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Endothelins are the most potent vasoconstrictors known

In a healthy individual, a delicate balance between vasoconstriction and vasodilation is maintained by endothelin and other vasoconstrictors on the one hand and nitric oxide, prostacyclin and other vasodilators on the other.

Overproduction of endothelin in the lungs may cause pulmonary hypertension, which can sometimes be treated by the use of an endothelin receptor antagonist, such as bosentan, sitaxentan or ambrisentan. The latter drug selectively blocks endothelin A receptors, decreasing the vasoconstrictive actions and allowing for increased beneficial effects of endothelin B stimulation, such as nitric oxide production. The precise effects of endothelin B receptor activation depends on the type of cells involved.

Disease involvement

The ubiquitous distribution of endothelin peptides and receptors implicates its involvement in a wide variety of physiological and pathological processes in the body. Among numerous diseases potentially occurring from endothelin dysregulation are

• several types of cancer^[6]
• cerebral vasospasm following subarachnoid hemorrhage^[7]
• arterial hypertension and other cardiovascular disorders
• pain mediation^[8]
• cardiac hypertrophy
• Dengue haemorrhagic fever
• Type II diabetes
• some cases of Hirschsprung disease

In insulin resistance the high levels of blood insulin results in increased production and activity of ET-1, which promotes vasoconstriction and elevates blood pressure.^[9]ET-1 impairs glucose uptake in the skeletal muscles of insulin resistant subjects, thereby worsening insulin resistance.^[

Production of Antihypertensive Peptides from food sources

Antihypertensive peptides have been mainly produced from dairy products such as milk, cheese, etc. However, production from food sources other than dairy has also been carried out recently. These peptides can be produced by one or a combination of following methods [11]:

1. Enzymatic hydrolysis
2. Fermentation of protein based food sources
3. Genetic recombination in bacteria

Enzymatic hydrolysis by digestive enzymes: According to literature the most common way of producing antihypertensive peptides from food proteins is enzymatic hydrolysis. Many of the Angiotensin Converting Enzyme (ACE) inhibitory peptides have been produced using gastrointestinal enzymes, usually pepsin and trypsin [12,13]. Enzymes from plant (e.g. Papain) and animal sources (e.g., pepsin and trypsin), have also been used in producing antihypertensive peptides [14].

A variety of food sources, antihypertensive peptides have been produced using enzymatic hydrolysis. Different enzymes have been used to digest milk proteins to yield antihypertensive peptides. The first study has been conducted in 2004 which yielded lactokin in Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) as ace inhibitory peptides [15]. The latest study on lactoferr in hydrolyzates (LFHs) generated by trypsin and proteinase K yield different antihypertensive peptides which acts on rennin-angiotensin system (RAS) and the endothelin (ET) system [16].

Black-bone silky fowl (Gallus gallus domesticus Brisson) muscle has been treated by multistage separation. The black-bone silky fowl muscle peptides (BSFP) was hydrolyzed with Alcalase and papain has been tested for ACE in hibitiory activities. From a total of 29 peptides two novel potent ACE inhibitory peptides Leu-Glu-Arg and Gly-Ala-Gly- have been found [17]. Hen eggs provide biological functions beyond basic nutrition. In a study, the antihypertensive effect of peptide RVPSL from egg protein decreased the blood pressure of SHRs in 4 weeks. The peptide influenced the expression of major RAS components by down-regulating the renin, ACE, Ang II, and AT1 receptor while upregulating the AT2 receptor in SHRs [18]. In an another study a tri-peptide IRW (Ile-Arg-Trp) from egg white protein ovotransferrin; demonstrated anti-hypertensive effects of IRW in vivo which is mediated through ACE inhibition and endothelial nitric oxide synthase [19]. ACE inhibitory peptides have also been isolated from fish sources. Suetsuna & Osajima [20] identified first ACE inhibitory peptide in sardine over thirty years ago. Since then many other peptides have been isolated from various fish species, including shellfish, tuna, bonito, salmon, etc. [21].

Amongst plant sources, Met-Arg-Trp (MRW) isolated from the pepsin-pancreatin digest of spinach lowers blood pressure via prostaglandin D(2)-dependent vasorelaxation in SHRs [22].

Pea protein isolate, hydrolyzed with alcalase, have proven inhibitory against ACE, renin, and calmodulin-dependent phosphodiesterase 1 (CaMPDE) [23].

Four peptides of sequence: ITP IIP GQY STYQT have been isolated by protease enzyme digestion of sweet potato protein. ITP peptide was found to be the most potent ACE inhibitor as concluded by in vivo study on rats [24].

