Plant Seed Storage Proteins As Potential Precursors of Bioactive Peptides

POLISH JOURNAL OF FOOD AND NUTRITION SCIENCES Pol. J. Food Nutr. Sei. 1995, Vol. 4/45, No 3

PLANT SEED STORAGE PROTEINS AS POTENTIAL PRECURSORS OF BIOACTIVE PEPTIDES

Jerzy Dziuba, Piotr Minkiewicz, Kamila Puszka, Slawomir D~browski1

Department of Food Biochemistry, Olsztyn University of Agriculture and Technology, Olsztyn; 1Depaitment of Microbiology, Gdari.sk Technical University, Gdari.sk

Key words: antihypertensive peptides, antithrombotic peptide:;, bioactive peptides, immuno modulating peptides, proteolysis, seed storage pro•;eins

Amino acid sequences ofproteins taken from the SWISS-PROT database were analysed using the computer programme PROTEIN searching for fragments identical to bioactive peptides in chains ofplant se,id protein and localizing bonds susceptible to enzymatic proteolysis. Sequences of:proteins from barley (Hordeum vulgare), rice (Oryzae sativa), sorghum (Sorghum bicolor milo), oat (Avena sativa), soybean (Glycine max), pumpkin (Cucurbita maxima), sunflower (Helianthus annuus) and vetch (Vicia pannonica) were analy:ied. Fragments with potential antihypertensive activity were present in proteins of all these plants with the exception ofvetch. Fragments with potential immunomodulating (pumpkin and sunflower) and antithrombotic (vetch) activity were also found. Proteolytic enzy rnes may liberate bioactive peptides from plant pr,Jteins. Inmost cases the action of two enzymes is necessary. The need occurs ei;pecially in the case of protein hydrolysis by prolyl endopeptidases ( EC 3.4.2U!6) or alkaline proteinase from Tritirachium called proteinase K (EC 3.4.21.14) and intracellular proteinase from Streptococcus thermophilus (EC 3.4.24.4). Proteolytic enzymes of the digestive tract, such as chyrnotrypsin (EC 3.4.21.1), tryp~,in (EC 3.4.21.4), elastase (EC 3.4.21.36) or pepsin (EC 3.4.23.4) can also take part in the liberation of some active fragments.

INTRODUCTION

Plant seeds, especially cereal seeds are one ofthe most important sources of protein [Anantharaman & Finot, 1993]. Utilization of proteins as food components, requires knowledge ofphysico-chemical and functional properties. The properties

Author's address: Jerzy Dziuba, Zaklad Biochemii iywnosci, Wydzial Technolgii iywnosci, Akademia Rolniczo -Techniczna, PI. Cieszynski 1, 10-718 Olsztyn 5 32 Jerzy Dziuba et al. of cereal [Tatharn et al., 19901 and soybean [Barraquio & van de Voort, 1988] proteins are best known. Proteolysis is an indispern,ible step of protein digestion in the alimentary tract [Britton & Koldovsky, 1989]. Enzymic hydrolysis is also an important part of many technological processes applied in food industry [Voragen, 1989; Arai & Fujimaki, 1991]. Some peptides being products ofprotein hydrolysis show biological activity, e.g. antihypertensive, immunomodulating, opioid and others. Milk proteins are best known precursors ofbioactive peptides [Paroli, 1988; Ariyoshi, 1993; Fiat et al., 1993]. Plant proteins arE known as precursors of opioid [Paroli, 1988] and antihypertensive [ Ariyoshi, Hl93] peptides. The knowledge ofbioactive protein sequences and possibility ofliberation of these fragments by proteinases of the digestive tract and also by other proteolytic enzymes may be useful in establishing a diet and designing technological production of food with desired biological properties. Computer methods are used more frequently and more often as support for experiments in the area of protein structure and function. The knowledge of amino acid sequences allows to model physico-chemical and biological properties ofpeptides and proteins [Belton, 1993; Johnson et al., 1994; Mewes et al., 1994]. Computer programs have been applied, e.g. to determine the localization of bioactive fragments in protein chains. The aim of the study was to determine the localization of sequences with potential biological activity in the peptide chains of selected plant seed storage proteins and to try to predict theoretically the possibilities ofliberation ofthese fragments by proteolytic enzymes.

