Scirpusin B isolated from Passiflora edulis Var. flavicarpa attenuates carbohydrate digestive enzymes, pathogenic bacteria and oral squamous cell carcinoma.

Passiflora edulis Var. flavicarpa (passion fruit) generates vast waste (60-70%) in the form of peel and seed after the juice extraction. The study aimed to isolate Scirpusin B (SB) from passion fruit (PF) seed waste collected from Northeast India and to analyse its anti-radical, antibacterial, anti-diabetic, and anti-oral cancer activities. Scirpusin B was isolated following hydro-alcoholic extraction, fractionation, and column chromatography. The isolated fraction was further identified through NMR and mass spectroscopy. SB exhibited significant antiradical activity against six standard antioxidant compounds, indicating its commercial application. SB inhibited α-amylase (IC50 Value: 76.38 ± 0.25 µg/mL) and α-glucosidase digestive enzymes (IC50 Value: 2.32 ± 0.04 µg/mL), signifying its antidiabetic properties. In addition, SB showed profound antibacterial activity against eight gram-positive and gram-negative bacteria reported for the first time. Furthermore, SB inhibited SAS and TTN oral cancer cell proliferation up to 95% and 83%, respectively. SB significantly inhibited colonies of SAS and TTn cells in the clonogenic assay, attributing to its anticancer properties. The PI-FACS assay confirmed the ability of SB (75 µM) to kill SAS and TTn cells by 40.26 and 44.3% in 72 h. The mechanism of SB inhibiting oral cancer cell proliferation was understood through western blot analysis, where SB significantly suppressed different cancer hallmark proteins, such as TNF-α, survivin, COX-2, cyclin D1, and VEGF-A. The present study suggests that SB isolated from PF seed can add noteworthy value to the waste biomass for various industrial and medical applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03876-6.

Extraction, phytochemical characterization and anti-cancer mechanism of Haritaki churna: An ayurvedic formulation.

Haritaki churna (HC), a single herb ayurvedic formulations is known to be prescribed for various gastro-intestinal disorders in Ayurveda. Haritaki churna aqueous extract (HCAE) has anti-cancer activity against different types of cancer cells with an IC50 in the range of 50-97 µg/ml. Bioavailability of Haritaki Churna is very high in digestive track and treatment of colorectal cancer cells HCT-116, DLD1, HT-29 with HCAE reduces its cellular viability with anti-cancer IC50 70µg/ml. HCAE consumption is safe for human as it didn't affect the cellular viability of primary human PBMCs or non-cancerogenic HEK-293 cells. Haritaki churna was found to be stable in biological gastric fluids and bioactive agents are not losing their anti-cancer activity under such harsh conditions. The HPLC Chromatogram of HCAE is giving 13 major peaks and 11 minor peaks. Exploiting LC-MS, IR and NMR spectroscopic techniques, a total of 13 compounds were identified from HCAE namely Shikimic acid, Chebulic acid, gallic acid, 5-hydroxymethylfurfural, Protocatechuic acid, 4-O-galloyl-shikimic Acid, 5-O-galloyl-shikimic Acid, Methylgallate, corilagin, 1, 2, 6, Tri-O-galloyl ß-D-glucose, chebulagic acid, chebulinic acid, and Ellagic acid. Reconstitution and subtraction of phytochemicals from the mixture indicate that Ellagic acid significantly contribute into anti-cancer effect of HCAE. Cancer cells treated with ellagic acid from HCAE were incapable of completing their cell-cycle and halted the cell-cycle at DNA synthesis S-Phase, as demonstrated by decreased cyclin A2 expression levels with increasing ellagic acid concentration. Halting of cells at S-phase causes induction of apoptosis in cancer cells. Cancer cells exhibiting DNA fragmentation, changes in expression of several apoptotic proteins such as Bcl2, cytochrome-c and formation of cleaved products of caspase 3 and PARP-1 suggests ellagic acid induces cell death via mitochondrial pathway of apoptosis.

Study into the chemical changes of tea leaf polyphenols during japanese black tea processing.

