Greater Celandine’s Ups and Downs -21 Centuries of Medicinal Uses of Chelidonium majus From the Viewpoint of Today’s Pharmacology

aC/ 1Pharmaceutical Biology and Botany, WrocAaw Medical University, WrocAaw, Poland

aC/ 2Botanical Garden of Medicinal Plants, WrocAaw Medical University, WrocAaw, Poland

aC/ 3Analytical Chemistry, Medical University of Lublin, Lublin, Poland

aC/ 4Pharmaceutical Microbiology and Parasitology, WrocAaw Medical University, WrocAaw, Poland

As antique as Dioscorides era are the first records on using Chelidonium as legal remedies to several maladies. Inspired by the” signatura rerum ” principle and an apparent ancient folk tradition, various clues were given, such as anti-jaundice and cholagogue, pain-relieving, and quite often mentioned- ophthalmological problems. Central and Eastern European folk medicine has always been using this herb extensively. In such regions, the plant is known under many unique vernacular names, especially in Slavonic speeches, associated or not with old Greek relation to “chelidon”- the swallow. Typically for Papaveroidae subfamily, yellow-colored latex is produced in abundance and leaks intensely upon injury. Major pharmacologically relevant components, most of which were first isolated over a century ago, are isoquinoline alkaloids- berberine, chelerythrine, chelidonine, coptisine, sanguinarine. Modern pharmacology took interest in this herb but it has not ended up in gaining an officially approved and evidence-based herbal medication status. On the contrary, the number of relevant studies and publishings tended to drop. Recently, some controversial reports and sometimes insufficiently proven analyses appeared, indicating anticancer properties. Anticancer potential was in line with anecdotical knowledge spread in East European countries, however, in the absence of directly-acting cytostatic compounds, some other mechanisms might be involved. Other properties that could boost the interest in this herb are antimicrobial and antiviral activities. Being a common synanthropic weed or ruderal plant, C. majus spreads in all temperate Eurasia and acclimates well to North America. Little is known about the natural fluctuation of bioactive metabolites, including several aforementioned isoquinoline alkaloids. In this review, we put together older and recent literature data on phytochemistry, pharmacology, and clinical analyses on C. majus is targeted at a critical evaluation of state-of-the-art from the viewpoint of historical and folk indications. The disputes around this herb, the safety and narcotic quality issues and a prospective role in phytotherapy are discussed as well.

Introduction

” No less extraordinary is the property of the herb Celandine; which, it is said, if any human shall have this herb, with the heart of a Mole, he shall overcome all his foes, all matters in suit, and shall put away all debate ,” and” if before named herb be put upon the head of a sick human, if he shall die, he shall sing anon with a loud voice, if not, he shall sob “; and” it bringeth the business begun to an end ,” so wrote Albertus Magnus in 13th century A.D.( Best and Brightman, 1999 ). Nowadays, humankind would definitely benefit from such a miraculous redres. Unfortunately, such claims about Chelidonium majus L.- the Greater Celandine, have not been verified according to the modern evidence-based approach( but no data on rigorous testing toward such properties actually exist in the literature ). However, through years of investigations, many other properties ascribed to this inconspicuous but characteristic plant have been confirmed or re-discovered. Several others could not be positively corroborated. Despite the widespread use in folk medicine and in official phytotherapy, both in Europe and in Traditional Chinese Medicine, the celandine herb did not join the most popular herbal remedies such as chamomile, valerian, St. John’s wort or ginseng. It has been listed in pharmacopeias and sold in pharmacies in parallel to the spontaneous collection by people trying narcotics against gastrointestinal disorders, cancer, infections, but especially against warts and any skin protuberances. This was the reason to combine the available historical and ethnobotanical data with the state-of-the art in pharmacology of C. majus and its components in the present review( Supplementary Figure 1 ). To date, only a couple of papers have its consideration of pharmacological and phytochemical knowledge with the EMA assessment report from 2011( European Medicines Agency, 2011) and the review( Gilca et al ., 2010 ), being the most recent and comprehensive ones. Biswas( 2013) has published a short review with overview of selected bioactivities but only encompassing a fraction of available literature and suggesting future directions of research. Similar approach to review the C. majus properties was used by Arora and Sharma( 2013) who summarized some activities based on selected literature and included pharmacognostic characteristics. Gilca et al.( 2010) has categorized the pharmacological proof into the categories related to traditional usage, also from the viewpoint of the TCM, such as anti-infectious, spasmolytic, gastric and hepatic, and anticancer. They also listed traditional indications that had not been confirmed by modern research, such as diuretic, anti-edema, expectorant and antitussive, pulmonary, and ophthalmological diseases. Some current info is also available in herbal volumes and compendium( Wichtl, 2004) or in the Internet. The latter source is of course difficult to verify.

Therefore, we chose to include a maybe high number of available literature, by selecting records from database search( PubMed, Scopus, Google Scholar) with the term “chelidonium” or “celandine” and manually eradicating newspapers pertaining to field botany, ecology and other aspects not relevant to medicinal employ of this plant. Some references that have been reviewed earlier( e.g ., in Colombo and Bosisio, 1996) is likewise not quoted directly if the information was redundant. Some information about the historical applications and folk medicine in Central and Eastern Europe were obtained from sources available in local libraries. In particular, we describe the phytochemical composition of various parts of the plant, the methods used for obtaining extracts and analysis of the herbal material. Further, we critically summarize the most credible research on bioactivity and clinical efficacy of various products and substances from C. majus. In addition, the highly debated and controversial issue of the patented, apparently semi-synthetic drug NSC-6 31570( UkrainA( r )) promoted chiefly as a cancer remedy was discussed( Ernst and Schmidt, 2005 ). With this review, we hope to encourage more research and attract interest to this quite common but not always adequately respected weed.

Botanical Description

C. majus L.( Papaveraceae) is a short-lived hemicryptophyte. It has up to 1 m high stem, branched and sparsely pubescent. The alternately placed foliages are light bluish at the bottom and green at the top. The basal leaves are long-petioled, with the obovated in contour, pinnatosected leaflets with 5-7 lobed segments. The apical leaves are short-petioled, with 3-lobed leaflet. From April to October the plant produces umbellate inflorescences with 2-6 flowers, which have 4 bright yellow petals and two whitish, early fell sepals. The fruit is an elongated( 3 cm ), pod-shaped, multiseeded capsule, dehiscent with two valves. The seeds are shiny, ovate and dark brown or black, with elaiosomes. The underground portion is a short tap root with numerous and elongated lateral roots. The whole plant contains yellow to orange latex. C. majus grows in the lowlands and foothills in leafy woodlands, in brushwood, parks, gardens, on the roadsides and around buildings. It opts moist soils rich in nitrogen and organic matter( Zarzycki et al ., 2002 ).

Distribution Area

C. majus is native in Europe, western and central part of Asia and in northern Africa. It occurs from Portugal in the West, to Central, Eastern to Northern Europe. The Asian range encompasses Turkey, Iran, Kazakhstan, Mongolia, Caucasus, and Siberia. In North America it is an introduced plant.

Taxonomy and Nomenclature

Until the mid-twentieth century the genus Chelidonium L. was monotypic with C. majus L. as the only species. In 1982 Krahulcowa based on cytotaxonomic study of C. majus L. sensu lato, proposed to divide the genus into two microspecies. She proposed C. majus L.( 2n= 12) distributed in Europe, Siberia, and China and a new species C. asiaticum( Hara) Krahulcova( 2n= 10 ), an East Asian vicariant( KrahulcovA !, 1982 ). Aside from the difference in chromosome numbers and distribution area, C. asiaticum slightly differs morphologically from C. majus. It is more hairy, with narrower and sharper foliage lobes. Within C. majus she distinguished European C. majus L. subsp. majus with more laciniate lobes of leaves, and C. majus. subsp. grandiflorum( DC .) Printz, in South Siberia and China( KrahulcovA !, 1982 ).

( KrahulcovA !, 1982) Name of the genus derives from Greek( IIuI >> I1I’ A3I1/ 2I1oI1/ 2) with chelidon( IIuI >> I1I’ A3I1/ 2) meaning swallow( a bird) for the plant usually blooms simultaneously with arrival of these birds. The specific epithet majus in Latin entails bigger.

Common English name: Greater celandine.

The name celandine originates from Medieval Latin word celidonia, a phonetic variant of Latin chelidonia, which was recorded by Pliny. Similarly, the German name, SchAPllkraut, comes from Schellkraut( Bauhin, 1651 ), which is derived from Latin-Greek chelidonium( Waniakowa, 2015 ).

