Antitumour effect of odoroside A and its derivative on human leukaemia cells through the ROS/JNK pathway.

Oleandrigenin-3-O-ß-D-diginoside (a derivative of odoroside A), isolated and purified by our group, has seldom been explored for its pharmacological activity. This study aimed at clarifying the mechanisms towards the leukaemia-suppressive role of odoroside A (compound #1) and its derivative, oleandrigenin-3-O-ß-D-diginoside (compound #2) isolated from Nerium oleander. Viability and nuclear morphology change were assessed by CCK-8 assay and fluorescence microscope, respectively. Then, the cell apoptosis and autophagy induced by the compounds were detected by flow cytometry and Western blot. Xenograft model of nude mice was also applied to measure the leukaemia-suppressive effects of compound #2 in vivo. The result displayed that compound #1 and compound #2 inhibited the proliferation of HL60 and K562 cells and stronger effects were found in HL60 than K562 cells. Both of the compounds induced a dose-dependent apoptosis and autophagy in HL60 cells, where compound #2 was more potent than compound #1. Compound #2 also demonstrated a time-dependent apoptosis and autophagy in HL60 cells. Furthermore, ROS generation and JNK phosphorylation occurred in a dose-dependent manner in the cells treated with compound #2. Mitochondria also played critical role, proved by the decrease of Bcl-2, the release of cyto c to cytosol and the activation of caspase-3 and caspase-9. Moreover, the antitumour effects of compound #2 were validated in the nude mouse xenograft model in vivo. Odoroside A and its derivative inhibited the growth of leukaemia by inducing apoptosis and autophagy through the activation of ROS/JNK pathway. These results suggest that the compounds can serve as potential antitumour agents against leukaemia, especially acute myeloid leukaemia (AML).

Cardenolides and pentacyclic triterpenes isolated from Acokanthera oblongifolia leaves: their biological activities with molecular docking study.

Pentacyclic triterpenes and cardenolides were isolated from Acokanthera oblongifolia leaves. Their chemical structures were determined based on comprehensive 1D and 2D NMR spectroscopy. Their MIC was determined against 12 microorganisms. Their exerted cytotoxicity on the immortalized normal cells, hTERT-RPE1 was assessed by the sulforhodamine-B assay. The viral inhibitory effects of compounds against Newcastle disease virus (NDV) and H5N1 influenza virus IV were evaluated. Four in vitro antioxidant assays were performed in comparison with BHT and trolox and a weak activity was exhibited. Acovenoside A was with potent against H5N1-IV and NDV with IC50 ≤ 3.2 and ≤ 2.1 µg/ml and SI values of 93.75 and 95.23%, respectively, in comparison to ribavirin. Its CC50 record on Vero cells was > 400 and 200 µg/ml, respectively. Acobioside A was the most active compound against a broad range of microbes while Pseudomonas aeruginosa was the most sensitive. Its MIC (0.07 µg/ml) was 1/100-fold of the recorded CC50 (7.1 µg/ml/72 h) against hTERT-RPE1. The molecular docking of compounds on human DNA topoisomerase I (Top1-DNA) and IV glycoprotein hemagglutinin were studied using MOE program. This study has introduced the cardenolides rather than triterpenoids with the best docking score and binding interaction with the active site of the studied proteins.

New Cardenolides from Biotransformation of Gitoxigenin by the Endophytic Fungus Alternaria eureka 1E1BL1: Characterization and Cytotoxic Activities.

Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin (yield; ~25%). After 21 days of incubation, five new cardenolides 1, 3, 4, 6, and 8 and three previously- identified compounds 2, 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

In vitro chemotherapeutic and antiangiogenic properties of cardenolides from Acokanthera oblongifolia (Hochst.) Codd.

