Purification, molecular docking and in vivo analyses of novel angiotensin-converting enzyme inhibitory peptides from protein hydrolysate of moth bean (Vigna aconitifolia (Jacq.) Màrechal) seeds.

The moth bean is a high-protein food legume. Enzymatic hydrolysates of food proteins demostrate health benefits. Search for diet related food protein hydrolysates is therefore within the scope of functional foods. Present study asertains to produce, screen and identify natural ACE-I inhibitory peptides derived from moth bean seed protein hydrolysates. The extracted protein was hydrolysed using alcalase, chymotrypsin, flavourzyme, papain, pepsin and trypsin respectively. Alcalase achieved the greatest degree of hydrolysis and ACE inhibition. The highest ACE-I inhibitory activity was exhibited by the peptide with the lowest molecular weight i.e. <3 kDa (IC50 11.19 ± 0.15 µg/mL). This was further separated by FPLC, followed by mass spectrometry. Molecular docking analysis showed the peptides IAWDFR and ADLPGLK bind to active sites whereas DKPWWPK and AVIPNAPNLR to non-active sites of the ACE molecule. In vivo administration of MBP hydrolysate to dexamethasone-induced hypertensive rats reduced their systolic blood pressure (125 ± 0.76 mmHg) compared with positive control (155 ± 3.13 mmHg). Moth bean protein peptides exhibit functional nutraceutical properties with adequate antihypertensive activity.

Chickpea Peptide: A Nutraceutical Molecule Corroborating Neurodegenerative and ACE-I Inhibition.

Chickpea seeds are the source of proteins in human nutrition and attribute some nutraceutical properties. Herein, we report the effects of chickpea seed bioactive peptide on albumin, insulin, lactoglobulin and lysozyme amyloid fibril formation. Employing thioflavin T (ThT) assays and circular dichroism (CD), amyloid structural binding transition was experimented to analyze the inhibition of amyloid fibril formation. The purified active peptide with a molecular mass of 934.53 Da was evaluated in vitro for its ACE-I inhibitory, antibacterial, antifungal and antidiabetic activities. Further, in vivo animal studies were carried out in wistar rats for blood pressure lowering action. In hypertensive rats, chickpea peptide decreased 131 ± 3.57 mm of Hg for systolic blood pressure and 86 ± 1.5 mm of Hg for diastolic blood pressure after 8 h intraperitoneal administration. Additionally, the peptide suppressed the fibrillation of amyloid and destabilized the preformed mature fibrils. Data emphasize efficacy of chickpea peptide vis-a-vis ACE-Inhibitory, antibacterial, antifungal, antidiabetic and anti-amyloidogenic activities, allowing us to propose this novel peptide as a suitable candidate for nutraceutical-based drugs and seems the first kind of its nature.

Effects of Prednisolone Derivative and Panaxydol: Biosurfactants on Cell Wall Integrity of Acne-Causing Resistant Bacteria.

Acne is one of the most common dermatological skin problem caused due to inflammation of the skin, leading to unfavorable growth of Propionibacterium acnes. It is a slow growing anaerobic, gram-positive bacterium that releases chemotactic factors and leads to the complex pathogenicity of acne. There are several acne treatments/therapies available, but topical therapy is usually the first choice for mild to moderate acne, and as the severity of the acne increases, the treatment modalities fail. There are many acne treatment options available, but topical therapy is best suited for mild - to - moderate skin problems, and then as the seriousness of the acne grows, the therapeutic approaches fall short. Biosurfactants are surfactants produced from plants or animals; Saponins are plant derived non-ionic biosurfactants which have steroidal and triterpenic glycosides distributed largely in plant kingdom. Numerous studies conducted by scientists have established the antimicrobial activity of and are considered more advantageous over synthetic precursors as they are eco-friendly, cheap and non-toxic. The present study was undertaken to investigate the antibacterial activity of saponins (bio-surfactants) characterized using mass spectroscopy against acne-causing bacteria. The discharge of cellular components including protein and UV-sensitive materials in the cell-free supernatant was provoked by saponin, confirming the cellular and membrane disturbances.. Furthermore, various morphological changes on the bacterial cell surface structure by Transmission electron microscopy and scanning electron microscopy revealed the disruption of the cell integrity leading to death. Results confirmed presence of non-ionic surfactants primarily affecting the disruption and destruction to the bacteria which indicates that saponins are efficient components with great potential applications in various pharmaceutical preparations. Effects of Prednisolone derivative and Panaxydol: Biosurfactants on cell wall integrity of Acne-Causing Resistant Bacteria.