During the processing of black tea, enzymatic oxidation converts tea catechins into a complex mixture of oxidative products, including theaflavins, and thearubigins. In this study, ultra-performance liquid chromatography was carried out to analyze the tea extracts collected after each processing stage to clarify the quantitative changes in these compounds during black tea processing. It was found that the catechin content was significantly reduced during processing, and in particular between the later rolling and fermentation periods. In contrast, the variation in the theaflavin content was negatively correlated with the catechin content. Twenty-three compounds containing the known catechins dimers and flavonol glycosides were identified by liquid chromatography-mass spectrometry (LC-MS). In addition, 45 components whose contents increased significantly between the late rolling and late fermentation stages were extracted using principal components analysis by LC-MS. It was considered that due to the catechin-like units present in these components, they likely contribute to thearubigin production during black tea processing.

4'-Iodo-α-Pyrrolidinononanophenone Provokes Differentiated SH-SY5Y Cell Apoptosis Through Downregulating Nitric Oxide Production and Bcl-2 Expression.

Abuse of pyrrolidinophenone derivatives (PPs) is known to cause severe damage to the central nervous system due to their high lipophilicity. In this study, we compared sensitivity to toxicity elicited by 4'-iodo-α-pyrrolidinononanophenone (I-α-PNP), one of the most potent cytotoxic derivatives among PPs synthesized previously, between SH-SY5Y cells differentiated by all-trans-retinoic acid (ATRA) and the undifferentiated cells, and found that the differentiated cells are more sensitive to I-α-PNP toxicity than the undifferentiated cells. Treatment with I-α-PNP elicited some apoptotic alterations (Bax expression, loss of mitrochondrial membrane potential, and activation of caspases) in the differentiated cells, whose patterns were similar to those in the undifferentiated cells. I-α-PNP treatment resulted in no significant alteration in Bcl-2 expression in the undifferentiated cells, whereas it considerably downregulated the protein expression in the differentiated cells, suggesting that the high I-α-PNP sensitivity of the differentiated cells is mainly due to downregulation of Bcl-2 expression. I-α-PNP treatment decreased nitric oxide (NO) production and neuronal NOS (nNOS) expression in the differentiated cells, and the patterns of I-α-PNP-evoked alterations in phosphorylation of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression were almost the same as that in nNOS expression. Additionally, the addition of an NO donor restored the I-α-PNP-evoked alterations in expressions of Bcl-2, BDNF, and nNOS in the differentiated cells. These findings suggest that the downregulation of Bcl-2 expression by I-α-PNP in differentiated cells is attributed to the acceleration of two negative feedback loops (nNOS/NO/CREB loop and CREB/BDNF loop) triggered by decreased NO production.

Lowering of brain endothelial cell barrier function by exposure to 4'-iodo-α-pyrrolidinononanophenone.

Overuse of pyrrolidinophenones (PPs) is known to cause damage to vascular and central nervous systems, but little is known about its effect on brain endothelial barrier function. In this study, we found that exposure to 4'-iodo-α-pyrrolidinononanophenone (I-α-PNP), one of the most potently cytotoxic PPs, at sublethal concentrations decreases trans-endothelial electrical resistance and increases paracellular permeability across a monolayer of human brain microvascular endothelial cells. Treatment with I-α-PNP also elevated the production of superoxide anion. Furthermore, the treatment reduced the expression and plasma membrane localization of a tight junction protein claudin-5 (CLDN5), which was almost restored by pretreatment with an antioxidant N-acetyl-l-cysteine. These results indicate that I-α-PNP treatment may down-regulate the plasma membrane-localized CLDN5 by elevating the production of reactive oxygen species (ROS). The treatment with I-α-PNP increased the nuclear translocation of Forkhead box protein O1 (FoxO1), an oxidative stress-responsive transcription factor, and pretreating with a FoxO1 inhibitor ameliorated the decrease in CLDN5 mRNA. In addition, I-α-PNP treatment up-regulated the expression and secretion of matrix metalloproteinase-2 (MMP2) and MMP9, and the addition of an MMP inhibitor reversed the degradation of CLDN5 by I-α-PNP. Moreover, I-α-PNP treatment facilitated the activation of 26S proteasome-based proteolytic activity and pretreatment with an inhibitor of 26S proteasome, but not autophagy, suppressed the CLDN5 degradation by I-α-PNP. Accordingly, it is suggested that the down-regulation of CLDN5 by exposure to I-α-PNP is ascribable to suppression of the gene transcription due to FoxO1 nuclear translocation through ROS production and to acceleration both of the MMPs (MMP2 and MMP9)- and 26S proteasome-based proteolysis.

