Hijacking of N-fixing legume albumin-1 genes enables the cyclization and stabilization of defense peptides.

The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.

Antiobesity and antidyslipidemic properties of Clitoria ternatea petals aqueous extract against rats induced by high-fat diet.

Background/aim: Obesity is a chronic metabolic disease involving dysregulation of fat metabolism that affects 13% of the world's population. Obesity has been linked to dyslipidemia with a lot of complication, including stroke, chronic kidney disease, fatty liver disease, and so on. One of the natural resources that have several potential effects including anticholesterol, antiobesity, and antidyslipidemia is the butterfly pea (Clitoria ternatea/CT). CT's petal has been found to contain high levels of anthocyanins and tannins that can inhibit the biosynthesis of cholesterol and lipid. This study aims to investigate the antiobesity and antidyslipidemic effects of Clitoria ternatea extract (CTE). Materials and methods: The CTE was obtained through the aqueous extract method and then was investigated using spectrophotometry to determine anthocyanin and tannin content. The effect of CTE against a high-fat diet (HFD)-induced rat model was measured by weight and obesity index, lipid profile (total cholesterol (TC), triglycerides (TG), and HDL-C), and histopathology analysis. Results: CTE showed total anthocyanin and tannin content of 78.0943 mg/100 g and 1424.90 mg/100 g, respectively. The data analysis also showed significantly different within groups (p < 0,05), especially between HFD and HFD + CT750 groups on the cholesterol (MD 111.12 mg/dL; 95% CI (99.57 to 122.67); p < 0.001), LDL (MD; 76.38 mg/dL; 95% CI (56.77 to 96.00); p < 0.001), VLDL (MD 0.37 mg/dL; 95% CI (0.18 to 0.57); p < 0.001), body weight (MD: 56.20 g; 95% CI (13.89 to 98.51); p = 0.012); and thickening of tunica layer in the thoracic aorta (MD 22.76 µm; 95% CI (20.11 to 24.4); p < 0.001). Conclusion: This study shows that Clitoria ternatea petals aqueous extract promotes amelioration of the lipid profile, body weight, and tunica thickness in rats with the high-fat diet.

Potato oxidized hydroxypropyl starch/pectin-based indicator film with Clitoria ternatea anthocyanin and silver nanoparticles for monitoring chilled beef freshness.

Potato oxidized hydroxypropyl starch (POHS)/pectin (P) functional and smart beef freshness indicator films were prepared using butterfly pea (Clitoria ternatea) anthocyanin (BA) and silver nanoparticles (AgNPs). BA exhibited significant pH-responsive color changes. BA and AgNPs were evenly distributed within a polymer matrix to create a compatible film with POHS/P. The films containing BA and AgNPs had good UV resistance and maintained strong mechanical strength, barrier properties, and color stability. The color of the indicator film changed from purple to green when exposed to ammonia, with the 1 % POHS/P/BA/AgNPs film showing the most sensitive response. The films also demonstrated strong antibacterial and antioxidant properties. The freshness of beef was monitored using 1 % POHS/P/BA/AgNPs films and was identified as sub-fresh and spoiled on days 4 and 7, respectively. The relationship between the color change of the indicator label and the freshness of chilled beef was established: purple for fresh meat, blue for less fresh meat, and green for spoiled meat. Thus, the new POHS/P/BA/AgNPs film can serve as a smart packaging material to indicate food freshness and extend shelf life. These results suggest that POHS/P/BA/AgNPs films have significant potential as an active and smart food packaging material.