Bacillus calmette-guerin as a quick and temporary solution to coronavirus disease-2019.

The coronavirus disease-2019 (COVID-19) pandemic is one of the most devastating things that happened in the world which has taken the lives of millions of people and has brutally shattered the world economy. This pandemic has instigated an urgent need for a vaccine to reduce the ongoing morbidity and mortality. Bacillus Calmette-Guerin (BCG) apart from being used as an effective and old vaccine against tuberculosis has some known off-target protection effect and is getting more attention in this scenario. BCG confers nonspecific innate immune-boosting effects called trained immunity against secondary infection. Various recent publications have proposed the inverse relationship between the COVID-19 morbidity and mortality with that of BCG coverage of that country on the basis of epidemiological studies. However, these studies have not considered the confounding factors, and a lot of recent articles are contradicting these epidemiological and observational data. Several random control trials for BCG on health-care workers and elderly people are ongoing worldwide and could depict the actual relation between COVID-19 and BCG protection. Although a recent trial has found a protective function of BCG against COVID-19 in health-care workers, more results of the trials can only give approval on this. There has been a shortage of BCG worldwide due to its use in bladder cancer and vaccination in neonates, and hence, its use should be carefully regulated. In this review, we have tried to summarize the various issue and conflicts on BCG to be used as a temporary solution to COVID-19.

Plant growth promoting bacteria induce anti-quorum-sensing substances in chickpea legume seedling bioassay.

Microorganisms and their hosts communicate through an array of signals. Many physiological processes regulated in quorum sensing (QS) are dependent on auto-inducers, like N-acyl-homoserine lactones (AHLs) as in numerous groups of both gram-positive and gram-negative bacteria. In vitro grown seven-day old chickpea seedlings treated with plant growth promoting bacteria (PGPRs) were used to screen the AHL mimicking and for phytochemical substances like phytohormones and secondary metabolites such as phenolics and flavonoids. Potential anti-quorum sensing (anti-QS) activity surrounding the roots on semi-solid agar lawn of Chromobacterium violaceum (ATCC12742) was observed. Crude protein (4.46-8.30 µg/mL) and methanolic extracts (100 µg/mL) of seedling gave moderate anti-QS activity against CV12742 anti QS bioassay, respectively. Crude protein and methanolic extract of Bacillus amyloliquefaciens (34.00 ± 2.23; 34.00 ± 4.33 mm) and B. subtilis A (27.00 ± 2.10; 3.29 ± 2.16 mm) treated samples showed higher zone of inhibition due to anti-QS activity. Phytohormone analysis using LC-MS for zeatin, auxin and methyl jasmonate (MeJA) indicated that phytohormones were significantly upregulated by 1909.80 ng/g FW, 669.67 ng/g FW and 244.55 ng/g FW, respectively in Pseudomonas brassicacearum treated seedlings compared to control. UHPLC of PGPR treated seedlings showed overly expressed gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid in B. amyloliquefaciens treated seedlings compared to others. Enrichment analysis identified significant pathways related to metabolism, biosynthesis of secondary metabolites. The present study indicates that chickpea neutralizes an extensive range of functional responses to AHLs that may play important role in legume host-microbe interactions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01034-x.

Bioactive peptide of Cicer arietinum L. induces apoptosis in human endometrial cancer via DNA fragmentation and cell cycle arrest.

