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Purification, characterization and antioxidant efficacy testing of trypsin inhibitor protein from Anonna squamosa seeds
Abstract
Plant derived proteins are receiving ample acceptance as therapeutic agents against oxidative injury throughout the progression of many human diseases. Plant trypsin inhibitors are compounds that help plants to exhibit defense on pests. Extensive investigations are proceeding globally to identify the natural trypsin inhibitors with therapeutic applications. The present study ventures to identify and isolate plant proteins having trypsin inhibitory activity and analyse their antioxidant potential. Seeds of four widely distributed plants- Anonna squamosa, Carica papaya, Manikara zapota and Passiflora edulis-were screened for the presence of trypsin inhibitor proteins. Anonna squamosa seed extract showed the highest trypsin inhibitory activity and was used for further analysis. Purification was done by ammonium sulphate precipitation, silica gel column chromatography and DEAE-Cellulose Anion-exchange chromatography. There was a significant trypsin inhibitory activity and considerable fold increase in purity after each step of purification. Molecular weight of the A. squamosa trypsin inhibitor was determined by SDS PAGE analysis. DPPH method was followed to determine the antioxidant activity of the purified trypsin inhibitor. It showed considerable radical scavenging activity with IC50 value of 108 µg/mL. The results indicate that A. squamosa seeds possess a trypsin inhibitory protein of molecular mass 43.5 kDa which has significant antioxidant properties. © 2022, The Author(s), under exclusive licence to Society for Plant Biochemistry and Biotechnology.
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Because the majority of bacterial species divide by binary fission, and do not have distinguishable somatic and germ-lines cells, they could be considered to be immortal. However, bacteria ‘age’ due to damage to vital cell components such as DNA and proteins. DNA damage can often be repaired using efficient DNA repair mechanisms. However, many proteins have a functional ‘shelf life’; some short lived, others are relatively stable. Specific degradation processes are built into the life span of proteins whose activities are required to fulfil a specific function during a prescribed period of time (e.g. cell cycle, differentiation process, stress response). In addition, proteins that are irreparably damaged or that have come to the end of their functional lifespan need to be removed by quality control proteases. Other proteases are involved in performing a variety of specific functions that can be broadly divided in to three categories: processing, regulation and feeding. This review presents a systematic account of the proteases of Bacillus subtilis and their activities. It reviews the proteases found in, or associated with, the cytoplasm, the cell membrane, the cell wall and the external milieu. Where known, the impacts of the deletion of particular proteases are discussed, particular in relation to industrial applications.
In cancer treatments, many natural and synthetic products have been examined; among them, protease inhibitors are promising candidates for anti-cancer agents. Since dysregulated proteolytic activities can contribute to tumor development and metastasis, antagonization of proteases with tailored inhibitors is an encouraging approach. Although adverse effects of early designs of these inhibitors disappeared after the introduction of next-generation agents, most of the proposed inhibitors did not pass the early stages of clinical trials due to their nonspecific toxicity and lack of pharmacological effects. Therefore, new applications that modulate proteases more specifically and serve their programmed way of administration are highly appreciated. In this context, nanosized drug delivery systems have attracted much attention because preliminary studies have demonstrated that the therapeutic capacity of inhibitors has been improved significantly with encapsulated formulation as compared to their free forms. Here, we address this issue and discuss the current application and future clinical prospects of this potential combination towards targeted protease-based cancer therapy.
Plant‐derived protease inhibitors (PI) are a promising tool for crop improvement and insect pest management. Although agronomist made significant efforts in utilizing PIs for managing insect pests, the potentials of PIs are still obscured. Insect ability to compensate nutrient starvation induced by dietary PI feeding using different strategies, i.e., overexpression of PI‐sensitive protease, expression of PI‐insensitive proteases, degradation of PI, has made this innumerable collection of PIs worthless. A practical challenge for agronomist is to identify potent PI candidates, to limit insect compensatory responses and to elucidate insect compensatory and resistance mechanisms activated upon herbivory. This knowledge could be then efficiently utilized to identify potential targets for RNAi mediated pest control. These vital genes of insects could be functionally annotated using the advanced gene‐editing technique, CRISPR/Cas9. Contemporary research is exploiting different in silico and modern molecular biology techniques, to utilize PIs in controlling insect pests efficiently. This review is structured to update recent advancements in this field, along with its chronological background.
