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Current Medicinal Chemistry, 2016, 23, 893-910

eISSN: 1875-533X ISSN: 0929-8673 Current

Impact Factor: Food Proteins as Source of -A Review 3.85 Medicinal Chemistry

The International Journal for Timely In-depth Reviews Swati Garg, Kulmira Nurgali and Vijay Kumar Mishra* in Medicinal Chemistry

BENTHAM SCIENCE

College of Health and Biomedicine, Victoria University, PO Box 14428, Melbourne, Victoria 8001, Australia

Abstract: Traditional , mainly , have been used in the clinical management of pain for a number of years but are often associated with numerous side-effects including sedation, dizziness, physical dependence, tolerance, addiction, nausea, vomiting, constipa- tion and respiratory depression which prevent their effective use. Opioid peptides derived from food provide significant advantages as safe and natural alternative due to the possibility of their production using animal and plant proteins as well as comparatively less side-effects. This review aims to discuss the current literature on food-derived opioid peptides focusing on their produc- tion, methods of detection, isolation and purification. The need for screening more dietary proteins as a source of novel opioid peptides is emphasized in order to fully understand their potential in pain management either as a drug or as part of diet complementing therapeutic prescription. Keywords: Opioids, , opioid-receptors, , , fermentation.

1. INTRODUCTION dicinal effects are predominantly due to the presence of polyphenols, antioxidants, probiotics, tannins, polyun- Food provides energy and essential nutrients to the saturated fatty acids or bioactive peptides. body in the form of carbohydrates, proteins, fats, vita- mins and minerals which are necessary for proper Bioactive peptides are inactive within native pro- growth, development and functioning of the body. Im- teins but exert physiological functions upon release proper eating habits and lifestyle changes increase the from animal or plant proteins (dairy, , vegeta- risk of a number of disorders, including obesity, heart bles, meat and their products) during gastrointestinal disease, hypertension, cancer, osteoporosis and arthri- (GI) digestion and/or after food processing. They may tis. Currently available clinical treatments are often exhibit , anti-oxidant, anti-thrombotic, anti- associated with serious side-effects. Search for natural, hypertensive, anti-cancerous or immune-modulatory healthy and safe alternatives with minimum side- activity thus benefiting cardiovascular, nervous, diges- effects is required for beneficial effects to patients and tive and immune systems (Fig. 1) [3-8]. Hence, food improving quality of life. Nutraceuticals are promising can contribute to health, modulate immunity and pre- in this regard, as they are derived naturally from food, vent and help in clinical management of specific dis- hence are part of diet and should have minimal side- eases. It may be possible to use these peptides as part effects. The term “nutraceutical” is coined from the of complementary therapy along with other drugs. words "nutrition" and "pharmaceutical", and is defined Most commonly clinically used drugs for pain man- as "a food (or part of a food) that provides medical or agement are opioids such as and [9- health benefits, including the prevention and/or treat- 12]. However, they are often associated with side- ment of a disease [1]. “Let food be thy medicine and effects like sedation, dizziness, nausea, vomiting, con- medicine be thy food”, quoted by Hippocrates stipation, physical dependence, addiction, tolerance and 2,500 years ago is certainly the need of today [2]. respiratory depression [13, 14]. Food-derived opioid These foods have potential health benefits beyond ba- peptides possess weaker activity making them less sic nutrition and are called functional foods. These me- likely to cause side-effects and are relatively inexpen- sive to produce. Herein we summarize research on *Address correspondence to this author at the College of Health and Biomedicine, Victoria University, PO Box 14428, Melbourne, opioids, their receptors and classification with particu- Victoria 8001, Australia; Tel: +61 3 99198130; lar emphasis on food-derived opioid peptides, methods E-mail: [email protected] of production, detection, isolation and purification from

1875-533X/16 $58.00+.00 © 2016 Bentham Science Publishers Current Medicinal Chemistry Current 894 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al.

