In Vitro Screening of Seed Extracts of Medicinal Plants for Protease Inhibitory Activity (original) (raw)
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Recent Updates on Pharmaceutical Potential of Plant Protease Inhibitors
TJPRC, 2013
Plants are rich source of chemicals that are helpful for human in many ways. The pharmacological properties of several different classes of secondary metabolic compounds isolated from different groups of plants are well known. But there are some other compounds that are different from so called secondary metabolic compounds, are also beneficial from pharmacological point of view. One of such compound is small molecular weight proteins that act as inhibitors of proteases. Proteases are essential in survival and maintenance of living organisms. But when the amount increases much higher than the optimum value, they can damage the living cells and tissues. Thus, the involvement of protease inhibitors is essentially required to regulate the amount of proteases within the living cells and tissues. In plants proteases inhibitors are helpful in protecting them from the deleterious effect of insect gut proteases and thus provide defense against insect pests. Several groups of plant protease inhibitors along with their sequences are reported. Many inhibitors are being over expressed in transgenic plants for better insecticidal property. Besides, plant protease inhibitors have enormous pharmaceutical value which is still less explored. The current knowledge of plant protease inhibitors and their pharmaceutical applications have been reviewed.
Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gelfiltration chromatography followed by preparative polyacrylamide gel electrophoresis. One of the inhibitors, Cajanus trypsin-chymotrypsin inhibitor (CTCI), inhibits both bovine trypsin and chymotrypsin while the other, Cajanus trypsin inhibitor (CTI), inhibits only bovine trypsin. The two inhibitors contained no carbohydrate and had an isoelectric point of 6. CTCI and CTI had average molecular weights of 15000 and 10500, respectively. The purified inhibitors in solution were stable to heat at 80°C for 15 min and pH 7-10. In the pH range 3-5, 80% of the activity was retained. Autoclaving totally destroyed the inhibitor activity. CTCI had two sites for trypsin binding and one site for chymotrypsin binding while CTI had only one site for trypsin binding. The inhibitors were very specific towards mammalian serine proteases and did not inhibit other proteases or serine proteases of bacterial origin.
Review on Plant Protease Inhibitors as Therapeutic Molecules
The Journal of Plant Science Research, 2022
Plants contain several phytochemical compounds with capacity to exert therapeutic effects on human beings. One such group of molecules are the Protease inhibitors (PIs) which exert inhibitory activity towards several classes of mammalian, bovine, insect or microbial proteinases/proteases. PIs are polypeptides or proteins capable of forming reversible stoichiometric protein-protein complexes with specific proteolytic enzymes. This inhibits the catalytic function of proteolytic enzymes. Study of PIs is important to control proteolysis as it is the key to control the onset of several disease associated phenomena. Although initially, PIs were considered only as protein degrading enzymes, recent studies point them to be signalling molecules in biological activities. In this article, various classes of plant protease inhibitors and their general mode of actions are discussed. The families belonging to the serine protease class of inhibitors (Serpins) are discussed in detail as these constitute an important class of therapeutically important molecules. Data available on the use of PIs for treating a wide range of human diseases and disorders including the still incurable cancers is reviewed. Literature review confirms wide and successful use of Proteasome inhibitors (with similar activities to PIs) in treatment of haematological malignancies. Synthetic proteasome inhibitors are employed to inhibit the proteasome cascades and thus control disease onset and proliferation. Sharing the similar biological activities with the proteasome inhibitors, gives potential for the plant-based PIs to be experimented for similar uses. An attempt is made to report PIs from several plant species and their ongoing clinical trials to study their therapeutic actions.
Aquaculture Research, 2007
The in vitro inhibitory e¡ect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) ¢ngerlings at two di¡erent pH ranges (8^8.5 and 10^11). That protein digestion occurs mainly in the alkaline condition in this ¢sh without a stomach is evident from very high trypsin activity (0.95 AE 0.04 benzoyl-DL-arginine-p-nitroanilide U mg protein À1 ) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 mg U À1 . More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 mg U À1 . Black gram at 11.0 mg U À1 and soybean seed proteins at 62.75 mg U À 1 resulted in 50% and more than 30% inhibition of digestive protease activity in rohu ¢ngerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in di¡erent dose^response curves for di¡erent protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned di¡erences in inhibition. Each seed extract produced a characteristic pro¢le of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely a¡ect the digestive proteases in ¢sh and hence there is a need to eliminate/ reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.
2016
The trypsin serine protease inhibitor from Albizia niopoides seeds was purified, characterized, and had its antimicrobial activity tested against different bacteria and fungi strains. The purification was performed in three chromatographic steps. The molecular mass estimated by polyacrylamideTricine-SDSPAGE-gel was confirmed by mass spectrometry as 19,479.071Da. Its biochemical characterization by the inhibition curve indicated the inhibition stoichiometry as 1:1(inhibitor/Enzyme). The Ki was calculated to be 4.5x10M. In the stability test, the inhibitor activity remained above95% at pH2.0 to 12.0. The A.niopoides trypsin inhibitor(AnTI) also showed high thermostability up to 60°C, with a gradual reduction in temperatures above 70oC. In the test of resistance against DTT, this protease inhibitor showed resistance to all three of the tested concentrations of the reducing agent(1mM, 10mM and 100mM), with a slight loss of residual activity(96%) at a concentration of 100mM DTT after 60m...
Journal of Biosciences, 1983
A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a M r of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0-12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a twofold increase in inhibitor potency.
