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International Journal of Medicine and Pharmaceutical Science (IJMPS) ISSN 2250-0049 Vol. 3, Issue 4, Oct 2013, 101-120 © TJPRC Pvt. Ltd.

RECENT UPDATES ON PHARMACEUTICAL POTENTIAL OF PLANT PROTEASE INHIBITORS DIBYENDU DUTTA MAJUMDAR Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Lower Saxony, Germany

ABSTRACT 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.

KEYWORDS: Plant Protease Inhibitors, Cystatins, Bowman Birk Inhibitors, Protease, Anticancer Compounds INTRODUCTION Plants are enriched with several active compounds which have pharmacological property. Plant natural products are being used in medicine from the ancient time, and till today they used in preparing medicines. In Ayurvedic, Homeopathy, Unani and even in Allopathic system, medicine practitioners and producers suggests and use several medicines that are prepared from plants or plant parts or plant derived chemicals. Plant secondary metabolites comprise an array of several thousands of compounds, most of which are used in human health care. Unlike primary metabolites, the secondary metabolites have no role in plant’s growth and survival, but are play a vital role in protecting plants from pathogen infection, herbivore attack and UV irradiation [1]. These groups of compounds are highly explored in biochemical and molecular genetics level and exploited enormously in human health care sector. But, there are some other compounds which are still less explored among plant natural products, are protein of small molecular weight. Among the small molecular weight proteins, protease and protease inhibitor (PIs) are two most important groups of proteins with fully opposite character. Plant Protease Proteases are enzymes which catalyzes the hydrolytic cleavage of peptide bond in the target protein. Some of them act on the peptide bond present near the end of polypeptide chains (exopeptidases) and others act on the peptide bond located within the polypeptide chain (endopeptidases) [2]. In this context it is worth mentioning that the term “protease” refers to both “endopeptidases” and “exopeptidases” while, the term “proteinase” only refers to “endopeptidases” [3].


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Proteases can be classified into six different classes based on the mechanism of catalysis: cysteine protease, serine protease, threonine protease, aspartic protease, glutamic protease and metalloproteases. Cysteine, serine and threonine proteases utilize Cys, Ser and Thr amino acid residue respectively as nucleophile to attack the peptide bond. The amino acids are located in the active site of the proteases and hence the names of the groups are coined accordingly. While the aspartic, glutamic and metalloprotease utilize an activated water molecule as a nucleophile to attack the peptide bond [4]. The metalloproteases have metallic ion in their active site. Proteases are involved in almost all aspect of plant growth and development [5]. The processes involving the mobilization of seed storage proteins during germination, initiation of senescence and programmed cell death [6], circadian rhythms and photo-morphogenesis during seedling development [7], all are influenced by plant proteases. Latex of several plant species from different families like asteraceae, asclepiadaceae, apocynaceae, caricaceae, moraceae and euphorbiaceae contain many important proteases that are used in industry [8]. Several physical, chemical and biological properties of proteins, like solubility, coagulation, emulsification and immunogenicity can fully be changed by the proteolysis process. The important characters of proteases have been exploited in industry and thus proteases get more importance in industrial sectors like in food, leather processing, detergent and pharmacological sector [9]. Among the several industrial use, the use of protease as a component of culture medium [10] and in isolating genetic material from living or stored tissue [11] are also reported. Another important character of plant protease that makes it more attractive is it’s stability over a wide range of temperature and pH. From the ancient time, plant extracts full with proteolytic enzymes have been in use in traditional medicine [12]. Proteases like papain (from Carica papaya) and bromelain (from Ananas comosus) have been used in different industrial processes including pharmaceutical process. An enzyme complexe of Ananas comosus, reported to be applied in food processing and also in therapeutic applications, contains bromelains [13]. Several other uses of plant proteases obtained from different sources are well reported [14]. Plant Protease/Proteinase Inhibitor On the other hand, protease inhibitors are such small proteins or peptides that fully or partially inhibit the proteolysis process of proteases. Similar to animals and microorganisms, plants also contain several different types of protease inhibitors. The widespread distribution of proteinase inhibitors throughout the plant kingdom is well known since 1938 [15, 16]. But most well studied proteinase inhibitors of plant origin are from three main families, leguminosae, graminae and solanaceae [17]. Although at the beginning, the proper function of the proteinase inhibitors in plant cell was unknown, but soon after the observation by Mickel and Standish (1947) [18] that some insect larvae were unable to grow on soybean products, it became clear that the proteinase inhibitors play role in plant defense. Studies revealed that the trypsin inhibitors present in soybean are toxic to the larvae of flour beetle, Tribolium confusum [19]. Later on in vitro assays with insect gut proteases [20] and further study by expressing protease inhibitor gene in transgenic plant [21] were conducted resulting in the conclusion that the proteinase inhibitors work against the insect gut proteases. Antifungal activity of plant protease inhibitor is also reported. Proteinase inhibitor from different plants and specially the trypsin inhibitor from potatoes can effectively neutralize the exogenous proteases found in the culture filtrates of phytopathogenic fungus Fusarium solani [22]. There are several examples citing the role of plant proteinase inhibitor in disease resistance. But controversies regarding their proper function also raised by the fact that the level of proteinase inhibitor (Inhibitor I) in Agrobacterium tumefaciens infected tissue of tobacco were increased while in the same condition, the amount of proteinase inhibitor in the tissues of tomato and potato remain unchanged [23]. It is well reported that the family solanaceae contain a wide range of proteinase inhibitors and in response to stimuli, they are well induced. Increase in the amounts of Inhibitor I in the leaves of young tomato and potato plants infected with Colorado potato beetles is well documented [24]. But


