The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Chelidonium majus Induces Apoptosis of Human Ovarian Cancer Cells via ATF3-Mediated Regulation of Foxo3a by Tip60.

Forkhead transcription factor 3a (Foxo3a) is believed to be a tumor suppressor as its inactivation leads to cell transformation and tumor development. However, further investigation is required regarding the involvement of the activating transcription factor 3 (ATF3)-mediated Tat-interactive protein 60 (Tip60)/Foxo3a pathway in cancer cell apoptosis. This study demonstrated that Chelidonium majus upregulated the expression of ATF3 and Tip60 and promoted Foxo3a nuclear translocation, ultimately increasing the level of Bcl-2-associated X protein (Bax) protein. ATF3 overexpression stimulated Tip60 expression, while ATF3 inhibition by siRNA repressed Tip60 expression. Furthermore, siRNA-mediated Tip60 inhibition significantly promoted Foxo3a phosphorylation, leading to blockade of Foxo3a translocation into the nucleus. Thus, we were able to deduce that ATF3 mediates the regulation of Foxo3a by Tip60. Moreover, siRNA-mediated Foxo3a inhibition suppressed the expression of Bax and subsequent apoptosis. Taken together, our data demonstrate that Chelidonium majus induces SKOV-3 cell death by increasing ATF3 levels and its downstream proteins Tip60 and Foxo3a. This suggests a potential therapeutic role of Chelidonium majus against ovarian cancer.

Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.

Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.

Comparative analysis of metabolic variations, antioxidant potential and cytotoxic effects in different parts of Chelidonium majus L.

Metabolic variations, antioxidant potential and cytotoxic effects were investigated in the different plant parts like the leaf, stem, flower, pod, and root of C. majus L. using spectroscopic and chromatographic methods. Total phenolics and flavonoids were studied in the different parts of C. majus L., leaf showed higher flavonoid content (137.43 mg/g), while the pod showed the highest phenolic (23.67 mg/g) content, when compared with the stem, flower and root. In the ABTS antioxidant assay, the flower extract showed 57.94% effect, while the leaf, pod and root extract exhibited 39.10%, 36.08% and 28.88% activity, respectively. The pod and leaf extracts demonstrated the potential effect, exhibiting 45.46 and 41.61% activity, respectively, for the DPPH assay. Similar to the phosphomolybdenum assay, the flower revealed higher antioxidant activity (46.82%) than the other plant parts. The in vitro SRB assay facilitated evaluation of the cytotoxic effect against the HeLa and CaSki human cervical cancerous cells. The extract displayed dose-dependent inhibitory effect on both the cell lines. The highest cytotoxic effect was observed in the pod and flower extracts post 48 h of exposure at 1000 µg/mL. The results of C. majus L. offered new insights in the preliminary steps regarding the development of a high value product for phytomedicine applications though promising metabolic variations with antioxidant and anticancer potentials.

Effects of Abiotic Elicitors on Expression and Accumulation of Three Candidate Benzophenanthridine Alkaloids in Cultured Greater Celandine Cells.

Efforts to develop the necessary biotechnologies in Greater Celandine (Chelidonium majus L.), a leading plant resource for the development of plant-derived medicines, have been hampered by the lack of knowledge about transcriptome and metabolome regulations of its medicinal components. Therefore, this study aimed to examine the effect of abiotic elicitors, methyl jasmonate (MJ) and salicylic acid (SA), at different time courses (12, 24, 48, and 72 h), on expression and metabolome of key benzophenanthridine alkaloids (BPAs) in an optimized in vitro culture. Gene expression analysis indicated the upregulation of CFS (cheilanthifoline synthase) to 2.62, 4.85, and 7.28 times higher than the control at 12, 24, and 48 h respectively, under MJ elicitation. Besides, MJ upregulated the expression of TNMT (tetrahydroprotoberberine N-methyltransferase) to 2.79, 4.75, and 7.21 times at 12, 24, and 48 h respectively, compared to the control. Investigation of BPAs revealed a significant enhancement in the chelidonine content (9.86 µg/mg) after 72 h of MJ elicitation. Additionally, sanguinarine content increased to its highest level (3.42 µg/mg) after 24 h of MJ elicitation; however, no significant enhancement was detected in its content in shorter elicitation time courses. Generally, higher gene expression and BPAs' level was observed through longer elicitation courses (48 and 72 h). Our findings take part in improving the understanding of transcription and metabolic regulation of BPAs in cultured Greater Celandine cells.