Effects of clitorienolactones from Clitoria ternatea root on calcium channel mediating hippocampal long-term potentiation in rats induced chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a common mechanism of acute brain injury due to impairment of blood flow to the brain. Moreover, a prolonged lack of oxygen supply may result in cerebral infarction or global ischemia, which subsequently causes long-term memory impairment. Research on using Clitoria ternatea root extract for treating long-term memory has been studied extensively. However, the bioactive compound contributing to its neuroprotective effects remains uncertain. In the present study, we investigate the effects of clitorienolactone A (CLA) and B (CLB) from the roots of Clitoria ternatea extract on hippocampal neuroplasticity in rats induced by CCH. CLA and CLB were obtained using column chromatography. The rat model of CCH was induced using two-vessel occlusion surgery (2VO). The 2VO rats were given 10 mg/kg of CLA and CLB orally, followed by hippocampal neuroplasticity recording using in vivo electrophysiological. Rats received CLA and CLB (10 mg/kg) significantly reversed the impairment of long-term potentiation following 2VO surgery. Furthermore, we investigate the effect of CLA and CLB on the calcium channel using the calcium imaging technique. During hypoxia, CLA and CLB sustain the increase in intracellular calcium levels. We next predict the binding interactions of CLA and CLB against NMDA receptors containing GluN2A and GluN2B subunits using in silico molecular docking. Our result found that both CLA and CLB exhibited lower binding affinity against GluN2A and GluN2B subunits. Our findings demonstrated that bioactive compounds from Clitoria ternatea improved long-term memory deficits in the chronic cerebral hypoperfusion rat model via calcium uptake. Hence, CLA and CLB could be potential therapeutic tools for treating cognitive dysfunction.

Development and optimization of Clitoria teratea synthesized silver nanoparticles and its application to nanogel systems for wound healing.

OBJECTIVE: The present research deals with sequential optimization strategy based on Central Composite Design to optimize the process variables for efficient production of Clitoria teratea (CLT) synthesized silver nanoparticles (AgNPs) using biological synthesis. METHODS: Two substantial factors influencing the dependent variables viz UV-visible absorbance, particle size, zeta potential and polydispersity index (PDI) were identified as NaOH concentration, RH concentration, temperature as independent variables. In-vitro and ex-vivo studies of prepared CLT-AgNPs gel and marketed gel were carried out using dialysis membrane and egg membrane, respectively. In addition, antimicrobial study was also performed on the bacterial strains. RESULTS: The particles size (114 nm), PDI (0.45), and zeta potential (-29.5 mV) of optimized formulation were found, respectively. In-vitro profile of AgNPs from prepared CLT-AgNPs gel was noted (95.6%) in 8 h. It was found that the prepared CLT-AgNPs gel stimulates fibroblast and agranulocytosis development resulting better and timely wound healing. CONCLUSIONS: The prepared CLT-AgNPs gel can be as a potential substitute in the management and treatment of acute and chronic wounds.

Starch-PVA based films with Clitoria ternatea flower extract: Characterization, phenolic compounds release and compostability.

The kinetic release of phenolic compounds from biodegradable films with Clitoria ternatea flower extract (ECT) in different food-simulant fluids and compostability were evaluated for the first time. This work aimed to incorporate ECT in starch-PVA-based film formulations, and the antioxidant capacity, total phenolic compounds, opacity, color, mechanical properties, compostability, and polyphenol release in different fluid simulants were determined. The results obtained showed that antioxidant activity and the total phenolic compounds were ECT dose dependent. Due to its antioxidant properties, ECT interfered with the film's composting process, reaching an average weight loss of 70 %. Additionally, the addition of ECT interfered with the mechanical properties, reducing the tensile strength, probably due to the plasticizer effect. The type of simulating fluid influenced the release of polyphenols from the films, and the presence of water favored the release because it hydrated and swelled the starch-PVA matrix, facilitating diffusion. The classic zero- and first-order models were the most effective in describing the release kinetics of polyphenols from the films. The results of this study demonstrate that the antioxidant potential and the release of polyphenols from starch-PVA-based films in different simulated fluids allow their application in active packaging, making them a sustainable alternative for food preservation.

Bio-enhanced fraction from Clitoria ternatea root extract ameliorates cognitive functions and in vivo hippocampal neuroplasticity in chronic cerebral hypoperfusion rat model.

