Greater Celandine's Ups and Downs−21 Centuries of Medicinal

[herbsandhelpers]

Greater Celandine's Ups and Downs−21 Centuries of Medicinal Uses of Chelidonium majus From the Viewpoint of Today's Pharmacology- Part 1

• 1Pharmaceutical Biology and Botany, Wrocław Medical University, Wrocław, Poland

• 2Botanical Garden of Medicinal Plants, Wrocław Medical University, Wrocław, Poland

• 3Analytical Chemistry, Medical University of Lublin, Lublin, Poland

• 4Pharmaceutical Microbiology and Parasitology, Wrocław Medical University, Wrocław, Poland

As antique as Dioscorides era are the first records on using Chelidonium as a remedy to several sicknesses. Inspired by the "signatura rerum" principle and an apparent ancient folk tradition, various indications 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 this region, the plant is known under many unique vernacular names, especially in Slavonic languages, 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 medicine status. On the contrary, the number of relevant studies and publications tended to drop. Recently, some controversial reports and sometimes insufficiently proven studies appeared, suggesting 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 variation of bioactive metabolites, including several aforementioned isoquinoline alkaloids. In this review, we put together older and recent literature data on phytochemistry, pharmacology, and clinical studies on C. majus aiming at a critical evaluation of state-of-the-art from the viewpoint of historical and folk indications. The controversies around this herb, the safety and drug 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 man shall have this herb, with the heart of a Mole, he shall overcome all his enemies, all matters in suit, and shall put away all debate," and "if before named herb be put upon the head of a sick man, if he shall die, he shall sing anon with a loud voice, if not, he shall weep"; and "it bringeth the business begun to an end," so wrote Albertus Magnus in thirteenth century A.D. (Best and Brightman, 1999). Nowadays, mankind would definitely benefit from such a miraculous remedy. 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 confirmed. 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 seeking drugs 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 provided review 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 covering 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 classified the pharmacological evidence 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 information is also available in herbal books and compendia (Wichtl, 2004) or in the Internet. The latter source is of course difficult to verify.

Therefore, we chose to include a possibly high number of available literature, by selecting records from database search (PubMed, Scopus, Google Scholar) with the term "chelidonium" or "celandine" and manually eliminating papers pertaining to field botany, ecology and other aspects not relevant to medicinal use of this plant. Some references that have been reviewed earlier (e.g., in Colombo and Bosisio, 1996) were also not cited 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-631570 (Ukrain®) promoted mainly as a cancer cure 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 leaves 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 dropping 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 part 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 forests, in brushwood, parks, gardens, on the roadsides and around buildings. It prefers 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 covers 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 (Krahulcová, 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 leaf 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 (Krahulcová, 1982).

(Krahulcová, 1982) Name of the genus derives from Greek (χελιδóνιoν) with chelidon (χελιδóν) meaning swallow (a bird) for the plant usually blooms simultaneously with arrival of these birds. The specific epithet majus in Latin means 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, Schöllkraut, 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 - vlaštovičník, celadona, celduně, cen dalie, dravnicovina, hadí mlíč, krkavník; Dutch - stinkende gouwe; English - Tetterwort, devils's milk, rock poppy; French - grande chélidoine Éclair, herbe aux boucs, herbe a l'hirondelle; German - Schöllkraut, Gilbkraut, Goldwurz, Schwalbenkraut, Warzenkraut; Italian - celidonia, cinerognola, Montenegrin - rusopas, rusa; Polish - glistnik jaskólcze-ziele, celidonia, cyndalia, cencylja, glistewnik, gliśnik, niebospad, złoty groszek, złotnik, zółte ziele, zółte kwiatki, roztopaść; Ukrainian - hladyshnyk, hnystnyk, zhovtyi molochay, smetannyk, chystotil; Romanian: rostopască; Russian - chistotel; Rusyn - rostopast'; Serbian - rusopas, rusa, rusomača; 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 nestlings 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 sixteenth century, was treating visual impairment and eye diseases. For such conditions, Dioscorides advised to use herb juice, boiled with honey in a brass vessel. 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 together with anise fruits was helpful in treating jaundice and dermatologic disorders such as herpes. Besides, chewing on a root relieved 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 Middle Ages, mostly used to cure eye diseases, for throat cleansing, treatment of ulcers and skin 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 poem 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 skin and liver conditions" (Mayer et al., 2003). Hildegard of Bingen wrote about celandine in her work Liber subtilitatum diversarum naturarum creaturarum created during 1150-1160 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 (Czekański, 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 using celandine to treat eye and skin conditions. According to Lonicer, to cure various dermatologic diseases, known in those days as "leprosy," the juice of the root of celandine had to be applied on the skin, 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 written in 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 diseases, 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 case of jaundice, root of celandine should be boiled 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, relieved the pain (Marcin of Urzedowo, 1595).