Acovenoside A (Acov-A) and acobioside A (Acob-A) were isolated from Acokanthera oblongifolia. Their anticancer properties were explored regarding, antiproliferative and antiangiogenic activities. The study included screening phase against six cancer cell lines followed by mechanistic investigation against HepG2 cancer cell line. The sulforhodamine-B (SRB) was used to determine their growth inhibitory power. In the other hand, flow cytometry techniques were recorded the cell death type and cell cycle analysis. The clonogenic (colony formation) and wound healing assays, enzyme-linked immunosorbent assay (ELISA) and molecular docking, were performed to evaluate the antiangiogenesis capability. Both compounds were strongly, inhibited four cancer cell lines at GI50 less than 100 nM. The in vitro mechanistic investigation against HepG2 resulted in cell accumulations at G2M phase and induction of apoptosis upon treating cells separately, with 400 nM Acov-A and 200 nM Acob-A. Interestingly, the same concentrations were able to activate caspase-3 by 7.2 and 4.8-fold, respectively. Suppressing the clonogenic capacity of HepG2 cells (20 and 40 nM) and inhibiting the migration of the colon Caco-2 cancer cells were provoke the results of vascular endothelial growth factor receptor2 (VEGFR2) kinase enzyme inactivation. The docked study was highly supportive, to the antiangiogenic approach of both cardenolides. The isolated cardenolides could orchestrate pivotal events in fighting cancer.

Anticancer Cardenolides from the aerial parts of Calortopis procera.

Column chromatography (CC) analysis of methanol and butanol extracts of the aerial parts of Calortopis procera as well as the methanol extract of its latex, led to the isolation of 8 cardenolides, of which the structures were elucidated by NMR and HRESIMS spectroscopy. They also revealed several triterpenes and flavonoid glycoside. Based on the antiproliferative activity reported for cardenolides, the activity of calotropin and calotoxin was tested against two common cancer cell lines, human triple-negative breast cancer cell line (MDA-MB-231) and human lung adenocarcinoma cell line (A549). The high toxicity of the latex also encouraged performing the same test on the same cancer cell lines. The anti-proliferative activity of calotropin and calotoxin was compared to the methanol extract and the wax of the latex. The results showed that calotropin and calotoxin have significant cytotoxicity against MDA-MB-231 and A549 cell lines ranging from 0.046 to 0.072 µM compared to the methanol extract and the wax of its latex ranging from 0.47 to 58.41 µM. Moreover, the results showed lower toxicity of all treatments to the human skin fibroblasts compared to the toxicity to both MDA-MB-231 and A549 cancer cells lines except the higher toxicity of Methanolic extracts of C. procera latex to the MDA-MB-231 cells. In conclusion, C. procera is a medicinal plant with a wide spectrum of cardinolides including calotropin and calotoxin, which are promising agents for targeted cancer phytotherapy.

Non-classical cardenolides from Calotropis gigantea exhibit anticancer effect as HIF-1 inhibitors.

Six new non-classical cardenolides (1-6), and seventeen known ones (7-23) were isolated from Calotropis gigantea. All cardenolides showed inhibitory effect on hypoxia inducible factor-1 (HIF-1) transcriptional activity with IC50 of 8.85 nM-16.69 µM except 5 and 7. The novel 19-dihydrocalotoxin (1) exhibited a comparable HIF-1 inhibitory activity (IC50 of 139.57 nM) to digoxin (IC50 of 145.77 nM), a well-studied HIF-1 inhibitor, and 11, 12, 14, 16 and 19 presented 1.4-15.4 folds stronger HIF-1 inhibition than digoxin. 1 and 11 showed a dose-dependent inhibition on HIF-1α protein, which led to their HIF-1 suppressing effects. Compared with LO2 and H9c2 normal cell lines, both 1 and 11 showed selective cytotoxicity against various cancer cell lines including HCT116, HeLa, HepG2, A549, MCF-7, A2780 and MDA-MB-231. Moreover, a comprehensive structure-activity relationship was concluded for these non-classical cardenolides as HIF-1 inhibitors, which may shed some light on the rational design and development of cardenolide-based anticancer drugs.

A new cardenolide glycoside from the roots of Streptocaulon juventas (lour.) merr. (Asclepiadaceae).

From remaining aqueous fraction of the roots of Streptocaulon juventas, one new cardiac glycoside named periplogenin 3-O-ß-gentiobioside (1) together with six known ones (2-7) were isolated. Their relative structures were elucidated based on NMR spectroscopic analysis. Compound 1 showed moderate cytotoxicity against non-small cell lung carcinoma NCI-H460 and ovarian cancer HeLa cells. Moreover, compounds 2 and 3 exhibited remarkable cytotoxicity against NCI-H460 cell with the IC50 values of 0.34 and 0.068 µM, respectively.