Research employing a bio-enhanced fraction of Clitoria ternatea (CT) to treat cognitive decline in the animal model has not yet been found. This study aimed to determine the neuroprotective effect of CT root bioactive fraction (CTRF) in chronic cerebral hypoperfusion (CCH) rat model. CTRF and its major compound, clitorienolactones A (CLA), were obtained using column chromatography. A validated HPLC-UV method was employed for the standardization of CTRF. CCH rats were given orally either vehicle or fraction (10, 20 and 40 mg/kg). Behavioural and hippocampal neuroplasticity studies were conducted following 4 weeks post-surgery. The brain hippocampus was extracted for proteins and neurotransmitters analyses. HPLC analysis showed that CTRF contained 25% (w/w) of CLA. All tested doses of CTRF and CLA (10 mg/kg) significantly restored cognitive deficits and reversed the inhibition of neuroplasticity by CCH. However, only CTRF (40 mg/kg) and CLA (10 mg/kg) significantly reversed the elevation of amyloid-beta plaque. Subsequently, treatment with CTRF (40 mg/kg) and CLA (10 mg/kg) alleviated the downregulation of molecular synaptic signalling proteins levels caused by CCH. The neurotransmitters level was restored following treatment of CTRF and CLA. Our finding suggested that CTRF improves memory and neuroplasticity in CCH rats which was mainly contributed by CLA.

Effect-Directed Profiling of Akebia quinata and Clitoria ternatea via High-Performance Thin-Layer Chromatography, Planar Assays and High-Resolution Mass Spectrometry.

Two herbal plants, Akebia quinata D. leaf/fruit and Clitoria ternatea L. flower, well-known in traditional medicine systems, were investigated using a non-target effect-directed profiling. High-performance thin-layer chromatography (HPTLC) was combined with 11 different effect-directed assays, including two multiplex bioassays, for assessing their bioactivity. Individual active zones were heart-cut eluted for separation via an orthogonal high-performance liquid chromatography column to heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) for tentative assignment of molecular formulas according to literature data. The obtained effect-directed profiles provided information on 2,2-diphenyl-1-picrylhydrazyl scavenging, antibacterial (against Bacillus subtilis and Aliivibrio fischeri), enzyme inhibition (tyrosinase, α-amylase, ß-glucuronidase, butyrylcholinesterase, and acetylcholinesterase), endocrine (agonists and antagonists), and genotoxic (SOS-Umu-C) activities. The main bioactive compound zones in A. quinata leaf were tentatively assigned to be syringin, vanilloloside, salidroside, α-hederin, cuneataside E, botulin, and oleanolic acid, while salidroside and quinatic acids were tentatively identified in the fruit. Taraxerol, kaempherol-3-rutinoside, kaempferol-3-glucoside, quercetin-3-rutinoside, and octadecenoic acid were tentatively found in the C. ternatea flower. This straightforward hyphenated technique made it possible to correlate the biological properties of the herbs with possible compounds. The meaningful bioactivity profiles contribute to a better understanding of the effects and to more efficient food control and food safety.

Theoretical and experimental study of the diastereoisomers (2S) and (2R)-naringenin-6-C-ß-D-glucopyranoside obtained from Clitoria guianensis.

In this work the diastereoisomers (2S) and (2R)-naringenin-6-C-ß-D-glucopyroside, isolated for the first time from Clitoria guianensis, were studied using the density functional theory. The frontier molecular orbitals and structural properties showed that the diastereoisomers exhibit the same energy gap 166.61 kcal mol-1 and structural properties different, where in the S diastereoisomer, the bond length between the chiral carbon and the phenolic group is greater (difference of 0.0126 Å). The HPLC data showed that the retention time of the S-diastereoisomer (16.7 min) is shorter than that of R, suggesting that the S compound is more polar than R. The HPLC results corroborates with the molecular electrostatic potential which showed that in the S configuration, the electronegative density was more intense overall, particularly in the glucose molecule. The reactivity indices showed that the diastereoisomers are good electrophiles and reactive species. Finally, the absolute configuration of the diastereoisomers were determined using electronic circular dichroism (ECD) spectroscopy and the theoretical spectra were similar to the experimental. METHODS : All calculations of Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TDDFT) were performed using the program Gaussian 09 and the structures of the diastereoisomers were generated and analyzed using the GaussView program. The optimization and vibrational frequency calculations were performed using the functional CAM-B3LYP and 6-311 + + G(2d,2p) basis set. Conformational searches were performed for R configuration, by molecular mechanics using the MM + , MMFF, and OPLS05 force fields; the entire molecular mechanics simulation was performed using the Maestro/MacroModel software. The calculations for the simulations of the ECD spectra were performed for the eight lowest energy conformers obtained in the geometric optimization step, and the TDDFT at the CAM-B3LYP/6-311 + + G(2d,2p) theory level used. The effects of methanol and chloroform were calculated using the SMD implicit solvent model.