Apoptotic mechanism in human brain microvascular endothelial cells triggered by 4'-iodo-α-pyrrolidinononanophenone: Contribution of decrease in antioxidant properties.

In this study, we newly synthesized four α-pyrrolidinononanophenone (α-PNP) derivatives [4'-halogenated derivatives and α-pyrrolidinodecanophenone (α-PDP)], and then performed the structure-cytotoxicity relationship analyses. The results showed the rank order for the cytotoxic effects, α-PNP < α-PDP < 4'-fluoro-α-PNP < 4'-chrolo-α-PNP < 4'-bromo-α-PNP < 4'-iodo-α-PNP (I-α-PNP), and suggest that cytotoxicities of 4'-halogenated derivatives were more intensive than that of elongation of the hydrocarbon chain (α-PDP). We also surveyed the apoptotic mechanism of I-α-PNP in brain microvascular endothelial (HBME) cells that are utilized as the in vitro model of the blood-brain barrier. HBME cell treatment with I-α-PNP facilitated the apoptotic events (caspase-3 activation, externalization of phosphatidylserine, and DNA fragmentation), which were almost completely abolished by pretreating with antioxidants. In addition, the immunofluorescent staining revealed the enhanced production of hydroxyl radical in mitochondria by the I-α-PNP treatment, inferring that the I-α-PNP treatment triggers the apoptotic mechanism dependent on the enhanced ROS production in mitochondria. The treatment with I-α-PNP increased the production of cytotoxic aldehyde 4-hydroxy-2-nonenal and decreased the amount of reduced glutathione. Additionally, the treatment decreased the 26S proteasome-based proteolytic activities and aggresome formation. These results suggest that decrease in the antioxidant properties is also ascribable to HBME cell apoptosis elicited by I-α-PNP.

Expression GWAS of PGIP1 Identifies STOP1-Dependent and STOP1-Independent Regulation of PGIP1 in Aluminum Stress Signaling in Arabidopsis.

To elucidate the unknown regulatory mechanisms involved in aluminum (Al)-induced expression of POLYGALACTURONASE-INHIBITING PROTEIN 1 (PGIP1), which is one of the downstream genes of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) regulating Al-tolerance genes, we conducted a genome-wide association analysis of gene expression levels (eGWAS) of PGIP1 in the shoots under Al stress using 83 Arabidopsis thaliana accessions. The eGWAS, conducted through a mixed linear model, revealed 17 suggestive SNPs across the genome having the association with the expression level variation in PGIP1. The GWAS-detected SNPs were directly located inside transcription factors and other genes involved in stress signaling, which were expressed in response to Al. These candidate genes carried different expression level and amino acid polymorphisms. Among them, three genes encoding NAC domain-containing protein 27 (NAC027), TRX superfamily protein, and R-R-type MYB protein were associated with the suppression of PGIP1 expression in their mutants, and accordingly, the system affected Al tolerance. We also found the involvement of Al-induced endogenous nitric oxide (NO) signaling, which induces NAC027 and R-R-type MYB genes to regulate PGIP1 expression. In this study, we provide genetic evidence that STOP1-independent NO signaling pathway and STOP1-dependent regulation in phosphoinositide (PI) signaling pathway are involved in the regulation of PGIP1 expression under Al stress.

Identification of peptides in blood following oral administration of ß-conglycinin to Wistar rats.

In this study, ß-conglycinin (100 mg/kg) was orally administered to Wistar rats in order to identify peptides that may be derived from the protein in the blood. Plasma samples taken from the tail vein up to 8 h after administration were analyzed by matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography-time-of-flight (LC-TOF) mass spectrometry (MS). In total, 126 signals were detected by MALDI-MS. Among the signals, nine oligopeptides (SEL, KGPL, SILGA, DSEL, GDANI, SYFV, CLQSC, GEQPRPF, and LVINEGDA) were successfully identified as ß-conglycinin-derived peptides by LC-TOF/MS at a plasma concentration of 0.75-756 pmol/mL. The results demonstrated that ß-conglycinin could be the dietary source protein for the oligopeptides produced prior to entering the circulating bloodstream of rats.

Identification and Characterization of a-Glucosidase Inhibition Flavonol Glycosides from Jack Bean (Canavalia ensiformis (L.) DC.