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Tetranychus urticae (two-spotted spider mite) is a striking example of polyphagy among herbivores with an extreme record of pesticide resistance and one of the most significant pests in agriculture. The T. urticae genome contains a large number of cysteine- and serine-proteases indicating their importance in the spider mite physiology. This work is focused on the potential role of the Kunitz trypsin inhibitor (KTI) family on plant defense responses against spider mites. The molecular characterization of two of these genes, AtKTI4 and AtKTI5, combined with feeding bioassays using T-DNA insertion lines for both genes was carried out. Spider mite performance assays showed that independent KTI silencing Arabidopsis lines conferred higher susceptibility to T. urticae than WT plants. Additionally, transient overexpression of these inhibitors in Nicotiana benthamiana demonstrated their ability to inhibit not only serine- but also cysteine-proteases, indicating the bifunctional inhibitory role against both types of enzymes. These inhibitory properties could be involved in the modulation of the proteases that participate in the hydrolysis of dietary proteins in the spider mite gut, as well as in other proteolytic processes.
Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical–scavenging activity in a concentration-dependent manner with IC50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC50) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.
The gastrointestinal barrier is - with approximately 400 m(2) - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.
Inhibitors from diverse sources are being explored for understanding their inhibitory potential. Among
several alternatives, protease inhibitors represent an attractive class of biopesticides with several important features. Such protease inhibitors are environmentally friendly and can work as defence molecules against plant pest and pathogens. Trypsin inhibitors CFTI-1 and CFTI-2 were purified from the seeds of the Cassia fistula and the prominent inhibitor CFTI-1 was studied for its effects on growth parameters and developmental stages of Helicoverpa armigera. CFTI-1 and CFTI-2 exhibited 92% and 78% inhibitory activity against midgut trypsin like proteases, respectively. Dixon and Lineweaver–Burk double reciprocal plots analysis revealed a competitive inhibition pattern with the Ki values 2.9x10-10 M and 4.2x10-10M for CFTI-1 and CFTI- 2 respectively. Lethal concentration (0.34% m/m) and concentration for weight reduction by 50% (0.24% m/m) determined for CFTI-1. CFTI-1 showed a concentration-dependent effect on mean larval weight, a decline of the fertility and fecundity of the moths, and an extended total developmental duration of H. armigera life cycle. These results suggest that CFTI-1 has toxic and adverse effect on the developmental physiology of H. armigera and could be useful in controlling the pest H. armigera.
Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesising a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor’s resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor’s potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor’s sequence and structure relate to its binding kinetics and function. Using enzyme assays, molecular dynamics simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show the geometry of the binding loop mainly influences the inhibitor’s potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor’s sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin, and two kallikrein-related peptidases, KLK5 and KLK14, that are implicated in various cancers and skin diseases.
The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.
Applications of antioxidants are increasing due to their multiple roles in minimising harmful effects of oxidative stress. 2,2-diphenyl-1-picrylhydrazyl (DPPH�) radical scavenging assay is routinely practiced for the assessment of antiradical properties of different compounds. A detailed literature survey revealed use of different materials and methods for DPPH� assay by different investigators resulted in variation in
the values of reference standards and measured parameters of new antioxidants. In the present work a detailed kinetic study of antioxidants has been performed and comprehensive results in terms of effective concentration which scavenges 50% radical (EC50), antioxidant reducing power (ARP), stoichiometry and second order rate constant (k2) values have been reported with DPPH� assay. Importance of selection of appropriate reference compounds and kinetic calculations are suggested. Few case studies of standard antioxidants have been discussed to emphasise the utilisation of appropriate methodology and reference compounds.