The influence of aqueous butterfly pea (Clitoria ternatea) flower extract on active and intelligent properties of furcellaran Double-Layered films - in vitro and in vivo research.

Innovative, intelligent and active double-layer films, based on furcellaran and with the addition of gelatin hydrolysates, have been obtained for the first time. An aqueous extract of clitoria flower in 3 concentrations was included in the 1st FUR layer. The films demonstrated strong antimicrobial effects, but did not exhibit fungicidal properties. The antioxidant properties of the films were within the range of 2.27-3.92 mM Trolox/mg (FRAP method) and 36.67-61.24 % (DPPH method). The films were used as active packaging materials in salmon fillets, which were stored for a period of 12 days in 4 °C. Analysis concerning microbiological properties of the stored fillets showed the possibility of extending their shelf-life by 6 days. Lipid oxidation, determined by TBARS has delayed. The obtained films are a promising material for the packaging industry. This is an important aspect within the context of global food waste and also the need to reduce synthetic materials.

Multivariate Analysis of Butterfly Pea (Clitoria ternatea L.) Genotypes With Potentially Healthy Nutraceuticals and Uses.

Butterfly pea (Clitoria ternatea L.) is a legume used as tea, forage, ornamental, salad, and medicinal plant. The flowers range from white to dark purple with little known about the variation for seed and flower color in the United States Department of Agriculture, Agricultural Research Service, Plant Genetic Resources Conservation Unit germplasm collection. Therefore, 26 butterfly pea accessions were analyzed using a principal component analysis (PCA) and average linkage cluster analysis (ALCA). These butterfly pea genotypes ranged from 56% to 99% for viabilities, 2.57 to 5.88 g for 100 seed weight, 34.07 to 226.26 g for total seed weight, and 1,326 to 3,874 for total seed numbers. PCA accounted for 40%, 57%, 70%, 79%, and 86% of the variation using principal components (PCs) 1 through 5, respectively. PC1 was most correlated with 100 and total seed weight, while PC2 correlated with blue, white, and purple flowers. PC3 correlated mostly with germination, purple flowers, and total seed weight. PCs 4 and 5 primarily correlated with blue and purple flowers, respectively. Several significant correlations were also observed. ALCA grouped the 26 butterfly pea genotypes into four distinct seed number-producing clusters. Clusters 1 to 4 represent the lowest to highest seed numbers produced by the butterfly pea genotypes. Several potential health benefits from butterfly pea flowers, leaves, seeds, and roots for human use were identified from the literature.

Chemical constituents and in vivo preliminary evaluation of the toxicological activity of Ouratea spectabilis (OCHNACEAE) and Clitoria guianensis (Fabaceae) leaves

Clitoria guianensis and Ouratea spectabilis, found in the Brazilian Cerrado, are used in folk medicine, despite the few chemical and biological studies reported in the literature. The present study aims to investigate the toxicity and effect of extracts from both species on the microcrustacean Artemia salina, and to determine the chemical composition of the hexane extract of O. spectabilis leaves and the EtOAc fraction of C. guianensis leaves. Kaempferitrin, a flavonoid isolated from of the EtOAc fraction of C. guianensis leaves, was identified by chemical analysis. Analysis of the hexane extract of O. spectabilis leaves using gas chromatography-mass spectrometry (GC-MS) suggested the presence of twenty-five known substances. The Hex, EtOAc, and EtOH crude extracts of C. guianensis leaves exhibited high and moderate toxicity against Artemia salina, with median lethal dose values (LD50) of 43.7, 25.4, and 233.4 mg.L−1, respectively. The acetone extract of O. spectabilis leaves showed moderate toxicity against Artemia salina with an LD50 value of 115.13 mg.L−1.

Clitoria ternatea blue petal extract protects against obesity, oxidative stress, and inflammation induced by a high-fat, high-fructose diet in C57BL/6 mice.