anti-h ypertensive

Milk and dairy products anti-oxidative Cardiovascular system

anti-thrombotic

Eggs hypocholesterolemic

Nervous

Meat and opioid system meat products Bioactive peptides

cytomodulatory

Immune system Cereals immunomodulatory

anti-microbial

Digestive Vegetables system mineral-binding

anti-apetizing

Fig. (1). Effects of food-derived bioactive peptides on various system. food, as well as their fate after digestion in order to and KOP receptors exert effects mainly in fully understand their potential use. peripheral tissues [9]. Activation of these receptors by opioid ligands leads to change in Ca++ and K+ channel 2. OPIOID RECEPTORS conductance and protein phosphorylation via inhibition Opioid receptors belong to the superfamily of G- of cyclic AMP (cAMP) [11]. cAMP acts as a second protein coupled receptors and are distributed within the messenger activating protein kinases and gene tran- central and peripheral nervous system [15, 16]. Interna- scription which can result in effects including analge- tional Union of Basic and Clinical Pharmacology Re- sia, respiratory depression, euphoria, release of hor- ceptor Nomenclature Committee (NC-IUPHAR) ap- mones, and reduction of GI transit [21]. proved nomenclature for receptors as μ 3. CLASSIFICATION OF OPIOID LIGANDS (mu or MOP), δ (delta or DOP) and κ (kappa or KOP) [15]. A fourth opioid peptide receptor is selectively Opioid ligand is any substance that binds specifi- activated by endogenous ligand, and termed cally to to produce morphine like ef- as NOP (nociceptin opioid peptide) and it is not an- fects and the activity is reversed by non-specific an- tagonized by unlike the other three receptors tagonist, naloxone [12]. Opioids were initially re- [15]. All of these receptors have close structural ho- stricted to be alkaloids by nature (morphine, codeine mology and are members of one family of proteins [17- and ), but now peptides having opioid activity 20], with differences between the receptor types arising (, , exogenous peptides) have also due to gene duplication events during evolution [15]. been identified (Fig. 2). The affinity for the specific receptors influences the differences in activity of opioids [11]. Activation of 3.1. Opioid Alkaloids MOP receptors mediates the most potent antinocicep- Naturally occurring opioids are obtained from tive effects, however, they are prone to induce depend- poppy plant, , and include ence; DOP receptors have lower efficacy in pain relief; morphine and structurally related alkaloids including Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 895 codeine, noscapine (narcotine), thebaine, and papaver- tanyl, meperidine, , ) and diphen- ine [12]. Out of them, morphine and codeine are most ylheptanes (propoxyphene, ) [11]. widely used opioid . Morphine is an ligand primarily binding to µ receptors and has less 3.2. Opioid Peptides affinity for δ and κ receptors [9]. Codeine has weak Opioid peptides were first identified in 1975 when affinity for µ opioid receptors and its analgesic activity met-enkephalin and leu-enkephalin from brain were is attributed to its principal metabolite, morphine [22, found to have opioid activity [24]. Further investiga- 23]. Semisynthetic opioids are produced from naturally tions confirmed that , and ex- occurring alkaloids and include , hydromor- ogenous peptides from food have also opioid activity phone, , and . which opened a new era in the history of opioid ligands Synthetic opioids are chemically synthesized in the [25-29]. Majority of peptide ligands are opioid laboratory from compounds unrelated to natural alka- except casoxins and lactoferroxins which are opioid loids and include , or methadone (Fig. antagonists [30, 31]. Opioid peptides can be classified 2) [12]. into endogenous or exogenous based on their origin as These opioid ligands have different affinity described below (Fig. 2). (strength of interaction) and efficacy (strength of activ- 3.2.1. Endogenous Opioid Peptides ity) against opioid receptors and are classified into agonist, partial agonist or antagonist based on their ac- Endogenous opioid peptides are produced naturally tion [16]. While an agonist has both affinity and effi- in the body and may function as hormones (secreted by cacy (morphine, and fentanyl); an an- gland and delivered to target tissues) or neuromodula- tagonist has affinity, but no efficacy (naloxone and tors (secreted by nerve cells and act in the central and ); and a partial agonist (agonist/antagonist) peripheral nervous system) [9, 32, 33]. These peptides has affinity, but only partial efficacy (, include β-endorphin, dynorphins and , , , ) (Fig. which are hydrolyzed by peptidases from precursor 3) [11]. proteins, pro-opiomelanocortin, pro-dynorphin and pro- By chemical structure, opioid alkaloids are classi- enkephalin respectively [9, 29, 34]. These peptides fied into phenanthrenes (morphine, codeine, hydro- have conserved YGGF sequence at their N terminus morphone, , oxycodone, buprenorphine), and are also called as typical opioid peptides (Table 1) benzomorphans (pentazocine), phenylpiperidines (fen-

Opioids

Alkaloids Peptides

Natural (morphine, Endogenous Exogenous codeine, noscapine, thebaine, papaverine)

Natural (casomorphins, Natural (enkephalins, gluten , Semisynthetic (heroine, dynorphins, endorphin) hydromorphone, soymorphins, ) oxymorphone, hydrocodone, oxycodone)

Semisynthetic Semisynthetic analogues analogues (, (DADLE, CTOP, DAGO, DPDPE ) valmuceptin, β-casorphin, α- Synthetic (fentanyl, lactorphin, casoxin-5 ,-6 and tramadol, methadone) lactoferroxin A, B, C)

Fig. (2). Classification of opioid ligands. 896 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al.

a) Agonists

CH3 N CH3 N CH3 N R H R H H R H H R R R

S S S RS R S RS H3 C O HO OH O H OH O H AcO O H OAc

morphine codeine heroin

CH3 N

R H

N R

S N R

HO O H O O

hydromorphone fentany1

b) Antagonists

N N

R OH R OH H S S

S S

R R

HO O O H HO O H O

naloxone naltrexone

Fig. (3). Chemical structures of opioid alkaloids

Table 1. Endogenous opioid peptides.

Opioid peptide Protein precursor Amino acid sequence Opioid receptor References selectivity

β-endorphin pro-opiomelanocortin YGGFMTSEKSQTPLVTLF µ [25] KNAIILNAYKKGE met-enkephalin pro-enkephalin YGGFM δ>> µ [24, 35, 160] leu-enkephalin YGGFL δ>> µ pro-dynorphin YGGFLRRIRPKLKWDNQ κ>> µ,δ [29, 35] YGGFLRRQFKVVT nociceptin /orphanin FQ pro-nociceptin/OFQ FGGFTGARKSARKLANQ ORL [161] -1 pro-endomorphin YPWF-NH2 µ [36] endomorphin-2 YPFF-NH2 µ