Screening of Promising Chemotherapeutic Candidates from Plants against Human Adult T-Cell Leukemia/Lymphoma (VI): Cardenolides from Asclepias curassavica.

In the course of our screening program for novel chemotherapeutic candidates from plants against adult T-cell leukemia/lymphoma, the extracts of Asclepias curassavica L. showed potent activity against MT-1 and MT-2 cells. Therefore, we attempted to isolate their active components. We identified a new cardenolide, 19-dihydrocalactinic acid methyl ester (1), along with 16 known cardenolides (2-17). Their structures were determined on the basis of spectroscopic data. Almost all of the isolated cardenolides inhibited the growth of both tumor cell lines. All the doubly linked cardenolides (11-17) except for 14 showed more potent activity than the other cardenolides. A comparison of the activities of 11, 14 and 16 revealed that the presence of hydroxy or acetoxy functional groups at C-16 led to a decrease in the activity. The 50% effective concentration (EC50) value of calotropin (11) against MT-2 cells was comparable to the potency of the clinical antineoplastic drug doxorubicin. The cytotoxic effect of 11 toward normal mononuclear cells obtained from the peripheral blood (PB-MNCs) was observed at a concentration 6 to 12 times higher than that used to induce growth inhibition against MT-1 and MT-2 cells. The proportions of annexin V-positive cells after 72 h of treatment with 11 were increased, indicating that it significantly induced apoptosis in MT-1 and MT-2 cells in a concentration-dependent manner. Cell cycle experiments demonstrated that 11 arrested MT-1 and MT-2 cells at the G2/M phase. Therefore, compound 11 may be a promising candidate for the treatment of adult T-cell leukemia/lymphoma.

Cardenolide-rich fraction of Pergularia tomentosa as a novel Antiangiogenic agent mainly targeting endothelial cell migration.

PURPOSE: Angiogenesis related abnormalities underlie several life-threatening disorders. Despite approved therapies, scientists have yet to develop highly efficient, low cost approaches with minimal side effects. METHODS: We evaluated the antiangiogenic activity of 50% hydroalcoholic extracts of Pergularia tomentosa L. root and aerial parts along with their EtOAc and water fractions, in vivo and in vitro. Transgenic zebrafish line Tg(fli1:EGFP) was used for in vivo assay and human umbilical vein endothelial cell (HUVEC) migration test along with possibility of tube formation were performed as in vitro tests. Furthermore, microvasculature in chicken chorioallantoic membrane (CAM) was assessed under P. tomentosa treatment. The fractionation of the 50% hydroalcoholic extracts was led to the identification of the best active fraction in this study. The metabolite profiling of the active fraction was also carried out using LC-HRESIMS analysis. RESULTS: Pergularia tomentosa markedly inhibited intersegmental vessel (ISV) formation at 48 h post-fertilization (hpf) embryos in zebrafish. The water fraction of root hydroalcoholic extract (PtR2), showed strong antiangiogenic effect with minimal adverse viability impacts. Over 80% of embryos showed more than 50% inhibition in their ISV development at 20 and 40 µg/mL. PtR2 at 20 µg/mL substantially reduced human umbilical vein endothelial cell (HUVEC) migration up to 40%, considerable destruction of the formed tubes in the tube formation and microvasculature in CAM assays. Immunocytochemistry showed a marked reduction in vascular endothelial cadherin (VE-cadherin) abundance at cell junctions concurrent with substantial reduction of phospho-Akt (p-Akt) and ß-catenin protein expressions. Phytochemical profile of PtR2 showed a rich source of cardenolide structures, including ghalakinoside, calactin and calotropin derivatives. CONCLUSION: Thus, the P. tomentosa cardenolide-rich fraction (PtR2) may hold a considerable promise for an antiangiogenic impact by impairment of endothelial cell (EC) migration and viability. Graphical abstract.

Calosides A-F, Cardenolides from Calotropis gigantea and Their Cytotoxic Activity.