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surprising in case of Agrobacterium tumefaciens infection, their level does not increase and the reason behind this is unknown. There are several proposals regarding the possible function of plant proteinase inhibitors. A positive correlation between the levels of PIs and plant’s association with symbiotic bacteria like Rhizobium were drawn and the possible role of PIs in plant’s protection was concluded [25]. The protective role of proteinase inhibitor is helpful in protecting the seeds travelling through animals’ alimentary canal, from the digestive enzymes. This helps in endozoic dispersal of seeds [26]. Although there are several contrasting examples and arguments, but the significance of plant PIs in determining the nutritional value of the product is well reported. It was observed that experimental rats and chickens fed with soybean products or partially purified trypsin inhibitors, showed pancreatic hypertrophy and excessive enzyme secretion due to the metabolic disturbances [25]. The nutritive value of leguminous seeds is reported to improve after controlled heat treatment or during germination. This is correlated with the fact that by both the processes, the level of PI decreases and many PIs are inactivated [27]. But there is no concrete proof weather the increase in nutritional value is due to the elimination of other growth inhibitors excluding protease inhibitor [28, 29]. On the other hand, most of plant protease inhibitors are resistance to heat and this can easily be noticed from their extraction procedure in which the temperature are raised up to 100˚ C and the plant extracts are heated for considerable long time [30, 31]. Therapeutic potential of plant PI in several inflammatory disorders, allergy and pancreatitis is well documented [25]. This makes it commercially more important.

PLANT PROTEASE INHIBITOR FAMILIES Plant proteinase inhibitors are widely distributed and interestingly the inhibitors of all most all proteases occur in nature [32]. The molecular weight of plant PIs vary from 4 to 85 kDa and majority of them are in the range of 8 to 20 kDa [33]. Generally most of the PIs interact directly with the protease by binding to their active site, resulting in the formation of proteinase-inhibitor complex which lacks enzymatic property [34]. Although the mechanism of inactivation of proteases by PIs is not always the same, but one unique thing about their activity is their enormous functional diversity [35]. Plant PIs are generally classified according to their sequence similarity and named after the prominent member of the particular family. All plant PIs contain an inhibitory domain consisting of a few amino acids and are directly involved in inhibitory activity. The number of the inhibitory domain within a protease inhibitor sequence may be one or more than one. Based on the sequence similarity of the inhibitor domain, a number of total 48 different families of PIs have been reported [36]. Among the 48 families, member of nearly 31 PIs family contain more than one inhibitor domain and they are known to be the complex type of inhibitor, while the rests contain one inhibitory domain and are known to be the simple type of inhibitor. Some reports of classification of PIs into four different classes, named as cysteine, serine, aspartate and metallocarboxy PIs, are also available [37, 38]. A database containing the information about all the families of plant protease inhibitors, related genes and isoinhibitors available till date, is framed and coordinated by Luigi R Ceci at Centro di studio sui Mitocondri and Metabolismo Energetico-CNR, via Amendola 165/A, Bari, Italy [37]. Detailed informations including the sequences and domain structure of plant protease inhibitors are available in database that can be at http://www.plantpis.ba.itb.cnr.it. As according to De Leo et al., (2002) [37], a total of 10 families of plant PIs are available: Bowman-Birk serine protease Inhibitors, Soybean Trypsin Inhibitor (Kunitz), Mustard Trypsin Inhibitors, Cereal Trypsin/α-amylase Inhibitors, Potato type I Inhibitors, Potato type II Proteinase Inhibitors, Serpin, Squash Inhibitors, Cysteine Protease Inhibitors and Metallocarboxypeptidase Inhibitors. Due to the functional diversity of PIs, some families contain inhibitors which are also active against other mechanistic classes of proteases. For example, serpin family and kunitz type PIs. The former group members are active against both serine proteases and cysteine proteases, while the later one includes members that are active against serine, cysteine and aspartate protease [39, 40].