Common and Folk Names in Some European Languages

Albanian- latrapeci, bar saraleku; Belarussian- padtynnik, barodaunik( wart herb ); Bosnian- rosopas; Bulgarian- zmiysko mlako; Croatian- zmijino mlijeko, rosopas; Czech- vlaA! toviAnAk, celadona, celdunA, cen dalie, dravnicovina, hadA mlAA, krkavnAk; Dutch- stinkende gouwe; English- Tetterwort, devils’s milk, boulder poppy; French- grande chA( c) lidoine Aclair, herbe aux boucs, herbe a l’hirondelle; German- SchAPllkraut, Gilbkraut, Goldwurz, Schwalbenkraut, Warzenkraut; Italian- celidonia, cinerognola, Montenegrin- rusopas, rusa; Polish- glistnik jaskA3lcze-ziele, celidonia, cyndalia, cencylja, glistewnik, gliAnik, niebospad, zAoty groszek, zAotnik, zA3Ate ziele, zA3Ate kwiatki, roztopaAA; Ukrainian- hladyshnyk, hnystnyk, zhovtyi molochay, smetannyk, chystotil; Romanian: rostopascA; Russian- chistotel; Rusyn- rostopast ‘; Serbian- rusopas, rusa, rusomaAa; Spanish- Golondrinera, Hierba de las verrugas( wart herb ).

Chelidonium majus in Folk Medicine

History of Usage

C. majus has been known as medicinal species since the very Antiquity. Medicinal properties of C. majus were described by Dioscorides and Pliny the Elder in the first century AD. Dioscorides in De Materia Medica states that celandine begins to blossom when the swallows arrive and withers when they depart. He also refers to a lore saying that swallows restore sight to their blind snuggles with use of celandine( Osbaldeston and Wood, 2000 ). Pliny the Elder repeats these accounts in Naturalis Historiae( Jones, 1966 ).

The foremost medicinal use of celandine, described since the ancient times until the 16th century, was treating visual impairment and eye illness. For such conditions, Dioscorides advised to use herb juice, simmered with honey in a brass ship. The juice could also be dried in the shade and the resulting small pellets were ingredients of other medicinal products. According to Dioscurides celandine soaked in wine along with anise fruits was helpful in treating jaundice and dermatologic disorders such as herpes. Besides, chewing on a root alleviated toothache( Osbaldeston and Wood, 2000 ). Pliny advised a kind of eye lotion, which takes its name, chelidonia, from the name of the plant( Jones, 1966 ).

Celandine was an admired medicinal plant during the course of its Middle Ages, largely used to cure eye cancers, for throat cleansing, therapy of ulcers and scalp eczema as well as against colic and jaundice( Mayer et al ., 2003 ). In 1080, French monk and physician Odo Magdunensis wrote De viribus herbarum, also known as Macer floridus, a botanical lyric describing medical effects of 77 plants. One of the chapters of M. floridus deals with medicinal application of celandine in” visual impairment as well as scalp and liver conditions”( Mayer et al ., 2003 ). Hildegard of Bingen wrote about celandine in her run Liber subtilitatum diversarum naturarum creaturarum created during 1150 -1 160 A.D. and finally published in 1533. Hildegard recommended celandine juice to enhance sight and juice mixed with tallow as a cure for skin ulcer( Mayer et al ., 2003 ). Moreover, celandine would be a strong aphrodisiacum, but causing infertility on ladies. Breathing the smell of the plant by spouses prevents arguments. Celandine was also recommended by to treat jaundice and against hair overgrowth( CzekaAski, 2007 ).

Since the sixteenth century, according to Paracelsus’s signature doctrine, celandine was used to treat jaundice and liver diseases( Rostanski, 1997 ). C. majus was described in comprehensive botanical medical works of scholars such as: Joannes Minoritanus, Marcello Vergili, Hieronymus Bock, Leonhard Fuchs, Pierandrea Matthioli, and Adam Lonicer. The authors refer to antique sources and recommend employing celandine to treat eye and skin conditions. According to Lonicer, to cure various dermatologic diseases, known in those days as “leprosy,” the juice of root causes of celandine had to be applied on the scalp, conjointly with drinking the juice mixed with syrup of common fumitory( Fumaria officinalis) twice a day for 9 days( Mayer et al ., 2003 ). At the turn of the sixteenth and seventeenth century two large works on herbs and their applications were writes to Poland. The first is Herbarz Polski by Marcin of Urzedowo, printed in 1595 and the second is Herbal by Simon Syrenius, published in 1613. Marcin of Urzedowo described, based on works of Dioscorides, uses of celandine to treat eye cancers, jaundice, wounds, toothache, and colic. To cure sight impairment, herb juice boiled with honey or pellets made of juice dried in shade should be used. Cataract, on the other hand, should be treated with juice draining from a broken stem or root of the plant. In instance of jaundice, root of celandine should be simmered in white wine and the decoction should be drunk for a few days. The crashed root with wine was drunk to treat colic and applied on wounds. Applying a piece of root of celandine to an aching tooth, alleviated the ache( Marcin of Urzedowo, 1595 ).

Simon Syrenius, described celandine-based recipes used in eye diseases. The main ingredient of such medications is fresh celandine juice. As one of the few authors, Syrenius considered the juice irritative and therefore recommended mixing it with small amounts of vinegar, milk, or rose water. He also advised drinking a decoction of celandine roots cooked in wine with anise or a mixture of pulverized root with vinegar before bedtime to treat jaundice, colic, and stomach ache. In case of toothache he advised to rub teeth with powdered root with vinegar. Body ulcers and scabs on the head scalp can be cured with a salve of powdered root mixed with pork fat and vinegar. Alternatively, the powdered root alone could be put directly on the ulcers. Syrenius also describes the diaphoretic and diuretic effects of the herb along with the roots or the root itself. The root simmered in wine has a diuretic impact. As diaphoretic remedy Syrenius recommended taking dry bath of celandine herb, which would cause extensive sweating and expulsion of excess water from the body as well as drinking decoction from roots simmered in rose vinegar or white wine with great water dock( Rumex hydrolapathum ). In addition, the celandine could be used to dye the hair yellow, and to lighten the freckles and hyperpigmentation on the face( Syrenius, 1613 ). The British Flora Medica( Barton and Castle, 1845) cites the traditional applications of C. majus in therapy of jaundice, visceral blockages, fevers, dropsies, scrofula, syphilitic affliction, gout, cataract, ophtalmia, and specks as described previously by Dioscorides and Galen.

In countries, where C. majus is the native species, it became one of the most widespread narcotic of folk herbal medication. The scope of its applications in folk medicine demonstrates high similarity among many areas of Central and Eastern Europe. It is worth to mention its prevalent application to treat warts, eczema and other skin disorder, gastrointestinal parasites, jaundice, and liver objections, inflammatory eye infections and other illness, including cancer.

Skin Diseases

In folk medicine, C. majus was most commonly used to remove warts. Herb juice or latex were used most frequently for this purpose, however, utilize of leaves and blooms was also noted. In Poland it was common to scratch the fresh juice from the broken stem of the celandine onto warts( De Verdmon, 1936; KuAoniewski and Augustyn-Puziewicz, 1999; Kujawska et al ., 2016 ). In the Bieszczady Mountains and in the Podkarpacie Region( S-E Poland) the juice was utilized immediately on warts, or they were first scrubbed off and then the juice was applied on the meander( Szary, 2013 ). A cataplasm made of blooms that was supposed to be changed every few days was used in the Kielce Region( Central Poland)( Szot-Radziszewska, 2012 ). Occasionally, fresh foliages were also used( Kujawska et al ., 2016 ). The juice of the aerial part of the plant was used in the Ukrainian Carpathians( Szary, 2010, 2013) and in Russia( Zevin et al ., 1997 ). The herb juice was applied to treat warts also in the Balkan countries( RedA3/ 4iA, 2007; Tita et al ., 2009; MenkoviA et al ., 2011; Mustafa et al ., 2012; Koleva et al ., 2015) as well as in Central Italy( Menale et al ., 2006) and Great Britain( Barton and Castle, 1845 ). Other dermatologic conditions were also treated. All around Poland it was common to apply fresh foliages or juice on wounds. In Podolia( Ukraine) corns were treated by rubbing with a root of celandine and by application of fresh foliages. After a week, corns softened and ruptured. A salve from celandine, olive oil, fir resin and beeswax was a remedy for pustules( Kujawska et al ., 2016 ). In the Bieszczady mountains( Polish-Slovakian-Ukrainian frontier ), juice of celandine was applied to eczema and cuts, and decoction of root was used for baths and rinse for dermatologic conditions( Szary, 2013 ). In the Rzeszowszczyzna( S-E Poland) region leaves were applied to ulcers to induce picking up and rupture( Wdowiak and Bielecka-Grzela, 2013 ). The juice was also are applied to lighten freckles( KuAoniewski and Augustyn-Puziewicz, 1999 ). In Russia the juice of aerial proportions was used in the treatment of skin wounds, skin irritation, allergic rashes and dermatitis, leaves, and blooms in the treatment of simmers( Mamedov et al ., 2004 ). The aerial parts of the plant were used by the people of Montenegro to cure blisters, rashes, and scabies( MenkoviA et al ., 2011 ). In Central Serbia, juice was applied directly on scalp in skin eruptions, psoriasis and eczema( JariA et al ., 2007 ).