Natural trypsin inhibitors from plant tissues are emerging with promising therapeutic uses. They have profound applications in medicine and biotechnology and are extensively used in the food and medicine industry. Their activities are affected by detergents, metal ions, and reducing or chelating agents that are commonly used in these industries. Artocarpus hirsutus and Garcinia gummi-gutta are two tropical trees wherein most of the plant parts except the seeds were extensively studied and proved to possess medicinal properties. In the present study, the seeds of these tropical plants are proved to possess trypsin inhibitory activity. We report here the partial purification of trypsin inhibitory proteins from mature seed extracts of A. hirsutus and G.gummi-gutta in 50 mM phosphate buffer (pH 7.6). The partial purification was done by ammonium sulphate precipitation. Modulation of activity of A. hirsutus and G. gummi-gutta TIs by thermal stabilisers, metal ions and detergents were analysed. There was a significant fold of purification, in both cases. The thermal stabilisers, metal ions and detergents modulate the activities of the two TIs in their way. The study effectively provides choices of optimal additives to be used, where industrial processing of these TIs is required for therapeutic applications. © 2021, National Institute of Science Communication and Information Resources. All rights reserved.
Mitochondria contain more than 1000 different proteins, including several proteolytic enzymes. These mitochondrial proteases form a complex system that performs limited and terminal proteolysis to build the mitochondrial proteome, maintain and control its functions or degrade mitochondrial proteins and peptides. During protein biogenesis presequence proteases cleave and degrade mitochondrial targeting signals to obtain mature functional proteins. Processing by proteases also exerts a regulatory role in modulation of mitochondrial functions and quality control enzymes degrade misfolded, aged or superfluous proteins. Depending on their different functions and substrates, defects in mitochondrial proteases can affect the majority of the mitochondrial proteome or only a single protein. Consequently, mutations in mitochondrial proteases have been linked to several human diseases. This review gives an overview of the components and functions of the mitochondrial proteolytic machinery and highlights the pathological consequences of dysfunctional mitochondrial protein processing and turnover.
Proteases have an important role in homeostasis, and dysregulation of protease function can lead to pathogenesis. Therefore, proteases are promising drug targets in cancer, inflammation, and neurodegenerative disease research. Although there are well-established pharmaceuticals on the market, drug development for proteases is challenging. This is often caused by the limited selectivity of currently available lead compounds. Proteinaceous plant protease inhibitors are a diverse family of (poly)peptides that are important to maintain physiological homeostasis and to serve the innate defense machinery of the plant. In this review, we provide an overview of the diversity of plant peptide- and protein-based protease inhibitors (PIs), provide examples of such compounds that target human proteases, and discuss opportunities for these molecules in protease drug discovery and development.
Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.
A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor,
named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed
by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of
AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine
trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity
assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition
mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation
constant (Ki) of 3.7 × 10−8 M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of
pH 4–8 and temperature 20–80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition
assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest.
Nephelium lappaceum L., commonly known as “rambutan,” is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N
. lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.
A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.1 M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion exchange chromatography on DEAE-Sephadex A50. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60°C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low Ki value of 0.2 μM. The inhibitor was found to be susceptible to varying concentrations of reducing agents like DTT and 2-mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth of Pieris brassicae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.
The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14kDa.
Protein extracts of 640 soybean cultivars and landraces, mainly from China and a few from the US, were analyzed for their
components and subunits based on distribution patterns of bands with varying molecular weights (MW) under SDS-PAGE (sodium
dodecyl sulfate-polyacrylamide gel electrophoresis). The number and molecular weight of the bands in SDS-PAGE varied among
materials and showed a tendency of continuous distribution. Accordingly, the SDS-PAGE patterns of the soybean protein extracts
were divided into two regions: the region of bands with MW<44KDa and that with MW≥44KDa. The first region containing
mainly 11S proteins was divided into four parts, called subunit groups, i.e. 11S-1 (14.4–22KDa), 11S-2 (22–26KDa), 11S-3
(26–34KDa) and 11S-4 (34–44KDa). The second region containing mainly 7S protein was divided into six subunit groups, i.e.
7S-1 (44–49KDa), 7S-2 (49–55KDa), 7S-3 (55–67KDa), 7S-4 (67–73KDa), 7S-5 (73–82KDa) and 7S-6 (82–91KDa). The sum of
relative contents of 11S-1–11S-4 was obtained as the relative content of 11S protein, those of 7S-1–7S-6 as that of 7S protein,
and therefore, the 11S/7S ratio obtained. The proposed criteria were demonstrated to be simple, stable and feasible. Among
the 640 tested materials, 39 lacked 11S-1 but none lacked the other 11S subunit groups, while deficiencies existed in all
the six subunit groups of 7S, indicating a great potential for the genetic variation of protein components and subunits for
breeding for the improvement of protein qualities.