Chickpea seed proteins are alleged source of nutraceuticals. These seed proteins were subjected to different proteases to produce peptides. The efficacy of these peptides was confirmed using six diverse human cancer cell lines (PA-1, Ishikawa cells, A549, MCF-7, HepG2, MDA-MB-231). Alcalase generated peptides exhibited the highest antagonistic inhibition of Ishikawa cells. Flow cytometric analysis revealed that chickpea peptide induced S and G2 phase arrest of cell cycle in a dose dependent manner. DNA fragmentation and apoptosis occurred by down regulation of Bcl-2 expression, upregulation of Bax expression and promotion of caspase-3 initiation. Chickpea peptides ascertain potential antiproliferative molecule that can be deployed in cancer treatment regimes.

Putative serum protein biomarkers for epsilon toxin exposure in mouse model using LC-MS/MS analysis.

Epsilon toxin (ETX), produced by Clostridium perfringens Type B or type D strains, is a potential biological and toxin warfare (BTW) agent, largely for its very high toxicity. The toxin is implicated in several animal diseases. Using LC-MS/MS analysis, we report here elucidation of putative serum maker proteins for ETX exposure with an objective of the early diagnosis of intoxication. Of 166 consensus proteins (488 peptides), showing ETX-induced alterations, 119 proteins exhibited increase and 47 proteins showed decreased abundance in serum, as revealed by SWATH (DIA) acquisition on LC-MS/MS and label free quantitative analysis of control and test samples. Complement and coagulation cascade, nitrogen metabolism, negative regulation of peptidase activity, and response to ROS were among the biological processes and pathways perturbed by the ETX exposure. Interaction network indicated enzyme inhibitor activity, detoxification of ROS, and steroid binding functions were the major interaction networks for the proteins with increased abundance, while, hemostasis and structural molecule activity were the prominent networks for the down-regulated proteins. Validation studies were carried out by immunoprecipitation, ELISA, and Western blot analysis of selected proteins to demonstrate diagnostic potential of the putative marker proteins of ETX exposure.

Phosphatase-defective DevS sensor kinase mutants permit constitutive expression of DevR-regulated dormancy genes in Mycobacterium tuberculosis.

The DevR-DevS/DosR-DosS two-component system of Mycobacterium tuberculosis, that comprises of DevS sensor kinase and DevR response regulator, is essential for bacterial adaptation to hypoxia by inducing dormancy regulon expression. The dominant phosphatase activity of DevS under aerobic conditions enables tight negative control, whereas its kinase function activates DevR under hypoxia to induce the dormancy regulon. A net balance in these opposing kinase and phosphatase activities of DevS calibrates the response output of DevR. To gain mechanistic insights into the kinase-phosphatase balance of DevS, we generated alanine substitution mutants of five residues located in DHp α1 helix of DevS, namely Phe-403, Gly-406, Leu-407, Gly-411 and His-415. For the first time, we have identified kinase positive phosphatase negative (K+P-) mutants in DevS by a single-site mutation in either Gly-406 or Leu-407. M. tuberculosis Gly-406A and Leu-407A mutant strains constitutively expressed the DevR regulon under aerobic conditions despite the presence of negative signal, oxygen. These mutant proteins exhibited ∼2-fold interaction defect with DevR. We conclude that Gly-406 and Leu-407 residues are individually essential for the phosphatase function of DevS. Our study provides new insights into the negative control mechanism of DevS by demonstrating the importance of an optimal interaction between DevR and DevS, and local changes associated with individual residues, Gly-406 and Leu-407, which mimic ligand-free DevS. These K+P- mutant strains are expected to facilitate the rapid aerobic screening of DevR antagonists in M. tuberculosis, thereby eliminating the requirement for hypoxic culture conditions.

Biochemical characterisation of lectin from wild chickpea (Cicer reticulatum L.) with potential inhibitory action against human cancer cells.