Liver Diseases

C. majus is one of the best-known folk medicine remedy for jaundice and liver diseases, such as inflammation, spastic conditions, and gallstones. In Poland, infusion made of young celandine leaves was used as a cholagogue and to regulate action of the digestive tract( KuAoniewski and Augustyn-Puziewicz, 1999 ). Jacques( De Verdmon, 1936) in case of jaundice advised infusion made of half of a teaspoon celandine per cup. All around Poland it was common to bath children with jaundice in celandine and to give celandine infusion to drink( Kujawska et al ., 2016 ). In the Bieszczady and in the Ukrainian Carpathians, herb infusion was drunk to relieve liver conditions( Szary, 2010 ). In Western Ukraine, infusion was used as relaxant in colic assaults( Szot-Radziszewska, 2007 ). Also in Balkan countries, celandine was employed in the treatment of liver disorders. In the Albanian Alps to treat hepatitis a decoction of fresh aerial portions has been drunk with sugar in small portions- half coffee cup( Pieroni et al ., 2005 ). In Serbia celandine was used internally for inflammation of the gallbladder, bile duct, jaundice, and hepatitis( JariA et al ., 2007; A avikin et al ., 2013 ). The use of celandine is similar in Gollak region in Kosovo( Mustafa et al ., 2012 ), in the Prokletije Mountains( MenkoviA et al ., 2011) and in Zagori in Epirus, North-West Greece( Vokou et al ., 1993 ).

Against Digestive Tract Parasites

Polish name of C. majus “glistnik”( roundworm herb) comes from a common traditional usage of this plant to expel roundworms. For this reason decoction of the herb had to be drunk for 12 days( De Verdmon, 1936 ). In the Bieszczady Mountains children were bathed in decoction of the celandine herb and were given celandine infusion to drinking( Szary, 2013 ). Decoction of seeds was also used in the Kielce Region( Szot-Radziszewska, 2012 ). In western Ukraine, infusion of the herb was prepared( Szot-Radziszewska, 2007 ).

Eye Diseases

Contrary to ancient phytotherapy, celandine was rarely used to treat eye conditions in folk medicine. Wdowiak( 2015) reports that in the Podolia Region( Ukraine) fells of celandine juice mixed with vodka were put into eyes. In west Ukraine a tincture made from celandine was applied. Furthermore, a popular belief among people of the Podolia Region, as well as the Lubelszczyzna and Podkarpacie Regions( Eastern Poland) said that feces of a swallow can cause sight loss, if they fall into the eye, which can be only cured by C. majus. In the small town Giby, of Polish-Lithuanian-Belarusian borderland, the celandine pollen was employed against eye infections( Kujawska et al ., 2017 ).

Other Usage

People of S-W Romania and Zagori in Greece were applying celandine as diuretic( Vokou et al ., 1993; Tita et al ., 2009 ). In the central Serbia and in Podolia( Ukraine ), celandine was considered a remedy for gout. Occasionally, it was used as tonic and stimulant of cardiac functions, also increasing blood pressure( Vokou et al ., 1993 ). In the Bieszczady Mountains people incensed aching teeth with the smoking from the burning herb( Szary, 2015 ). In Poland, juice was used internally to cure hydropsy( Kujawska et al ., 2016 ). In Romania it was esteemed as an antidote for snake venom( Tita et al ., 2009 ). People of Russian descent, called Russlanddeutschen, living in S-W Germany used celandine as depurative( Pieroni and Gray, 2008 ). Among the Hutsuls living on the Ukrainian side of Bukovina( S-W Ukraine ), tea from aerial parts of celandine was employed in the treatment of cancer ( SCukand and Pieroni, 2016 ). In Bosnia and Herzegovina it was used to cure cancer of lungs( RedA3/ 4iA, 2007 ). In veterinary treatments, herb decoction was given to kine suffering from inflammation of the udder, in case of dermatologic conditions animals were scratched with foliages. Furthermore, kine were given root to eat to cause vomit to relieve bloat. Besides, herb overcooked in milk was applied to ulcers( Kujawska et al ., 2016 ). In the Bieszczady Mountains celandine was a emblem of purification of living world from threatening demise, it was used as talismen to protect from demons( Szary, 2015 ). Dried herb was used to incense the interiors of huts to deter flies and mosquitos as well as during plague and other epidemics. Grains soaked in celandine juice were used as fish and bird poison( Szary, 2013 ).

Phytochemistry

For the therapeutic purposes, dried herb of C. majus is used( European Pharmacopeia ). In some regions( Central and Eastern Europe) roots are also exploited. European Pharmacopeia calls for total alkaloid content as chelidonine[ 1 ], assayed spectrophotometrically with additional TLC screening and microscopic authentication.

Alkaloids

Pharmacologically relevant substances of C. majus are isoquinoline alkaloids( Figures 1-7, Table 1 ). These are the components of latex produced in all plant parts, but flowers. Latex is stored in special secretory cells called laticifers. Presence of articulated laticifers with yellowish content is also used as an authentication microscopic mark in pulverized herb by pharmacopoeial monographs. The composition of latex is plant organ specific( TomA and Colombo, 1995; Sowa et al ., 2018 ). Generally, five groups of alkaloids were found in C. majus. These are the derivatives of phenanthridine( 3,4 -benzoisoquinoline ), protoberberine, protopine[ 37 ], quinolizidine, aporphine( Kadan et al ., 1990, 1992; Pavao and Pinto, 1995; TA! borskA! et al ., 1995; Petruczynik et al ., 2002; NeAas et al ., 2005; SArkAPzi et al ., 2006; Zhou et al ., 2012; Kedzia et al ., 2013; Grosso et al ., 2014; Poormazaheri et al ., 2017 ). More than forty alkaloids of different types were isolated and identified from C. majus( Figures 1-7 ). Major phenanthridine derivatives that were found in aerial and underground proportions are (+) -chelidonine[ 1 ], chelerythrine[ 9 ],( Kadan et al ., 1990; SArkAPzi et al ., 2006; Zhou et al ., 2012 ).

Protoberberine derivatives that amass in higher amounts are coptisine[ 31 ], berberine[ 28 ], stylopine[ 33]( Slavik and Slavikowa, 1977; Fulde and Wichtl, 1994; Shafiee and Jafarabadi, 1998; SArkAPzi et al ., 2006 ). Aporphine alkaloids like corydine[ 39] also seem( Slavik and Slavikowa, 1977; Shafiee and Jafarabadi, 1998; Kopytko et al ., 2005 ). Two protopines were found in C. majus, allocryptopine[ 36] and protopine[ 37]( Fulde and Wichtl, 1994; Shafiee and Jafarabadi, 1998; Kopytko et al ., 2005 ). Sparteine[ 38] is the only representative of quinolizidine alkaloids( Kopytko et al ., 2005) but no other publications report its presence. Moreover, new unusual turkiyenine-type alkaloid named( -) -turkiyenine[ 42] was found in C. majus from Turkey( Kadan et al ., 1990 ).