Edible mushrooms have also yielded ACE inhibitory oligo peptides.

One such peptide with the sequence LSMGSASLSP was isolated in water extracts of mushroom Hypsizygus marmoreus (brown cultivar). The extract from its fruiting body was purified and proven to have antihypertensive action on SHRs [25]. In an another study, two new ACE in hibitiory peptides from the fruiting body of Pleurotus cornucopiae were purified. Their sequences were determined to be RLPSEFDLSAFLRA and RLSGQTIEVTSEYLFRH with the molecular mass of 1622.85 and 2037.26Da, respectively [26]. In vivo study on SHRs validated the antihypertensive activity of both the peptides.

Macro-algae have been part of the staple diet in east Asia for centuries and has wide applications in food and pharmaceutical industries. Papain hydrolysates of the crude Palmaria palmata protein release renin inhibitory peptide of the sequence: IRLIIVLMPILMA. The bioactivity of this peptide was confirmed by renin inhibitory assay [27].

Amongst cereals Wheat gliadin hydrolysates can act as ACE inhibitors. The peptide Ile-Ala-Pro prepared with acid protease decreased the blood pressure in SHRs significantly with intra-peritoneal administration [28]. Arginine-rich peptides from Flaxseed protein isolate (FPI) obtained by enzymatic hydrolysis with trypsin and pronase were observed to produce in vivo vasodilatory effects. The in vivo study on SHRs suggested that the rate of peptide absorption is rapid as compared to amino acids and thus it provides a fast relief from hypertension [29]. Rapeseed protein isolate (RPI) digested with proteinase, thermolysin, flavourzyme and pepsin produced rapeseed protein hydrolyzates which can be separated into different anti hypertensive peptides [30].

At large scale the serine type protease Alcalase is most widely used endo-protease in digests of various plant proteins such as rapeseed, canola, sunflower seed protein, soy protein legumes, rice as well as mung and chick beans showing high potency for ACE inhibition [31].

Antihypertensive peptides produced by fermentation: Many industrially utilized dairy starter cultures are highly proteolytic in nature and can be used for production of antihypertensive peptides by fermentation of dairy products. The proteolytic system of lactic acid bacteria (LAB) such as Lactococcus lactis, Lactobacillus helveticus and L. delbrueckii ssp. Bulgaricus consists of a cell wall-bound proteinase and a number of intracellular peptidases, including endo peptidases, amino peptidases, tri peptidases and dipeptidases [32]. Based on these peptidases, a number of commercial products have been synthesized for clinical trials for efficacy testing using different hypertensive subjects.

Many dietary proteins, especially milk proteins, contain physiologically active peptides encoded in the protein sequence. These peptides may be released during gastrointestinal digestion or food processing and once liberated, cause different physiological functions. Milk-derived bioactive peptides are shown to have antihypertensive, antimicrobial, immune modulatory, anti oxidative and mineral-binding properties [33]. Thus fermentation can yield peptides that are ACE-inhibitory and thus blood pressure–lowering, to be derived from milk proteins. Some of these peptides have also been found to have opioid receptor binding properties [34,35].

A fermented milk product with the biologically active peptides valyl-prolyl-proline (Val-Pro-Pro) and isoleucyl-prolyl-proline (Ile-Pro-Pro) was shown to lower blood pressure in spontaneously hypertensive rats [36]. It was suggested that small peptides are absorbed from the gastrointestinal tract without being degraded further by digestive enzymes [37]. Two other peptides (Tyr-Pro and Lys-Val-Leu-Pro-Val-Pro-Gln) that were purified and characterized from fermented milk were also shown to have ACE-inhibitory activity in SHRs [38,39]. Nurminen et al. [40] found that alpha-lactorphin (Tyr-Gly-Leu-Phe) also reduced blood pressure in normotensive and SHRs.

Seppo et al. [41] reported that fermented milk product named Evolus or Kaiku Vitabrand, lowered the blood pressure in hypertensive human volunteers in 8 weeks by -14.9mm.

Another controlled trial in hypertensive men lowered the systolic blood pressure in two weeks by -4.3mm Hg and -5.2mm Hg in 4 weeks [42]. A study on casein hydrolysate (Ameal Peptide) lowered the blood pressure by -6.3mm Hg in 6 weeks [43].