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Bowman Birk Inhibitors (BBIs) The name of the family is coined after D. E. Bowman and Y. Birk who identified and characterized the typical member of this family, the soybean inhibitor from soybean (Glycine max) [41, 42]. The members of this family are cysteine-rich proteins having inhibitory activity against a wide range of proteinases found in monocots as well as in dicots [43]. The inhibitors which are found in dicotyledonous plants are generally double headed with two homologous domains, each containing a separate catalytic site [44]. The active site is stabilized by seven disulfide bonds which are generally conserved in this group [45]. While the inhibitors found in monocotyledonous plants consist of a single polypeptide chain and may be in monomeric form containing one single catalytic site or may be in dimeric form with two catalytic sites [46, 47]. Odani et al., (1986) [48], suggested the origination of larger inhibitors from the smaller one by gene duplication. The representative members of this family are generally found in cereals, legumes [49, 50] and in grasses (members of poaceae family) [48]. Soybean inhibitor, sunflower trypsin inhibitor-1 [51] and peanut (Arachis hypogoea) inhibitor [52] are typical example of BBI. Soybean Trypsin Inhibitor (Kunitz) Kunitz-type PIs are widespread in nature and the most of the family members are reported from cereals, legumes and in several members of solanaceae family [53]. The inhibitors contain one catalytic site and the amino acids lining the catalytic site are reported to be arginine (R) and isoleucine (I) at position 63 and 64 respectively. Several experiments have been performed substituting the amino acids at positing 63 and 64 with other amino acids to check the significance of the presence of these two amino acids in active site. It has been reported that the exchange of amino acid isoleucine at position 64 either with alanine (I64A), leucine (I64L) or glycine (I64G) has no effect on the catalytic property of the inhibitor [54]. Although, replacing arginine at position 63 with lysine (R63K) has no effect on the catalytic property of the inhibitor, but if the arginine is replaced with tryptophan (R63W), then the protein turns to be a good inhibitor of chymotrypsin [26]. Insertion of amino acid residue like Ile, Ala, Glu in between the Arg (63)-Ile (64) pair, surprisingly eliminates the inhibitory property of the protein and instead of being a trypsin inhibitor, it becomes a trypsin substrate [54]. Stress inducible trypsin inhibitor found in potato tubers (Solanum tuberosum) [55, 56] and antifungal trypsin inhibitor found in the roots of punce ginseng (Pseudostellaria heterophylla) [57] are typical example of this class of inhibitors. Mustard Trypsin Inhibitors This type of proteinase inhibitors have been isolated and sequenced from the seeds of some members of the family cruciferae like mustard (Sinapis alba). These inhibitors are generally serine protease inhibitors rich in cysteine and glycine residues and have no structural similarity with other known families of plant serine protease inhibitors [58, 59]. Similar type of protease inhibitors have been isolated and characterized from rapeseed (Brassica napus) [60, 61]. This supports the presence of this type of trypsin inhibitors in cruciferae as a discrete group from the other serine proteinase inhibitors. The mustard trypsin inhibitor MTI2 expressed in the yeast Pichia pastoris and the recombinant protein was reported to be active against Spodoptera exigua gut proteases [62]. Cereal Trypsin/ Îą -Amylase Inhibitors Members of this family are widely distributed in most cereals like wheat, barley, rye, maize, ragi, sorghum, triticale, pearl millet, oats and several other species. Mostly all members of this family have only Îą-amylase inhibitory activity. But some members like inhibitors from barley (Hordeum vulgare) and rye (Secale cereale), also exhibit trypsin inhibitory activity [63]. A few reports are available regarding the dual inhibitory activity (both serine proteinases and Îąamylase inhibitory activity) of some inhibitors from maize (Zea mays) and ragi (Elusine coracana) [64, 65].