Antitumor Potential of Plant Protease Inhibitors
One of the most important issues facing health care is the prevention and treatment of different types of cancer. Among the most frequently encountered types of cancer leading to lethality are lung cancer, breast cancer and colon cancer. The search for new drugs for cancer cells treatment and clarifying the mechanisms of their anti-tumor effect is a scientific challenge and a necessary basis for new more effective methods of cancer treatment. A major role in tumor growth, invasion, angiogenesis and metastasis have different types of proteases whose activities are inhibited by some synthetic drugs. Protease inhibitors of plant origin have the potential to be an alternative or supplement to the treatment with synthetic drugs, but the mechanisms of their anti-tumor effects are poorly understood. In this review the current knowledge of plant protease inhibitors as tumor preventive and suppresive agents, and the perspectives of their use as antitumour drugs are discussed. Abbreviations: ...
Protease inhibitor from Moringa oleifera active against therapeutic proteases
Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine
Aspartic acid proteases participate in many physiological processes and their activities are associated with the spreading of diseases such as Alzheimer's disease, hypertension, cancers, and inflammatory, cardiovascular, viral, and other parasitic diseases. Aspartic proteases have become a widely spoken topic due to human immunodeficiency virus protease and malaria parasite protease that are targeted as key therapeutic intervention points in treating AIDS and malaria, respectively. But, relatively few studies have been reported on natural aspartic inhibitors. Therefore inhibition of proteases is one of the most promising approaches as therapeutic drugs for such conditions. In this case, plants have become a good source of proteinaceous and non-proteinaceous inhibitors. The present study is aimed at the identification, characterization, and partial purification of potent aspartic protease inhibitor/s from several medicinal plants in Sri Lanka. For this study plant samples were collected from Kandy district, Sri Lanka. Aqueous extracts of equal concentrations were prepared separately using fresh mature bark samples from Annona muricata, Garcinia quaesita Pierre, Bauhinia tomentosa, Caesalpinia bonduc, Crotalaria laburnifoloria, Crotalaria micans, Entada zeylanica (kosterm), Erythrina suberosa, Azadirachta indica, and Limonia acidissima L. To our knowledge to recent, there are no aspartate inhibitors identified from these plants. Aspartate inhibitory activity was determined by spectrophotometric stop rate determination method using 0.1mg ml-1 pepsin enzyme, 2.5% bovine hemoglobin substrate with plant extracts (inhibitors). The reaction was terminated by adding 5% TCA solution. The absorbance of the acid-soluble peptides was measured at 280 nm. The inhibitor was purified by ammonium sulfate precipitation and ion exchange chromatography methods. Molecular weight was estimated by dialysis. All the experiments were conducted in duplicate three times. Maximum inhibition of pepsin was shown by Garcinia quaesita Pierre while other species didn't show any significant inhibition of pepsin. Hence for further studies, Garcinia quaesita bark extract was selected. The thermal stability of the inhibitor in the crude extract was studied by incubating the extract at different temperatures and determining the remaining activity. The inhibitors were subjected to ammonium Sulphate precipitation and Ion exchange chromatography, in an attempt to purify the inhibitor/s. Molecular weight was estimated by dialysis which implies that the inhibitors comprise small and large molecules with different molecular weights ranging from less than 3.5 kDa to more than 12 kDa. Crude extract of Garcinia quaesita retained more than 50% of inhibitory activity over a wide range of temperatures (4-95 o C) for 30 minutes and also at 4 o C for one month. But the remaining inhibitory activities of the crude extract incubated at room temperature and 37 o C for one month were 20% and 10%, respectively. This suggests that inhibitor molecules are moderately thermostable. This assay procedure provided a quantitative measurement of the inhibitory activity of the inhibitor/s present in the crude bark extract. Fractions obtained from ammonium sulfate precipitation and ion exchange chromatography didn't show inhibition towards pepsin suggesting that inhibitors could be non-proteinaceous. Further studies on purified inhibitors and necessary to characterize and elucidate the structure of the inhibitors
Antioxidant and Protease Activities of Seven Native Plant Sources
International Journal of Bio-resource and Stress Management
The in-vitro antioxidant activity and protease activity of the latex of different plant sources, namely Tabernaemontana divericata, Croton bonplandianum, Plumeria rubra, Nerium oleander, Cascabela thevetia, Alstonia scholaris, Allamanda cathartica were analysed. The antioxidant activity was analyzed using DPPH (1-1, dipenyl-2-picrylhydrazyl) reduction assay and protease activity was analyzed by the cleavage of milk protein, casein. From the results, it was observed that Croton bonplandianum latex has exhibited very strong and highest antioxidant activity (76±5.4%) followed by Allamanda cathartica (71±3.8%). Nerium oleander (62±4.2%) and Plumeria rubra latexes (58±2.9%) have showed good antioxidant activity. Cascabela thevetia (35±4.4%) and Tabernaemontana divericata latexes (28±3.0%) have showed moderate antioxidant activity. However, Alstonia scholaris latex has showed the least and negligible antioxidant activity (0.19±0.05%). The protease activity was found to be significantly highest in the Tabernaemontana divericata latex (4461.55±230 µg ml-1) followed by Cascabela thevetia (3307.7±284 µg ml-1). Good amount of the protease activity was found in Allamanda cathartica latex (1205.15±155 µg ml-1) followed by Croton bonplandianum (923.1±213 µg ml-1). Moderate amount of the protease activity was found in Nerium oleander (333.35±84 µg ml-1) and Plumeria rubra latexes (179.5±38 µg ml-1). However, least protease activity was observed in Alstonia scholaris latex (51.3±14 µg ml-1). The results revealed that almost all plant latex samples except A. scholaris have showed from moderate to strong antioxidant and protease activities. C. bonplandianum latex has exhibited significantly highest antioxidant activity and T. divericata latex has exhibited significantly highest protease activity.