[9]. Apart from these, endomorphin-1 and -2 which ases by incorporation of unnatural amino acids fol- lack conserved YGGF sequence have also been identi- lowed by cyclization [14, 37-40]. Changes in leu- fied as endogenous opioids [35, 36] (Fig. 4). enkephalin (δ selective) by substitution and cyclization These endogenous peptides have been modified into have modified it to analogues which are µ selective semisynthetic analogues using amino acid substitution, with known analgesic effects [41]. Modifications of addition, deletion, cyclization or hybridization of two leu-enkephalin by amino acid substitution, addition and ligands to incorporate conformational constraints and deletions have resulted in several agonists with en- make them more potent to be used as clinical analge- hanced δ receptor selectivity [9]. Extensive research sics [9, 14]. have been modified into has been done to synthesize analogues with desired analogues which have increased stability against prote- characteristics which are not discussed in this review, Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 897

a) Endogenous Opioids

H H S N O S N S O H3 C

HO O HO O

O O O O H H N N S S S N N N N S H H H H

NH2 O NH2 O HO HO

met-enkephalin leu-enkephalin

H O N S

HO H O NH2 OH N NH2 NH2 O S O S O S NH N H N N S S S N H O S O O NH2

endomorphin-1 endomorphin-2

b) Exogenous opioids

HO H O N NH2 S

O S O H N O OH N N O O S S N S H H N S O S N N OH H H O N H O NH2 O OH

b -5 gluten exorphin b5

H O N S HO

NH2 O NH O HO O OH S O HO NH S 2 H N O N O S S S N O S H O OH N O N S S H S N H O

soymorphin-5 rubiscolin-5

Fig. (4). Chemical structures of opioid peptides. however, this information is available in more recent Majority of these atypical opioid peptides contain tyro- reviews [14, 42]. sine residue at the amino terminal end and another aromatic amino acid at the 3rd or 4th position (Fig. 4) 3.2.2. Exogenous Opioid Peptides [31]. This structural motif fits into the binding site of Exogenous peptides are hydrolyzed from animal or opioid receptors [30, 44]. However, peptide lacking plant precursor proteins and are also called as atypical at amino terminus RYLGYLE from bovine α- opioid peptides since they carry various amino acids at casein, has also shown opioid activity [30, 45]. Thus their N terminus with conserved tyrosine residue [9, far, it has been identified that animal protein derived 43]. They were discovered in 1979, and term exorphin opioid peptides bind to µ receptors and those from was used for food-derived peptide for the first time plant proteins to δ receptors [46] except, soymorphins. (exogenous origin and morphine like activity) [28]. This is further described below. 898 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al.

3.2.2.1. Animal Proteins as Source of Opioid Peptides. muceptin (βh-casein 51-54 amide, YPFV-NH2), β - Dietary proteins from animals (milk and their prod- casorphin (βh-casein 41-44 amide, YPSF-NH2) and α- ucts, egg, poultry and fish) have been a source of bio- lactorphin (αh-lactalbumin 50-53 amide, YGLF-NH2) active peptides and opioid peptides have been identi- and β-casomorphin-4 and β-casomorphin-5 amides [55, fied in milk and dairy products (Table 2). 56]. Lactoferroxin A (YGSGY-OCH3), B(RYYGY- OCH3) and C(KYLGPQY-OCH3) are the opioid an- Milk contains caseins (α, β and κ casein) and whey tagonists derived from methyl esterified peptic digest proteins (albumin and globulin). Hydrolysis of these of human lactoferrin [57]. proteins either inside or outside the body produces pep- tides that may have opioid activity. Depending on the Studies have identified release of β h-casomorphin protein they are derived from, opioid peptides have from human milk during different gestational stages. been named α-casein exorphins or casoxin D (α- Large precursor fragments of β h-casomorphin (51–73, casein), β -casomorphins or β-casorphin (β-casein), ca- 48–73 and 35–73) were present both in preterm and soxins or casoxin A, B, or C (κ-casein), α-lactorphins normal term milk but the shorter sequences (including (α-lactalbumin), β-lactorphin (β-lactoglobulin) or lac- βh-casomorphin–8) were only detected in milk from toferroxins (lactoferrin) [30, 31, 47]. women delivering after normal gestation of 39 and 40 weeks [58]. The concentration of β h-casomorphin-5 In 1979, milk was proposed to have opioid activity and βh-casomorphin-7 in colostrum was higher than in [28, 48] and morphine like substance was isolated form mature milk [59]. These studies confirm that the size of milk at concentrations of 200 to 500 ng/L [49]. Opioid the peptide decreases with an increase in gestation and activity was due to the presence of peptides corre- lactation periods [21, 58, 59]. sponding to f60-66 (YPFPGPI, β-casomorphin-7) of β- casein [50] and f90-96 (RYLGYLE) and f90-95 Differences between genetic variants of β-casein (RYLGYL) of α-casein [45]. Sequence corresponding have also been investigated. Three genetic variants to f91-96 (YLGYLE) and f91-95 (YLGYL) amino acid (A1, A2 and B) of β-casein were tested for release of residues also showed opioid activity and RYLGYLE βb-casomorphin-7 on simulated GI digestion, and con- being the most potent [45]. centration of peptide released was more from A1 and B variants as compared to A2 variant [60, 61]. This could β-casomorphin-7 (YPFPGPI), corresponding to f60- be explained due to the difference between variants at 66 of bovine β-casein (βb-casein) was the most potent position 67; being histidine in A1 and B and proline in opioid peptide among β b-casomorphin-6, -5 and -4 A2 [62]. Peptide bond between proline and isoleucine [51]. Based on primary structure of human β-casein has more enzymatic resistance than that between his- (βh-casein) and sequence comparison with βb-casein, 10 tidine and isoleucine which gets hydrolyzed by GI en- residue shifted alignment relationship and 47 % iden- zymes, resulting in the release of β-casomorphin-7 tity have been revealed [52, 53]. Accordingly, βh- from A1 and B variants [63]. casomorphin-4, -5, -6 and -8 with Tyr-Pro-Phe- amino- The effects of administration of these opioid pep- termini were tested for opioid activities [52]. β h- casomorphin-7 (YPFVEPI) was found at position 51- tides in different doses in various animal models are presented in Table 4. It is difficult to compare the doses 57 and have Val-Glu in position 4-5 as opposed to βb- casomorphins-7 which was found at position 60-66 and and effects as different animal models and routes of administration were used by various researchers. β- have Pro-Gly [43, 53]. However, both βh-casomorphins casomorphins have shown analgesic activity in mice and β b-casomorphins bind particularly to µ receptors, with highest affinity for µ receptors and lowest for κ which is reversed by naloxone, thus confirming that the effects are mediated by opioid receptors [64, 65]. receptors [53]. β h-casomorphin-4 and -5, have higher Intraperitoneal administration of β-casomorphins-5 and affinity to δ receptors than corresponding β b- casomorphins (Table 3) [53]. Enzymatic digestion of -7 has positive effects on learning and memory [66, human κ-casein/α-casein complex released opioid an- 67]. Orally administered opioid peptides from milk tagonist peptide casoxin D (YVPFPPF) corresponding stimulated release of postprandial pancreatic polypep- tide [68]. β-casomorphins stimulate release of soma- to f158-164 of αS1-casein [30, 54]. tostatin and insulin [69, 70]. They have shown to pro- A number of opioid peptide analogues have been long gastrointestinal transit time [71-73] and modulate identified by amidation or esterification (by methyl intestinal mucus secretion [74-76]. Apart from these group) of carboxyl terminal of the peptide such as effects on central and peipheral nervous system medi- morphiceptin (β-casein amide, YPFP-NH2), val- ated via opioid receptors, β-casomorphins-7 increased Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 899