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Potato Type I Inhibitors Members of this family of inhibitors are generally monomeric, single headed type. The reactive site chemistry of this type of inhibitors has been studied in details. The reactive site contains the amino acids like leucine (LEU), methionine (MET), aspartic acid (ASP) and asparagine (ASN). The peptide bond in reactive site is reported to be LEU-ASN [66] and in some cases MET-ASP [67]. The members of this type of inhibitor are reported in potato tubers [68], tomato fruit [69] and also in squash [70]. Being widespread in nature, the inhibitors are also wound inducible as reported in tomato leaves [71]. Potato Type II Proteinase Inhibitors The members of this family of inhibitors are found in solanaceae family. The Proteinase inhibitor II, a potato type II proteinase inhibitor was isolated and characterized by Bryant et al., (1976) [72]. Potato proteinase inhibitors IIa and IIb were isolated and reported in Japanese potatos [73, 74] and they are different from the potato inhibitor II reported by Byrant et al., (1976) in amino acid composition and their iso-electric point are also different. This supports the hypothesis of the existence of intervarietal and intravarietal differences among potato tuber inhibitor II [72]. Other reports of this type of inhibitors from potato are available and one of such example is the potato inhibitors II from potato tubers [75]. Proteinase inhibitors of this family have also been found in tobacco leaves [76] and reported to be wound inducible. Constitutively expressed proteinase inhibitors of this type has also been reported in tobacco flower (Nicotiana alata) [77]. Serpin The name ‘serpins’ derived from serine protease inhibitor [78]. Mostly, all the members of this family inhibit serine proteases and some cysteine protease inhibitors and iso-inhibitors are also reported. Members of this family of inhibitor are widespread in nature [79]. They are reported to be present in green algae, the bryophytes, the pteridophytes (e.g. Picea glauca) [80], cereal seeds [81, 82], phloem sap of Cucurbita maxima [83] and other flowering plants from monocots and eudicots [79]. According to Fluhr et al., (2012) [80], Arabidopsis and rice (Oryza sativa cv. Nipponbare) genome contains eight and 14 genes encoding full-length serpins, respectively, while a single full length serpin gene is reported to be present in unicellular green alga, Chlamydomonas reinhardtii. Plant serpins are relatively large proteins (340–440 aa) [80] which usually forms a covalent, irreversible complexes with proteases. After the cleavage of the peptide bond of the target protease, it undergoes a rapid conformational change which stops the further steps in the catalysis process [84]. The insecticidal role of plant serpins is complex to understand. The fact that a variety of insects from the order Diptera and Lepidoptera use serine proteases as digestive enzymes [85] and these proteases are the target of plant serpins. It can then be postulated that serpins have role in plant defense. But the contrasting evidence comes from the observation of Yoo et al., (2000) [82]. The feeding of purified serpin, CmPS-1 from pumpkin (Cucurbita maxima) to the green peach aphid, Myzus persicae, showed no effect on the survival of the insect, instead the survival of the aphid and the level of the serpins were negatively correlated. On the other hand, Arabidopsis serpin, AtSerpin1 has been reported to have negative effect on the growth of Spodoptera littoralis [86]. Squash Inhibitors The squash inhibitors are the smallest one in size and the members are reported in cucurbit families. The inhibitors of this family generally contain 28 to 30 amino acids, cross-linked with three disulfide bridges with the average molecular mass of 3.0 to 3.5 kDa [87]. Proteinase inhibitors isolated from pumpkin seeds have been reported to be the inhibitors of bovine trypsin and Hageman factor [88, 89]. Inspired from this findings, several proteinase inhibitors from the plants of cucurbit family have been isolated and characterized. The members of this family have been reported from the


