it, much more joined and interdisciplinary efforts are necessary. Further, the plant's diversity on different levels must be thoroughly evaluated and optimal combination of the complex profile should be established for various target applications. As one of the oldest proverbs from Aristotle's book, also referring to C. majus original Greek name, "one swallow does not make a spring," even if we observe a lot of "first swallows," we urgently need more, to ensure rational exploitation of the huge potential hidden in the inconspicuous and common weed.
Author Contributions
SZ: Wrote phytochemical and bioactivity parts of the manuscript and critically reviewed and corrected the final manuscript; AJ-D: Wrote the ethno botanical and historical section of the manuscript; MW-K and IS: Contributed to the phytochemical and analytical parts of the manuscript; AJ: Contributed to the antimicrobial activity section of the manuscript; AM: Developed the entire concept of this review, wrote bioactivity and pharmacology sections, and critically read and corrected all parts of the paper.
Funding
The scientific activity of Botanical Garden of Medicinal Plants is supported by the Ministry of Science and Higher Education of Poland, grant No. 215259/E-394/SPUB/2-16/1. AM and SZ get support from the Wroclaw Medical University grant No. ST.D030.17.028.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
Assistance in graphical abstract preparation by Miss Hanna Zielinska is kindly acknowledged. The support of Wroclaw Medical University is acknowledged.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10 ... y-material
Supplementary Figure 1. Chelidonium majus has been highly valued by ancient physicians. Nowadays, it is still attracting researchers' and clinicians' attention for its unique composition of the yellow latex, rich in alkaloids and proteins.
Supplementary Table 1. The examples of conditions used to isolate alkaloids from C. majus.
Supplementary Table 2. The examples of application the separation techniques in analysis of alkaloids from C. majus.
Footnotes
Source: Frontiers