Simon Syrenius, described celandine-based recipes used in eye diseases. The main ingredient of such medicines 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 powdered 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 skin 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 boiled in wine has a diuretic effect. 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 boiled 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 treatment of jaundice, visceral obstructions, 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 drug of folk herbal medicine. The scope of its applications in folk medicine shows high similarity among many regions of Central and Eastern Europe. It is worth to mention its prevalent application to treat warts, eczema and other skin diseases, gastrointestinal parasites, jaundice, and liver complaints, inflammatory eye infections and other diseases, 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, use of leaves and flowers was also noted. In Poland it was common to rub the fresh juice from the broken stem of the celandine onto warts (De Verdmon, 1936; Kuźniewski and Augustyn-Puziewicz, 1999; Kujawska et al., 2016). In the Bieszczady Mountains and in the Podkarpacie Region (S-E Poland) the juice was used directly on warts, or they were first scrubbed off and then the juice was applied on the wound (Szary, 2013). A cataplasm made of flowers that was supposed to be changed every few days was used in the Kielce Region (Central Poland) (Szot-Radziszewska, 2012). Occasionally, fresh leaves 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 (Redžič, 2007; Tita et al., 2009; Menković 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 leaves or juice on wounds. In Podolia (Ukraine) corns were treated by rubbing with a root of celandine and by application of fresh leaves. 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 stimulate picking up and rupture (Wdowiak and Bielecka-Grzela, 2013). The juice was also used to lighten freckles (Kuźniewski and Augustyn-Puziewicz, 1999). In Russia the juice of aerial parts was used in the treatment of skin wounds, skin irritation, allergic rashes and dermatitis, leaves, and flowers in the treatment of boils (Mamedov et al., 2004). The aerial parts of the plant were used by the people of Montenegro to cure blisters, rashes, and scabies (Menković et al., 2011). In Central Serbia, juice was applied directly on skin in skin eruptions, psoriasis and eczema (Jarić 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 (Kuźniewski 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 attacks (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 parts 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 (Jarić et al., 2007; Šavikin et al., 2013). The use of celandine is similar in Gollak region in Kosovo (Mustafa et al., 2012), in the Prokletije Mountains (Menković 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 drink (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) drops of celandine juice mixed with vodka were put into eyes. In west Ukraine a tincture made from celandine was applied. Moreover, 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 used 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 smoke 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 (Sǒukand and Pieroni, 2016). In Bosnia and Herzegovina it was used to cure cancer of lungs (Redžič, 2007). In veterinary treatments, herb decoction was given to cows suffering from inflammation of the udder, in case of dermatologic conditions animals were rubbed with leaves. Furthermore, cows 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 symbol of purification of living world from threatening death, it was used as talismans 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 articulate laticifers with yellowish content is also used as an authentication microscopic mark in powdered herb by pharmacopoeial monographs. The composition of latex is plant organ specific (Tomē 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; Táborská et al., 1995; Petruczynik et al., 2002; Nečas et al., 2005; Sārközi 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 parts are (+)-chelidonine [1], chelerythrine [9], (Kadan et al., 1990; Sārközi et al., 2006; Zhou et al., 2012).

Protoberberine derivatives that accumulate in higher amounts are coptisine [31], berberine [28], stylopine [33] (Slavik and Slavikowa, 1977; Fulde and Wichtl, 1994; Shafiee and Jafarabadi, 1998; Sārközi et al., 2006). Aporphine alkaloids like corydine [39] also appear (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; Tomē 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 (Tomē and Colombo, 1995). Day light seems to be the crucial factor influencing alkaloid biosynthesis in C. majus, especially in underground parts of the plants (Kustrak et al., 1982; Tomē and Colombo, 1995). Diurnal changes of alkaloid content seem to be less dependent on temperature, what was observed during winter time, when the alkaloid content was low and stable, due to the reduced metabolism and the senescence of the aerial parts. According to Tomē and Colombo (1995) total content of alkaloids in leaves was lower than in underground parts. 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 they are stored. Moreover, the number of laticifers is probably organ specific. Laticifers in C. majus are unbranched (without anastomoses), articulated with perforated transverse cells (Hagel et al., 2008). Articulated 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 elements, unlike that of Greater celandine (Hagel et al., 2008). From wounded laticifers, a matrix emerges with various 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 concern protein determination in latex and confirm its complex composition (for more information see 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 found mostly in fruits and herb (Sārközi et al., 2006; Kedzia et al., 2013; Sowa et al., 2018), whereas berberine [28] showed no significant difference between aerial parts and roots (Tomē and Colombo, 1995). Larger amounts of phenanthridine alkaloids like chelidonine [1], chelerythrine [9], sanguinarine [12] were observed in roots rather than in aerial parts of the plants (Tomē 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 (Cirić et al., 2008).