Phytochemical analysis of the roots of Calotropis gigantea led to the isolation of six new cardenolide glycosides, calosides A-F (1-6), and five known cardenolides (7-11). The structures of 1-6 were elucidated based on NMR and ECD spectroscopic data interpretation. Caloside D (4) is the first naturally occurring example of a cardenolide containing a C-8/C-19 oxygen bridge. In turn, calosides E (5) and F (6) represent the first naturally occurring 3-epi-cannogenol diglycosides having potent cytotoxicity against the PANC-1 cell line (IC50, 0.081 and 0.070 µM, respectively) and HeLa (IC50, both 0.17 µM) cells, under normoglycemic conditions.

Cytotoxic Cardenolides from the Leaves of Acokanthera oblongifolia.

Three previously undescribed cardenolides, acovenosigenin A 3-O-α-L-acofriopyranoside (1: ), 14-anhydroacovenosigenin A 3-O-[ß-D-glucopyranosyl-(1″→4')-O-α-L-acofriopyranoside] (2: ), and 14-anhydroacovenosigenin A 3-O-[ß-D-glucopyranosyl-(1″→4')-O-α-L-acovenopyranoside] (3: ), together with the two already known ones, 14-anhydrodigitoxigenin 3-O-ß-D-glucopyranoside (4: ) and acospectoside A (5: ), were isolated from the leaves of Acokanthera oblongifolia. The influence of cardenolides 1:  - 3: and acovenoside A (found in the Acokanthera genus) on three cancer cell lines (HT29, HCT116, and AGS) was also investigated. The most promising results, in comparison with oxaliplatin, were obtained for compound 1: , which was found to be highly cytotoxic for all tested cell lines, HT29 (IC50 = 63.49 nM), HCT116 (IC50 = 67.35 nM), and AGS (IC50 = 80.92 nM). Unfortunately, 1: also showed similar toxicity towards normal lymphocytes (IC50 = 98.03 nM).

New knowledge about old drugs; a cardenolide type glycoside with cytotoxic effect and unusual secondary metabolites from Digitalis grandiflora Miller.

Phytochemical investigation of the aerial parts of Digitalis grandiflora Miller (Plantaginaceae) led to the isolation of an undescribed cardenolide type glycoside digigrandifloroside (1) along with five known compounds, rengyoside A (2), rengyoside B (3), cleroindicin A (4), salidroside (5), and cornoside (6), from its aqueous fraction of methanolic extract. Structures of the isolated compounds were determined by means of spectroscopic techniques. 1-6 were isolated for the first time from D. grandiflora. 2 and 3 are being reported for the first time from Digitalis genus and Plantaginaceae family with this study. This is the second report for occurrence of 4 from a Digitalis species. Cytotoxic activity of the aqueous fraction was also tested against HEp-2 (Human larynx epidermoid carcinoma) and HepG2 (Human hepatocellular carcinoma) cancer cell lines and L929 (Mouse fibroblast cell) non-cancerous cell line. Aqueous fraction showed stronger cytotoxicity on HEp-2 cells than HepG2. Therefore, the cytotoxic activity of 1, 2, 4, and 6 were tested against HEp-2 and L929 cell lines. 3 and 5 couldn't be tested due to their insufficient amount. 1 showed the highest cytotoxicity against HEp-2 cells with IC50 value 10.1 µM when compared with the positive control, etoposide and 2-6 (IC50 of etoposide; 39.5 µM).

Antiproliferative Cardenolides from the Aerial Parts of Pergularia tomentosa.

The LC-MS analysis of the MeOH extract of the aerial parts of Pergularia tomentosa led to the isolation of 23 compounds, of which the structures were elucidated unambiguously by NMR spectroscopic data analysis. Three new doubly linked cardenolides (4, 13, 14) along with several known cardenolides (1-3, 5, 7, 8, 15-23) and flavonol glycosides (6, 9-12) were identified. LC-HRESIMS analysis, in the negative-ionization mode, showed the absence of flavonoids in a methanol extract of the roots of P. tomentosa. On the basis of the antiproliferative activity reported for cardenolides, the isolated compounds were tested for their ability to decrease the cell viability of five different human cancer cell lines, PC3, HeLa, Calu-1, MCF-7, and U251MG, exhibiting IC50 values ranging from 0.2 to 8.0 µM. Moreover, an S-phase entry assay was performed to investigate if the compounds could affect the cell cycle progression of PC3 prostate carcinoma cells. The results obtained demonstrated that the compounds 4, 7, and 14 at 1 µM considerably reduced the number of cells in the S-phase.