Alkaloid content in different plant organs was found to be unstable( Kustrak et al ., 1982; TomA and Colombo, 1995 ). Daily variations were probably due to the alkaloid degradation rather than translocation, because of similar time-course of the compounds accumulation in all plant parts. Significant increase of sanguinarine[ 12 ], chelerythrine[ 9 ], chelidonine[ 1 ], and coptisine[ 31] was observed during the day, with the highest content in the evening, whereupon the alkaloids diminished during the night( TomA and Colombo, 1995 ). Day illuminated seems to be the crucial factor influencing alkaloid biosynthesis in C. majus, especially in underground parts of the plants( Kustrak et al ., 1982; TomA and Colombo, 1995 ). Diurnal changes of alkaloid content seem to be less dependent on temperature, what was observed during winter day, when the alkaloid content was low and stable, due to the reduced metabolism and the senescence of the aerial portions. According to TomA and Colombo( 1995) total content of alkaloids in foliages was lower than in underground components. In latex the content was 32 times higher than in leaves and 9 times higher than in roots. These results suggest that the amount of alkaloids in plant organs depend on the number of laticifers in which the objective is stored. Moreover, the number of laticifers is likely organ specific. Laticifers in C. majus are unbranched( without anastomoses ), enunciated with perforated transverse cells( Hagel et al ., 2008 ). Enunciated laticifers develop from multiple cells. The structures form longitudinal rows composed of series of superimposed cells with perforated end walls. The type of laticifers can differ even within the same plant family. In another Papaveraceae species- opium poppy, the perforation of lateral walls leads to the formation of anastomoses( connections) between adjacent laticifer components, unlike that of Greater celandine( Hagel et al ., 2008 ). From wounded laticifers, a matrix emerges with different organic substances suspended in it. This excretion is called latex and depending on the plant species, it contains proteins, organic acids, alkaloids, sterols, tannins, and mucilage. The growth and development of laticifers runs close to the surrounding phloem, which affects the composition of latex. A major site of alkaloid accumulation in the protoplast of laticifer cells are vesicular organelles, that had been found in opium poppy between early 70′ s and 80′ s of the last century( Dickenson and Fairbairn, 1975; Roberts et al ., 1983 ). Unfortunately, research on the site of alkaloid biosynthesis in Greater celandine has not been continued since then. Recent reports fear protein determination in latex and confirm its complex composition( for more information consider the separate” Protein subsection” underneath ).

The presence and number of laticifers is due to the physiological functions of aerial parts and underground parts of the plant, such as responses to environmental factors, defense against herbivory, metabolic reserves, and energy store( Agrawal and Konno, 2009 ). Coptisine[ 31] was received mostly in fruits and herb( SArkAPzi et al ., 2006; Kedzia et al ., 2013; Sowa et al ., 2018 ), whereas berberine[ 28] proved no significant difference between aerial parts and roots( TomA and Colombo, 1995 ). Larger quantities of phenanthridine alkaloids like chelidonine[ 1 ], chelerythrine[ 9 ], sanguinarine[ 12] were observed in roots rather than in aerial parts of the plants( TomA and Colombo, 1995; Sowa et al ., 2018 ).

Total alkaloid content (%) in in vitro shoots and embryos expressed as chelidonine[ 1] was 1.53 and 1.58%, respectively( CiriA et al ., 2008 ).

Phenolic Compounds

Several flavonoids were found in aerial parts of C. majus in low quantities. Four diglycosides and five monoglycosides were identified as derivatives of kaempferol, quercetin, and isorhamnetin( kaempferol-3-O-rutinoside, quercetin-3-O-rutinoside, isorhamnetin-3-O-glucoside ). The identification was based on the mass spectra of the compounds( Grosso et al ., 2014 ). In stems, leaves, and flowers, 5a2-methoxy-flavonol[ 43] and 6a2- methoxy-flavonol[ 44] were also saw( Stancic-Rotaru et al ., 2003 ).

Hydroxycinnamic acids( caffeic, p-coumaric, ferulic) and their derivatives (( -) -2-( E) -caffeoyl-D-glyceric acid[ 46 ],( -) -4-( E) -caffeoyl-L-threonic acid[ 47 ],( -) -2-( E) -caffeoyl-L-threonic acid lactone, (+) -( E) -caffeoyl-L-malic acid[ 48 ]), as well as hydroxybenzoic acids( genistic, p-hydroxybenzoic) were identified in aerial parts( Hahn and Nahrstedt, 1993; WojdyAo et al ., 2007 ). Later, another three hydroxycinnamic acids were identified use HPLC-DAD-ESI/ MS: caffeoyl threonic acid, caffeoyl glyceric acid, caffeoylmalic acid( Grosso et al ., 2014 ), that have been detected previously by Hahn and Nahrstedt( 1993 ). Two caffeoyl acid derivatives isomers with precursor ions at m/ s 359 corresponding to rosmarinic acid were also found in aerial proportions( Grosso et al ., 2014 ).

Proteins

A phytocystatin- chelidocystatin are members of the first proteins isolated from latex and characterized( Rogelja et al ., 1998 ). Cystatins, a class of thiol protease inhibitors are involved in defense and stress-response mechanisms and could also contribute to the antimicrobial and antiviral activity of C. majus latex( Benchabane et al ., 2010 ). Whether or not the presence of cystatin is relevant to the medicinal properties and such traditional folk uses as anti-warts is yet to be found out.

In a series of papers, Nawrot et al.( 2007 a, b, 2008, 2013, 2014, 2016, 2017 a, b) described a number of proteins from root and leaf latex. Proteomic analysis using LC-ESI-MS/ MS system uncovered the presence of three categories of proteins according to their functions: proteins involved in disease and defenses answers( i.e ., superoxide dismutases, lactoylglutathione lyases ), nucleic acid binding proteins( i.e ., glycine-rich proteins, nucleic acid binding, DN-Abinding, or RN-Abinding proteins ), and these that are involved in general metabolism( acyl-CoA binding protein, malate dehydrogenase, flavodoxin-like quinone reductase, ubiquitin, polyubiquitin, serine/ threonine protein kinases, rubber elongation factor ). A total of 21 proteins were identified in C. majus latex, although in several suits the identification was based on correlation between experimental and the theoretical pI/ molecular mass, due to their low score outcomes. The results indicated less complexity of latex proteins in this species compared to opium poppy. Their contribution to the traditional use of C. majus as antiviral and antimicrobial remedy has to be further explored and may, in combination with highly active alkaloids, render unique synergistic consequences a multifaceted tool for combat against troublesome infections. In living plants, these proteins are likely also serving as a chemical defense against pathogens( Nawrot et al ., 2007 a, b, 2014, 2017 a, b ). Protein-bound polysaccharide( CM-AIa) bearing immunomodulatory and cytotoxic activity was isolated by Song et al.( 2002 ).

Other Compounds

Organic acids: chelidonic[ 45 ], malic, citric, succinic,( Kopytko et al ., 2005 ); Biogenic amines: histamine, methyloamine, tyramine( Kwasniewski, 1958 ); choline in fruit( Kwasniewski, 1958 ); essential petroleum constituents( Hansel et al ., 1992; KohlmA1/ 4nzer, 2000) triterpenoids( Hahn and Nahrstedt, 1993; Deng et al ., 2016 ); saponins( Kwasniewski, 1958; Kopytko et al ., 2005 ); Resin( Hahn and Nahrstedt, 1993 ); vitamins A, C, nicotinic acid( Hahn and Nahrstedt, 1993; Kopytko et al ., 2005 ).

Flowers contain xanthophyll pigments like lutein, violaxanthin, flavoxanthin, chrysanthemoxanthin( HorvA! th et al ., 2010 ).