METHODS

Amino acid sequences of plant seed storage proteins were taken from the SWISS-PROT database. Our own "PROTEIN" computer program was applied to localize bioactive peptides in protein sequences. The programme compares primary structures of bioactive peptides (over 250) treated as standard peptides and protein primary structures and searches for fragments with amino acid sequences identical to the sequences ofthese peptides in protein chains. lt also searches for the sites susceptible to the action ofproteolytic enzymes with determined specificity and gives sequences ofpredicted proteolysis products. The program contains a list ofproteolytic enzymes [Arai & Fujimaki, 1991] and database of amino acid sequences of peptides and proteins. The list of proteolytic enzymes is given in Table 1, ofproteins analysed - in Table 2 and of bioactive peptides - in Appendix. The investigations were made by using IBM Computer 386DX (SVGA-512 graphic board and hard disc Seagate 340 MB). Bioactive sequences uf plant proteins 33

Table 1. Proteolytic enzyme data used in program

Enzyme EC Number Specificity*

Chymotrypsin 3.4.21.1 Y-, W-, F-, L-

Trypsin 3.4.21.4 K-, R-

Prolyl endopeptidase 3.4.21.26 P-

Elastase 3.4.21.36 G-, A-, V-, L-, I-

Ficin 3.4.22.3 A-, Y-, G-, N-, L-, V-

Bromelain 3.4.22.4 K-, A-, Y-, G-

Pepsin 3.4.23.4 F-, L-

Proteinase from 3.4.24.4 -L, -F, -Y, -A Streptococcus thermophilus

Proteinase K 3.4.21.14 V-, L-, I-, F-, Y-, W-, P-, M-

* - Amino acid sequences are presented in single-letter code: A -Ala, C - Cys, D - Asp, E - Glu, F - Phe, G - Gly, H - His, I - Ile, K - Lys, L - Leu, M - Met, N - Asn, P - Pro, Q - Gin, R - Arg, S- Ser, T-Thr, V - Val, W -Trp, Y -Tyr; (X-) : Bonds formed by carboxylicgroup ofamino acid, hydrolysed by enzyme; (-X): Bonds formed by amine group of amino acid, hydrolysed by enzyme

RESULTS

The localization ofpotentially bioactive sequences in the chains ofplant seed storage proteins is presented in Table 2. Amino acid sequences and activity of particular fragments are given in Table 3. Thirteen fragments with sequences identical to bioactive peptides have been found in peptide chains ofthe proteins analysed. Twelve ofthem contain three amino acid residues. One bioactive fragment is a tetrapeptide. Ten fragments found correspond with antihypertensive tripeptides which are inhibitors of angiotensin-converting enzyme (EC 3.4.15.1). The enzyme catalyses hydrolysis ofinactive peptide - angiotensin I to angiotensin II which acts as a vasoconstrictor and causes an increase in blood pressure [Fiat et al., 1993; Ariyoshi, 1993]. Nine out of ten antihypertensive tripeptides contain C-temtinal proline residues. The 34 Jerzy Dziuba et al.