Cardenolides and dihydro-ß-agarofuran sesquiterpenes from the seeds of Salacia staudtiana.

Phytochemical studies of the seeds of the Cameroonian medicinal plant, Salacia staudtiana, resulted in the isolation and identification of five new cardenolides (1-5) as well as a new dihydro-ß-agarofuran (9), along with eight known compounds. The structures of all compounds were elucidated by 1D/2D NMR, ESI-HRMS data and comparison with literature data. The relative configurations of the new compounds were defined by X-ray crystallography analysis, NOESY correlations and coupling constants. We evaluated their antibacterial efficacy against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, and two pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa, compared to the standard antibiotics, streptomycin and gentamicin. Moreover, we assessed the antibacterial activity of the crude extract of the seeds in parallel to evaluate the plausible synergistic effects of the compounds in chemical defense of the seeds during germination and plant reproduction. The isolated compounds showed moderate antibacterial activities against the tested organisms. Compounds 1 and 3 and the crude extract exhibited distinct antibacterial activities against B. subtilis and S. aureus. The isolated compounds showed weak DPPH radical scavenging properties compared to the reference standard (Trolox). Our study lends evidence to the antibacterial chemical defense of S. staudtiana seeds by seed-borne compounds.

The Effects of Milkweed Induced Defense on Parasite Resistance in Monarch Butterflies, Danaus plexippus.

Many plants express induced defenses against herbivores through increasing the production of toxic secondary chemicals following damage. Phytochemical induction can directly or indirectly affect other organisms within the community. In tri-trophic systems, increased concentrations of plant toxins could be detrimental to plants if herbivores can sequester these toxins as protective chemicals for themselves. Thus, through trophic interactions, induction can lead to either positive or negative effects on plant fitness. We examined the effects of milkweed (Asclepias spp.) induced defenses on the resistance of monarch caterpillars (Danaus plexippus) to a protozoan parasite (Ophryocystis elektroscirrha). Milkweeds contain toxic secondary chemicals called cardenolides, higher concentrations of which are associated with reduced parasite growth. Previous work showed that declines in foliar cardenolides caused by aphid attack render monarch caterpillars more susceptible to infection. Here, we ask whether cardenolide induction by monarchs increases monarch resistance to disease. We subjected the high-cardenolide milkweed A. curassavica and the low-cardenolide A. syriaca to caterpillar grazing, and reared infected and uninfected caterpillars on these plants. As expected, monarchs suffered less parasite growth and disease when reared on A. curassavica than on A. syriaca. We also found that herbivory increased cardenolide concentrations in A. curassavica, but not A. syriaca. However, cardenolide induction in A. curassavica was insufficient to influence monarch resistance to the parasite. Our results suggest that interspecific variation in cardenolide concentration is a more important driver of parasite defense than plasticity via induced defenses in this tri-trophic system.

Cardenolides from the stem bark of Salacia staudtiana.

Seven new cardenolides, staudtianoside A-F (1-6) and staudtianogenin A (8), were isolated along with six known compounds from the stem bark of the Cameroonian medicinal plant Salacia staudtiana Loes. ex Fritsch. The structures were elucidated by means of ESI-HRMS and NMR spectroscopic methods and by comparison with literature data. The relative configurations of the new compounds were determined by X-ray diffraction analysis, NOESY correlation and coupling constants. We evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compounds 1, 2 and 8 exhibited marked antibacterial potencies against the clinically relevant P. aeruginosa that were comparable to the standard antibiotics. Compound 2 was also active against S. aureus and E. coli. Further, compounds 5 and 8 demonstrated efficacy against E. coli as well as B. subtilis. The structure-activity relationship of the tested compounds is discussed.

Toxicity of the spiny thick-foot Pachypodium.