Antimicrobial activity of protease inhibitor isolated from Coccinia grandis (L.) Voigt. has been reported. A 14.3 kDa protease inhibitor (PI) was isolated and purified to homogeneity by ammonium sulfate precipitation (20-85% saturation), sephadex G-75, DEAE sepharose column and trypsin-sepharose affinity chromatography from the leaves of C. grandis. The purity was checked by reverse phase high performance liquid chromatography. PI exhibited marked growth inhibitory effects on colon cell lines in a dose-dependent manner. PI was thermostable and showed antimicrobial activity without hemolytic activity. PI strongly inhibited pathogenic microbial strains, including Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus subtilis and pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus and Cryptococcus neoformans. Examination by bright field microscopy showed inhibition of mycelial growth and sporulation. Morphologically, PI treated fungus showed a significant shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. Results reported in this study suggested that PI may be an excellent candidate for development of novel oral or other anti-infective agents.
During the germination of legume seeds, enzymes become active in order to degrade starch, storage-protein and proteinaceous antinutritional factors. The degradation of storage-protein is necessary to make peptides and amino acids available in order to stimulate seed growth and early plant growth. Proteinaceous antinutritional factors such as amylase inhibitors, lectins and trypsin inhibitors are present in legume seeds and protect them against predators. However, during germination, they degrade to a lower level by the action of several enzymes. The effect of germination on the content and activity of amylase inhibitors, lectins, tannins and trypsin inhibitors is discussed.
Unlabelled:
The popular medicine Passiflora edulis has been used as a sedative, tranquilizer, against cutaneous inflammatory diseases and intermittent fever. Most of the pharmacological investigations of Passiflora edulis have been addressed to its Central Nervous System activities, such as anxiolytic, anticonvulsant and sedative actions. Otherwise, there are few reports about the anti-inflammatory activity of the Passiflora species. The aim of this study was to investigate the mechanism of the anti-inflammatory effect of aqueous lyophilized extract obtained from leaves of Passiflora edulis var. flavicarpa Degener (Passifloraceae) in the mouse model of pleurisy induced by carrageenan (Cg), bradykinin, histamine or substance P, observing the effects upon leucocytes migration, myeloperoxidase (MPO), nitric oxide (NO) concentrations and tumor necrosis factor-alpha (TNFalpha) and interleukin-1 beta (IL-1beta) levels.
Results:
Passiflora edulis (250mg/kg) administered by intraperitoneal route (i.p.) inhibited the leukocyte, neutrophils, myeloperoxidase, nitric oxide, TNFalpha and IL-1beta levels (P<0.01) in the pleurisy induced by carrageenan. Passiflora edulis (250-500mg/kg, i.p.) also inhibited total and differential leukocytes in the pleurisy induced by bradykinin, histamine or substance P (P<0.05).
Conclusion:
Several mechanisms, including the inhibition of pro-inflammatory cytokines (TNFalpha, IL-1beta), enzyme (myeloperoxidase) and mediators (bradykinin, histamine, substance P, nitric oxide) release and/or action, appear to account for Passiflora edulis's actions.
Garcinia cambogia L. seeds as a source of oil and a trypsin inhibitory protein having microbicidal and antioxidant potentials
- R K Merin
- R Jiji
- D D Gayathri
- RK Merin
Identification of Artocarpus hirsutus and Garcinia gummi-gutta as the sources of trypsin inhibitory proteins
- R K Merin
- K P Lijith
- C Chanchitha
- D D Gayathri
- RK Merin
Analytical and phytochemical exploration of bioinsecticide granules mixed betel leaf extract (Piper betle) and Srikaya seed extract (Annona squamosa)
- D A Susanti
- A Soesetijo
- D Wahyuni
- DA Susanti
Development of quality standards of Carica papaya Linn leaves
- A Varisha
- SH Ansari
- KJ Naquvi
- A Poonam
- A Adil