Enantioselective Syntheses of Strychnos and Chelidonium Alkaloids through Regio- and Stereocontrolled Cooperative Catalysis.

We describe enantioselective syntheses of strychnos and chelidonium alkaloids. In the first case, indole acetic acid esters were established as excellent partner nucleophiles for enantioselective cooperative isothiourea/Pd catalyzed α-alkylation. This provides products containing indole-bearing stereocenters in high yield and with excellent levels of enantioinduction in a manner that is notably independent of the N-substituent. This led to concise syntheses of (-)-akuammicine and (-)-strychnine. In the second case, the poor performance of ortho-substituted cinnamyl electrophiles in the enantioselective cooperative isothiourea/Ir catalyzed α-alkylation was overcome by appropriate substituent choice, leading to enantioselective syntheses of (+)-chelidonine, (+)-norchelidonine, and (+)-chelamine.

Green Synthesis of Ag-MnO2 Nanoparticles using Chelidonium majus and Vinca minor Extracts and Their In Vitro Cytotoxicity.

Medicinal plants are often used as reducing agents to prepare metal nanoparticles through green-synthesis due to natural compounds and their potential as chemotherapeutic drugs. Thus, three types of eco-friendly Ag-MnO2 nanoparticles (Ag-MnO2NPs) were synthesized using C. majus (CmNPs), V. minor (VmNPs), and a 1:1 mixture of the two extracts (MNPs). These NPs were characterized using S/TEM, EDX, XRD, and FTIR methods, and their biological activity was assessed in vitro on normal keratinocytes (HaCaT) and skin melanoma cells (A375). All synthesized NPs had manganese oxide in the middle, and silver oxide and plant extract on the exterior. The NPs had different forms (polygonal, oval, and spherical), uniformly distributed, with crystalline structures and different sizes (9.3 nm for MNPs; 10 nm for VmNPs, and 32.4 nm for CmNPs). The best results were obtained with VmNPs, which reduced the viability of A375 cells up 38.8% and had a moderate cytotoxic effect on HaCaT (46.4%) at concentrations above 500 µg/mL. At the same concentrations, CmNPs had a rather proliferative effect, whereas MNPs negatively affected both cell lines. For the first time, this paper proved the synergistic action of the combined C. majus and V. minor extracts to form small and uniformly distributed Ag-MnO2NPs with high potential for selective treatments.

Comparative analysis of the root and leaf transcriptomes in Chelidonium majus L.

Chelidonium majus is a traditional medicinal plant, which commonly known as a rich resource for the major benzylisoquinoline alkaloids (BIAs), including morphine, sanguinarine, and berberine. To understand the biosynthesis of C. majus BIAs, we performed de novo transcriptome sequencing of its leaf and root tissues using Illumina technology. Following comprehensive evaluation of de novo transcriptome assemblies produced with five programs including Trinity, Bridger, BinPacker, IDBA-tran, and Velvet/Oases using a series of k-mer sizes (from 25 to 91), BinPacker was found to produce the best assembly using a k-mer of 25. This study reports the results of differential gene expression (DGE), functional annotation, gene ontology (GO) analysis, classification of transcription factor (TF)s, and SSR and miRNA discovery. Our DGE analysis identified 6,028 transcripts that were up-regulated in the leaf, and 4,722 transcripts that were up-regulated in the root. Further investigations showed that most of the genes involved in the BIA biosynthetic pathway are significantly expressed in the root compared to the leaf. GO analysis showed that the predominant GO domain is "cellular component", while TF analysis found bHLH to be the most highly represented TF family. Our study further identified 10 SSRs, out of a total of 39,841, that showed linkage to five unigenes encoding enzymes in the BIA pathway, and 10 conserved miRNAs that were previously not detected in this plant. The comprehensive transcriptome information presented herein provides a foundation for further explorations on study of the molecular mechanisms of BIA synthesis in C. majus.