Phenolic Compounds

Several flavonoids were found in aerial parts of C. majus in low amounts. 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, 5′-methoxy-flavonol [43] and 6′- methoxy-flavonol [44] were also detected (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; Wojdyło et al., 2007). Later, another three hydroxycinnamic acids were identified using 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 parts (Grosso et al., 2014).

Proteins

A phytocystatin - chelidocystatin was one 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. (2007a,b, 2008, 2013, 2014, 2016, 2017a,b) described a number of proteins from root and leaf latex. Proteomic analysis using LC-ESI-MS/MS system revealed the presence of three categories of proteins according to their functions: proteins involved in disease and defenses responses (i.e., superoxide dismutases, lactoylglutathione lyases), nucleic acid binding proteins (i.e., glycine-rich proteins, nucleic acid binding, DNA-binding, or RNA-binding 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 cases the identification was based on correlation between experimental and the theoretical pI/molecular mass, due to their low score results. The results shown 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 effects providing a multifaceted tool for combat against troublesome infections. In living plants, these proteins are probably also serving as a chemical defense against pathogens (Nawrot et al., 2007a,b, 2014, 2017a,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 fruits (Kwasniewski, 1958); essential oil constituents (Hansel et al., 1992; Kohlmünzer, 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 (Horváth 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 occur 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 solution was alkalized with ammonia or sodium hydroxide to obtain base forms followed by liquid-liquid extraction with organic solvents such as dichloromethane, butanol or chloroform (Sārközi et al., 2006; Sárközi 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-Łozykowska et al., 2016), in water bath (Sārközi et al., 2006) or Soxhlet apparatus (Bugatti et al., 1987; Stuppner and Ganzera, 1995) as well as ultrasound assisted (UAE) (Kursinszki et al., 2006; Sárközi 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) (Gañán et al., 2016) was also applied. Moreover, 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-μm membrane (Gu et al., 2010) or additionally purified using solid phase extraction (SPE) on C18 cartridge (Stuppner and Ganzera, 1995; Kursinszki et al., 2006; Sārközi et al., 2006). Sārközi et al. (2006) developed ion-pair SPE with n-heptanesulfonic acid (HS). Dried residue obtained after 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 C18 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 mostly 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 modernized 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 time) used already in studying of other species (Móricz et al., 2018) should be useful also for C. majus alkaloids.

Silica was the most common stationary phase, often impregnated with salts (Ni, Zn, Cr, Co) to improve the selectivity (Wagner et al., 1984; Then et al., 2000; Waksmundzka-Hajnos et al., 2000; Sārközi et al., 2006; Sárközi et al., 2007; Petruczynik et al., 2008; Jesionek et al., 2016) Silica modified with octadecyl (C-18) and cyanopropyl (CN) groups and aqueous solutions 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) combined various types of stationary phases e.g., cyanopropyl silica with silica or octadecyl silica to obtain adsorbent gradient.