The whey from milk fermented by LAB, Streptococcus thermophilus and Lactobacillus bulgaricus along with protease treatment was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction showed the highest inhibitory efficiency ratio (IER) and contains the inhibitory peptide Tyr-Pro-Tyr-Tyr, of which the IC50 was 90.9 lM. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced by 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml). Tri peptides IPP and VPP released from casein inhibit ACE and reduce arterial stiffness in humans at micro molar concentrations produced during the fermentation of milk with lactobacilli [33].

Several novel antihypertensive peptides have been reported in milk fermented with Enterococcus faecalis CECT 5727. Two of the identified peptides LHLPLP and LVYPFPGPIPNSLPQNIPP, showed angiotensin converting enzyme-inhibitory (ACEI) activity with IC50 values as low as 5mM and showed antihypertensive activity in hypertensive rats. In particular, b-casein f (133–138), yielded a significant antihypertensive effect in these animals [44].

A total of 75 peptides included in the fraction with molecular mass below 3000 from an 8-month-old Manchego cheese could be identified using HPLC coupled on line to an ion trap mass spectrometer. Some previously described peptides with antihypertensive and/or angiotensin-converting enzyme (ACE)-inhibitory activity were detected. The formation of five active sequences was followed during cheese ripening in four different batches of Manchego cheese. Two experimental batches of Manchego cheese elaborated with selected bacterial strains with the aim of improve the organoleptic characteristics demonstrated also a good performance in the formation of peptides with ACE-inhibitory activity during cheese ripening [45].

Over the past decade, there has been a growing interest in the use of food sources other than dairy, for production of bioactive peptides with antihypertensive activity. Cereal based fermented products contain peptides which have a BP lowering effect [46]. Studies have shown that common foods from animal and plant origin are important sources of bioactive peptides.

Plant sources usually include cereals (wheat, barley, corn, rice) pseudocereals (buckwheat and amaranth), legumes (soybean, bean, pea), brassica species and others (sunflower).

The presence of bioactive peptides in cereals and legumes can contribute to increase their food protein quality value and add “functionality” to fermented functional foods consumed on a daily basis [47].

Soy protein foods are fast category products in the food industry with demand for soy ingredients with improved processing characteristics. Fermented soy products, traditionally consumed in Eastern countries, have been also found to be an important source of ACE inhibitory and antihypertensive peptides [48-50].

A potent antihypertensive peptide has been identified and characterized in a Korean soy product denominated “chunggugjang” and obtained by soy fermentation with Bacillus subtilis CH-1023 [51]. Other ACE-inhibitory and antihypertensive peptides have been identified in soy paste [52], soy sauce [53,54], natto and tempeh [55], and other fermented soy products [56-58].

Miso paste is a Japanese traditional fermented food, prepared from soy beans mixed with rice fermented with Aspergillus oryzae (rice koji).

Since A. oryzae protease based casein hydrolysate results in production of two antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, Casein was added to miso paste during miso paste fermentation in order to release angiotensin-I converting enzyme (ACE) inhibitory peptides. Casien miso paste had a higher ACE inhibitory activity as compared to casein-free miso paste after 7 days of fermentation. Further, a significant antihypertensive activity of casein miso paste was observed in spontaneously hypertensive rats compared to water and the general miso paste at a dosage of 1.8 g of the casein miso paste/kg of BW [59].

The two ACE inhibitory peptide fractions F2 and F3 were isolated after fermentation of soy protein with Lactobacillus casei spp. pseudoplantarum.

The peptide analogues of LIVTQ were synthesized based on N-terminal sequence of peptide (F2) Leu-Ile-Val-Thr-Gln (LIVTQ) and effect of individual residues on ACE enzyme were studied. The study determined the importance of glutamine (Q) and threonine (T) residues in ACE inhibition [60].

Genetic recombination in bacteria: Apart from the above methods for the production of peptides recombinant DNA techniques have also been explored. As compared to the enzymatic method, the advantages of microbiological genetic engineering techniques to prepare AHP include higher peptide yield and lower cost [61]. The first study involved expression of recombinant human alpha 1-casein in Escherichia coli and its purification [62].

Another antihypertensive peptide multimer (AHPM) was designed and cloned in an expression vector in E. coli. The release of high active fragments was confirmed by the simulated gastrointestinal digestion from the AHPM [63]. Antihypertensive peptides with sequences HHL, HVLPVP, FFVAPFPEVFGK, and GHIATFQER have been expressed successfully in E .coli [64]. Peptides expressed in Escherichia coli by a high throughput recombinant expression system have been designed and used widely, though other bacterial system could be used for the production of peptides at similar yields [65].

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