A wild chickpea lectin (WCL) from the seeds of Cicer reticulatum L. was chromato-purified using DEAE-Cellulose and SP-Sephadex ion exchange chromatography. WCL was thermostable upto 60°C with broad pH optima (pH 5-9) and various divalent metal ions did not influence its activity. WCL demonstrated DNA protection in a dose-dependent manner. The lectin exerted antifungal activity against diverse fungal pathogens. WCL augmented the mitogenic response of mouse spleen cells at 10 µg/ml concentration and showed an inhibition of HIV-1 reverse transcriptase at IC50 of 200 µM. Against human cancer cell lines, lectin demonstrated anticancer potential. The cell viability assay in HepG2, Ishikawa, MCF-7 and MDA-MB-231 cell line demonstrated IC50 values of 61.8, 54.4, 37.5 and 44.2 µg/ml respectively. PRACTICAL APPLICATIONS: WCL exhibited distinct medicinal properties vis-à-vis antiproliferative, mitogenic, antifungal/bacterial and HIV-1 reverse transcriptase inhibiting activities. The potential of WCL can be a subject of exploration from a pharmacological standpoint.

Proteomic analysis of chickpea roots reveal differential expression of abscisic acid responsive proteins.

Seeds of chickpea are nutritious and alleged to be a preferred source of protein next only to milk. Some of the biotic and abiotic factors reduce chickpea production worldwide. Plant roots are the first to perceive stress signals. The wild root free radical scavenging activity measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was 28.06 ± 1.43% and 25.12 ± 0.95% in cultivated chickpea type. The root proteins were resolved on 7 cm IPG strip having a pH gradient 5-8 and subsequently separated on the basis of mass using SDS-PAGE in second dimension. A total of eight representative spots were subjected for identification by MALDI-TOF-MS. A protein-protein association network analysis using STRING software permitted to build an interactomic map of all detected proteins, characterised by 16 interactions. The findings may provide a better understanding of the biochemical mechanism of different root pathways and stress-responses in chickpea. PRACTICAL APPLICATIONS: Information pertaining to stress resistance is essential from breeder's perspectives. Chickpea is prone to high yield losses due to recurring droughts. MALDI-TOF-MS coupled with MASCOT query search found significant correlations with abscisic acid responsive proteins associated to drought stress using comparative proteomics. This report will assist researchers a ready reference for executing further studies concerning chickpea root proteins. The findings may provide a better understanding of the biochemical mechanism of different root pathways and stress-responses in chickpea.

Variations in the antioxidant and free radical scavenging under induced heavy metal stress expressed as proline content in chickpea.

This study pertains to the effects of heavy metal salts viz., copper (Cu), manganese (Mn), lead (Pb) and zinc (Zn) on the chickpea accession ICC-4812. The salts were given as treatments to the chickpea seeds at various ascending levels of doses till proving toxic. The treatment of 24 h soaked and swollen seeds were then extended to 7 days duration from the date of treatment. Atomic absorption spectrophotometric analysis of bioassay tissue Cicer, showed maximum uptake of 9.41 mg/g and minimum of 1.65 mg/g tissue dry weight for Pb and Zn respectively. The study reveals that enhanced antioxidant responses are associated with substantial proline accumulation indicating induced stress. Ferric reducing antioxidant power assay measuring antioxidant activity was highest in the chickpea seedling treated with Zn, whereas, free radical scavenging activity was highest in the treatments with Mn. The total phenolic and flavonoid contents ranged between 0.24-0.97 and 0.27-1.00 mg/g of dry matter content respectively. Higher Pb and Zn doses seem to elicit higher proline levels therefore, suggesting an extreme condition of induced abiotic stress. Dose dependent protein oxidation coupled with DNA degradation was observed in all treatments, depicting genotoxicity. Unweighted pair-group method arithmetic average analysis presented similarity coefficients between the treatments.

Elucidation of protein biomarkers in plasma and urine for epsilon toxin exposure in mouse model.