Methods for Analysis of Active Components from Chelidonium majus

Alkaloids

First isolation of Chelidonium alkaloids was achieved in nineteenth century with an important contribution from Mr. Emanuel Merck’s company at Darmstadt( Henschke, 1888; Schmidt, 1888) and obtaining and characterization of pure compounds( chelidonine[ 1 ], chelerythrine[ 9 ], protopine[ 37 ]) was successful in the following years( Selle, 1890; Wintgen and Schmidt, 1901 ). Alkaloids of C. majus result as salts or bases depending on pH of medium; thus, their extraction was mostly carried out in acidic condition to convert all compounds to water-soluble salts. Methanol or ethanol often with addition of water ( Bugatti et al ., 1987; Han et al ., 1991; Koriem et al ., 2013) and hydrochloric( Kursinszki et al ., 2006; Gu et al ., 2010) or acetic acid( Paulsen et al ., 2015) were used as extractants. The isolation from plant material was also achieved with the use of pure acidified water; further, the answer was alkalized with ammonia or sodium hydroxide to procure base forms followed by liquid-liquid extraction with organic solvents such as dichloromethane, butanol or chloroform( SArkAPzi et al ., 2006; SA! rkAPzi et al ., 2007; Migas et al ., 2012; Jesionek et al ., 2016; Bogucka-Kocka and Zalewski, 2017 ). The isolation was conducted by percolation( Capistrano et al ., 2015 ), maceration( Koriem et al ., 2013; Borghini et al ., 2015 ), heating under reflux( Gu et al ., 2010; Yao et al ., 2011; Seidler-Aozykowska et al ., 2016 ), in water bath( SArkAPzi et al ., 2006) or Soxhlet apparatus( Bugatti et al ., 1987; Stuppner and Ganzera, 1995) as well as ultrasound assisted( UAE)( Kursinszki et al ., 2006; SA! rkAPzi et al ., 2007; Paulsen et al ., 2015; Jesionek et al ., 2016) or microwave energy( MAE)( Then et al ., 2000; Zhou et al ., 2012 ). Supercritical fluid extraction( SFE)( Then et al ., 2000) or SFE combined with enhanced solvent extraction( ESE) and low pressure solvent extraction with water ( LPSE)( GaA +- A! n et al ., 2016) was also applied. Furthermore, in situ solvent formation microextraction( ISFME) with the use of ion-pairing agent( KPF6) and a water-miscible ionic liquid ([ C6MIM ][ Br ]) which formed a hydrophobic ionic liquid extraction phase ([ C6MIM] PF6) for the pre-concentration of sanguinarine[ 12] and chelerythrine[ 9] was elaborated by Wu and Du( 2012 ). After extraction, the solution was usually filtrated through a 0.22 -I1/ 4m membrane( Gu et al ., 2010) or additionally purified use solid stage extraction( SPE) on C18 cartridge( Stuppner and Ganzera, 1995; Kursinszki et al ., 2006; SArkAPzi et al ., 2006 ). SArkAPzi et al.( 2006) developed ion-pair SPE with n-heptanesulfonic acid( HS ). Dried residue obtained since extraction was dissolved in methanol with 0.05 M hydrochloric acid, diluted with 0.05 M aqueous solution of HS and loaded on an SPE C1 8 microcolumn. Further, 70% HS( 0.05 M) in methanol was used to remove the matrix and 5% HS( 0.05 M) in methanol to elute analytes. The examples of conditions used to isolate alkaloids from C. majus are presented in Supplementary Table 1.

Thin Layer Chromatography( TLC)

TLC was largely employed for screening purposes or for qualitative analysis of alkaloid composition in C. majus extracts. This technique has been largely limited to the screening and multiple sample fingerprinting, but usually does not enable high-sensitivity or high-resolution insight into the minor components of the phytochemical profile. Despite of being less intensively modernise in comparison to column-based techniques like( U) HPLC, this method is still favored when cost-effectiveness and simplicity of sample preparation is important, for example in herbal industries and educational use. Some recent developments in mass spectrometry hyphenation in form of matrix transfer or DART( direct analysis in real day) utilized already in studying of other species( MA3ricz et al ., 2018) should be useful also for C. majus alkaloids.

Silica was the more common stationary stage, often impregnated with salts( Ni, Zn, Cr, Co) aimed at improving selectivity( Wagner et al ., 1984; Then et al ., 2000; Waksmundzka-Hajnos et al ., 2000; SArkAPzi et al ., 2006; SA! rkAPzi et al ., 2007; Petruczynik et al ., 2008; Jesionek et al ., 2016) Silica modified with octadecyl( C-1 8) and cyanopropyl( CN) groups and aqueous answers of methanol or isopropanol with ammonia or diethylamine which prevented tailing of chromatographic bands were sporadically applied( Petruczynik et al ., 2007 ). Petruczynik et al.( 2008) blended various types of stationary phases e.g ., cyanopropyl silica with silica or octadecyl silica to procure adsorbent gradient.

Apart from the routine isocratic elution with solvent concoctions, using different modes of gradient elution improved the separation, e.g ., as a two-dimensional or unidimensional multiple growth( SzumiAo and Flieger, 1999; Waksmundzka-Hajnos et al ., 2000; Migas et al ., 2012 ), bivariant multiple growth( Bogucka-Kocka and Zalewski, 2017 ), or stepwise gradient elution( Matysik and Jusiak, 1990; Waksmundzka-Hajnos et al ., 2000; Bogucka-Kocka and Zalewski, 2017 ). TLC separation of alkaloids was supported by employ of magnetic field( Malinowska et al ., 2017) or forced flowing of mobile stage( OPLC- overpressure layer chromatography or optimum performance laminar chromatography; Pothier et al ., 1993; Malinowska et al ., 2005 ).

TLC was also applied for direct bioautography( TLC-DB) to test the antibacterial activity of C. majus extracts( SA! rkAPzi et al ., 2007; MA3ricz et al ., 2015; Jesionek et al ., 2016) be included with densitometry for quantitative analysis( Then et al ., 2000; SArkAPzi et al ., 2006) and it demonstrated useful as preparative technique for isolation of alkaloids( Waksmundzka-Hajnos et al ., 2002; Koriem et al ., 2013 ).

High Performance Liquid Chromatography( HPLC)

High performance liquid chromatography( HPLC) and ultra-fast liquid chromatography( UFLC) have been the most often applied analytical techniques. For many years, separation of C. majus extracts was largely carried out in reversed stage( RP) system utilizing long( 150 or 250 mm) C18 columns with 4.6 mm of diameter and 5 I1/ 4m of particle size( Niu and He, 1994; Petruczynik et al ., 2002; Kursinszki et al ., 2006; Borghini et al ., 2015; Paulsen et al ., 2015; GaA +- A! n et al ., 2016 ). More recently, adsorbents with smaller particles( a $? 3 I1/ 4m) or shorter columns( Prosen and Pendry, 2016; Seidler-Aozykowska et al ., 2016) is likewise applied to achieve shorter separation and saving solvents. Mobile phases are usually composed of the representatives of water and acetonitrile or/ and methanol with different additives e.g ., ammonium formate/ acetate( Borghini et al ., 2015; Seidler-Aozykowska et al ., 2016 ), organic amines( triethyl-, tetrabutylamine)( Paulsen et al ., 2015 ), ion-pair reagents( sodiumdodecylsulfate, alkylsulfonic acids)( GaA +- A! n et al ., 2016 ). The additives were necessary to reduce peak tailing forming as a results of interaction of alkaloid cationic sorts with residual silanol group of stationary stage. Eluents were acidified with acetic or formic acid to pH <4 to avoid the co-occurrence of ionic and uncharged forms.

Normal-phase (NP) chromatography set ups were used rather seldom in analysis of alkaloids. Here, silica columns was eluted with sodium acetate in methanol, dioxane and acetic acid mixture (Bugatti et al., 1987) or chloroform and methanol with trifluoroacetic acid (Rey et al., 1993) Also, cyanopropyl stationary phase was eluted with acetonitrile, tetrahydrofuran, dioxane or methanol with phosphate buffer and octane-1-sulfonic acid sodium salt or di-(2-ethyl hexyl) orthophosphoric acid (HDEHP) (Petruczynik et al., 2002).

Alkaloids have a strong absorption in UV region and have ability to fluorescence; thus both spectrophotometric and fluorescence detector (Wu and Du, 2012) were applied. Additionally, ESI-MS and/or NMR were coupled with HPLC to confirm the identity and structure elucidation of new alkaloids (Paulsen et al., 2015). Moreover, preparative separation of particular alkaloids from C. majus extract using silica gel and sequentially elution with petroleum ether, ethyl acetate and methanol (Yao et al., 2011) or CaCO3 and different compositions of toluene-hexane and acetone-hexane mixtures (HorvA!th et al., 2010) was performed with use of LC system.

Capillary Electrophoresis CE

CE method with spectrophotometric (DAD) and fluorescence detection (UV-LEDIF ultraviolet light-emitting diode-induced native fluorescence or LED-fluorescence) was also used for C. majus analysis (Stuppner and Ganzera, 1995; A evAik et al., 2000; Kulp et al., 2011; Zhou et al., 2012; Kulp and Bragina, 2013); however, this technique has not yet won high popularity and it is also limited to charged or polar compounds. Recently, Sun et al. (2015) elaborated the microchip variant of CE with laser-induced fluorescence detection and 50% formamide as a run buffer and this technique was applied for separation of chelerythrine [9] and sanguinarine [12]. The examples of application the separation techniques in analysis of alkaloids from C. majus are presented in Supplementary Table 2.