Although the intake of jack bean (Canavalia ensiformis (L.) DC.), an underutilized tropical legume, can potentially decrease the risk of several chronic diseases, not much effort has been directed at profiling the polyphenolics contained therein. Hence, this work aimed to identify and quantify the dominant jack bean polyphenolics, which are believed to have antioxidant and other bioactivities. Four major compounds were detected and identified as kaempferol glycosides with three or four glycoside units. Their structures were established based on UV-visible, 1d, 2D NMR, and HR-ESI-MS analyses. Specifically, kaempferol 3-O-a-l-rhamnopyranosyl (1®6)- b-d-glucopyranosyl (1®2)-b-d-galactopyranosyl-7-O-[3-O-o-anisoyl]-a-l-rhamnopyranoside was detected for the first time, while the other three compounds have already been described in plants other than jack bean. This new compound was found to have a higher a-glucosidase inhibition activity compared to acarbose.

Redox balance, metabolic fingerprint and physiological characterization in contrasting North East Indian rice for Aluminum stress tolerance.

Aluminum (Al) toxicity is a serious problem for rice crop productivity in acidic soils worldwide. The present work was conducted to look out for the alteration in ROS homeostasis; metabolic fingerprint; and morphology in two contrasting Indica rice cultivars of North East India (NE India) to Al toxicity. Al stress led to excess accumulation of ROS (H2O2 and O2-), and this in turn induced ROS mediated cellular damage, as indicated by lipid peroxidation both qualitatively as well as quantitatively. This excessive ROS production also led to significant reduction in chlorophyll content and stomatal conductance. This was followed by the loss of photosynthetic efficiency as detected by chlorophyll fluorescence. This excessive damage due to ROS prompted us to check the anti-oxidative machinery. Antioxidants, especially enzymes (SOD, APX, POX, GR, CAT, DHAR, MDHAR) are very important players in maintenance of ROS homeostasis. In tolerant variety Disang, higher activity of these enzymes and vice versa in sensitive variety, was observed in response to Al treatment. The non-enzymatic antioxidants (proline, ascorbate and glutathione) also showed similar trend. Though the tolerant variety showed strong anti-oxidative machinery, it was unable to completely nullify the stress experienced by the seedlings. Organic acids are also important players in detoxification of Al stress through efflux in the rhizosphere. In tolerant genotype, citrate exudate was found to be more when compared to sensitive genotypes on exposure to high dose of Al. This is supported by higher abundance of FRDL4, a citrate transporter. Not only FRDL4, other stakeholders for Al stress response like ART1 and ALS1 depicted prominent transcript abundance in the tolerant variety. In conclusion, through this study detailed physiological and metabolic characterisation of two contrasting Indica rice varieties Disang and Joymati, native to NE India for Al tolerance was performed for the very first time.

Molecular Mechanism by Which Tea Catechins Decrease the Micellar Solubility of Cholesterol.

Tea polyphenols lower the levels of cholesterol in the blood by decreasing the cholesterol micellar solubility. To clarify this mechanism, the interactions between taurocholic acid and (-)-epigallocatechin gallate (EGCg) and its derivatives were investigated. 13C NMR studies revealed remarkable chemical-shift changes for the carbonyl carbon atom and the 1″- and 4″-positions in the galloyl moiety. Furthermore, 1H NMR studies using (-)-EGCg derivatives showed that the number of hydroxyl groups on the B ring did not affect these interactions, whereas the carbonyl carbon atom and the aromatic ring of the galloyl moiety had remarkable effects. The configuration at the 2- and 3-positions of the catechin also influenced these interactions, with the trans-configuration resulting in stronger inhibition activity than the cis-configuration. Additionally, a 1:1 component ratio for the catechin-taurocholic acid complex was determined by electrospray ionization-mass spectrometry. These molecular mechanisms contribute to the development of cholesterol-absorption inhibitors.

Structural characterization of proanthocyanidins from adzuki seed coat.

Adzuki bean seed coats contain a variety of pigments with potential bioactivity, including polyphenols, such as proanthocyanidin. To clarify the structure of the pigments from adzuki bean seed coats, the MeOH extract of the seed coats and the extraction residue were subjected to various instrumental analyses and degradation by thiolysis. The MeOH extract was found to contain two types of polymeric red pigments. One of these pigments was assumed to be a simple proanthocyanidin, whereas the other was a complicated polyphenol produced by several modifications and conversions. Direct thiolysis of adzuki seed coat yielded three products as extension subunits. From their chemical structures, a glyoxylic acid-bridged catechin skeleton was suggested as a partial structure of adzuki pigments.