PREMISE OF THE STUDY: Pachypodium (Apocynaceae) is a genus of iconic stem-succulent and poisonous plants endemic to Madagascar and southern Africa. We tested hypotheses about the mode of action and macroevolution of toxicity in this group. We further hypothesized that while monarch butterflies are highly resistant to cardenolide toxins (a type of cardiac glycoside) from American Asclepias, they may be negatively affected by Pachypodium defenses, which evolved independently. METHODS: We grew 16 of 21 known Pachypodium spp. and quantified putative cardenolides by HPLC and also by inhibition of animal Na+ /K+ -ATPase (the physiological target of cardiac glycosides) using an in vitro assay. Pachypodium extracts were tested against monarch caterpillars in a feeding bioassay. We also tested four Asclepias spp. and five Pachypodium spp. extracts, contrasting inhibition of the cardenolide-sensitive porcine Na+ /K+ -ATPase to the monarch's resistant form. KEY RESULTS: We found evidence for low cardenolides by HPLC, but substantial toxicity when extracts were assayed on Na+ /K+ -ATPases. Toxicity showed phylogenetic signal, and taller species showed greater toxicity (this was marginal after phylogenetic correction). Application of Pachypodium extracts to milkweed leaves reduced monarch growth, and this was predicted by inhibition of the sensitive Na+ /K+ -ATPase in phylogenetic analyses. Asclepias extracts were 100-fold less potent against the monarch compared to the porcine Na+ /K+ -ATPase, but this difference was absent for Pachypodium extracts. CONCLUSIONS: Pachypodium contains potent toxicity capable of inhibiting sensitive and cardenolide-adapted Na+ /K+ -ATPases. Given the monarch's sensitivity to Pachypodium, we suggest that these plants contain novel cardiac glycosides or other compounds that facilitate toxicity by binding to Na+ /K+ -ATPases.

Cardenolides from the leaves of Nerium oleander.

Six new cardenolides (1-6), including three 14-hydroxylated cardenolides and three 14-carbonylated cardenolides were isolated from the dried aerial parts of Nerium oleander Linn in addition to twenty-seven known compounds (7-33). Their structures were elucidated on the basis of extensive spectroscopic evidences and single-crystal X-ray diffraction analysis. Compounds 1, 4, 7-10 and 13 exhibited significant cytotoxicity against four colon cancer cell lines (HCT116, HT29, SW620, RKO), one gastric cancer cell line (GT) and one cervical cancer cell line (HeLa) in vitro.

An Overview of Cardenolides in Digitalis - More Than a Cardiotonic Compound.

The genus Digitalis L. containing species, commonly known as the "foxglove", is the main source of cardenolides, which have various pharmacological properties effective against certain pathological conditions including myocardial infarction, arterial hypertension, cardiac dysfunction, angina, and hypertrophy. Togehter with a prime effect of controlling the heart rhythm, many workers demonstrated that lanatoside C and some other cardiac glycosides are effective in several cancer treatments such as prostate and breast cancers. Due to digoxigenin derivatives of cardenolides, which are mainly used for medicinal purposes, such as digoxigenin, D. lanata as a main source is of great interest for commercial scale production of cardenolides in Europe. Phytochemical studies on cardenolides, naturally occurring plant secondary metabolites, have mainly focused on the species of the genus Digitalis L., as the members of this family have a high level and diverse content of cardenolides. During the last few decades, plant tissue culture techniques have been optimised for many plant species including Digitalis, however, the production capacity of cardenolides somehow failed to reach a commercially desired extent. In this review paper, the genus Digitalis is evaluated in terms of its main botanical and physiological features, traditional uses, molecular genetics and metabolomics, cellular mechanism of action, medicinal uses, clinical pharmacology, drug interactions, therapy in the management of cardiovascular disorders, potential utility of therapy in extracardiac conditions, and toxicity.

Identification of BMI1 Promoter Inhibitors from Beaumontia murtonii and Eugenia operculata.

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of Beaumontia murtonii and Eugenia operculata were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (1-3) and one new compound (4) from B. murtonii and two known triterpenoids (5 and 6) and one new compound (7) from E. operculata. These seven compounds inhibited BMI1 promoter activity (IC50 range 0.093-23.0 µM), and the most active compound, wallichoside (1), was further evaluated. Western blot analysis revealed that wallichoside (1) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (1) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs.