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Angiotensin I-converting enzyme inhibitors from nature source as a major target for

antihypertensive therapy

Le Hoang Lama, Benjabhorn Sethaboupphab, Hiroyuki Ukedac aDepartment of Quality Management, School of Biotechnology and Food Technology, Hanoi Uiversity of

Science and Technology, 1 Dai Co Viet Rd., Hanoi, Vietnam

bDepartment of Pharmaceutical Chemistry and Technology, Faculty of Pharmaceutical Sciences, Ubon

Ratchathani University, 85 Sathonlamark Rd., Warin Chamrap, Ubon Ratchathani 34190, Thailand

cDepartment of Bioresources science, Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku

783-8502, Kochi, Japan

2014

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Hypertension and related diseases

Angiotensin I-converting enzyme (ACE)

ACE kit for screening ACE inhibitors

ACE inhibitors for antihypertension

Contents

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Hypertension and related diseases

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• Hypertension, another word for high blood pressure, is defined as systolic pressure greater than 140 mmHg and/or diastolic pressure greater than 90 mmHg.

• More than 1 billion individuals worldwide are hypertensive and 4 million individuals die annually as a direct result of hypertension.

Hypertension

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Hypertension

http://providencewellness.info

• Although it is a “silent” disease in that patients usually have no specific symptoms, hypertension poses as a major risk factor for heart attack, stroke, and kidney failure.

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Causes of hypertension

Most cases of hypertension are polygenic and combined with environmental factors, such as obesity, inactivity, stress, alcohol.

The interaction of genetics and environment affects sodium, insulin, cell membrane function, and especially the renin-angiotensin system, causing elevation of blood pressure.

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Angiotensin I-converting enzyme

(ACE)

• N-domain (Leu1–Pro601)

• linker region (Pro602–Asp612)

• C-domain (Leu613–Pro1193)

• stalk region (Gln1194–Arg1227)

• transmembrane segment (Val1228–Ser1248)

• intracellular domain (Gln1249–Ser1277)

• the membrane-bound ACE sheddase

• Arg1203: C-terminal residue of soluble ACE

•Kenneth E.B., Frank S.O., Wendell-Lamar B.B., Kandarp

H.S., Jorge F.G., Romer A.G., Xiao Z.S., Sebastien F.,

Rhian M. Touyz, Pharmacol Rev 65:1–46, 2013.

•Chen et al., 2010; Danilov et al., 2011.

•Acharya et al., 2003; Corradi et al., 2006.

Model of human somatic ACE

9 Decrease of blood pressure

Angiotensinogen

Activity of ACE

X: Leu or Val

amino

acids Asp Arg Val Tyr Ile His Pro Phe His Leu X Val

Angiotensin I

Asp Arg Val Tyr Ile His Pro Phe His Leu

Angiotensin II VASOCONSTRICTOR

Asp Arg Val Tyr Ile His Pro Phe

Bradykinin

VASODILATOR

Arg Pro Pro Gly Phe Ser Pro Phe Arg

Inactive peptide

Arg Pro Pro Gly Phe Ser Pro

Elevation of blood pressure

Renin–Angiotensin system

Kininogen

Ser Leu Met Lys Arg Pro Pro Gly Phe Ser Pro PheArg Ser

Renin

Kallikrein–Kinin system

Kallikrein

ACE Angiotensin I-

converting enzyme

Inhibitor

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ACE inhibitors

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Pharmaceutical ACE inhibitors

ACE inhibitors have been developed as orally active drugs that are used in

the treatment of hypertension and congestive heart failure.

A. Gumieniczek, Chem. Anal.(Warsaw), 2002, 47, 1.

Captopril Enalapril Lisinopril

Benazepril Cilazapril Trandolapril Perindopril Quinapril Ramipril

Fosinopril

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Proposed binding model for interaction between substrates

or competitive inhibitors and the active site of ACE

S1, S1′, and S2

′ represent subsites that can potentially interact with the

corresponding amino acid residues of peptide substrates or inhibitors.

G.H. Li, G.W. Le, Y.H. Shi and S. Shrestha, Nutr. Res., 2004, 24, 469.

Natesh R., Schwager SLU,

Sturrock ED, Acharya KR,

Nature (2003) 421 p.551

Crystal structure of the

human ACE-lisinopril

complex

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Functional foods with

antihypertensive effect

(peptide-based ACE

inhibitory foods)

ACE inhibitors

Pharmaceutical drugs:

captopril, enalapril,

lisinopril, alacepril,

trandolapril…

Food-derived ACE

inhibitory compounds:

peptides (Val-Tyr, Val-

Pro-Pro, Ile-Pro-Pro),

flavonoids…

Side-effect

Coughing,

taste

disturbances,

skin rashes

Tokuho

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ACE inhibitors from nature

SNAKE VENOM

5–13 amino acid residues

Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro

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ACE inhibitors from nature

ACE INHIBITORY PEPTIDES

Source Amino acid sequence Enzyme for hydrolysis IC50 (M)

Bonito LKP, ALPHA, IVGRPR… Thermolysin 0.32–300

Salmon VW,WA…… Thermolysin 2.5–277.3

Sardine KY, RFH… Alcalase 1.63–330

Tuna PTHIKWGD Acid

Pollack GPL, GPM… Alcalase+pronase+collagenase 2.65, 17.13

Pork RMLGQTPTK, PPK Thermolysin, pepsin 34–1000

Chicken IKW, FQKPKR… Thermolysin 0.21–14

Antartic krill KLKFV Pepsin+trypsin 30

Oyster LF Denazyme AP 126

Squid YALPHA 9.8

Sake lees VW Thermoase 2.5

Fish sauce VW 2.5

Human serum AW, YLYEIAR Trypsin 15.4, 16

concentration giving 50% inhibition

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ACE inhibitors from nature

ACE INHIBITORY PEPTIDES

Source Amino acid sequence Enzyme for hydrolysis IC50 (M)

Wheat germ IVY, IY -Amylase+alkaline protease 0.48

Corn LRP, LSQ… Thermolysin 0.61

Rice TQVY, RDHP Thermolysin 18200

Bovine GKKVLQ, FQAVVK Alcalase 1.9, 5.8

Egg NIFYCP, FFGRCVSP Pepsin 0.4, 15

Seaweed VW, IY, YNKL.. Protease S, pepsin 2.5–90.5

Microalgae IAPG, AFL, FAL… Pepsin 11.4, 26.3

Red alga IY 3.8

Broccoli YPK

Soybean NWGPLV

Sesame LKY, IVY… 0.78–14.74

Mung bean KLPAGTLF,VTPALR 13.4–82.4

Pea LRY 0.15, 0.23

Mushroom GEP 320

concentration giving 50% inhibition

ACE inhibitors from nature

ACE INHIBITORY PEPTIDES

(1), paracellular; (2), passive diffusion;

(3) endocytosis; (4) carrier-mediated transport

Mechanisms for intestinal transport of peptides

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FLAVONOIDS AS ACE INHIBITORS

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Group of Flavonoids Compound IC50 Value

Anthocyanins

Delphinidin-3-O-sambubioside

Cyanidin-3-O-sambubioside

Cyanidin-3-O-β-glucoside

142 μM

118 μM

139 μM

Flavones

Apigenin

Luteolin

Luteolin-7-O-glucopyranoside

280 μM

290 μM

280 μM

Flavonols

Quercetin glucuronide

Isorhamnetin-3-β-glucopyranoside

Quercetin-3-β-glucopyranoside

Quercetin-3-α-arabinopyranoside

Kaempferol-3-α-arabinopyranoside

200 μ M

409 μM

709 μM

310 μM

393 μM

Flavan-3-ols

Flavan-3-ols Epicatechin – dimer

Epicatechin - tetramer

Epicatechin - hexamer

97 μM

4 μM

8 μM

Chalcones Butein 730 μM

Flavonoid metabolites Quercetin-3-O-glucuronic acid 27 μM

FLAVONOIDS AS ACE INHIBITORS

IC50 values of ACE inhibitory flavonoids and their metabolites

B.W. Nileeka Balasuriya and H.P. Vasantha Rupasinghe, Functional Foods in Health and Disease 2011; 5:172-188

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Group of Flavonoids IC50 Value

Hibiscus sabdariffa (Hibiscus) 91 μg/mL

Camelia synensis (green tea) 125 μg/mL

Vaccinium ashei reade (Blueberry leaf extract) 46 μg/mL

Vaccinium myrtillus (Bilberry) Log -2.6 mg/mL

Senecio inaequidens

(A perennial herb)

192 μg/mL

S. ambiguous subsp. Ambigus

(ethyl acetate extract) 219 μg/mL

S. ambiguous subsp. Ambigus

(n-hexane extract)

307 μg/mL

Cryptomeria japonica (Japanese Cedar) 16 μg/mL

Malus domestica (Apple skin ethanol extract) 49 μg/mL

FLAVONOIDS AS ACE INHIBITORS

ACE inhibition (IC50 Values) by various plant extracts

B.W. Nileeka Balasuriya and H.P. Vasantha Rupasinghe, Functional Foods in Health and Disease 2011; 5:172-188

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Bioavailability is “the rate and extent to

which the active ingredient or active moiety

is absorbed from a drug product and

becomes available at the site of action” –

U.S. FDA

low bioavailability, lack of stability, poor

membrane penetration, lack of site specific

distribution and rapid elimination

K. S. Shivashankara and S. N. Acharya, The Open Nutraceuticals Journal, 2010, 3, 227-241.

B.W. Nileeka Balasuriya and H.P. Vasantha Rupasinghe, Functional Foods in Health and Disease 2011; 5:172-188

Bioavailability ranking of polyphenols

Flavonoids are shown more effective beneficial

pharmacological properties in vitro than in vivo

FLAVONOIDS AS ACE INHIBITORS

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Foods with health claims

Foods with health claims

Medicines

FOSHU

(Foods for specific health

uses)

Foods with nutritional

health claims

General

foods

“Functional foods are foods and beverages with claimed health benefits based

on scientific evidences”. *

Tokuho = 特定保健用食品

In 1991, a system regulating FOSHU (Tokuho) was introduced by the Ministry of Health

and Welfare. FOSHU system was initiated in Japan, followed by U.K., Sweden, and China. **

In April 2001, Japanese government established a new system regulating functional

foods, the new category “food with health claims” was created.

* H. Verhagen, S. Coolen, G. Duchateau, M. Hamer, J. Kyle and A. Rechner, Mutat. Res., 2004, 551, 65.

** H. Ohama, H. Ikeda and H. Moriyama, Toxicology, 2006, 221, 96.

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Hitachi: Tochu 120

• Product Category: Non Carbonated Drink

• Functional Component: Eucommia Leaf (Tochu)

Glucoside

• Recommended Daily Intake: 150ml

• Flavor: Plain

• Volume/Retail Price: 50ml/200yen

• Manufacturer/Location: Hitachi

Zousen/Hiroshima

Eucommia ulmoides Oliv.

Product name: Eucommia Ulmoides P.E. Eucommia Leaf Extract (Chlorogenic acid)

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Calpis: AmealS

• Product Category: Lactic Acid Bacteria Drink

• Functional Component: Lactotripeptide

• Recommended Daily Dose: 160g

• Volume/Retail Price/Sales: 160g/190yen

• Manufacturer/Location: Calpis/Tokyo

• Note: Calpis developed Lactotripeptide (Val-Pro-Pro,

Ille-Pro-Pro) AmilS from their original fermented milk

drink, Calpis.

According to the company, taking 95 ml of Ameal S for 12

weeks resulted reduction of 12-15 mmHg on Calpis Ameal

low calorie high and 5-7 mmHg on low blood pressure. The

company claims that 100 thousands people are taking

Ameal S every day

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Tokiwa: Rapid Support

• Product Category: Non carbonated Drink

• Functional Component: Sardine Peptide

• Recommended Daily Intake: 200ml

• Volume/Retail Price: 100ml/190yen

• Manufacturer/Location: Tokiwa Yakuhin/Osaka

• Note: Tokiwa Pharmaceuticals has developed

peptide from sardine. Valyl-Tyrosine which found in sardine

peptide effectively control high blood pressure.

The research reports after 3 weeks of taking Lapis Support,

high blood pressure went down 145 mmHg to 125 mmhg.

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Nippon Supplement: Peptide ACE 3000

• Product Category: Dietary supplement/Tablets

• Functional Component: Sardine Peptide

• Recommended Daily Intake: 2500 mg

• Volume/Retail Price: 250 mgx300/3800 yen

• Manufacturer/Location: Nippon

Supplement/Osaka

• Note: Peptide Ace 3000 is the first dietary

supplement that has been approved by the MHLW

as FOSHU.

It contains 250 mg of sardine peptide that may

help for people who has high blood pressure.

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ACE kit for screening of ACE

inhibitors

28 in vitro ACE

inhibition assay Derivation of

peptides

in vivo ACE

inhibition assay

Manufacture of

functional foods

Significance of ACE inhibitory activity assay

Functional foods with

antihypertensive effect Foods possess ACE inhibitory

activity

Analysis Kit

Effective

Convenient

?

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Principle of common ACE inhibition assay

Activity of ACE

in vitro assay

Activity of ACE

in human body

Angiotensin I

Asp Arg Val Tyr Ile His Pro Phe His Leu

ACE

Synthetic substrate

S aa aa

ACE

Cleaved

substrate

S

Angiotensin II

Asp Arg Val Tyr Ile His Pro Phe

Dipeptide

aa aa

Detected

spectrophotometrically…

ACE

inhibitor

Decrease in signal

intensity

Detected

spectrophotometrically…

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His Leu

Ethyl acetate

Hip His Leu

Hip

Conventional assay:

- Low selectivity

- Time-consuming

- Tedious handling

- Interferences by

unhydrolyzed substrate

and extracting solvent

Principle of conventional and novel ACE inhibition assays

CH3-CH-CH2-C- NH-CH2-C-OH

OH O O

3HB - - - - - G

OH

CH3-CH-CH2-C-OH

O

3HB

max = 228 nm

Novel assay:

- High selectivity

- Rapidity

-Convenience

- Less interference

Hip

OH O O O

CH3-CH-CH2-C- NH-CH2-C-NH-CH2-C-NH-CH2-C-OH

O

3HB - - - - G - - - - - G - - - - - - - G

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Commercial ACE kit

3HB-GGG

3HB-G Gly-Gly

Gly

ACE

aminoacylase

3HB

H2O

Acetoacetate

NAD+

NADH WST-1

3HBDH

H2O

WST-1

formazan

Mediator

Mediator (reduced form)

= 450 nm

Amount of each solution for sample and blanks

Sample Blank 1 Blank 2

Sample solution 20 L - -

Deionized water - 20 L 40 L

Substrate buffer 20 L 20 L 20 L

Enzyme working solution 20 L 20 L -

Indicator working solution 200 L 200 L 200 L

Compact

Convenience

high selectivity

great throughput

Mediator: phenazinium methylsulfate

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Thank You for your attention

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ACE • located in a variety of tissues (the

surface of the vascular endothelium)

• is a glycoprotein containing a

carbohydrate moiety composed of

mannose, galactose, fructose, N-

acetylneuraminic acid, and N-

acetylglucosamine.

• molecular masses = 130–170 kDa

• an overall ellipsoidal shape with a

central groove that extends for about

30 angstroms and divides the protein

into two homologous domains (N and C

domains) . Schematic structure of the membrane

bound form of ACE

(-NH2), N-terminal active site; (-COOH),

C-terminal active site;

(Zn2+), zinc ion; (), site of glycosylation

B.A. Murray and R.J. FitzGerald, Curr. Pharm. Des., 2007, 13, 773.