Table 2. Exogenous opioid peptides from food.

Opioid peptide Protein precursor Amino acid se- Opiod Agonist(A)/ Reference quence receptor Antago-

Source Source selectivity nist(AN)

βb-casomorphin-4 βb-casein (60-63) YPFP µ A [51] βb-casomorphin-5 βb-casein (60-64) YPFPG µ A βb-casomorphin-6 βb-casein (60-65) YPFPGP µ A βb-casomorphin-7 βb-casein A2 (60-66) YPFPGPI µ A

βb-casomorphin-8 βb-casein (60-67) YPFPGPIP/H µ>δ>>κ A [30, 162]

αb-casein exorphin (1-7) αb-casein (90-96) RYLGYLE µ/δ<<κ A [30, 45] αb-casein exorphin (2-7) αb-casein (91-96) YLGYLE µ/δ A

casoxin A κb-casein (35-41) YPSYGLN µ>>δ,κ AN [163] casoxin B κb-casein (57-60) YPYY µ AN casoxin C κb-casein (25-34) YIPIQYVLSR µ AN

neocasomorphin-6 βb-casein (114-119) YPVEPF µ A [63]

morphiceptine βb-casein YPFP-NH2 µ>>δ>>κ A [164, 165]

αb-lactorphin αb-lactalbumin (69-72) YGLF-NH2 µ A [30, 166] βb-lactorphin βb-lactoglobulin (118-121) YLLF-NH2 µ A

βh-casomorphin-4 βh-casein (51-53) YPFV µ>δ>κ A [30, 52, 53] βh-casomorphin-5 βh-casein (51-55) YPFVE µ>δ>κ A Animal protein βh-casomorphin-7 βh-casein (51-57) YPFVEPI µ>δ>κ A

βh-casomorphin-8 βh-casein (51-58) YPFVEPIP µ>δ>κ A [30, 53]

casoxin D α S1-casein (158-164) YVPFPPF µ/δ AN [54]

αh-casomorphin α h-casein (158-162) YVPFP µ/δ<<<κ A/AN [167] αh-casomorphin amide αh-casein (105-111) YVPFP- NH2 µ<<δ/κ A/AN

βh-casorphin βh-casein (41-44) YPSF-NH2 µ A [30, 55] valmuceptin βh-casein (51-54) YPFV-NH2 µ A αh-lactorphin αh-lactalbumin (50-53) YGLF-NH2 µ A

lactoferroxin A lactoferrin(318-323) YLGSGY-OCH3 µ AN [57] lactoferroxin B lactoferrin (536-540) RYYGY-OCH3 µ AN lactoferroxin C lactoferrin (673-679) KYLGPQY- µ AN OCH3 gluten exorphin A5 HMW glutenin GYYPT µ<<<δ A [46, 81] gluten exorphin A4 HMW glutenin GYYP µ<<δ A gluten exorphin B5 HMW glutenin YGGWL δ A gluten exorphin B4 HMW glutenin YGGW δ A gluten exorphin C HMW glutenin YPISL µ<δ A [46, 82] rubiscolin-5 rubisco (103-108) YPLDL δ A [87] rubiscolin-6 rubisco (103-109) YPLDLF δ Plant proteins proteins Plant soymorphin-5 soy β-conglycinin (323-327) YPFVV µ A [93] soymorphin-6 soy β-conglycinin (323-328) YPFVVN µ A soymorphin-7 soy β-conglycinin (323-329) YPFVVNA µ A plasma insulin and superoxide dismutase and catalase tem and have role in regulation of blood sugar in dia- activities in diabetic rats, thus, protecting them from betics (Table 4). There are also reports indicating that hyperglycaemia and free radical-mediated oxidative β-casomorphin-7 stimulates the intake of high-fat diet stress [77]. In most cases, casomorphins have signifi- and blocks the inhibitory effect of enterostatin on high- cant effects on the central and peripheral nervous sys- fat intake leading to obesity [78, 79]. 900 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al.

Table 3. Opioid activity of peptides derived from food proteins.

Opioid Peptide Opioid activity (IC50 in µM) µ/δ ratio References Guinea-pig ileum Mouse vas deferens

gluten exorphin A5 1000 60 60.7 [81] gluten exorphin A4 >1000 70 - [81] gluten exorphin B5 0.05 0.017 2.9 [81] gluten exorphin B4 1.5 3.4 0.44 [81] gluten exorphin C 110 30 3.7 [82]

βb-casomorphin-7 57 >200 <0.29 [51]

βb-casomorphin-6 27.4 >150 <0.18 [51]

βb-casomorphin-5 6.5 40 0.16 [51]

βb-casomorphin-4 22 84 0.26 [51]

βh-casomorphin-4 19 750 0.025 [55]

βh-casomorphin-5 14 nd - [55]

βh-casomorphin-6 25 350 0.071 [55]

βh-casomorphin-8 25 540 0.047 [55] rubiscolin-5 1110 51 21.8 [87] rubiscolin-6 748 24.4 30.7 [87] soymorphin-5 6 50 0.12 [93] soymorphin-6 9.2 32 0.2875 [93] soymorphin-7 13 50 0.26 [93] nd- not determined

3.2.2.2. Plant Proteins as Source of Opioid Peptides. consolidation process of learning and memory [46, 83]. Despite a large variety of available dietary proteins Gluten exorphin B5 stimulated prolactin secretion [84, from plants, release of opioid peptides has been re- 85] and gluten exorphin C increased exploratory ported in gluten from wheat, D-ribulose-1,5- activity, decreased anxiety and improved learning bisphosphate carboxylase/oxygenase (RUBISCO) from (Table 4) [86]. These studies suggest the potential spinach, and β-conglycinin from soyabean. effect of gluten exorphins on the central and peripheral nervous system. Gluten Exorphins Rubiscolin Gluten is the storage protein of wheat and consists of two different proteins, gliadin and glutenin. The Rubiscolin-5 (YPLDL) and -6 (YPLDLF ) peptides presence of opioid peptides was first described in pep- identified in spinach RUBISCO have opioid activity tic digest of wheat gluten based on adenylate cyclase attributed to the presence of YP at amino end. The IC50 and mouse vas deferens (MVD) assays [28, 80]. These values of rubiscolin-5 and -6 using δ receptor binding peptides were identified as gluten exorphins A5 assay were 2.09 µM and 0.093 µM, respectively. These (GYYPT), A4 (GYYP), B5 (YGGWL), B4 (YGGW) are selective for δ opioid receptor with significant antinociceptive activity [87]. Rubiscolin–6 enhances and C (YPISL) and showed lower IC50 in MVD as compared to guinea-pig ileum (GPI) assay, (Table 3) memory consolidation [88], exerts anxiolytic [89] and [81, 82]. So, these gluten exorphins bind specifically to orexigenic effects [90, 91] and supresses high-fat intake (Table 4) [92]. δ opioid receptors and B5 is most potent having IC50 value 0.017 µM in MVD among the known sequences Soymorphins [46]. The associated effects of the use of gluten exor- Soymorphins are synthesized from spinach β- phin A5 are mild antinociception and facilitation of the conglycinin. Soymorphin-5 (YPFVV), -6 (YPFVVN), Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 901

Table 4. Dose-effects relationship of opioid peptides in animal models

Opioid Peptide Dosage Animal Route of ad- Effect Reference model ministration

β-casomorphin-5 0.166–166 nM rat i.v analgesic, naloxone reversible [64] β-casomorphin-4,- 60–2000 nM rat i.c.v analgesic, naloxone reversible [65] 5,-6,-7 β-casomorphin-5 1–100 mg/kg rat pups i.p no effect on walking, decreased active [168] sleep at a dose of 100mg/kg β-casomorphin-7 0.1–20 nM rat i.c.v stimulated food intake of high fat meal [79] β-casomorphin-7 1mg/kg albino rat i.p negative effect on passive avoidance [67] pups conditioning β-casomorphin-5 1mg/kg mice i.p improves learning and memory [66] β-casomorphin-7 7.5 × 10−8 diabetic rats oral increased plasma insulin and superoxide [77] mol/day dismutase and catalase activity gluten exorphin A5 30 & 300 mg/kg mice oral no antinociception and morphine analge- [169] 30 & 300 i.c.v sia effects µg/mouse mild antinociception but no effect on morphine analgesia gluten exorphin B5 3 mg/ kg rats i.v stimulated prolactin secretion [85] gluten exorphin C 5mg/kg i.p increased exploratory activity, decreased [86] anxiety, and improved learning rubiscolin-5 3 nM/mouse mice i.c.v antinociception [87] rubiscolin-6 1 nM/mouse rubiscolin–6 100 mg/kg mice oral enhanced memory consolidation [88] 3 nM/mouse i.c.v rubiscolin–6 0.3–1 mg/kg mice oral stimulated food intake (orexigenic effect) [170] in non-fasted mice rubiscolin–6 1 mg/kg mice oral supressed high fat intake [91] soymorphin-5 10-30 mg/kg mice oral anxiolytic effect [93] soymorphin-5 30 mg/kg mice oral suppressed food intake and small intesti- [94] nal transit soymorphin-5 10 mg/kg mice oral increased β-oxidation and energy expendi- [95] ture soymorphin-5amide 5 mg/kg rats i.p stimulated locomotion in adults and [96] decreased anxiety in juvenile rats i.c.v- intracerebroventricular; i.p- intraperitoneal; i.v- intravenous; nM- nano mol and -7 (YPFVVNA) were found to have lower IC50 4. PRODUCTION OF OPIOID PEPTIDES FROM value using GPI assay compared to those obtained by FOOD PROTEINS MVD assay [93]. They were selective for the µ opioid Food proteins themselves do not have opioid activ- receptor and have shown anxiolytic activities (Table 4) ity and therefore need to be hydrolyzed to release pep- [93]. Soymorphin-5 suppresses food intake and small tides that have opioid activity through enzymes in the intestinal transit [94], increases β-oxidation and energy GI tract or during fermentation [4, 5, 97]. To assess expenditure [95]. Analogue synthesized by amidation their formation during GI digestion, GI enzymes are of soymorphin-5 (YPFVV-NH2), stimulated locomo- required. Hence, fermentation and enzymatic hydroly- tion in adult rats and decreased anxiety in juvenile rats sis is a requirement for the production of opioid pep- [96]. These activities have shown potential of soymor- tides. phin-5 in management of obesity, diabetes and anxiety [94-96]. 902 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al.