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seeds of summer squash, cucumber, zucchini [83, 90], watermelon, spaghetti squash, red bryony, figleaf gourd [90, 91], Momordica repens [92], wild cucumber (Cyclanthera pedata) [93], wax gourd [Benincasa hispida (Thumb) cogn] [94] and in several other plants of cucurbit family. The small size, structural rigidity and stability of the members of the squash inhibitor family serve as potential materials for studying serine protease-protein inhibitor interaction [95]. Cysteine Protease Inhibitors The members of this family inhibit the activity of cysteine proteases and are widely distributed in plants as well as in animals and microorganisms [96]. The members of the cysteine protease inhibitors have insecticidal activity and are involved in plant defence. Several natural and synthetic cysteine proteinase inhibitors have been reported to inhibit the cysteine proteinases isolated from insect larvae [97]. They are reported to be encoded by gene families [98] and exhibit differential expression pattern during development and in response to biotic and abiotic stresses [99]. According to Barrett et al., (1986) [100], the members of this family are subdivided into three classes: the stefins, the cystatins and the kininogens. Ritonja et al., (1989) [101] reported a new cysteine proteinase inhibitor, cathelin which inhibits cathepsin L and after this report it had been proposed that a separate family of cysteine protease inhibitors may exist. Another reported family of cysteine protease inhibitor is phytocystatins which has similar sequences like stefins and cystatins, but has no free cysteine residue [102, 103]. The stefin family of cysteine protease inhibitors has been reported to be a potent reversible and competitive inhibitor of cysteine proteases and lack the disulfide bonds and carbohydrates [104]. They are found within the cells as intracellular proteins and also in extracellular fluid [105]. The cystatin family of cysteine protease inhibitors is characterized to have two disulfide bonds without any carbohydrate group [106]. They contain some conserved sequences which are important in binding to the cognate proteases. These conserved sequences include the dipeptide sequence Phe-Tyr near N-terminus and Phe-Ala-Val near Cterminus end [107, 108]. The kininogen family of cysteine protease inhibitors contain carbohydrate group and this makes it different from the other two families. The molecular weight of this family of inhibitors is generally quite high. The members of this family contain domains produced by tandem repeats of gene sequences and are supposed to be evolved by gene duplication of the cystatins [103]. The family phytocystatins includes mostly all the cysteine protease inhibitors reported in plants. Majority of the members of this family are single domain proteins [109], but a few members with multiple domain structure are also reported [110]. The examples of the members of this family include oryzacystatin found in rice [111, 112], inhibitors isolated from soybean [113], maize [114], apple fruit [115] and several other monocotyledonous as well as dicotyledonous plants [109, 116]. Metallocarboxypeptidase and Aspartyl Protease Inhibitors The members of this family of inhibitors bind to metallocarboxypeptidases and have been identified in the plant family solanaceae. This type of inhibitors has been identified in tomato and potato plants [117]. The metallocarboxypeptidase inhibitors that are found in potato tubers have been reported to accumulate during development. They are also present in potato leaves along with potato type I and type II inhibitors and are also reported to be wound inducible [3]. This type of proteases inhibitors are reported to be active against carboxypeptidases found in animals and microorganisms, but has no activity against serine carboxypeptidases of plants and yeast [118]. The carboxypeptidase


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inhibitors isolated from potato (Solanum tuberosum), tick (Rhipicephalus bursa), leech (Hirudo medicinalis) and Ascaris (Ascaris suum) have been studied in detail. These small sized inhibitors contain several disulfide bridges and thus they are more stabilized. Besides, they show similar mode of inhibition [119, 120]. There are a few reports of aspartic proteinase inhibitors in plants as compared to serine and cysteine protease inhibitors. The cathepsin D inhibitor reported in potato tubers [121] is an aspartic protease inhibitor. It inhibits cathepsin D which is an aspartic protease, but surprisingly it does not inhibit other aspartyl proteases like pepsin, rennin, or cathepsin E. It also inhibits serine proteases like trypsin and chymotripsin and has considerable amino acid sequence similarity to soybean trypsin inhibitor [103]. Other examples include the protease inhibitors reported from barley [122], tomato [123], squash [124], sunflower [125], etc. Aspartic protease inhibitor reported from Anchusa strigosa [126] and squash inhibit pepsin which is a digestive aspartic protease.