Table 2. Localization of bioactive fragrnents in plant seed storage proteins

Active fragments Plant; protein sequence; localization 1 Barley (Hordeum vulgare); LQP; 110-112, PYP; 17-19, LVL; y-hordeina (sequence 305 AA), 252-254 [Cameron-Mills & Brandt, 1988]

Rice (Oryza sativa); prolarnin (136 AA), LSP; 30-32 clone PPROL 4A [Kirn & Okita, l!J88]

Rice (Oryza sativa); prolarnin (135 AA), LSP; 30-32 clone PPROL 7 [Kirn & Okita, 1988]

Rice (Oryza sativa); prolarnin (13~, AA), LSP; 24-26 clone PPROL 14 [Kirn & Okita, Ul88]

Rice (Oryza sativa); prolarnin (134 AA), LSP; 30-32 clone PPROL 17 [Kirn & Okita, 1988]

Sorghum (Sorghum bicolor milo); kafirin IPP; 18-20 PG K 1 (284 AA) [De Rose et al., 1989]

Sorghum (Sorghum bicolor milo); kafirin LQP; 216-218

PSKR 2 (246 AA) [De Rose et al., 1989] LPP; 18-20

Sorghum (Sorghum bicolor milo); kafirin IPP; 18-20 PSK 8 (248 AA) [De Rose et al., 1989]

Oat (Avena sativa); 12S globulin SSG 1 LSP; 60-62 in basic chain (518 AA), [Schubert et al., 1990]

Oat (Avena sativa); 12S globulin SSG 2 LSP; 60-62 in basic chain (494 AA), [Schubert et al., 1990]

Soybean (Glycine max); 7S globulin (403 AA), VAP; 47-49inlightsubunit [Kagawa & Hirano, 1989)

Pumpkin (Cucurbita maxima); II subunit of EAE; 42-44 in y-chain;LSP; 174-176 in llS globulin (459 AA), [Hayashi et al., 1988] ö-chain; VAP; 60-62 in ö-chain

Sunflower (Helianthus annuus); llS globulin VRP; 250-252 in acidic chain, LL Y; (4 73 AA), [van der Haar et al., lll88] 71-73 in acidic chain, LRP; 54-56 in basic chain, FAP; 174-176 in basic chain

Vetch (Vicia pannonica); fragrnent 1-17 of KRDS; 12-15 narbonin (MW 33500), [Schlesier et al., 1992]*

* -The sequence was not taken from the SWISS-PROT database. Bi.oactive sequences ofplant pmteins 35

Table 3. Characteristics ofbioactive fragments ofplant seed storage proteins

Sequence Activity Re ferences

LQP An tihypertensi ve Miyoshi et al., l!l91 a; Ariyoshi, 1993*

pyp Antihypertensive Maruyama et al., 1987; Meise!, 1992; Fiat et al., 1993'; Ariyoshi, 1993*

IPP An tihypertensi ve Kohmura et al., 1990; Fiat et al., 1993*; Ariyoshi, 1993*

LPP An tihypertensi ve Maruyama et al., 1989; Ariyoshi, 1993*

LVL An tihypertensi ve Hazato and Kase, 1986; Ariyoshi, 1993*

VAP An tihyperten si ve Maruyama et al, 1987; Meise!, 1992; Fiat et al., 1993*; Ariyoshi, 1993*

VRP An tihypertensi ve Kohmura et al., 1990; Fiat et al., 1993*; Ariyoshi, 1993*

LRP Antihypertensive Miyoshi et al., 1991a; Ariyoshi, 1993*

LSP An tihypertensi ve Miyoshi et al., 1991a; Ariyoshi, 1993*

FAP Antihypertensive Maruyama et al, 1987; Meise!, 1992; Fiat et al., 1993"; Ariyoshi, 1993*

EAE Immunomodulating Mokotoff et al., 1990

LLY Im munomodulating Migliore-Samour et al., 1992

KRDS An tithrom botic Mazoyer et al., l 990; Wu et al., 1992; Fiat et al., 1993"

* - Review

EAE immunomodulating tripeptide is a fragment of thymosin-a1 (residues: 25-

27). This tripeptide shows activity similar to the activity of whole thymosin-a1 particle. lt stimulates the secretion of lymphokines such as a- and y-interferones, T-cell growth factor and migration inhibiting factor [Mokotoff et al., 1990]. The LLY tripeptide stimulates phosphoinositide metabolism and oxidative processes in leucocites [Migliore-Samour et al., 1992]. The KRDS tetrapeptide is an inhibitor of platelet aggregation and also an inhibitor of serotonin release [Mazoyer et al., 1990; Wu et al., 1992]. 36 Jerzy Dziuba et al.