This study examined the chemical compounds and bioactivity of the aqueous extract of Clitoria ternatea blue petals and investigated its beneficial effects in vivo on a mouse model of obesity and metabolic syndrome. The extract mainly contained flavonoids, and nine compounds were tentatively identified. Male C57BL/6J mice were either fed a standard diet (SD) or a high-fat, high-fructose diet (HFFD) for 16 weeks, and HFFD-fed animals were treated with 0.25%, 0.5%, and 2% (w/w) of the aqueous extract in drinking water. The aqueous extract ameliorated oxidative stress and inflammation mediators. Furthermore, the aqueous extract reduced plasma leptin, free fatty acid, low-density lipoprotein cholesterol levels and hepatic malondialdehyde content. The aqueous extract significantly reduced total cholesterol and ameliorated insulin resistance. The results demonstrated that the aqueous extract of C. ternatea blue petals contains bioactive anthocyanins that exert substantial hypolipidemic and anti-inflammatory effects by promoting reverse cholesterol transport in HFFD-fed mice.

Phytochemical Properties and In Vitro Biological Activities of Phenolic Compounds from Flower of Clitoria ternatea L.

Phenolic compounds from the flower of Clitoria ternatea L. (PCFCTL) were extracted using a high-speed shearing extraction technique and purified by AB-8 macroporous resins, and the phytochemical composition of the purified phenolic compounds from the flower of Clitoria ternatea L. (PPCFCTL) was then analyzed. Subsequently, its bioactivities including antioxidant properties, enzyme inhibitory activities, and antiproliferative activities against several tumor cell lines were evaluated. Results indicated that the contents of total phenolics, flavonoids, flavonols, flavanols, and phenolic acids in PPCFCTL were increased by 3.29, 4.11, 2.74, 2.43, and 2.96-fold, respectively, compared with those before being purified by AB-8 macroporous resins. The results showed PPCFCTL have significant antioxidant ability (measured by reducing power, RP, and ferric reducing antioxidant power method, FRAP) and good DPPH, ABTS+, and superoxide anion radical scavenging activities. They can also significantly inhibit lipase, α-amylase, and α-glucosidase. In addition, morphological changes of HeLa, HepG2, and NCI-H460 tumor cells demonstrated the superior antitumor performance of PPCFCTL. However, the acetylcholinesterase inhibitory activity was relatively weak. These findings suggest that PPCFCTL have important potential as natural antioxidant, antilipidemic, anti-glycemic and antineoplastic agents in health-promoting foods.

Antioxidant, cytotoxic, and antibacterial activities of Clitoria ternatea flower extracts and anthocyanin-rich fraction.

Clitoria ternatea flower is a traditional medicinal herb that has been used as a natural food colourant. As there are limited studies on investigating the bioactivities of the anthocyanin-rich fraction of Clitoria ternatea flower, this study aimed to determine an efficient column chromatography method to obtain the anthocyanin-rich fraction from this flower and characterise its composition, antioxidant, antibacterial, and cytotoxic activities. Amberlite XAD-16 column chromatography was more efficient in enriching the total anthocyanin content (TAC) of the fraction with the highest TAC to total phenolic content (TPC) ratio of 1:6 than that using C18-OPN. A total of 11 ternatin anthocyanins were characterised in the anthocyanin-rich fraction by LC-MS analysis. The antioxidant activity of the anthocyanin-rich fraction was more potent in the chemical-based assay with an IC50 value of 0.86 ± 0.07 mg/mL using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay than cellular antioxidant assay using RAW 264.7 macrophages. In vitro cytotoxicity assay using human embryonic kidney HEK-293 cell line showed the anthocyanin-rich fraction to be more toxic than the crude extracts. The anthocyanin-rich fraction had more potent antibacterial activity than the crude extracts against Bacillus cereus, Bacillus subtilis, and Escherichia coli. The anthocyanin-rich fraction of C. ternatea has the potential to be used and developed as a functional food ingredient or nutraceutical agent.

Active-intelligent and biodegradable sodium alginate films loaded with Clitoria ternatea anthocyanin-rich extract to preserve and monitor food freshness.

The aim of this study was to develop and characterize sodium alginate films loaded with 10-40 % Clitoria ternatea extract (CTE) and apply to monitoring the quality of milk, pork and shrimp. Films loaded with CTE showed high light barrier capacity and improved tensile strength by 3.8 times over control films. The incorporation of CTE in alginate films improved the thermal stability of the materials due to intermolecular interactions and crosslinking of polymeric networks. The addition of 40 % of CTE generated films with antibacterial action against E. coli. The alginate films showed biodegradable characteristics in soil and beach sand in 15 days. The food simulant test revealed that the loaded films show good compatibility with aqueous and acidic foods due to the release of higher levels of polyphenols and anthocyanins. The films showed great colorimetric potential due to their ability to change color at different pH (pink-green), ammonia gas (blue-green) and sterilization process (blue-yellow). When the film loaded with 40 % CTE (F40) was applied to monitor the freshness of milk and meat products (shrimp and pork), its blue color changed to purple and green, respectively. Therefore, the F40 has great potential to be used as a biodegradable indicator of freshness.

Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

A vicilin-like protein extracted from Clitoria fairchildiana cotyledons was toxic to Callosobruchus maculatus (Coleoptera: Chrysomelidae).

Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.

Study on activation mechanism and cleavage sites of recombinant butelase-1 zymogen derived from Clitoria ternatea.

Asparagine endopeptidases (AEPs) were synthesized as a zymogen and were known to undergo pH-dependent autoproteolytic activation using their endopeptidase activity. Butelase-1, one of the few AEPs with ligation activity, can also be synthesized as a zymogen and activated at acidic pH in vitro, but the detailed activation process and potential activation sites of its zymogen are not fully understood. In this study, recombinant butelase-1 exhibited high ligation activity and ineffective endopeptidase activity, and its activities were strictly pH-dependent. The endopeptidase activity caused the activation of butelase-1 zymogen at acidic pH, which was autocatalytic, required sequential removal of C- and N-terminal pro-peptides, and was a bimolecular reaction. The pro-peptides were critical to the stability of butelase-1. Once the pro-peptides left the active domain, butelase-1 was quickly inactivated at pH 7.0. Based on the LC-MS/MS sequencing of activation products, Asp319 and Asn322 were identified as potential C-terminal pro-region hydrolysis sites of the butelase-1 zymogen, which was validated by site-directed mutagenesis. Our results provided a reasonable explanation for the self-activation of butelase-1 zymogen in vitro and provided supplementary information for the activation of AEP ligase zymogen.

Clitoria ternatea (Linn.) flower extract attenuates vascular dysfunction and cardiac hypertrophy via modulation of Ang II/AT1 R/TGF-ß1 cascade in hypertensive rats.

BACKGROUND: Clitoria ternatea (CT) (the Fabaceae family) has been reported to elicit several biological responses, such as anti-inflammation and anti-depression effects. This study evaluated the effect of CT flower extract on blood pressure, vascular function, and left ventricular hypertrophy in a two-kidney, one-clip (2K-1C) rat model. Hypertensive rats were treated with CT extract at various doses (100, 300, or 500 mg kg-1 day-1 ) or losartan (10 mg kg-1 day-1 ) for 4 weeks (n = 8/group). RESULTS: CT extract reduced blood pressure in a dose-dependent manner, and CT extract at a dose of 300 mg kg-1 was an effective concentration (P < 0.05). Augmentation of contractile responses to electrical field stimulation and impairment of vascular responses to acetylcholine in mesenteric vascular beds and aortic rings of 2K-1C rats were suppressed by treatment with CT extract or losartan (P < 0.05). Serum angiotensin-converting enzyme activity and plasma angiotensin II concentration were high in 2K-1C rats but alleviated by CT extract or losartan treatment (P < 0.05). Increases in superoxide production and lipid peroxidation were attenuated in 2K-1C rats treated with CT extract or losartan compared with the untreated group (P < 0.05). Increased plasma concentration of nitric oxide metabolites was found in hypertensive rats that received CT extract or losartan. CT extract or losartan suppressed the overexpression of Ang II receptor subtype I (AT1 -R) and transforming growth factor-ß1 (TGF-ß1) in 2K-1C rats. CONCLUSION: CT extract had antihypertensive effects that were associated with improving vascular function and cardiac hypertrophy in 2K-1C rats. The mechanisms involved suppression of the renin-angiotensin system, of oxidative stress, and of the AT1 R/TGF-ß1 cascade. © 2021 Society of Chemical Industry.