Impact of drought and salt stress on the biosynthesis of alkaloids in Chelidonium majus L.

When plants are exposed to various stress situations, their alkaloid concentration frequently is enhanced. This well-known phenomenon is presumably due to a passively enhanced rate of biosynthesis, caused by greatly elevated concentrations of NADPH in stressed plants. Here, we used Chelidonium majus L. plants, which accumulate high concentrations of dihydrocoptisine in their leaves, to study the impact of drought and salt stress on the biosynthesis and accumulation of alkaloids. In comparison to well-watered controls, in the transcriptome of the gene encoding the key enzyme in alkaloid biosynthesis, stylopine synthase, is enhanced in stressed C. majus plants. If we presuppose that increased transcript levels correlate with increased enzymatic activity of the gene products, these data indicate, for the first time, that stress-related increases in alkaloid concentration might not only be caused by the well-known stress-related passive shift, but may also be due to an enhancement of enzymatic capacity.

Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.

Proteomic comparison of Chelidonium majus L. latex in different phases of plant development.

Chelidonium majus L. (Papaveraceae) latex is used in traditinonal folk medicine to treat papillae, warts, condylomas, which are visible effects of human papilloma virus (HPV) infections. The aim of this work was to provide new insights into the biology and medicinal use of C. majus milky sap in the flowering and fruit ripening period of the plant by comparing the protein content between samples collected on respective developmental stages using LC-MS-based label-free proteome approach. For quantification, the multiplexed LC-MS data were processed using comparative chemometric approach. Progenesis LC-MS results showed that in green fruit phase (stage IV), comparing to flowering phase (stage III) of plant development, a range of proteins with higher abundance were identified as stress- and defense-related. On the other hand at stage III very intense protein synthesis, processes of transcription, protein folding and active transport of molecules (ABC transporters) are well represented. 2-DE protein maps showed an abundant set of spots with similar MWs (about 30-35 kDa) and pIs (ca. 5.5-6.5), which were identified as major latex proteins (MLPs). Therefore we suggest that biological activity of C. majus latex could be related to its protein content, which shifts during plant development from intense biosynthetic processes (biosynthesis and transport of small molecules, like alkaloids) to plant defense mechanisms against pathogens. Further studies will help to elucidate if these defense-related and pathogenesis-related proteins, like MLP, together with small-molecule compounds, could inhibit viral infection, what could be a step to fully understand the medicinal activity of C. majus latex.

Combination of transcriptomic and proteomic approaches helps to unravel the protein composition of Chelidonium majus L. milky sap.

MAIN CONCLUSION: A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.

Screening of As-accumulating plants using a foliar application and a native accumulation of As.

The discovery of novel accumulating plants is useful for efficient phytoremediation due to the demands of various conditions of impacted sites such as land use, soil properties, concentration of pollutants, and climate. In the present study, we investigated foliar application or a field with highly bioavailable arsenic (As) to screen As-accumulating plants. Plants grown in the downstream of a hot springs area were analyzed for native As accumulation and As foliar application, and the rhizosphere soils were collected. The water-soluble As in the rhizosphere soils had a high average, 144 microg/kg, whereas total As was similar to normal soil in Japan. Among 34 herbaceous plants and 17 woody plants, Chelidonium majus var. asiaticum accumulated a relatively high As level, 8.07 mg/kg DW (93.6% of As added), that was not revealed by native accumulation. In a further pot experiment, C. majus accumulated a moderately high As level (314 mg/kg DW) in the roots but not in the shoot (30.1 mg/kg DW), and exhibited a low transfer factor (TF = 0.096). Thus, a foliar application would be a simple and high-throughput method to screen plants that accumulate and tolerate As. C. majus would be useful as a tool for phytostabilization of As.

Alkaloid metabolism in thrips-Papaveraceae interaction: recognition and mutual response.