PHARMACEUTICAL APPLICATIONS OF PLANT PI In animal and human tissues, the ratio of proteinases and proteinase inhibitors is always maintained in such way that they remain in a balanced equilibrium state. It has been observed that this equilibrium is disturbed in case of carcinogenesis, infection and in other pathological disorders. In several clinical disorders like skin lesions produced by fungi and bacteria, burn, eye infection and most importantly in carcinogenesis, the ratio of proteinase and their inhibitors is reported to be changed [127, 128]. There are several different routes for the development of cancer, but in all the cases the cellular biochemical and metabolic steps are changed leading to uncontrolled growth of the cells. Positive correlation between tumor progression and the amount of cellular proteinase is well reported. The invasion of cancer cells into surrounding tissues from the primary tumor mass requires the dissolution of cell membrane barrier and also the junctions between the cells and all these steps require the involvement of several proteases [129]. Trypsin, a member of serine proteases, activates matrix metalloproteases (MMPs) which are helpful in promoting the invasion and metastasis process [130, 131]. A positive correlation between the elastase level and mortality rate in breast cancer patient has been reported [132]. This indicates the possible role of elastase in tumor progression and metastasis in breast cancer [133, 134]. Hence inhibition of the protease activity by using protease inhibitors might be one of the possible ways to prevent carcinogenesis. The investigation to search for plant protease inhibitors to combat several clinical disorders like allergy, inflammatory disorders, started in early 1950’s [25]. The therapeutic potential of protease inhibitors were more clearly evidenced from the use of thrombin, plasmin and kallikrein inhibitors in blood clotting and fibrinolysis related to kinogen-kinin system [135]. The search for this type of inhibitors in plants began. Extensive research to find out possible candidates of protease inhibitors having therapeutic importance from plant has become an interesting field of study. One of the rich sources of plant protease inhibitors is the potato tubers. Among the other inhibitors reported from potato tubers, the inhibitors of human plasma kallikreins is the most important from therapeutic point of view. It has also been reported that the inhibitors present in potato tubers has weaker activity against the glandular kallikreins and other serine proteases [136]. The proteinase inhibitors isolated from cultivated cells of Scopolia japonica shows broad spectrum activity on proteases. The inhibitor has been reported to active against several different proteases like trypsin, chymotrypsin, plasmin, kallikrein and pepsin [137]. This is the only plant protease reported to be active against acidic protease like pepsin. Inhibitors of the pancreatic metalloexopeptidases, carboxypeptidases A and B have been isolated and characterized from potato tubers [120] as well as from ripe tomato fruit [138]. The mechanism of inhibition via the formation of inhibitor-enzyme complexes [139] and the cDNA sequence of the mature metalloexopeptidases inhibitors have also been reported from tomato fruit [140]. Plant seeds are rich source of protease inhibitors. It has been reported that the occurrence of breast, colon and prostate cancer rates are lower in


