Alkaloids and flavonoids from ficus, artocarpus and macaranga species : structure and anti-cancer activity

Plant natural products have played a pivotal role in the discovery and development of many new drugs for the treatment of various infectious diseases and cancers. The present study was therefore aimed at isolation and identification of novel bioactive compounds from selected plants collected in Malaysia with anti-cancer activity. Plant crude extracts were obtained using solvent extraction methods, while various chromatographic and spectroscopic methods were employed for compound isolation and structure elucidation. Evaluation of pure compounds and crude extracts for anti-cancer activity involved the use of Neutral Red assay (NR), acradine orange – ethidium bromide (AO/EB) staining and cell cycle analysis. Phytochemical investigations of five Malaysian plants, namely, Ficus hispida, F. fistulosa, F. schwarzii, Artocarpus heterophyllus x integer and Macaranga hypoleuca, with the focus on alkaloids and flavonoids, have resulted in the isolation of a total of 24 compounds, of which six are new. The leaf and stem-bark extracts of Ficus hispida yielded two new alkaloids, hispidacine (1) and hispiloscine (2), and a known alkaloid, 13a(S)-(+)-deoxypergularinine (3). Hispidacine represents the first example of an 8,4'-oxyneolignan incorporated an unusual 2-hydroxyethylamine moiety, while hispiloscine represents the first naturally occurring phenanthroindolizidine alkaloid with acetoxy substitution. The leaf and bark extracts of Ficus fistulosa provided two new septicine alkaloids, fistulopsines A and B (4 and 5), together with four known phenanthroindolizidine alkaloids, 13a(R)-(–)-3,6-didemethylisotylocrebrine (6), 13a(S)-(+)-tylocrebrine (7), 13a(S)-(+)-tylophorine (8) and 13a(S)-(+)-septicine (9), and one known non-alkaloid (vomifoliol, 10). The leaves of Ficus schwarzii gave two novel tri-nor-sesquilignan alkaloids, schwarzinicines A and B (11 and 12). The bark of Artocarpus heterophyllus x integer yielded five known compounds, of which four are prenylated flavonoids, namely, cudraflavone C (13), artocarpetin A (14), cycloheterophyllin (15) and artonin J (16), and one natural xanthone, lichexanthone (17). The leaves of Macaranga hypoleuca provided seven known compounds, of which three are flavonoid glycosides, namely, quercetin-3-O--L-arabinopyranoside (18), quercetin-3-O--L-arabinofuranoside (19) and quercetin-3-O-β-D-galactoside (20), three are flavonoid aglycones, namely, quercetin (21), kaempherol (22), and 5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)chroman-4-one (23), and one sterol 3-epi-taraxerol (24). Preliminary screening by NR assay found that the crude extracts (except for Artocarpus heterophyllus x integer) showed growth inhibitory activities against human breast (MDA-MB-231 and MCF-7), lung (A549), and colon (HCT-116) cancer cell lines. Of the 24 pure compounds obtained, hispiloscine (2), fistulopsine A (4), fistulopsine B (5) and cudraflavone C (13) were found to show growth inhibitory activity against colon (HCT-116) and breast (MCF-7) cancer cells. Furthermore, 4 and 5 were found to dominantly arrest cells in G1 phase of the cell cycle without the induction of apoptosis. Cell cycle perturbation of these compounds was found to be reversible for HCT-116 cells at the onset of 72 hours. These results suggest that 4 and 5 have the potential to be further exploited for the development of new anti-cancer agents

Cudraflavone C induces tumor-specific apoptosis in colorectal cancer cells through inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumorselective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110?/p85? PI3K activity, followed by p120?, p110?/p85?, and p110?/p85? PI3K activities. The inhibition by Cud C on p110?/p85? PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NF?B activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M&gt;70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0&lt;e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Alkaloids and flavonoids from ficus, artocarpus and macaranga species : structure and anti-cancer activity