Spectrophotometry

Spectrophotometric method given in European Pharmacopeia (monograph of Greater celandine) may be useful to estimate of total alkaloids in C. majus extract. The sample is mixed with sulphuric and chromotropic acid, and heated 10 min. at 100AdegC in water-bath. After cooling to 20AdegC, the absorption of sample is measured at 570 nm and the amount of alkaloid is expressed as chelidonine [1]. This approach was applied by (Then et al., 2000; Seidler-Aozykowska et al., 2016). However, for research purposes, a total-content approach should be discouraged as too inaccurate and sometimes misleading about contribution of each component of an actual alkaloid profile.

In summary, most of the published preparative and analytical approaches were rather routine and typical for phytochemical studies. However, the efficiency of standard methods often appeared to be insufficient. The properties that influenced the separation and analysis processes depend mostly on the tertiary or quaternary character, oxygenation and secondary cyclization pattern. Among the most important features are thermo- and photo-sensitivity, so the procedures should be carried out in mild conditions which has not always been considered. Thus, due to the diversity in physicochemical properties, even among compounds from a single subclass, various specialized modifications were applied which in most cases significantly improved the separation.

Another issue that should be addressed in future analytical studies is that numerous minor alkaloids have been frequently missed or overlooked whereas some of them have profound activity. The focus should be on comprehensive qualitative and quantitative profiling under non-destructive conditions that would reveal the full phytochemical complexity and allow to understand the intraspecific and environmental variation of C. majus.

Other Components

Carotenoids

Carotenoids were determined in aerial parts (Varzaru et al., 2015) and flowers (HorvA!th et al., 2010) of greater celandine. The components were hydrolyzed with alcoholic solution of potassium hydroxide and extracts were analyzed on RP-HPLC using C18 column in isocratic mode using 13% of water in acetone( Varzaru et al ., 2015) or in gradient mode with concoction of three eluents: 12%( v/ v) water in methanol( A ), methanol( B ), and 50%( v/ v) acetone in methanol( C) in different proportions( HorvA! th et al ., 2010 ).

Phenolic Compounds

Grosso et al.( 2014) tested different combinings: time, temperature type of extraction, and eluent composition and used Box-Behnken design to establish the effective extraction conditions for phenolic compounds. 76.8% of methanol, 150.0 min and 60 AdegC were found to be optimal. Further, hydroxycinnamic acids and flavonoids were characterized by HPLC-DAD-ESI/ MSn( C18 column: 150 A 1.0 mm, 3 I1/ 4m) and quantified using RP-HPLC DAD( C18 column: 250 A 4.6 mm, 5 I1/ 4m ). For both methods, gradient elution with mobile stage composed of 1% acetic acid in water and methanol was applied( Grosso et al ., 2014 ). Hence, the method for phenolic analysis is similar to some which were used for alkaloids and there would be reasonable to include phenolic compounds in any future HPLC or LC-MS profiling of this herb.

Proteins

The composition of proteins in C. majus milky sap was studied by Nawrot et al.( 2007 a, b, 2014, 2017 a, b ). The compounds were pre-separated employing two-dimensional gel electrophoresis and further, HPLC on BEH C18 column( 100 mm A 100 I1/ 4m, 1.7 I1/ 4m particle diameter)( Nawrot et al ., 2017 b) or nano-HPLC on 75 I1/ 4m analytical column( Nawrot et al ., 2007 a, 2014, 2017 a) and gradient elution with mobile phase containing 0.1%( v/ v) formic acid in water and in acetonitrile were performed. Proteins were identified by tandem mass spectrometry( MS/ MS ).

Micro and Macro-Elements

Mineral composition of greater celandine herb and root, aqueous solutions( infusion, decoction) and alcoholic extracts were determined with the use of inductively coupled plasma atomic emission spectrometry( Then et al ., 2000; SA! rkAPzi et al ., 2005 ).

Pharmacological Activities and Clinical Evidence

Obviously enough, the great majority of published pharmacological properties of C. majus pertains to the complex concoction or individual alkaloids. Recently, more and more attention has been also get paid to proteins from latex, that could significantly contribute to the observed activities. Other compounds, such as various phenolics and chelidonic acid were rarely considered. The versatile pharmacological activities of coptisine[ 31 ], one of the major constituents of C. majus as well as the less abundant berberine[ 28 ], have been largely described in the literature but usually as compounds obtained from other sources, such as Coptis( Ranunculaceae)( Tan et al ., 2016) or Berberidaceae( Imanshahidi and Hosseinzadeh, 2008 ).

However, these compounds are also contributing significantly to many of the pharmacological properties of C. majus.

From the growing proof obtained both in vitro and in vivo, with several examples of ex vivo analyses on isolated organs, it is clear that four types of medicinal properties are predominating: antimicrobial and antiviral, hepatic and gastric, anti-inflammatory, and finally anticancer. Several other activites have been also reported but less extensively.

Activites in Vitro and in Vivo

Antibacterial and Antifungal

The antimicrobial activity of C. majus is attributed largely to the alkaloids and flavonoids( Zuo et al ., 2008 ). This types of activities was reported already in the early research on chelidonium alkaloids, e.g ., by Stickl( 1928) who proved the bactericidal properties against Gram-positive stress( Staphylococcus aureus and Bacillus anthracis) with chelerythrine[ 9] and sanguinarine[ 12] being more potent than chelidonine[ 1] and berberine[ 28 ].

In experiments with multidrug resistant bacteria existing in surgical meanders and infections of critically ill patients, C. majus ethanol extract affected Gram-positive bacteria. Ethanolic extracts of C. majus also showed antimicrobial activity against Bacillus cereus, E. coli, Pseudomonas aeruginosa, S. aureus,( Kokoska et al ., 2002 ). The complex composition of alkaloids can manifest wide spectrum of antimicrobial activity, arising from different chemical structures of the compounds. Hence, antimicrobial activity of C. majus was also tested with a utilize of various types of solvent extraction. Antibacterial and antifungal tests were performed utilizing 96% methanol extracts from leaves and petioles of plants grown in nature as well as in vitro shoots and embryo( CiriA et al ., 2008 ). Methanolic extracts were examined against Gram-positive( Bacillus subtilis, Micrococcus luteus, Sarcinia lutea, and S. aureus ), Gram-negative bacteria( E. coli, Proteus mirabilis, Salmonella enteritidis ), plant pathogens- Agrobacterium rhizogenes, A. tumefaciens ), and clinically isolated C. albicans. Both, in vivo and in vitro derived plant material extracts indicated similar bioactivity, with a slight advantage of in vitro shoots. Only few extracts were equally active (8 0 mg/ ml) against E. coli, S. enteritidis, and C. albicans, when compared to the commercially available antibacterial and antifungal narcotics( streptomycin, bifonazole, respectively ), while the rest of them showed low or no activity( CiriA et al ., 2008 ).

Apart from plant extracts, as it was previously summarized by Kedzia and HoAderna-Kedzia( 2013 ), individually tested compounds presented different antimicrobial activity. Chelerythrine[ 9] and sanguinarine[ 12] were significantly more potent than chelidonine[ 1] against Gram-positive( S. aureus, S. epidermidis, B. subtilis, B. anthracis ), Gram-negative( P. aeruginosa, E. coli, Klebsiella pneumoniae, Salmonella gallinarum, S. typhi, S. paratyphi, Proteus vulgaris, Shigella flexneri ), and acid-fast mycobacteria( Mycobacterium tuberculosis, M. smegmatis ). Moreover, chelerythrine[ 9] exhibited antimicrobial activity against S. aureus and M. smegmatis, while chelerythrine[ 9] derivatives: 8-hydroxydihydrosanguinarine[ 14 a ], 8-hydroxydihydrochelerythrine[ 10 a ], dihydrosanguinarine[ 10 ], and dihydrochelerythrine[ 14] actively engaged against methicillin-resistant S. aureus( MIC5 0= 0.49, 0.98, 23.4, 46.9 I1/ 4g/ ml, respectively ). The strongest activity was presented by 8-hydroxydihydrosanguinarine at MIC9 0= 1.95 I1/ 4g/ ml, comparable to that of vancomycin( 2.23 I1/ 4g/ ml)( Zuo et al ., 2008 ). In the ethanol extract of C. majus aerial components there are several other alkaloids that present potent antimicrobial inhibitory effect. For instance, berberine[ 28] was effective against Gram-negative bacteria- Vibrio cholerae and E. coli. It injury bacterial fimbria, thereby impeded adhesion to the mucosal surface( Imanshahidi and Hosseinzadeh, 2008; Wongbutdee, 2009 ).

Enzymes is to be found in latex, like extracellular peroxidases, DNases, and lectin-like-active glycoproteins can also exhibit antimicrobial activity. Moreover, chelidocystatines protect plant against pests and are among the components of latex which presumably contribute to removal of warts resulting from human papilloma virus infection( Rogelja et al ., 1998; Song et al ., 2002; GerenAer et al ., 2006; Nawrot et al ., 2007 b; CiriA et al ., 2008 ).

The antifungal activity was tested with the use of plant latex, and different solvent extracts from aerial and underground parts like ethanol/ methanol ether, chloroform, acetone, and water ( Kokoska et al ., 2002; Kedzia et al ., 2003 ). Chelerythrine[ 9 ], sanguinarine[ 12 ], chelidonine[ 1 ], and their derivatives were also individually tested against large number of human and plant pathogenic fungi, i.e ., Aspergillus fumigatus, A. niger, Candida sp. Cladosporium herbarum, Cryptococcus neoformans, Epidermophyton floccosum, Fusarium sp. Keratinomyces ajelloi, Microsporum sp. Penicillium notatum, Rhodotorula rubra, Scopulariopsis brevicaulis, Torlopsis utilis, Trichophyton sp. The effect of C. majus extracts on pathogenic fungi was significantly weaker compared to the effect on pathogenic bacteria. For example, the MIC of ethanol and methanol extracts was ranging between 1.5 and 8 mg/ ml against the most resistant bacteria, whereas the MIC of ethanol herb extract against the most pathogenic fungi was 20 -4 0 mg/ ml( Pepeljnjak et al ., 2003 ). Chelerythrine[ 9 ], sanguinarine[ 12] and their derivatives( e.g ., 8-hydroxydihydrochelerythrine[ 10 a ]) were up to several times stronger than chelidonine[ 1]( Ma et al ., 2000; Meng et al ., 2009; Kedzia and HoAderna-Kedzia, 2013 ). Chelerythrine[ 9] was also able to inhibit spore germination of several plant pathogenic fungis: Sphaerulina juglandis, Septoria microspora, Fusarium oxysporum, and Curvularia lunata. It indicates the actual biological- antipathogenic function of isoquinoline alkaloids in the plant( Wei et al ., 2017 ). However , no particular structural feature seems to be pinpointed as a determinant of antimicrobial potency despite some variation in activity toward different stress between individual alkaloids. It is rather the combination of them and other compounds targeting multiple sites of action that result in the observed final effect. This hypothesis still needs experimental verification for potential synergies or other interactions.

Antiviral

The glycosaminoglycan present in the latex inhibits intracellular human immunodeficiency virus HIV viral migration and blocks reverse transcriptase( GerenAer et al ., 2006 ). Moreover, separately tested five C. majus alkaloids: chelidonine[ 1 ], chelerythrine[ 9 ], sanguinarine[ 12 ], coptisine[ 31 ], and berberine[ 28] were able to hinder the development of HIV-1. The first two reduced the activity of the virus reversal transcriptase at the concentrations 150 -2 00 I1/ 4g/ ml, while sanguinarine[ 12 ], berberine[ 28 ], and coptisine[ 31] were already active at concentrations of 50-150 I1/ 4g/ ml( Tan et al ., 1991 ).

The chloroform extract in the concentration of 35 I1/ 4g/ ml lessened the number of adenoviruses responsible for inducing acute fevelitis of the upper respiratory tract and conjunctiva in humans( KA( c) ry et al ., 1987 ). The experimentations 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 I1/ 4g/ ml inhibited influenza virus kind A and B in chicken embryos with 33 -9 9.97% 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 I1/ 4g/ ml) were highly effective in the treatment of trichomoniasis caused by Trichomonas vaginalis. After 8-10 days of treatment there was no protozoa identified in genitals of virtually 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” regulation 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 analyzes, caution is necessary for alleged hepatotoxicity. Also, clinical evidence is not possible 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 therapy) reaction to C. majus tincture indicated 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 provides information 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 concoction 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 A 10 a4 g/ ml organ bath) against( ACh) -induced contraction compared to caffeoylmalic acid[ 48]( 6.9%; 2.5 A 10 a5 g/ ml) and coptisine[ 31]( 16.5%; 1.0 A 10 a5 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 reaction, initially inducing 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 motion toward fallopian tubings. However, this indication was rather unknown in traditional utilization and would be a fiction 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, is a lack 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 100 x and 1,000 x 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 recognise hepatoprotective herb- Silybum marianum. The putative the system 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 mouse, 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. Furthermore, the expression of cell demise 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 demise and inflammation markers( decreased protein level of TNF-I +-, IL-6, NFIoB, p65, cas-3, iNOS)( Paul et al ., 2013 ).

Notwithstanding, the literature evidence supporting beneficial properties in hepatobiliary ailments from both in vitro, in vivo, or ex vivo analyzes is still less abundant than lawsuit reports on liver toxicity. This, quite surprising inequality, 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 ailments, the antitumor properties have been indicated since relic. Today, this kind of activity belongs to the most intensely analyse. 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 detected, albeit most examines 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 ., 2016 a ).

The selected examples of cell line-based analyses 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-6 0, U-9 37, HEL-9 2.1.7, CCRF/ CEM, CCRF/ ADR5 000 ), colon carcinomas( Caco-2, HT-2 9, HCT1 16, SW480 ), breast cancer ( MCF-7, MD-AMB2 31 ), pancreatic cancer ( human PANC-1, murine PANC0 2 ), lung cancer (( A549, H460 ), prostate cancer ( DU-1 45 ), cervical cancer ( HeLa ), ovarian carcinoma( A2780 ), liver cancer ( HepG2 ), gastric cancer ( SGC-7 901 ), vulvar squamous cell carcinoma( A431 ), oesophageal squamous carcinoma( WHCO5 ), and mouse melanoma( B16F10 ). Non-cancerous lines, such as lung fibroblasts( MRC-5, WI-3 8), scalp fibroblasts( Hs27) immortalized cells from mice( 3T3 ), green monkey( Vero ), humans( 293 N3S, HS-2 7, HaCaT ), or SV-4 0 transformed bronchial epithelium( BEAS-2B) were also used.

From most of the published mechanistic surveys 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 bind 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 merely a weak DNA intercalating agent and does not induce lethal mutants or DNA damage. Its mechanism of action is suggested to rely on interactions with spindle microtubules leading to cell cyle arrest and mitotic misfortune, 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 links with multidrug resistance( e.g ., ABC transporters and CYP) were significantly downregulated( from 50% in case of CYP3A 4 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/ ADR5 000 cells. Upon low-dose treatment with chelidonine[ 1]( 20 I1/ 4M ), the LC50 decreased from 3.67 I1/ 4M( Caco-2) and 32.16 I1/ 4M( CCRF/ ADR5 000) to 0.42 and 7.4 I1/ 4M, respectively. Similar outcomes were obtained with an appropriate concentration of extract( 5 I1/ 4g/ ml) containing( in decreasing order) chelidonine[ 1 ], coptisine[ 31 ], stylopine[ 33 ], and protopine[ 37] as four major compounds and smaller sums of other alkaloids. In the most resistant CCRF/ ADR5 000 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 strengthening of caspase activity and annexin staining.

These outcomes 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 utilizing planarian stem cell model( Isolani et al ., 2012) in which the gene expression of mcm2( essential DNA replication factor) or inx-1 1( a gap-junction protein essential for regeneration) was decreased by 20 I1/ 4M of the alkaloid. The stem cells were affected but not differentiated populations such as neuronal or intestinal.

Quite a specific 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-6 31570 or UkrainA( r ). 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 producer claims, it couldn’t be positively confirmed that it is indeed a thiophosphoric acid complex and at least in some batches, it seemed as a rich mixture of native alkaloids, that have been identified utilizing reliable chromatographic and spectroscopic methods.

Several newspapers additionally report cytotoxic activity of UkrainA( r) 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 ( insure 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 UkrainA( r) 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 UkrainA( r)( 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 combining would be responsible for this property. Also, other studies reporting UkrainA( r) as an active principle require re-evaluation with native alkaloids as probably constituting major component in the patented preparation. Furthermore, UkrainA( r) remains the only preparation from C. majus that has a vast literature documenting its anti- cancer and also other properties in clinical( assure 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 answer. Both have been confirmed in many studies use in vitro cellular models, as well as in vivo.

The ability to impede 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) utilized human colon cell line( NCM4 60) 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 replies by reducing levels and gene expression of several mediators and enzymes in colon tissues( COX-2, HIF1I +-, PGE2) and in allergic mice( IL-4, IL-1I 2, COX-2, caspase-1, and strengthening of IFN-I3 ). In human mast cell line HMC-1 induced for inflammatory reaction by the phorbol ester( TPA) and calcium ionophore A23187, chelidonic acid[ 45] inhibited IL-6 expression by blocking NFIoB( Shin et al ., 2011 ).