Apart from the routine isocratic elution with solvent mixtures, using different modes of gradient elution improved the separation, e.g., as a two-dimensional or unidimensional multiple development (Szumiło and Flieger, 1999; Waksmundzka-Hajnos et al., 2000; Migas et al., 2012), bivariant multiple development (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 use of magnetic field (Malinowska et al., 2017) or forced flow of mobile phase (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 (Sárközi et al., 2007; Móricz et al., 2015; Jesionek et al., 2016) combined with densitometry for quantitative analysis (Then et al., 2000; Sārközi et al., 2006) and it proved 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 mostly carried out in reversed phase (RP) system using long (150 or 250 mm) C18 columns with 4.6 mm of diameter and 5 μm of particle size (Niu and He, 1994; Petruczynik et al., 2002; Kursinszki et al., 2006; Borghini et al., 2015; Paulsen et al., 2015; Gañán et al., 2016). More recently, adsorbents with smaller particles (≤ 3 μm) or shorter columns (Prosen and Pendry, 2016; Seidler-Łozykowska et al., 2016) were also applied to achieve shorter separation and saving solvents. Mobile phases are usually composed of water and acetonitrile or/and methanol with various additives e.g., ammonium formate/acetate (Borghini et al., 2015; Seidler-Łozykowska et al., 2016), organic amines (triethyl-, tetrabutylamine) (Paulsen et al., 2015), ion-pair reagents (sodiumdodecylsulfate, alkylsulfonic acids) (Gañán et al., 2016). The additives were necessary to reduce peak tailing forming as a results of interaction of alkaloid cationic forms with residual silanol group of stationary phase. 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 (Horváth 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; Ševčik 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 100°C in water-bath. After cooling to 20°C, 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-Łozykowska 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 (Horváth 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 mixture of three eluents: 12% (v/v) water in methanol (A), methanol (B), and 50% (v/v) acetone in methanol (C) in different proportions (Horváth et al., 2010).

Phenolic Compounds

Grosso et al. (2014) tested different combinations: 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°C were found to be optimal. Further, hydroxycinnamic acids and flavonoids were characterized by HPLC-DAD-ESI/MSn (C18 column: 150 × 1.0 mm, 3 μm) and quantified using RP-HPLC DAD (C18 column: 250 × 4.6 mm, 5 μm). For both methods, gradient elution with mobile phase 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. (2007a,b, 2014, 2017a,b). The compounds were pre-separated using two-dimensional gel electrophoresis and further, HPLC on BEH C18 column (100 mm × 100 μm, 1.7 μm particle diameter) (Nawrot et al., 2017b) or nano-HPLC on 75 μm analytical column (Nawrot et al., 2007a, 2014, 2017a) 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; Sárközi et al., 2005).

Pharmacological Activities and Clinical Evidence

Obviously enough, the great majority of published pharmacological properties of C. majus pertains to the complex mixture or individual alkaloids. Recently, more and more attention has been also 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 widely 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 evidence obtained both in vitro and in vivo, with several examples of ex vivo studies 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 mostly to the alkaloids and flavonoids (Zuo et al., 2008). This kind of activity was reported already in the early research on chelidonium alkaloids, e.g., by Stickl (1928) who proved the bactericidal properties against Gram-positive strains (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 wounds 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 use of different solvent extraction. Antibacterial and antifungal tests were performed using 96% methanol extracts from leaves and petioles of plants grown in nature as well as in vitro shoots and embryos (Cirić 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 showed similar bioactivity, with a slight advantage of in vitro shoots. Only few extracts were equally active (80 mg/ml) against E. coli, S. enteritidis, and C. albicans, when compared to the commercially available antibacterial and antifungal drugs (streptomycin, bifonazole, respectively), while the rest of them showed low or no activity (Cirić et al., 2008).

Apart from plant extracts, as it was previously summarized by Kedzia and Hołderna-Kedzia (2013), individually tested compounds showed 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 [14a], 8-hydroxydihydrochelerythrine [10a], dihydrosanguinarine [10], and dihydrochelerythrine [14] were active against methicillin-resistant S. aureus (MIC50 = 0.49, 0.98, 23.4, 46.9 μg/ml, respectively). The strongest activity was presented by 8-hydroxydihydrosanguinarine at MIC90 = 1.95 μg/ml, comparable to that of vancomycin (2.23 μg/ml) (Zuo et al., 2008). In the ethanol extract of C. majus aerial parts there are several other alkaloids that show potent antimicrobial inhibitory effect. For instance, berberine [28] was effective against Gram-negative bacteria - Vibrio cholerae and E. coli. It damaged bacterial fimbria, thereby inhibited adhesion to the mucosal surface (Imanshahidi and Hosseinzadeh, 2008; Wongbutdee, 2009).

Enzymes included 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; Gerenčer et al., 2006; Nawrot et al., 2007b; Cirić 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 20-40 mg/ml (Pepeljnjak et al., 2003). Chelerythrine [9], sanguinarine [12] and their derivatives (e.g., 8-hydroxydihydrochelerythrine [10a]) were up to several times stronger than chelidonine [1] (Ma et al., 2000; Meng et al., 2009; Kedzia and Hołderna-Kedzia, 2013). Chelerythrine [9] was also able to inhibit spore germination of several plant pathogenic fungi: Sphaerulina juglandis, Septoria microspora, Fusarium oxysporum, and Curvularia lunata. It suggests 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 strains 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.

Continued ... See Part 2