Table 4. Potentialities for liberation of bioactive peptides from plant seed storage proteins by proteolytic enzymes

Enzymes• Protein - fragment 1 Nt - Str.th. proteinase Barley g-hordein - LQP Ct - Proteinase K*, prolyl endopeptidase, Str.th. proteinase

Nt - Str.th. proteinase, proteinase K*, elastase*, Rice prolamin, clone PPROL 4A - LSP ficin*, chymotrypsin* Rice prolamin, clone PPROL 7 - LSP Ct - Str.th. proteinase , proteinase K*, prolyl Rice prolamin, clone PPROL 17 - LSP endopeptidase

Nt - Str.th. proteinase, proteinase K* Rice prolamin, clone PPROL 14 - LSP Ct - Proteinase K*, prolyl endopeptidase*

Nt - Chymotrypsin, proteinase K*, pepsin Sorghum kafirin PGK 1 - IPP Ct - Proteinase K*, prolyl endopeptidase Sorghum kafirin PSK 8 - IPP

Nt - Chymotrypsin*, proteinase K*, pepsin* Sorghum kafirin PSKR 2 - LPP Ct - Proteinase K*, prolyl endopeptidase, Str.th. proteinase

Nt - Proteinase K*, Str.th. proteinase, elastase* Basic chainofoat llS globulin SSG 1 Ct - Proteinase K*, prolyl endopeptidase - LSP Basic chainofoat llS globulin SSG 2 -LSP

Nt - Proteinase K*, Str.th. proteinase, elastase*, 8 Chain of ß subunit of purnpkin llS ficin* Globulin - LSP Ct - Proteinase K*, prolyl endopeptidase

Nt - Proteinase K* yChainofßsubunitofpumpkin llS Ct - Proteinase K*, prolyl endopeptidase Globulin - V AP

Nt - Elastase*, ficin*, bromelain* yChain ofß subunit ofpurnpkin llS Ct - Str.th. proteinase Globulin - EAE

Nt - Proteinase K*, elastase* Acidic chain of sunflower l lS Ct - Proteinase K*, prolyl endopl,ptidase Globulin - VRP

Nt - Str.th. proteinase* Acidic chain of sunflower llS Ct - Proteinase K*, ficin*, bromelain* Globulin - LLY

Nt - Str.th. proteinase Basic chain ofsunflower llS Ct - Proteinase K*, prolyl endopeptidase Globulin - LRP

Nt - Proteinase K*, elastase*, ficin, Str.th. Basic chain of sunflower 1 lS proteinase, chymotrypsin*, pepsin* Globulin - F AP Ct - Proteinase K*, prolyl endopeptidase

• Nt- enzyme hydrolyses bond at the N-terminus ofbioactive fragment; Ct- enzyme hydrolyses bond at the C-terminus of bioacti':e fragment; * - enzyme may hydrolyse bonds within the bioactive fragment. Bioactive sequences ofpw.nt proteins 37

Theoretical possibilities ofliberation ofbioactive fragments from plant seed storage proteins are presented in Table 4. Bioactive peptides, LQP, LSP, IPP, LPP, VAP, EAE, VRP, LRP, FAP and LLY, can be liberated from these proteins by several proteolytic enzymes (Table 1) with the exception ofbioactive fragments of proteins from soybean (SVAPF) and vetch (VKRDSP). Most peptides can be liberated by joint action of more than one enzyme. The greatest possibilities of liberation of antihypertensive peptides (up to five peptides) have been found for: proteinase K (EC 3.4.21.14) , prolyl endopeptidases (EC 3.4.21.26) and intra cellular proteinase from Streptococcus thermophilus (E!C 3.4.24.4). Some of the enzymes (e.g. chymotrypsin, elastase, proteinase K, fi .cin) may also hydrolyse bonds within bioactive fragments.

DISCUSSION

Plant seed storage proteins contain bioactive fragments. Some short fragments of chains may be repeated in many plant, animal and human proteins even if homology between these proteins is low. Especially angiotensin-converting enzyme inhibitors are broadly proliferated. The antihypertensive peptides, (LQP, FAP, LSP, IPP, LPP, LRP, VAP, VRP) contain C-terminal proline residues. Apart from many important physiological roles reviewed by Williamson (1994] proline-rich proteins or protein fragments can be precursors of antihypertensive peptides. Such bioactivity ofknown proline -rich food proteins was reviewed by Ariyoshi (1993]. All the prolamins analysed are potential precursors of antihypertensive peptides. Proteins belonging to the group of globulins contain fragments with different activity. Bioactive fragments may be liberated by proteolytic enzymes. Most of the enzymes considered hydrolyse bonds formed only by amine group ofN-terminal amino acid or carboxylic group of C-terminal amino add ofbioactive fragment. The liberation of antihypertensive fragments from plant seed storage proteins requires thejoint action ofmore than one enzyme. Proteolytic enzymes specific for the carboxylic groups ofproline residues play an important role in the liberation of antihypertensive fragments [Maruyama et al., 198fi; Ariyoshi, 1993]. On the other hand, many proline-containing peptides may be inhibitors of the action of such enzymes [Asano et al., 1991, 1992; Maruyama et al., 1992]. According to theoretical predictions based on specificity [Arai & Fujimaki, 1991], most of the enzymes presented should hydrolyse bonds within bioactive fragments, but there are some experimental results showing that bonds within bioactive peptides may be resistant to proteolysis. Oral administration of enzymatic hydrolysates of cx-zein [Miyoshi et al., 1991 b] or fermented milk [Yamamoto et al., 1994 a, b] influenced a decrease in rat blood pressure. lt can be stated that antihypertensive peptides from a-zein (Sequences: LQP, LRP and LSP) and milk (e.g. sequences: IPP, VAP, VRP and LRP) contain bonds theoretically susceptible to proteolysis by enzymes ofthe digestive tract such as chymotrypsin, trypsin, elastase or pepsin. 38 Jerzy Dziuba et al.

The results by Miyoshi et al. [1991 b] and Yamamoto et al. [1994 a, b] show that antihypertensive substances can pass from the digestive tract to blood. These components should be resistant to the action of digestive enzymes. One of the explanations of this phenomenon is an assumption that proteinases of rat digestive tract do not hydrolyse antihypertensive peptides. The possibility ofliberation of bioactive plant fragments depends probably on the relative hydrolysis rate of peptidic bonds at the N- and C-terminus and also within these fragments. The protein chain folding and hence the accessibility of particular bonds for particles of proteolytic enzymes may be another factor affecting the kind of proteolysis products.

CONCLUSIONS

1. Plant seed storage proteins can be a source of bioactive peptides such as EAE, FAP, IPP, LLY, LPP, LRP, LSP, LVL, VAP, VRP and KRDS. 2. The active sequences are only tripeptides with antihypertensive or immuno modulating activity and tetrapeptide with antithrombotic activity. 3 • The methods of computer analysis of amino acid sequences guarantee accurate localization of bioactive peptides in protein chains. At the present stage, specificity ofproteolytic enzymes does not allow for such accurate theoretical predictions ofliberation ofparticular protein fragments. Results concerning the possibilities ofliberation of bioactive peptides can be a valuable support for experimental investigations but they cannot replace the experiment itself.

REFERENCES

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Received February, 1995. Revision received and accepted April, 1995.

APPENDIX The list ofbioactive peptides inserted into program database

The appendix covers the names or sequences ofpeptides. Peptide sequences are given in single-letter code (See Table 1). l. Blood pressure-regulating peptides 1.1 Angiotensins and their derivatives: human angiotensin I; (V5)-angiotensin I; des-D 1-angiotensin I; human angiotensin II; (V5)-angiotensin II; (N1,V5) angiotensin II; APG-(P ,V5 )-angiotensin II; human angiotensin III; (!7) angiotensin III; (V4)-angiotensin III; (V4,I7)-angiotensin III 1.2 Antihypertensive peptides (Inhibitors of angiotensin-converting enzyme): CE! 5; CE! 6; CE! 12; fragments of human ß-casein: 43-52 and 63-65; LRP; LSP; LQP;FGK; FPQVFGK; FFVAP; VAP; DVAP; FAP; AAP; PFPQ; A VPYPGR; AVP; PYP; IKPLQY; YQ(;!PVLGPVR; PAQLPWGSSQV; GHLIATYQER; !KP; Bioad:ive sequences ofpl.ant pmteins 41

GLLIATYQER;A VNPIR; LYPVK; LVR; LHLPLP; 'I'AP; YAQPAVVRP; VRP; IPP; MYY; FVAP; PSKIKWGD; VHLPPP; VHLPP; LPP; PLIYP; SPQPQPLIYP; LKVGKQY; VKAGF; LVLAGM; LVL; HQAAGW; PANIKWGD; LKL; PTHIKWGD

2. Neuropeptides 2.1 Endorphins and their derivatives: human a-endorphin; human ß-endorphin; R-ß-endorphin; fragment of human ß-endorphin: 1-27; (L5)-ß-endorphin; human y-endorphin; des-Y1-y-endorphin; a-neoendorphin; ß-neoendorphin; kyotorphin; adrenorphin; L-enkephalin; des-Y1-L-enkephalin; L-enkephalin R; L-enkephalin-K; M-enkephalin 2.2 Exorphins: cow's ß-casomorphins: 1-4, 1-5, 1-7, 1-8, 1-9; human ß• casomorphins: 1-4, 1-5, 1-7, 1-8, 1-9; fragments of cow's as1-casein: 90-96, 194-199; cow's ß-lactorphin; fragments ofa-lactalbumin (a-lactorphins): 50- 53, 51-53 2.3 Fragments of cow's K-casein (casoxins): 35-41, 58-61, 25-34 2.4 Fragments ofsubstance P: 1-4, 1-7, 1-9 2.5 Human endothelins: big endothelin 2; big endothelin 38; endothelin l; endothelin 2; endothelin 3 2.6 Bradykinin and related peptides: kinin T; kallidin; K-(A3)-bradykinin; MK bradykinin; Y-bradykinin; (K1)-bradykinin; ('1'6)-bra.dykinin; (T8)-bradykinin; des-R9-(L8)-bradykinin; des-R9-bradykinin; fragments of bradykinin: 1-5, 1- 6, 1-7, 2-7 2.7 Tuftsin 2.8 Kentsin 2.9 Catacalcin and Y-catacalcin

3. Chemotactic peptides: RGSE; MLF; VGDE; VGSE

4. Peptides affecting blood coagulation 4.1 Fragment ofhuman fibrin: 1-4 4.2 Fragments ofhuman fibrinogen: 400-411; 572-575 4.3 Fibrinopeptides: fibrinopeptide A; (E 1)-fibrinopeptide B 4.4 Antithrombotic fragments of milk proteins: fragments of cow's k-casein: 106- 116, 106-112, 113-116; fragment ofhuman lactotransferrin: 39-42 4.5 Fibrin polymerization inhibitor: GPRP 4.6 Fragments of hirudin: 54-65, 55-65 4.7 Fragments offibronectin: RGD; RGDSPASSKP 4.8 Fibronectin analogue: GRGDTP