Structure Determination of Novel Oxidation Products from Epicatechin: Thearubigin-Like Molecules.

Following the oxidation of epicatechin (EC), three novel compounds and two known compounds were isolated. The chemical structures of these oxidation products were determined by mass spectrometry (MS) and various nuclear magnetic resonance (NMR) experiments, and the A-ring-B-ring linkage that is characteristic of catechin was found in each molecule. Three compounds showed similar ultraviolet-visible (UV-Vis) spectra to EC, whereas two compounds showed different spectral absorption in the region between 300 and 500 nm. A similar spectrum was obtained for the thearubigin fraction prepared from a black tea infusion. This result suggests that the condensation reaction between the A-ring and B-ring is more important than reaction between B-rings for thearubigin formation.

Interaction between Tea Polyphenols and Bile Acid Inhibits Micellar Cholesterol Solubility.

The molecular mechanism by which tea polyphenols decrease the micellar solubility of cholesterol is not completely clear. To clarify this mechanism, this study investigated the interaction between tea polyphenols (catechins and oolongtheanins) and cholesterol micelles. A nuclear magnetic resonance (NMR) study was performed on a micellar solution containing taurocholic acid and epigallocatechin gallate (EGCg), and high-performance liquid chromatography (HPLC) analysis was carried out on the precipitate and the supernatant that formed when EGCg was added to a cholesterol-micelle solution. The data indicated a regiospecific interaction of EGCg with taurocholic acid. Therefore, the ability of EGCg to lower the solubility of phosphatidylcholine (PC) and cholesterol in micellar solutions can be attributed to their elimination from the micelles due to interaction between taurocholic acids and EGCg.

Synthesis of oolongtheanins and their inhibitory activity on micellar cholesterol solubility in vitro.

The synthesis of oolongtheanins (1a-d) was accomplished from EGC and/or EGCg in three steps. Oolongtheanin-3'-O-gallate (1b) showed more potent inhibitory activity on micellar cholesterol solubility than did EGCg.

Characterization of dihydro-A2PE: an intermediate in the A2E biosynthetic pathway.

Bisretinoid lipofuscin pigments that accumulate in retinal pigment epithelial cells are implicated in the etiology of several forms of macular degeneration, including juvenile onset Stargardt disease, Best vitelliform macular degeneration, and age-related macular degeneration. One of these compounds, A2E, is generated by phosphate hydrolysis of a phosphatidyl-pyridinium bisretinoid (A2PE) that forms within photoreceptor outer segments. Here, we demonstrate that the formation of the aromatic pyridinium ring of A2PE follows from the oxidation of a dihydropyridinium intermediate. Time-dependent density functional theory calculation, based on the structure of dihydro-A2E, produced a simulated UV-visible absorbance spectrum characterized by maxima of 494 and 344 nm. Subsequently, a compound exhibiting similar UV-visible absorbance maxima (lambdamax 490 and 330 nm) was identified in the A2E biomimetic reaction mixture. By liquid chromatography-mass spectrometry (LC-MS) this bischromophore had the expected mass of the dihydro-pyridinium bisretinoid. The compound also exhibited the behavior of a biosynthetic intermediate since it formed in advance of the final product A2E and was consumed as A2E accumulated. Moreover, under deoxygenated conditions, conversion to the aromatic pyridinium bisretinoid was inhibited. Taken together, these findings indicate that A2E biosynthesis involves the oxidation of a dihydropyridinium intermediate dihydro-A2PE. An understanding of the biosynthetic pathways of retinal pigment epithelial lipofuscin pigments is critical to the development of therapies for macular degeneration that are based on limiting the formation of these damaging compounds.

Oligomerization of N-tosylindole with aluminum chloride.

The reactivity of N-tosylindole (4) in the presence of aluminum chloride was studied, and two types of oligomerization of 4 were observed. One type was condensation between both pyrrole parts (dimers 5 and 6 and trimer 7) and the other was between a pyrrole part and a benzene part of each indole nucleus (dimers 8 and 9).