4.1. Fermentation lactoferroxin A were also detected in semi-hard cheeses (Edamski, Gouda and Kasztelan) along with β- Fermentation is used for the production of yogurt, casomorphins [106]. These studies suggest possibility sauerkraut, kimchi, salami, bread and beverages (wine, of formation of opioid sequences from food proteins beer, cider). Depending on the type of fermentation, fermented by fungus as compared to bacteria because proteolytic enzymes of the microorganism contribute to of specificity of enzymes present within them. the release of peptides which may have opioid activity. Fermentation of milk by Pseudomonas aeruginosa 4.2. Enzymatic Hydrolysis or Bacillus cereus leads to formation of β-casomorphin Enzymatic hydrolysis uses GI enzymes (pepsin, immunoreactive material [98]. Enzymatic (pepsin and trypsin, chymotrypsin and pancreatin) and microbial trypsin) proteolysis of Lactobacillus(L.) GG fermented enzymes thermolysin and alcalase to determine produc- milk released opioid sequences (YPFP, RYLGYLE, tion of opioid peptides. Opioid activity was detected in YGLF, YPFPGPIPNSL) [99]. Whey fermentation by pepsin hydrolysate of food proteins [28]. Further hy- Kluyveromyces marxianus var. marxianus released drolysis of pepsin hydrolysate with other enzymes in- YLLF which corresponds to β-lactorphin [100]. β - creased opioid activity. Gluten exorphins A5, A4, B5 casomorphin-4 was detected in milk fermented with and B4 were identified in pepsin-thermolysin hydro- PepX-deficient mutant strains of L.helveticus L89 and lysate [81] and gluten exorphin C in pepsin-trypsin- absent in milk fermented with wild strain [101]. This chymotrypsin hydrolysate [82]. Concentration of gluten suggests possible degradation of β-casomorphin by the exorphin A5 was higher in hydrolysate prepared with Pep X (X-prolyl dipeptidyl aminopeptidase) which di- pepsin-elastase (250 µg/g) than pepsin-thermolysin (40 gests the peptide bonds between proline and other µg/g) [108]. Recently, gluten exorphins A5 (0.747– amino acid residues [102]. 2.192mg/kg) and C (3.201–6.689 mg/kg) were detected Yoghurt fermented with L. delbrueckii ssp. bulgari- in bread and pasta after simulated in vitro GI digestion cus and S. salivarius ssp. thermophiles has been source (using pepsin-trypsin-chymotrypsin) [109]. Opioid ac- of β -casomorphin precursors corresponding to f57-68 tivity was detected in the hydrolysates produced by and f57-72 [103], but not β-casomorphin. This can be alcalase (6 hr) and pepsin (24 hr) with IC50 values of due to incapability of these starter cultures to hydrolyze 1.21 and 1.57 mg/ml, respectively and not in hydro- β-casein into β-casomorphin. Moreover, the stability of lysate produced by protamex and neutrase [110]. Thus, β-casomorphin in yoghurt may be influenced by the release of these gluten exorphins has been confirmed symbiotic growth of L. delbrueckii ssp. bulgaricus and during GI digestion. Similarly, release of casomorphins S. thermophilus. β-casomorphin may be released by L. after simulated GI digestion by various enzymes have delbrueckii ssp. bulgaricus protease activity but are been studied in milk and its products [107] and differ- degraded by S. thermophilus, and vice versa. ences have been detected in genetic variants of β-casein Earlier studies were not able to detect β- [60-62]. casomorphin-7 in Brie and Cheddar which could be 5. DETERMINATION OF OPIOID ACTIVITY due to lower threshold of analytical techniques em- ployed for detection of these peptides [104]. It was also Opioid activity of the hydrolysate can be tested by proposed that β-casomorphins are degraded in these using one of the several avaialable assays. Commonly cheeses during ripening as suggested by the fact that used assays for testing opioids from food are naloxone- enzymes derived from L. lactic ssp. cremoris can re- reversible inhibition of adenylate cyclase activity duce concentration of β-casomorphin-7 by 50 % (pH [111], naloxone-reversible inhibition of electrically 5.0 and 1.5 % NaCl) after 6-15 weeks [104]. stimulated contraction of isolated organ preparation The presence of β-casomorphin-7 was first detected either GPI or MVD [112], receptor binding assay or in Brie [105]. Its concentration ranged 6.48 µg/g [106] radioreceptor assay [113, 114]. to 0.15 µg/g [107]. β-casomorphin-7 was also detected Opioid receptors belong to the family of G protein- in the range of 0.01 - 0.11 µg/g in Gorgonzola, Gouda, coupled receptors and their activation results in Fontina and Cheddar cheeses. Upon in vitro simulated adenylate cyclase inhibition, K+ channel activation or GI digestion the concentration increased up to 15.22 Ca2+ channel inactivation [115, 116]. Inhibition of µg/g and 21.77 µg/g in Cheddar and Gouda cheeses, adenylate cyclase has been frequently used for respectively [107]. Along with β-casomorphin (opioid identification of opioid peptides from wheat gluten and agonist), opioid antagonists, casoxin-6, casoxin-C and α-casein [28, 45, 46]. Binding of opioid ligand to Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 903 opioid receptors in hybrid cells (tranformed with co-elution of peptides with similar physico-chemical specific opioid receptor) or brain membrane inhibits and spectrophotometric absorption properties. adenylate cyclase which decreases intracellular cAMP To overcome the sensitivity problem during identi- which can be detected through availabale asays [111, fication and quantification, HPLC has been coupled to 115, 11]. mass spectrometry such as tandem mass spectrometry, In addition, opioid activity of the peptide involves quadrupole ion-trap mass spectrometry (QIT-MS) and binding of ligand to specific opioid receptor present on time of flight mass spectrometry (TOF-MS). Using GPI longitudinal muscle myenteric plexus or MVD tandem mass spectrometry several potentially bioactive preparations [9, 10]. These tests are based on inhibition peptides, including β-casomorphin-7 have been identi- of electrically evoked contractions of the GPI and fied from cheddar cheese prepared using a neutral pro- MVD and have been used for determination of opioid tease produced from Bacillus subtilis [122]. It has been activities of food-derived peptides (Table 3). The used for detection of β-casomorphin-9 in water-soluble opioid effect in the GPI and MVD preparations are extracts of an Italian goat cheese [123], gluten exor- mediated by µ and δ receptors, repectively. Thus these phins A5 and B5 in cerebrospinal fluid [124, 125] and tests are used to screen µ and δ receptor, ligands from gluten exorphins B4 and B5 in blood [126]. It has been different sources [9, 28]. used for detection of β-casomorphins -5 and -7 from The receptor binding assays may involve saturation infant milk and casein variants [60] and dairy products and competition studies. While, in the saturation after simulated GI digestion [60]. QIT-MS is sensitive, binding studies, the affinity of different compounds to easy and time efficient technique that allows structural opioid receptors is characterized, competition studies elucidation of peptides. This technique gives informa- can be performed subsequently or independently to tion about the major product ions obtained which can confirm these results [9]. Binding assays require the be used to construct fragmentation pathways and has use of radiolabeled ligands, which are expensive, may been used for detection of β-casomorphin in cheese and not be available for the receptors being studied and milk [127]. TOF-MS has high resolving power and is generate radioactive waste that needs to be carefully highly accurate technique for identification and