Frankliniella occidentalis (Pergande), the Western Flower Thrips (WFT), is a polyphagous and highly adaptable insect of the order Thysanoptera. It has a broad host range but is rarely found on Papaveraceae, which might be due to deterrent effects of alkaloids present in most species of this family. In order to test the adaptive potential of WFT, we investigated its interaction with two Papaveraceae offered as sole feeding source. We found that WFT are able to live and feed on leaves of Eschscholzia californica and Chelidonium majus. Both plants respond to thrips feeding by the enhanced production of benzophenanthridine alkaloids. Furthermore, cell cultures of E. californica react to water insoluble compounds prepared from adult thrips with enhanced alkaloid production. During feeding, WFT take up benzophenanthridine alkaloids from either plant and from an artificial feeding medium and convert them to their less toxic dihydroderivatives. This was shown in detail with sanguinarine, the most cytotoxic benzophenanthridine. A similar conversion is used in plants to prevent self-intoxication by their own toxins. We conclude that WFT causes a phytoalexin-like response in Papaveraceae, but is able to adapt to such host plants by detoxification of toxic alkaloids.

6-Acetonyl-5,6-dihydrosanguinarine (ADS) from Chelidonium majus L. triggers proinflammatory cytokine production via ROS-JNK/ERK-NFκB signaling pathway.

Chelidonium majus L. is an herbal plant that is commonly used in Western phytotherapy and traditional Chinese medicine for diuretic, antitussive, eye-regenerative, anti-osteoporotic, and radioprotective purposes. In this study, we purified 6-acetonyl-5,6-dihydrosanguinarine (ADS) from C. majus and investigated its immune-stimulatory effect. We found that ADS has the potential to induce the inflammatory cytokines TNF-α, IL-6, and IL-8 in macrophages and dendritic cells (DCs), that NFκB activation is a critical mediator of ADS-induced cytokine production, and that the activation of NFκB was dependent on reactive oxygen species (ROS). ADS induced phosphorylation of ERK and JNK, which was also associated with NFκB activation; phosphorylarion and cytokine production were inhibited by ROS scavenger and by specific MAPK inhibitors. Taken together, the results suggest that ADS from C. majus, as a positive immune modulator, induces inflammatory cytokines that might improve immunity, via the ROS-ERK/JNK-NFκB pathway.

Precocious progression of tissue maturation instructs basipetal initiation of leaflets in Chelidonium majus subsp. asiaticum (Papaveraceae).

PREMISE OF THE STUDY: On a compound leaf, leaflet primordia are repetitively formed along the apical-basal axis, with the direction varying among taxa. Why and how the directions vary among species is yet to be solved, although a change in a single factor was proposed to cause the variation. In this study, we compared two species in the Papaveraceae with different directions of leaflet initiation, Chelidonium majus subsp. asiaticum (basipetal) and Eschscholzia californica (acropetal). Because E. californica has been studied in some detail, we focused on C. majus and asked how basipetal pattern is achieved. • METHODS: Since only immature leaf primordial tissue has leaflet-generating competency, we performed histological and gene expression analyses on markers of the tissue maturation state. In addition, we performed a time-course analysis of leaf primordial growth. • KEY RESULTS: Quantitative reverse transcription-PCR analysis demonstrated that a putative regulator of tissue maturation in C. majus, the CINCINNATA homolog, had higher expression in apical parts than in basal parts during the organogenetic phase. In contrast, expression of the CIN homolog was not elevated in either the apical or basal parts in E. californica during the organogenetic phase. • CONCLUSIONS: In C. majus, apical parts of leaf primordia have already lost leaflet-generating competency during the organogenetic phase. We propose that precocious progression of the maturation process instructs basipetal progression of leaflet initiation in C. majus. This is not the mirror image of data on E. californica, which shows the opposite direction in leaflet formation, indicating that variation in direction is not attributable to a change in a single factor.

[Characteristics of 23 species of weed in northeast of China hyperaccumulating PAHs in contaminated soils].