Epsilon toxin (ETX) is the major virulence determinant of C. perfringens type B or type D strains, causing diseases in animals, besides being a listed biological and toxin warfare (BTW) agent. Keeping in mind the high lethality and the rapid onset of clinical manifestations, early diagnosis of epsilon toxin exposure is of paramount importance for implementation of appropriate medical countermeasures. Using a 2DE-MS approach, the present study is the first comprehensive proteomic elucidation of ETX-induced protein markers in the mouse model, providing putative targets for early diagnosis of ETX exposure. A total of 52 unique proteins showing ETX-induced modulations were identified in plasma and urine samples. Fibrinogen, apolipoprotein, serum amyloid protein, plasminogen, serum albumin, glutathione peroxidase, transferrin, major urinary protein 2, haptoglobin, transthyretin, and vitamin D-binding protein were among the proteins observed in more than one dataset with altered abundance after the ETX-intoxication. The predicted localization, function, and interaction of the ETX-modulated proteins in the plasma and urine indicated involvement of multiple pathways; extracellular proteins, followed by macromolecular complexes associated with blood coagulation and plasminogen activating cascade, being the most prominent among others. The putative markers elucidated here warrants further validation and can be of immense value for the early diagnosis of ETX exposure.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity.

BACKGROUND: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. METHODS: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. RESULTS: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 µg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. ß-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. CONCLUSION: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Biochemical diversity evaluation in chickpea accessions employing mini-core collection.

The seeds of chickpea provide an exceptional source of dietary proteins and is one of the important legumes in both developed and developing countries over the world. The available germplasm of cultivated chickpea is deficient in desired biochemical signatures. To identify new sources of variations for breeding, reduced subsets of germplasm such as mini-core collection can be explored as an effective resource. In the present investigation, mini-core collections consisting of 215 accessions of chickpea were extensively evaluated for tapping biochemical diversity. Analysis included ten biochemical parameters comprising total protein, total free amino acids, phytic acid, tannin, total phenolics, total flavonoids, lectin, DPPH radical scavenging activity, in vitro digestibility of protein and starch. The spectrum of diversity was documented for total protein (4.60-33.90%), total free amino acids (0.092-9.33 mg/g), phytic acid (0.009-4.06 mg/g), tannin (0.232-189.63 mg/g), total phenolics (0.15-0.81 mg/g), total flavonoids (0.04-1.57 mg/g), lectin (0.07-330.32 HU/mg), DPPH radical scavenging activity (26.74-49.11%), in vitro protein digestibility (59.45-76.22%) and in vitro starch digestibility (45.63-298.39 mg of maltose/g). The principal component analysis revealed association of chickpea higher protein content to the lower level of total phenolics and flavonoid contents. The dendrogram obtained by unweighted pair group method using arithmetic average cluster analysis grouped the chickpea accessions into two major clusters. This is the first comprehensive report on biochemical diversity analysed in the mini-core chickpea accessions. The ultimate purpose of conducting such studies was to deliver information on nutritional characteristics for effective breeding programmes. Depending on the objectives of the breeding aforesaid accessions could be employed as a parent.

Chickpea Lectin Inhibits Human Breast Cancer Cell Proliferation and Induces Apoptosis Through Cell Cycle Arrest.

BACKGROUND: Breast cancer demands safe adjuvant to overcome the side effects of standard drug tamoxifen. Diet derived bioactive compounds are reported to exhibit modulation of cancer growth leading to cell death. Chickpea is a protein rich edible legume with several bioactive compounds that includes lectin as well. Characterization of chickpea lectin for its effect against cancer cells has been investigated in this study. METHOD: Cicer arietinum L. lectin (CAL) agglutinating trypsin-treated rabbit blood cells was purified employing DEAE-cellulose and SP-sephadex ion exchange chromatography. The lectin was characterized for its biological activity vis-à-vis antiproliferative and apoptotic effects through cell cycle arrest in MCF-7 human breast cancer cells. RESULT: There is a significant inhibition of the survival of breast cancer cells due to chickpea lectin in a dose dependent manner for 24 hr. Lectin treated cells revealed distinct features of apoptosis. Flow cytometric analysis at 80 µg/ml of lectin induced S and G2 phase cell cycle arrest. CAL induced apoptosis in MCF-7 cells associated with lactate dehydrogenase leakage, cell cycle arrest and reactive oxygen species generation. CONCLUSION: Our studies show that chickpea lectin exerted anticancer activity and could be exploited as an essential source for medicine leading to the treatment of breast cancer.