Cysteine Rich Peptides for Improving Thiol Homeostasis

Abstract

Described is the use of a mixture of peptides, the peptides comprising at least 6.5% wt cysteine, for the manufacture of a medicament, supplement, beverage or food product for restoring thiol homeostasis. Furthermore, a method is described for restoring thiol homeostasis in a subject in need thereof, said method comprising administering to said subject an effective amount of a mixture of peptides, the peptides comprising at least 6.5% wt cysteine.

Description

EXAMPLES

Example 1

Process For The Preparation Of Cysteine Rich Peptides

A 5% wt dispersion of whey protein isolate (WPI; typically Bipro, Davisco) was produced by adding the WPI into pre-heated demineralised water followed by heating to process temperature (50° C.). The pH was adjusted to pH 3 by adding 30% sulphuric acid. The hydrolysis was initiated by adding ENZECO fungal acid protease (EDC, U.S.). The enzyme/protein ratio was typically 2% wt, based on protein dry matter. After an appropriate hydrolysis time, typically 20 h, NaOH (33%) was added until the pH reached 6.5, followed by heating the mixture to 104° C. and holding the temperature for 3 min. The hydrolysate was subjected to diafiltration (typically 300%, optionally 200%) with demineralised water at 50° C., following volume reduction and nanofiltration, using a Nadir SS NF-PES-10 3838 B membrane module. The nanofiltration proceeded at 50° C. When a dry matter of typically 25% was reached, the retentate was spray-dried.

Example 2

Typical Analysis

The analysis was performed by a certified commercial laboratory (CCL Nutricontrol, Veghel, The Netherlands). Method: oxidation of the cysteine in the sample with performic acid prior to HCL hydrolysis of the peptides, ion-exchange chromatography of the free amino acids following post-column derivatisation with ninhydrine, according to EC Guideline 98/64/EG of Mar. 9, 1998; Publication L257/14-23 dated 19 Sep. 1998)

	typical
	Total solids	%	95.1
	Protein (N*6.38)	%	83.4
	Protein (N*6.62)	%	86.0
	Cysteine	g/kg	65.50
	Cysteine	%/Prot	7.9%
	Amino acids
	Alanine	g/kg	37.0
	Arginine	g/kg	18.6
	Aspartic acid	g/kg	146.9
	Glutamic acid	g/kg	186.4
	Glycine	g/kg	17.7
	Histidine	g/kg	19.6
	Isoleucine	g/kg	43.0
	Leucine	g/kg	59.4
	Lysine	g/kg	89.8
	Methionine	g/kg	12.2
	Phenylalanine	g/kg	18.4
	Proline	g/kg	37.6
	Serine	g/kg	43.3
	Threonine	g/kg	47.3
	Tryptophan (after enzymatic hydrolysis)	g/kg	11.4
	Tyrosine	g/kg	18.6
	Valine	g/kg	45.6
	Total a.a.		915.29

Free thiol groups in the peptides were determined with DTNB (Ellmann's reagent) in the presence of urea, with reduction by NaBH₄. The ratio of SH/SS was calculated by the concentration of available thiols without reduction/total concentration of thiols after reduction and was found to be 6.5%

Example 3

Molecular Weight Distribution

Method:

HPLC-system: isocratic HPLC system with UV detector, autosampler, Waters Millenium Data Acquisition Software

column: Progel TSK-G2000SWXL 7.8 mm×30 cm (Supelco), guard column: Progel TSK SWXL (Supelco)eluent: 30% actetonitrile/H₂O/0,1% TFA

Flow: 1 mL/min

Detection: 214 nm

Calibration: HPLC standards (Sigma): Carbonic Anhydrase, Ribonuclease A, Aprotinin, Insulin, Bacitracin, Phenylalanine,

The molecular weight distribution was found to be as follows:

MW-range (D) Area %
>10.000      17.5
10.000-5.000 3.7
5.000-2.000  6.6
2.000-1.000  12.1
1.000-500    20.2
<500         39.9

Example 4

Preparation of Tablets Comprising The Cysteine Rich Peptides

Dosage: 4 tablets/day, delivering 200 mg L-cysteine/day in the form of a peptide mixture.

Per 100 g
Cysteine rich peptides       88.24 g
Microcristalline cellulose 1 10.59 g
Silicon Dioxide 2            0.47 g
Magnesium Stearate           0.35 g
Stearic Acid                 0.35 g
1 Avicel PH-102 - FMC
2 CAB-O-SIL M-5

The powder were premixed whilst the Mg stearate was withheld for the last minutes of mixing. The tablets were prepared by direct compression (compression pressure 20 kN, hardness: 160 N).

Example 5

Preparation Of A Chocolate Caramel Bar Comprising Cysteine Rich Peptides

One bar delivers 200 mg L-cysteine in the form of a cysteine rich peptide mixture.

	Per 100 g	per Serving (40 g)
	Cysteine rich peptides	8.30	g	3.32	g
	Maltitol Syrup 1	66.01	g	26.41	g
	Calcium Caseinate	10.65	g	4.26	g
	Chocolate Liquor	8.52	g	3.41	g
	Sodium Caseinate, granular	4.26	g	1.70	g
	Cocoa Butter	2.13	g	0.85	g
	Butter, Unsalted	0.11	g	0.04	g
	Lecithin	0.01	g	0.0043	g
	Vanilla Flavor	0.01	g	0.0043	g
	1 Lycasin 85% solution

Example 6

Preparation Of A Heat-Treated Yoghurt Drink Comprising Cysteine Rich Peptides

	Per 100 g	L-cysteine/serving
	Cysteine peptide	0.86	g	0.05 g
	Skimmed milk	55	g
	Sugar	6	g
	Maltitol 1	3	g
	Lactic acid	0.002	g
	Pectin 2	0.3	g
	Flavor	0.055	g
	Water	34.6	g
	Inoculum 3	0.2	g
	1 C*maltidex 16385 Cerestar
	2 Genu Pectine YM-115-H CP Kelco
	3 YC-X11 Christian Hansen

Milk was mixed with water. The cysteine rich peptides, sugar and maltitol were added and dissolved with continuous stirring followed by pasteurisation (90° C., 5 min). After cooling to the fermentation temperature (42° C.), the inoculum was added. Fermentation was proceeded until the pH reached 4.3. The pH was lowered to 3.8-4.0 using lactic acid. The pectin was added under vigorous stirring. The mixture was heated to 70° C., homogenised at 120/20 bar and flavour was added. After filling, the product was pasteurised (80° C./3 min).

Example 7

Preparation Of A Liver Cleansing Drink Comprising Cysteine Rich Peptides

		per Serving
	Per 100 g	(250 ml)	L-cysteine/serving
Cysteine peptide	0.43	g	1.08		0.06 g
Dextrose	4.4	g	11	g
Fructose	3.6	g	9	g
Flavor apple	0.055	g	0.14
Flavor banana	0.037	g	0.09
Pectin 1	0.15	g	0.37
Water	91.33	g	228.36
1 Genu Pectine YM-115-H CP Kelco

All dry ingredients were dissolved in water and the pH was adjusted first till pH 3,8 with citric acid (+/−0,14% on total), than till pH 3,5 with malic acid (+/−0,66% on total). The solution was preheated to 70° C. followed by addition of the pectin premix (4% in water). After homogenisation (150 bar) the product was filled and pasteurised or pasteurised and filled aseptically.

Example 8

Reduced Hepatotoxicity Of Paracetamol After Administration Of Cysteine Rich Peptides

Rats were fed an isocaloric and isonitrogenic diet containing the maintenance concentration sulphur containing amino acids (38 mg/kg) or the respective diet enriched with cysteine rich peptides (62 mg/kg) for 14 days. After 14 days, the animals in each diet group were challenged with acetaminophen (orally, 300 mg/kg body weight, in corn oil) and food was deprived for the following 12 h. Immediately after the challenge as well as 12 and 24 hours, 6 (t=0) and 9 rats (t=12 h and t=24) of each group were sacrificed and the livers withdrawn for biochemical analysis and histological examination. Blood samples were taken for analysis of plasma liver marker enzymes. Paracetamol led to a liver tissue damage both in the control group and the group receiving cysteine rich peptides, but continued supply of cysteine rich peptides improved the capacity of the rat liver to restore its structural integrity significantly as compared with the control group.

Liver aspartate aminotransferase (ASAT)[U/L].

The specific activity of the liver enzyme aspartate aminotransferase is a relevant indicator for liver damage. If the level of activity of the enzyme is increased, this indicates liver damage.

Hours after		Cysteine rich peptides
challenging	Casein diet	enriched diet
0	69 +/− 2	78 +/− 6
12	126 +/− 14	93 +/− 15
24	120 +/− 16	109 +/− 16

The table above clearly indicates that liver damage by paracetamol is substantially reduced by taking a diet enriched in cysteine rich peptides.

Necrotic cells [cells/20 fields] Microscopic histological examination

		Cysteine rich peptides
Hours after	Casein diet	enriched diet
challenging	Median	Min	Max	Median	Min	max
0	2.5	0	4	4	1	14
12	51	2	229	61	1	376
24	16	0	89	3	1	9

From the table shown above it can be observed that the number of necrotic cells, after an initial rise, eventually comes down in the cysteine rich peptides enriched diet group to much lower levels compared to the control (casein) group, indicating a quicker restoration of the tissue integrity, as a result of restored biothiol homeostasis.

Vacuolated cells [cells/20 fields] Microscopic histological examination.

		Cysteine rich peptides
Hours after	Casein diet	enriched diet
challenging	Median	Min	Max	Median	Min	max
0	16	1	146	4	0	13
12	80	16	824	419	10	618
24	11	2	141	7	0	32

Vacuolated cells signify the beginning of cell damage, eventually leading to cell necrosis. Vacuolation of cells is a reversible process. From the table above it can be concluded that a cysteine rich peptides enriched diet leads to a quicker reversal to normal state.