con- discarded. Also, molecules that will allosterically firmation of unknown compounds in a complex matrix. change the binding of a chosen compound will not It combines elemental formula information from high necessarily lead to an increase in the signaling potency mass accuracy experiments with structural information [116]. Moreover, data obtained in different laboratories from fragmentation experiments. It has been used for are not comparable due to the differences in detection of β-casomorphins in the water-soluble ex- synaptosomal preparations, the concentration, type of tract of a matured Gouda cheese [128] and opioid radioligand used, and the method of reporting binding. fragment from bovine β-casein [129, 130]. Apart from Thus, bioassay involving adenylate cyclase and tissue these chromatographic techniques, enzyme–linked im- preparation offer certain advantages over receptor munosorbent assay (ELISA) is more precise and spe- binding assays since they are less expensive, more cific for detection of low quantities of peptide from feasible and have minimum impact on environment. different sources. It has been used for determination of β-casomorphin -7 and -5 from cheese [106] and milk 6. IDENTIFICATION AND DETECTION OF [131]. OPIOID PEPTIDES Thus, HPLC coupled to ranges of spectrometry Reversed-phase high performance liquid chroma- technologies can be used for identification and purifi- tography (RP-HPLC) is one of the most frequently cation of opioid peptides. However, more advance- used techniques for identification and purification of ments in analytical techniques are needed to enable bioactive peptides [117-121] because it is well- researchers to screen complex food materials with con- established, relatively inexpensive, and user-friendly. It fidence as interferences from constituents can lead to has also been used widely for identification and separa- misleading results. tion of β-casomorphins [59, 63, 101, 104]. Despite 7. IN SILICO APPROACH FOR PREDICTING these advantages, RP-HPLC is comparatively less sen- PRESENCE OF OPIOID SEQUENCES sitive to detect the relatively small amount of opioid peptide present in food and it can also lead to overesti- In order to screen bioactive peptides, protein is hy- mation of the peptide concentration [104] because of drolyzed and hydrolysate is tested for the presence of desired bioactivity, followed by isolation and purifica- 904 Current Medicinal Chemistry, 2016, Vol. 23, No. 9 Garg et al. tion of peptide and the whole process is time consum- and A4 can cross the intestinal epithelium [109, 141- ing and tedious. Moreover, hydrolysates will also have 145]. However, the mechanisms of transfer of opioid additive and synergistic effect of various components peptides across the intestinal epithelium have not been [132]. So, there is need for more precise and direct clearly established. Transportation of DADLE (a syn- methods for identification of bioactive peptides. Re- thetic opioid) occurs through sodium coupled intestinal cently, computer-based simulation has been used for transporter [146]. It has been reported that opioid pep- predicting the presence of bioactive peptides in food tides are not transported by translocation across cellular proteins [133-136]. In this approach, bioinformatics membranes [147], diffusion or paracellular pathways peptide databases BIOPEP and PEPBANK are used to [148]. predict the presence of bioactive peptides in the pri- Intestinal absorption of opioid peptides has been mary sequence of food protein, which can be obtained studied using in vitro systems and in vivo animal mod- from databases UniProtKB, SwissProt and TrEMBL els. In vitro systems use monolayer of Caco-2 cell line [133]. Proteolysis tools such as BIOPEP, ExPASy Pep- or intestinal mucosa mounted in Ussing-type chambers. tideCutter (http://web.expasy.org/peptide_cutter) and Umbach et al. have shown the presence of β- PoPS (http://pops.csse.monash.edu.au) are then used to casomorphin-7 immunoreactive material in the plasma find the specificities of various enzymes to liberate de- of newborn calves after milk intake. Morphiceptin acti- sired peptide from food. This approach has been used vates opioid receptor only when added to serosal side for prediction of converting enzyme inhibi- of rabbit ileum as compared to mucosal addition due to tory peptides [137, 138] and dipeptidyl peptidase-IV mucosal degradation of morphiceptin by di-peptidyl- inhibitors [135, 139] from different food proteins and peptidase-IV (DPP-IV) and pre-treatment of ileum with confirmed using in vitro experiments. protein diisopropylfluorophosphate (inhibitor of DPP-IV) pre- (wheat, oat, barley and rice) sequences clearly show vents mucosal degradation [149]. The trans-epithelial high occurrence frequencies of peptides having angio- transport of μ opioid receptor agonists, human β- tensin-converting enzyme-inhibitory, dipeptidyl pepti- casomorphin -5 and -7 and antagonist, lactoferroxin A dase-inhibitory, anti-thrombotic, anti-oxidant, hypoten- has been confirmed [148]. sive, and opioid activities [140]. Yet the sequences cor- responding to opioid peptides and their bioactivity need Food-derived opioids absorbed in the GI tract first further research [140]. interact with receptors present on the enteric nervous system (ENS) thus affecting GI functions. The ENS is 8. ABSORPTION AND FATE OF OPIOID PEP- a network of nerve cells found in the wall of the GI TIDES IN THE BODY tract controlling motility and secretion and regulating digestion, absorption and immunomodulation [150]. β- During digestion, food proteins are hydrolyzed to casomorphin fragments modulate mucus secretion by peptides and amino acids by digestive enzymes. Sev- acting on the intestinal mucin producing cells [74-76], eral factors affect the transport and absorption of pep- gluten exorphins [80] and β-casomorphin prolong GI tides in the GI tract including pKa, size of the peptide transit time [71, 72]. β-casomorphins have been shown and pH microclimate. Gastric emptying and intestinal to stimulate insulin release [69], inhibit gastric motility transit affect site where peptide is present along the GI and emptying [151] and attenuate the suppression of fat tract thereby impacting absorption. In healthy adults, intake via enterostatin [78]. peptides larger than di-tripeptides are not readily ab- sorbed except during stress, certain diseases or aggres- 9. TRANSPORT IN THE BLOOD STREAM AND sion when increased intestinal permeability occurs ACROSS THE BLOOD BRAIN BARRIER (BBB) [21]. Half-life of opioid peptides in blood is short due to Peptides formed during digestion can cross the in- peptidase activity. Endogenous opioids, dynorphin (1- testinal epithelium via transcellular or paracellular 13) and leu-enkephalin have half-life of less than one pathways. During transcellular transportation, peptides minute and 6.7 minutes, respectively [152, 153]. Der- are subjected either to carrier mediated transport or morphin displays a longer half-life as compared to transcytosis and/or endocytosis and during paracellular enkephalins [154]. Half-life of these peptides can be transportation, peptides are transported by passing extended by binding them to carrier proteins such as through intercellular spaces. It has been shown that albumin [155] or transferrin [156]. To our knowledge, intestinal mucosa does not present an absolute barrier in vivo half-lives of exogenous opioid peptides in blood and various peptides including gluten exorphins A5 have not been measured and needs investigation. Food Proteins as Source of Opioid Peptides-A Review Current Medicinal Chemistry, 2016, Vol. 23, No. 9 905