5. Immunomodulating peptides 5.1 Immunomodulating peptides from caseins: fragments of cow's ß-casein: 54- 59, 60-62, 63-68, 191-193; fragments of human ß-casein: 1-18, 54-59, 60-62, 105-117 42 Jerzy Dziuba

5.2 Fragments ofCD4 protein: 25-58, 37-53 5.3 Lymphocyte-activating pentapeptide: LPPSR 5.4 Peptides regulating phosphoinositide metabolism in lymphocytes: GLF; LLY; GFW;LGW

5.5 Fragments ofthymosin-a1 : 25-27, 25-28, K-24-28

6. Others 6.1 Human adrenocorticotropic hormone and its fragments: 1-4, 1-10, 1-13, 1-14, 1-16, 1-17, 1-24, 4-9, 4-11, 7-38, 11-24, 18-39, Y-4-9 6.2 Fragment ofinsulin B chain 22-30 6.3 Melanocyte-stimulating hormones: -ß, -y, -ö 6.4 Somatostatin and its derivatives: Y-somatostatin; (Y)-somatostatin; (Y11 )- somatostatin 6.5 Human urodilatin 6.6 Fragment of gonadotropin-liberating hormone 14-26 6. 7 Corticostatin 6.8 Fragments ofhuman parnthyroid hormone: 1~34, 1-38, 1-44, 13-34, 28-48,44- 68, 68-84

BIAIKA ZAPASOWE NASION ROSLIN JAKO POTENCJALNE PREKURSORY BIOARTYWNYCH PEPTYDOW

Jerzy Dziuba, Piotr Minkiewicz, Kamila Puszka, Slawomir D~browski 1

Zaklad Biochemii Zywnosci, Akademia Rolniczo-Techniczna, Olsztyn, 1Zaklad Mikrobiologii, Politechnika Gdanska, Gdansk

Sekwencje aminokwasowe bialek pochodz11ce z bazy danych SWISS-PROT analizo wano za pomoc11 wlasnego programu komputerowego PROTEIN, wyszukuj11cego w sekwen cjach aminokwasowych bialek odcinki identyczne z sekwencjami bioaktywnych peptyd6w oraz miejsca podatne na dzialanie enzym6w proteolitycznych. Analizowano sekwencje bia lek pochodz11cych zji;czmienia, ryzu, sorga, owsa, soi, dyni, slonecznika oraz wyki. W bial kach wymienionych roslin (opr6cz wyki) wysti;puj11 sekwencje o potencjalnym dzialaniu przeciwnadcisnieniowym. Ponadto stwierdzono wysti;powanie fragment6w o dzialaniu im munomodulacyjnym (dynia oraz slonecznik) a takze przeciwkrzepliwym_ (wyka). W szystkie znalezione bioaktywne sekw•~ncje (poza jednym wyj11tkiem - przeciwkrzepliwym tetra peptydem z wyki, KRDS) byly tripeptydami (EAE, FAP, IPP, LLY, LPP, LRP, LSP,LVL, VAPi VRP). Te bioaktywne peptydy mog11 byc uwalniane z badanych bialek przez enzymy proteo lityczne. Taka mozliwosc istnieje szczeg6lnie w przypadku hydrolizy bialek przez proteinaz~ 8tr.th. (EC 3.4.24.4), endopeptydaz(; prolilow11 (EC 3.4.21.26) a takze proteinazi; K (EC 3.4.21.14). W uwalnianiu niekt6rych peptyd6w mog11 uczestniczyc enzymy przewodu pokar mowego takie, jak chymotrypsyna (EC 3.4.21.1), trypsyna (EC 3.4.21.4), elastaza (EC 3.4.21.36) oraz pepsyna (EC ,L4.23.4) a takze inne proteinazy, takie jak katepsyna B (EC 3.4.22.1) i D (EC 3.4.23.5), ficyna (EC 3.4.22.3) i bromelaina (EC 3.4.22.4)