Pot experiments were conducted to investigate the 23 species of weed accumulation characteristics of phenanthrene, as a representative of polycyclic aromatic hydrocarbons (PAHs), from soil in the northeast of China. The result indicated that among the 23 species, the bioconcentration factors of Taraxacum ohwianum K., Potentilla aiscolor B. and Chelidonium majus L. were all higher than 1, which were 1.01, 4.98, 38.24 respectively. The phenanthrene concentrations in roots were 2.83, 16.34 and 9.66 mg/kg which were lower than those in aboveground part with phenanthrene concentrations were 15.18, 74.70 and 573.62 mg/kg, respectively. The hyperaccumulators were indicated by strong conveyance of phenanthrene from root to aboveground part. The analysis of phenanthrene concentration in aboveground weed and aboveground plant biomass showed that the accumulation of phenanthrene in plant were not correlated with their biomass. It concluded that Taraxacum ohwianum K., Potentilla aiscolor B. and Chelidonium majus L. had hyperaccumulative characteristics of phenanthrene, and it is possible to screen out plants with high biomass and hyperaccumulation capability.

Proteomic analysis of Chelidonium majus milky sap using two-dimensional gel electrophoresis and tandem mass spectrometry.

Milky sap, a milky-like orange fluid, isolated from the Greater Celandine (Chelidonium majus L.), family Papaveraceae, serves as a rich source of various biologically active substances such as alkaloids, several flavonoids, phenolic acids and proteins. The objective of this study was to separate Ch. majus milky sap extract proteins using two-dimensional gel electrophoresis (2-DE) to demonstrate for the first time the protein composition in the sap and to identify them using liquid chromatography-tandem mass spectrometry analysis (LC-ESI-MS/MS). It was possible to identify 21 proteins, which comprise disease/defence-related, signalling, Krebs cycle, nucleic acid binding and other proteins. The majority of the identified proteins can be linked to direct and indirect stress and defence reactions, e.g. against different pathogens. The specific protein composition of the milky sap suggests an important role of these proteins for the whole plant physiology and development.

Expression patterns of STM-like KNOX and Histone H4 genes in shoot development of the dissected-leaved basal eudicot plants Chelidonium majus and Eschscholzia californica (Papaveraceae).

Knotted-like homeobox (KNOX) genes encode important regulators of shoot development in flowering plants. In Arabidopsis, class I KNOX genes are part of a regulatory system that contributes to indeterminacy of shoot development, delimitation of leaf primordia and internode development. In other species, class I KNOX genes have also been recruited in the control of marginal blastozone fractionation during dissected leaf development. Here we report the isolation of class I KNOX genes from two species of the basal eudicot family Papaveraceae, Chelidonium majus and Eschscholzia californica. Sequence comparisons and expression patterns indicate that these genes are orthologs of SHOOTMERISTEMLESS (STM), a class I KNOX gene from Arabidopsis. Both genes are expressed in the center of vegetative and floral shoot apical meristems (SAM), but downregulated at leaf or floral organ initiating sites. While Eschscholzia californica STM (EcSTM) is again upregulated during acropetal pinna formation, in situ hybridization could not detect Chelidonium majus STM (CmSTM) transcripts at any stage of basipetal leaf development, indicating divergent evolution of STM gene function in leaves within Papaveraceae. Immunolocalization of KNOX proteins indicate that other gene family members may control leaf dissection in both species. The contrasting direction of pinna initiation in the two species was also investigated using Histone H4 expression. Leaves at early stages of development did not reveal notable differences in cell division activity of the elongating leaf axis, suggesting that differential meristematic growth may not play a role in determining the observed dissection patterns.

Ukrain - a new cancer cure? A systematic review of randomised clinical trials.

BACKGROUND: Ukrain is an anticancer drug based on the extract of the plant Chelidonium majus L. Numerous pre-clinical and clinical investigations seem to suggest that Ukrain is pharmacologically active and clinically effective. We wanted therefore to critically evaluate the clinical trial data in the form of a systematic review. METHODS: Seven electronic databases were searched for all relevant randomised clinical trials. Data were extracted and validated by both authors, tabulated and summarised narratively. The methodological quality was assessed with the Jadad score. RESULTS: Seven trials met our inclusion criteria. Without exception, their findings suggest that Ukrain has curative effects on a range of cancers. However, the methodological quality of most studies was poor. In addition, the interpretation of several trials was impeded by other problems. CONCLUSION: The data from randomised clinical trials suggest Ukrain to have potential as an anticancer drug. However, numerous caveats prevent a positive conclusion, and independent rigorous studies are urgently needed.