Stylopine[ 33] added to the cell culture of in lipopolysaccharide-stimulated RAW2 64.7 macrophages was impeding production of several proinflammatory molecules such as nitric oxide, PGE2, TNF-I +-, IL-1I 2, 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[ 14 a] in the same model impeded NO production and iNOS and COX-2 gene transcription( Park et al ., 2011 ). These outcomes indicate the underlying inhibition of NFIoB as potential mechanism but it wasn’t investigated in this study. However, in HCT-1 colon cancer cell line treated with chelidonine[ 1 ], the NFIoB activation was blocked by inhibition of IIoBI +- degradation and nuclear translocation of p65( an NFIoB 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] demonstrated lower activity( Lanfeld et al ., 1981 ). However, in the later study by MikoAajczak et al.( 2015 ), various fractions of water extract at relatively high dosages of 200 mg/ kg body weight failed to alleviate the inflammation in the similar model. The crude water extract treatment actually worsened the paw rednes. Conversely, the extracts containing mainly coptisine[ 31] and chelidonine[ 1] were effective in hot plate exam for antinociceptive properties that suggests a supramedullary route of action.

Chelerythrine[ 9] inhibited inflammatory and ache 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 I1/ 4g/ ml chelerythrine[ 9] had dose dependently reduced PGE2 and COX-2 expression.

In NC/ Nga mouse model for atopic dermatitis induced by DNCB( 1-chloro-2, 4-dinitrobenzene ), the hydroethanolic extract from aerial proportions alleviated several measures of dermatitis such as itching behavior and scalp seriousnes( 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 therapy. Also, IL-4 and TNF-I +- serum levels were significantly reduced but remained higher than in control animals.

In an animal model of ovoalbumine-provoked asthma, chelidonine[ 1] squelched 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 transcript 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 narcotic. It indicates a specific mechanism involving STAT6 and Foxp3 transcription pathways( Kim et al ., 2015 ). Yet another inflammation-based condition absent from listing of traditional clues, 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, squelched progression of joint injury as well as a situated 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, tnfi+-, ifni3 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 I1/4g/ml and BuChE by only 13%. The isolated alkaloid 8-hydroxydihydrochelerythrine [10a] was the most active and AChE-selective (IC50 0.61 I1/4M 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 (CahlAkovA! 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 (MikoAajczak 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 I1/ 4M) while berberine[ 28] depicted no inhibition in transfected HEK2 93 T cells. GlyT1 inhibition was time-dependent , noncompetitive and increased with glycine concentration. Interestingly, chelerythrine[ 9] consequence was reversible while sanguinarine[ 12] persisted through rinsing 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 require rigorous verification by appropriately designed and supervised clinical studies. At present, most of the allegedly curative properties toward some significant objections remain unconfirmed, even if there is strong pre-clinical proof. The millennia long tradition of use becomes confirmed in a significant proportion even if some mythical clues turned out to be invalid or perhaps have been misconstrue or distorted when passed through generations of practitioners and authors.

Several clinical surveys 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 ailments 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 mechanism behind these effects were also studied in vitro use cell lines proving anti-inflammatory activity( Schneider et al ., 2016 ). IFN-I3 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 objections and gall stones. These surveys, reviewed in detail in the EMA report, suggest significant improvement of many clinical parameters. These included: subjective objections( ache 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 grievances, one can estimate the number of human subjects to participate in to date published clinical literature as surpassing 1,500.

An impressive number of studies suggest antitumor properties of the apparently semisynthetic product- UkrainA( r) that demonstrated efficiency in several in vitro analyzes on an smorgasbord of neoplastic and non-transformed cell lines( Capistrano et al ., 2015) but it by big failed to demonstrate clinically relevant activity in humen. Several occurrence 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 UkrainA( r) were reviewed over a decade ago( Ernst and Schmidt, 2005) with a conclusion that despite the intensive publishing activity of several groups testing UkrainA( r) against miscellaneous malignacies with very promising outcome, most of these data are full of shortcomings that avoid unequivocal credibility. Since then, only a few more examines have been realized but it is still far away from definite resolving of this issue, especially in the situation that the chemical analysis, some of which were even co-authored by the discoverer( Jesionek et al ., 2016) proved that it is not the compound which was initially claimed.

Most of the RTCs published were full of incompatibilities or even seemed unreliable due to the insufficient information regarding the methodology. Despite the spectacular outcomes in such malignancies as pancreatic or terminal colorectal cancer, serious doubts remain about the evidence are available in some of these trials. Some of the studies are so poorly documented that it would undermine the validity of the results. For instance, missing or suspect randomization technique, missing methods for tumor dimensions measuring, unclear protocols, subjective outcome evaluation, absence of proper statistics. Also, a very large majority of the clinical trials and lawsuit reports were published in one periodical. Nonetheless, these few provides information on UkrainA( r) remain the only available clinical data focusing specifically on C. majus and strongly indicating 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 medication for clinical practise 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, farther analyses are mandatory that would focus on using individual alkaloids as lead structures for medications that would target multiple anticancer mechanisms. On the other hand, clinical data on efficacy of various types of 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 restrictions 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, examines and lawsuit reports occur that indicate hepatic trauma/ hepatotoxicity. It is especially important because one of the main the indicators 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 lately( Pantano et al ., 2017 ). Specifically, the findings from animal surveys are ambiguous and suggest a quite 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 humen, 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 medication 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 survey on HepG2 cells treated with various solvent extracts( Orland et al ., 2014 ), the biotransformation and toxicity-related gene expres was improved, 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 dosages( up to 3g/ kg body weight; Mazzanti et al ., 2009) did not elicits any symptoms of hepatic injury and did not alter liver function.

Despite proved interaction of sanguinarine[ 12] and other alkaloids with DNA , no genotoxicity was observed utilizing well established methods such as Ames tests or in vivo DNA damage( EMA report, 2011 ).

Nonetheless, uncontrolled internal employ 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 types of sorts and preparations as well as individual constituents.

The European Medicine Agency hence published the following recommendation πŸ˜› TAGEND ” Two possible therapeutic shows were proposed for the monograph πŸ˜› TAGEND Traditional herbal medicinal product πŸ˜› TAGEND

1) For symptomatic relief of digestive disorders such as dyspepsia and flatulence( oral intake)

2) For therapy of warts, callus and corns( cutaneous employ )”( EMA, 2011 ).

However, based on the reported undesirable effects, including cases of liver damage, special precautions are necessary, especially during pregnancy and lactation, those suffering from liver cancers 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 grievances, such as gastrointenstinal and hepatobiliary ailments 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 utilize of herbal redres 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 therapy 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 hazard of carcinogenesis and hepatotoxicity is still not well documented.

Finally, we conclude that the millenia long history of Chelidonium in folk, traditional, and official medication is far from coming to the end. On the contrary, recent years witnessed a resurgence of advanced pharmacological and mechanistic approaches employing 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. Furthermore, new discoveries should broaden the scope of traditional utilization and deliver better preparations, especially blending 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 proverb from Aristotle’s book, also referring to C. majus original Greek name,” one swallow does not make a spring ,” even if we find 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 segment 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 idea of its consideration of the, wrote bioactivity and pharmacology segments, 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-3 94/ SPUB/ 2-16/ 1. AM and SZ get support from the Wroclaw Medical University award No. ST.D0 30.17.028.

Conflict of Interest Statement

The authors declare that the research was conducted in the is a lack of any commercial or fiscal relationships that could be construed as a potential conflict of interest.

Acknowledgments

Assistance in graphical abstract preparation by Miss Hanna Zielinska is kindly recognise. The supporting of Wroclaw Medical University is acknowledged.

Supplementary Material

The Supplementary Material for this article can be found online at: https :// www.frontiersin.org/ articles/ 10.3389/ fphar. 2018.00299/ full #supplementary-material

Supplementary Figure 1. Chelidonium majus has been highly valued by ancient physicians. Nowadays, it is still attracting researchers’ and clinicians’ attention for its own unique composition of the yellow latex, rich in alkaloids and proteins.

Supplementary Table 1. The examples of conditions are applied 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

The post Greater Celandine’s Ups and Downsa2 1 Centuries of Medicinal Uses of Chelidonium majus From the Viewpoint of Today’s Pharmacology seemed first on Herbs and Helpers.

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