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Introduction: Inflammatory bowel diseases (IBDs) include Crohn's disease, and ulcerative colitis. Cannabis sativa preparations have beneficial effects for IBD patients. However, C. sativa extracts contain hundreds of compounds. Although there is much knowledge of the activity of different cannabinoids and their receptor agonists or antagonists, the cytotoxic and anti-inflammatory activity of whole C. sativa extracts has never been characterized in detail with in vitro and ex vivo colon models.Material and Methods: The anti-inflammatory activity of C. sativa extracts was studied on three lines of epithelial cells and on colon tissue. C. sativa flowers were extracted with ethanol, enzyme-linked immunosorbent assay was used to determine the level of interleukin-8 in colon cells and tissue biopsies, chemical analysis was performed using high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance and gene expression was determined by quantitative real-time PCR.Results: The anti-inflammatory activity of Cannabis extracts derives from D9-tetrahydrocannabinolic acid (THCA) present in fraction 7 (F7) of the extract. However, all fractions of C. sativa at a certain combination of concentrations have a significant increased cytotoxic activity. GPR55 receptor antagonist significantly reduces the anti-inflammatory activity of F7, whereas cannabinoid type 2 receptor antagonist significantly increases HCT116 cell proliferation. Also, cannabidiol (CBD) shows dose dependent cytotoxic activity, whereas anti-inflammatory activity was found only for the low concentration of CBD, and in a bell-shaped rather than dose-dependent manner. Activity of the extract and active fraction was verified on colon tissues taken from IBD patients, and was shown to suppress cyclooxygenase-2 (COX2) and metalloproteinase-9 (MMP9) gene expression in both cell culture and colon tissue.Conclusions: It is suggested that the anti-inflammatory activity of Cannabis extracts on colon epithelial cells derives from a fraction of the extract that contains THCA, and is mediated, at least partially, via GPR55 receptor. The cytotoxic activity of the C. sativa extract was increased by combining all fractions at a certain combination of concentrations and was partially affected by CB2 receptor antagonist that increased cell proliferation. It is suggested that in a nonpsychoactive treatment for IBD, THCA should be used rather than CBD.

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Introduction: Colorectal cancer remains the third most common cancer diagnosis and fourth leading cause of cancer-related mortality worldwide. Purified cannabinoids have been reported to prevent proliferation, metastasis, and induce apoptosis in a variety of cancer cell types. However, the active compounds from Cannabis sativa flowers and their interactions remain elusive.Research Aim: This study was aimed to specify the cytotoxic effect of C. sativa-derived extracts on colon cancer cells and adenomatous polyps by identification of active compound(s) and characterization of their interaction.Materials and Methods: Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography and gas chromatograph/mass spectrometry and their cytotoxic activity was determined using alamarBlue-based assay (Resazurin) and tetrazolium dye-based assay (XTT) on cancer and normal colon cell lines and on dysplastic adenomatous polyp cells. Annexin V Assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle, and RNA sequencing was used to determine gene expression.Results: The unheated cannabis extracts (C2F), fraction 7 (F7), and fraction 3 (F3) had cytotoxic activity on colon cancer cells, but reduced activity on normal colon cell lines. Moreover, synergistic interaction was found between F7 and F3 and the latter contains mainly cannabigerolic acid. The F7 and F7+F3 cytotoxic activity involved cell apoptosis and cell cycle arrest in S or G0/G1 phases, respectively. RNA profiling identified 2283 differentially expressed genes in F7+F3 treatment, among them genes related to the Wnt signaling pathway and apoptosis-related genes. Moreover, F7, F3, and F7+F3 treatments induced cell death of polyp cells.Conclusions: C. sativa compounds interact synergistically for cytotoxic activity against colon cancer cells and induce cell cycle arrest, apoptotic cell death, and distinct gene expression. F3, F7, and F7+F3 are also active on adenomatous polyps, suggesting possible future therapeutic value.

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10 µ g mL -1, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5-10 µ g mL -1, also as a drench) reduced the rate of infection by 75-85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.

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Genetic transformation mediated by bombardment with microscopic metal particles carrying target genes is the preferred method for the introduction of foreign genes into monocotyledonous plants. The fact that most flower bulbs are monocotyle-donous and that almost all commercial cultivars are propagated vegetatively makes them good candidates for molecular breeding through microprojectile bombardment. We report here on a development of a reliable method for an efficient genetic transformation of both Lilium longiflorum and Ornithogalum dubium using a particle inflow gun to deliver gene constructs into the target plant tissue, followed by a prolonged selection in the dark in liquid medium supplemented with kanamycin. The system was first optimized for Lilium longiflorum 'Snow Queen'. Based on the level of transient GUS expression, liquidgrown cell clumps are more competent than leaves. Large cell clusters (2-10mm) maintain their organogenic potential while smaller clusters (<2mm) cease to grow and die. The liquid-grown tissue cultures have a level of competence for transformation about 50-70 times greater than that of solid-grown callus cultures, and compact cell clusters are more competent than loose clusters. The cells were bombarded with a pCAMBIA2301 vector, carrying nptII gene conferring kanamycin resistance and GUS reporter gene. Following selection for 4-6 months in a liquid medium supplemented with 80 mg l-1 kanamycin in the dark, the cell clusters were transferred to a regeneration medium in the light where hundreds of transgenic plantlets developed. The plants retained their stable transgenic state when grown in the greenhouse for two seasons. The transformation of O. dubium was similar in principle to that of L. longiflorum with three major differences: lily liquid-grown cultures grew more rapidly and had a higher potential for somatic embryo development. Ornithogalum cultures under selection took longer to develop into semi-organized cell clumps of sufficient size to allow continued shoot regeneration, were mostly organogenic, and the regenerated plantlets had higher rate of vitrification.

Ephedra is likely one of the oldest medicinal plants still currently in use. In folk medicine, extracts of Ephedra foeminea are commonly used to treat cancer patients. In relation to its traditional use, the aim of the present study was to determine the cytotoxic activity in vitro of E. foeminea extracts on cancer and non-cancer cells. Cell viability was determined using XTT assay, induction of apoptosis by cell sorting and caspase-3 inhibition, and the effects on cell cytoskeleton structure were detected using cell transfection utilizing different cytoskeleton markers. Chemical profiling, analysis of active extracts and identification of compounds was done using high pressure liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GCMS). E. foeminea leaf ethanol extract and E. foeminea fruit juice reduced cancer cell viability in vitro, whereas the water extract reduced cytotoxic activity in all cell lines. The extract's cytotoxic activity was conveyed at least partially via the induction of caspase 3-dependent cell apoptosis, and enhanced by the addition of Taxol. Both E. foeminea ethanol leaf extract and fruit juice affected actin-stained but not tubulin-stained filaments. Ethanol extract promoted the formation of invadopodia-like structures and fruit juice promoted the formation of large focal adhesion points in the treated cells. Active sub-fractions of E. foeminea extracts were found to contain several compounds including trans-sinapyl alcohol and trans-sinapaldehyde derivative.