Example 9

ACE Inhibition Activity Of Cysteine Rich Peptides

The ACE inhibition assay is based on the hydrolysis of furylacryloyl-phenylalanyl-glycyl-glycine (FAPGG) as a substrate according to Maguire and Price (Ann. Clin. Biochem. 1985, vol. 22: 204-210), adapted for a microtiter procedure. ACE from rabbit lung (nr. A6778), FAPGG (nr F7131) and Captopril® (nr. C 8856) were obtained from Sigma. Different concentrations of the test substances (inhibitor) were added to the substrate solution (0.145 mmol), the reaction was started by adding the enzyme (ACE, 0.145 U). The decrease in absorbance at 340 nm was measured during 10 minutes at 1 minute intervals with a μQuant plate reader from Bio-Tek instruments. The IC50 was obtained from the plot of the inhibitor concentration vs. ACE inhibition (%). Whey protein isolate (Bipro, Davisco) was taken as a negative control. The IC 50 is defined as the inhibitor concentration causing 50% inhibition of ACE.

Results:

	IC 50
Cysteine rich peptides:
300% diafiltration of the hydrolysate (ex. 1)	203.23	mg/L
200% diafiltration of the hydrolysate (ex. 1)	133.47	mg/L
Captopril ®	<2	mg/L
Whey protein isolate (Bipro, Davisco)	no inhibition
CE 90 B	80	mg/L

Captopril® is a well known ACE inhibitor. Reference CE 90 B is a casein hydrolysate from DMV International (The Netherlands) showing ACE inhibitory activity.

Example 10

Human Study Of Effects Of Cysteine Rich Peptides

13 people aged 50+ were asked to take cysteine peptide pills for 4 weeks in a dose of 3.3 g of the micture of cysteine rich peptides corresponding to 200 mg cysteine/day). Before and after the test, the health concerns and status were studied by means of questionnaires and interviews.9 people finished the study. 7 of them observed positive effects which were described as follows:

• • increased natural level of energy
  • better sleep quality (more refreshed in the morning)
  • improved metal alertness, better attention
  • three people in the study with elevated blood pressure reported a decrease, which is consistent with the results of example 8.

The effects of increased energy were documented 4-5 days after the start of the intake, and energy levels were reported to have declined after having stopped taking the pills.

10 people of different age (30-50) but exposed to a high stress level took the same dose of the mixture of cysteine rich peptides but without the thorough examination.The observations reported by the test persons were: better sleep (more refreshed in the morning), and more energy during the day.5 adult people of Asian origin, which all described themselves as being hangover-sensitive, took 0.8-1.6 g of the mixture of cysteine rich peptides (comprising 6.5% wt cysteine) directly preceding alcohol consumption. All subjects reported that they neither suffered from a hangover, nor were they experiencing any embarrassing face-flushing.2 adult people of Asian origin took 0.8-1.6 g of the mixture of cysteine rich peptides (comprising 6.5% wt cysteine) during at least 4 weeks. The subjects reported that their skin condition and skin pattern had improved (reduced acne). Moreover they reported to have more energy, and slept better.

Claims

1-28. (canceled)

29: A method for restoring thiol homeostasis in a subject in need thereof, comprising administering to said subject an effective amount of a mixture of peptides comprising at least 6.5% wt cysteine.

30: The method according to claim 29, wherein said mixture of peptides is administered to prevent and/or reduce effects of alcohol consumption in a subject in need thereof.

31: The method according to claim 30, wherein said mixture of peptides is administered to prevent and/or reduce a hangover.

32: The method according to claim 30, wherein said mixture of peptides is administered to prevent and/or reduce face flushing.

33: The method according to claim 29, wherein said mixture of peptides is administered to boost vitality.

34: The method according to claim 33, wherein said mixture of peptides is administered to prevent and/or reduce fatigue.

35: The method according to claim 29, wherein said mixture of peptides is administered to improve sleeping.

36: The method according to claim 29, wherein said mixture of peptides is administered to prevent development of symptoms of Metabolic Syndrome such as non-insulin dependent diabetes.

37: The method according to claim 36, wherein said mixture of peptides is administered to prevent and/or reduce the development of cardiovascular diseases such as atheroscleropathy.

38: The method according to claim 29, wherein said mixture of peptides is administered to lower blood pressure.

39: The method according to claim 29, wherein said mixture of peptides is administered to prevent and/or treat drug-induced toxicity.

40: The method according to claim 29, wherein said mixture of peptides is administered to lighten skin.

41: The method according to claim 29, wherein said mixture of peptides is administered to reduce inflammation.

42: The method according to claim 29, wherein said mixture of peptides comprising at least 6.5% wt cysteine is obtained by a method comprising the steps of: a) cleaving proteins of a protein source into peptides;b) digesting the peptides obtained in step a) by an exopeptidase, the action of which is at least attenuated at a position of a cysteine in the peptide, therewith forming digested peptides having a terminal cysteine; andc) purifying the digested peptides.