Plant natural products have played a pivotal role in the discovery and development of many new drugs for the treatment of various infectious diseases and cancers. The present study was therefore aimed at isolation and identification of novel bioactive compounds from selected plants collected in Malaysia with anti-cancer activity. Plant crude extracts were obtained using solvent extraction methods, while various chromatographic and spectroscopic methods were employed for compound isolation and structure elucidation. Evaluation of pure compounds and crude extracts for anti-cancer activity involved the use of Neutral Red assay (NR), acradine orange – ethidium bromide (AO/EB) staining and cell cycle analysis. Phytochemical investigations of five Malaysian plants, namely, Ficus hispida, F. fistulosa, F. schwarzii, Artocarpus heterophyllus x integer and Macaranga hypoleuca, with the focus on alkaloids and flavonoids, have resulted in the isolation of a total of 24 compounds, of which six are new. The leaf and stem-bark extracts of Ficus hispida yielded two new alkaloids, hispidacine (1) and hispiloscine (2), and a known alkaloid, 13a(S)-(+)-deoxypergularinine (3). Hispidacine represents the first example of an 8,4'-oxyneolignan incorporated an unusual 2-hydroxyethylamine moiety, while hispiloscine represents the first naturally occurring phenanthroindolizidine alkaloid with acetoxy substitution. The leaf and bark extracts of Ficus fistulosa provided two new septicine alkaloids, fistulopsines A and B (4 and 5), together with four known phenanthroindolizidine alkaloids, 13a(R)-(–)-3,6-didemethylisotylocrebrine (6), 13a(S)-(+)-tylocrebrine (7), 13a(S)-(+)-tylophorine (8) and 13a(S)-(+)-septicine (9), and one known non-alkaloid (vomifoliol, 10). The leaves of Ficus schwarzii gave two novel tri-nor-sesquilignan alkaloids, schwarzinicines A and B (11 and 12). The bark of Artocarpus heterophyllus x integer yielded five known compounds, of which four are prenylated flavonoids, namely, cudraflavone C (13), artocarpetin A (14), cycloheterophyllin (15) and artonin J (16), and one natural xanthone, lichexanthone (17). The leaves of Macaranga hypoleuca provided seven known compounds, of which three are flavonoid glycosides, namely, quercetin-3-O--L-arabinopyranoside (18), quercetin-3-O--L-arabinofuranoside (19) and quercetin-3-O-β-D-galactoside (20), three are flavonoid aglycones, namely, quercetin (21), kaempherol (22), and 5,7-dihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-en-1-yl)chroman-4-one (23), and one sterol 3-epi-taraxerol (24). Preliminary screening by NR assay found that the crude extracts (except for Artocarpus heterophyllus x integer) showed growth inhibitory activities against human breast (MDA-MB-231 and MCF-7), lung (A549), and colon (HCT-116) cancer cell lines. Of the 24 pure compounds obtained, hispiloscine (2), fistulopsine A (4), fistulopsine B (5) and cudraflavone C (13) were found to show growth inhibitory activity against colon (HCT-116) and breast (MCF-7) cancer cells. Furthermore, 4 and 5 were found to dominantly arrest cells in G1 phase of the cell cycle without the induction of apoptosis. Cell cycle perturbation of these compounds was found to be reversible for HCT-116 cells at the onset of 72 hours. These results suggest that 4 and 5 have the potential to be further exploited for the development of new anti-cancer agents

IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.

<p>IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.</p

Differential gene expression regulated by cudraflavone C in Caco-2 cells.

<p>(A) Heatmaps generated based on the genes regulated by Cud C. Caco-2 cells were exposed to 10 μM Cud C for 48 hours. GeneChip^® Human Transcriptome Array 2.0 (Affymetrix, USA) was applied. Gene expression changes that ≥2-fold were considered significant. Control 1 and Control 2 represent gene expression from cells treated with vehicle control (1% DMSO); Cud C 1 and Cud C 2 were gene expression from cells treated with Cud C (10 μM). (B) qPCR was used to validate the microarray data. Caco-2 cells were exposed to 10 μM Cud C for 12, 24, 48 or 72 hours The left and right panels present genes that are up and down-regulated respectively. All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” indicates the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

Cudraflavone C inhibits PI3K activity.

<p>The effect of negative control (1%DMSO), Cud C or LY-294002 (100 μM) on p110α/p85α, p110β/p85α, p110δ/p85α, and p120γ PI3K activity were quantified using the PI3K-Glo^™ Class I Profiling Kit. All data represents the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p