Transfer of peptides, including food-derived pep- tion and interaction of these peptides with receptors tides, from peripheral circulation to the central nervous present in the central and peripheral nervous system system via the BBB can be done by four different pep- will open possible uses of these peptides for treatment tide transport systems PTS-1, PTS-2, PTS-3 and PTS-4 of various diseases. However, some studies have [157]. PTS-1 is responsible for the transport of opioid shown the effect of these peptides on the nervous sys- peptides including Tyr-MIF-1 met-enkephalin, and leu- tem (analgesia, antinociception and enhanced mem- enkephalin [157]. Transport of opioid peptides, DPDPE ory), GI functions (increased intestinal transit time, and II can also be done through organic an- enhanced appetite and suppression of high fat intake), ion transporting polypeptides which are expressed at and increase β-oxidation and energy expenditure sug- the BBB [158]. Transport of , a potent opioid gesting possibility of their use as nutraceuticals for analgesic involves neutral amino acid carrier [159]. management of pain, blood sugar and obesity. Further Glycosylation of peptides has shown promising result research is needed to develop methods of producing for transportation of peptides via glucose carrier opioid peptides from food proteins in significant GLUT1 including glycosylated analogues of met- en- amounts for pharmaceutical use and device strategies cephalin and [159]. to assure their targeted delivery.

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Received: September 13, 2015 Revised: January 22, 2016 Accepted: February 16, 2016