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population consuming higher amount of seeds like beans, maize and rice [141]. Similar reports of lower incidence of oral and pharyngeal cancers among populations with higher intake of cereals and bread products [142] as well as decreased rate of colorectal and breast cancers formation among individuals with higher intake of protease inhibitors from plant sources [143] are also available. Soybean and soybean products are rich in protease inhibitors and reports indicate the low occurrence of cancer and reduced mortality rate among populations consuming higher level of soybean products [144]. Although soybean and soybean products are rich in several anti-carcinogenic natural products like isoflavonoids, saponins and several other polyphenolic compounds [145], but the main component is the Bowman-Birk family of protease inhibitors that are more effective than the other anticancer compounds [146]. BBI from soybean has been reported to prevent radiation induced transformation in vitro [147], while the BBI isolated from chickpea prevents the malignant transformation of cells [148]. Several other properties of BBIs related to the anti-carcinogenicity have been review [149]. The mechanism behind the anti cancer activity of BBI has also been elucidated. BBI act on the MCF7 breast cancer cell and suppresses the proteasomal chymotrypsin like activity within the cell leading to the inhibition of cellular proliferation through accumulation of MAP kinase phosphatase-1 [150]. BBI from soybean is reported to inhibit M5067 ovarian sarcoma through enhanced rate of expression of tumor suppressor component connexin 43 [151]. BBI and BBI concentrate has also been reported to induce apoptosis in LNCaP prostate cancer cells [152] and rat colon cancer cells [153]. Recently reported cereal BBI has been shown to induce apoptosis in human colon cancer cells [154]. A member of Bowman-Birk protease inhibitor family, the Black-Eyed Pea Trypsin/Chymotrypsin Inhibitor isolated from Vigna unguiculata seeds has been reported to induce apoptosis and lysosome membrane permeabilization in human breast cancer cell MCF7 [155]. The member of the serpin family of plant protease inhibitors isolated from barley (Hordeum vulgae) inhibits trypsin and chymotrypsin and can also inhibits thrombin, plasma kallikrein, Factor VIIa and Factor Xa [156]. The trypsin inhibitor from sweet potato (Ipomoea batatas L.) is reported to have antiproliferative effect on NB4 promyelocytic leukemia cells and induces apoptosis involving p53, Bcl-2, Bax, and cytochrome c protein resulting in the activation of pathways of caspase-3 and -8 cascades [157]. Trypsin inhibitor isolated from Peltophorum dubium seeds and soybean kunitz type trypsin inhibitors have been reported to have anti-proliferative effect on human leukemia cells (JURKAT) and induce apoptosis in the particular cells [158]. Kunitz-type protease inhibitor from Bauhinia bauhinoides is reported to reduce the edema formation in isolated perfused rabbit lung [159]. Antifungal property of the protease inhibitor has also been reported. Kunitz-type trypsin inhibitor found in the roots of Pseudostellaria heterophylla has antifungal activity [57]. The subtilism inhibitor isolated from barley (Hordeum vulgare L cv. Kikaihadaka) is a good example of plant proteinase inhibitor active against microbial protease [160]. The buckwheat (Fagopyrum sculentum) protease inhibitor BWI-1, a member of the potato inhibitor family I can inhibit trypsin, chymotrypsin, and subtilisin, while another unique protease inhibitor BWI-2a can only inhibit trypsin. Both of these inhibitors have anti-proliferative effect on T-acute lymphoblastic leukemia cells, like JURKAT, CCRF-CEM and human normal blood lymphocytes. These two inhibitors induce apoptosis in these cells with DNA fragmentation [161]. The molecular mechanism of apoptosis induction by buckwheat trypsin inhibitor in the human solid tumor cells (EC9706, HepG2 and HeLa) with the loss in mitochondrial transmembrane potential and caspase activation has been reported [162]. Kobayashi et al., (2004) [163] reported that soybean kunitz type trypsin inhibitor suppresses the cellular invasion of ovarian cancer cell by urokinase upregulation [163]. Purified ragi (Elusine coracana) bifunctional inhibitor (RBI), a member of cereal Trypsin/Îą-amylase inhibitor family, is reported to reduce cellular proliferation and induced apoptosis of chronic myeloid leukemia cell, K562 [164]. Four different protease inhibitors isolated and purified from the seeds of Lavatera cashmeriana have been reported to be active against the pathogenic bacteria like Klebsiella pnuemoniae and Pseudomonas aeruginosa [165] and capable of inhibiting proteases like trypsin, chymotrypsin and elastase in vitro [166]. Further, their application as antitumor agents has been explored and


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the results shows that they are able to suppress the growth of several different cancer cell lines like THP-1, NCIH322, Colo205 and HCT-116 [167].

CONCLUSIONS There is no second opinion on the fact that protease inhibitors are the key players in regulating the protease activity. Proteases are needed in human body in optimum amount and any kind of imbalance in proteolytic activity can result severe pathological disorders. With the availability of the three dimensional structure of many proteases responsible for several pathological disorders, in silico structure based inhibitor designing techniques has been well developed. Many synthetic protease inhibitors are already in use for the therapy in several human diseases. Many pharmaceutical companies are taking keen interest and several inhibitors are in human trial. A large number of protease inhibitors have been isolated and characterized from different plants. The sequences and crystal structure of many of them are available. But, still only a few are used in medicine and are in clinical trial. The Bowman-Birk inhibitor is one of the most promising candidates, that is under human trial. There are several advantages of using protease inhibitors of natural source than the synthetic one. Plant protease inhibitors can also be supplied through diet (e.g. rice, legumes, soybean, etc.). Thus the opportunity of preventing the development of many diseases (e.g. cancer) can be achieved only by adding some extra plant based food preparations which will have no side effects in human body. This review illustrates the current status of plant protease inhibitors in pharmaceutical sector. Several scientific investigations till date, aiming to explore the pharmaceutical potential have been cited. The area is emerging and needs to be explored in more details. The present day knowledge supports the enormous potential of plant protease inhibitors in medicine. Continued research to find out more suitable protease inhibitors with strong efficiency leading to drug development may prove fruitful in near future.

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