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Collagen Peptides from Hyaline Cartilage for the Treatment and Prevention of Joint Diseases: Isolation and Characteristics

Authors:
  • Institution of Cell Biophysics, Russian Academy of Sciences, Russia, Pushchino

Abstract

We studied the effects of Chymopsin and Caripazim on the proteolysis of collagen proteins from cattle tracheal hyaline cartilage. Homogenization of the cartilage under conditions of high pressure and temperature facilitated subsequent enzymatic hydrolysis: the degree of hydrolysis increased upon elevation of pressure from 40 to 80 mPa and temperature from 60 to 70°C. Proteolysis with Chymopsin yielded collagen peptides with molecular weights from 900 to 7000 Da, while Caripazim processing yielded collagen peptides with lower molecular weights from 250 to 780 Da consisting of 2-8 amino acids, which could be easily absorbed and intensely incorporated in the formation of the joint tissue structures.
COLLAGEN PEPTIDES MADE OF HYALINE CARTILAGE TO TREAT AND PREVENT
JOINT DISEASES: OBTAINING AND THEIR PROPERTIES
T.I. Nikolaeva, K.S. Laurinavichus *, V.V. Kaptsov **, R.A. Chernyi ***, P.V. Shekhovtsov
Federal Governmental Budgetary Scientific Organization (FGBSO) Institute of Theoretical and Experimental
Biophysics RAS, Pushchino, Moscow region, RF; *FGBSO Institute of Biochemistry and Physiology of
Microorganisms RAS, Pushchino, Moscow region, RF; ** FGBSO Institute of Cell Biophysics RAS, Pushchino,
Moscow region, RF; *** FGBSO Lebedev’s Physical Institute RAS, Moscow, RF.
E-mail: tomivnik@yandex.ru, Ph. D. Tamara I. Nikolaeva
The development of nutraceuticals made of enzymatic hydrolyzates of cartilages facilitates to solve the problem
of supplementing of missing collagen tissue components. The influence of himopsin and caripazime on the
proteolysis of collagen proteins of hyaline cartilages of bovine trachea was studied. It was demonstrated that
homogenization of cartilages under the conditions of high pressure and temperature activates the subsequent
enzymatic hydrolysis: the degree of hydrolysis increases with increasing pressure from 40 to 80 MPa and
temperatures from 60 to 70°C. Under the influence of caripazim, collagen peptides having molecular weights
from 200 to 600 Da and consisting of 2-7 amino acids are obtained, which can be easily assimilated and
intensively included in the structure formation of the joint tissues.
Key words: hyaline cartilages, homogenization, collagen, enzymatic hydrolysis, low molecular weight peptides, himopsin,
caripazime.
Diseases of human musculoskeletal system represent both medical and biological problem.
Pharmaceutical treatment of arthritis and arthrosis of joints is complicated. Synthetic amino acids and
chondroprotectors can regenerate the cartilage tissue by no more than 50%. Other medicines,
including steroid and nonsteroid drugs, are aimed only at reducing pain and normalizing the mobility
of the joints. These medicines also have harmful side effects, they disturb the functioning of liver,
kidneys, heart and other internals. Therefore, there is basis to search for biologically active substances
so that they can replace drugs. In many countries nutraceuticals (dietary supplements or biologically
active additives) are used in medicine for the prevention and treatment of human diseases [9, 12].
Diseases of joints are accompanied by destruction of connective tissues of cartilages, ligaments,
tendons and bones. The extracellular matrix of these tissues contains collagen fibrils that determine
their mechanical strength. Pathological processes in the joints correlate with lower concentrations of
collagen compared to the norm. Changes in joint diseases primarily begin in the hyaline cartilage.
Thinning of collagen fibrils, their dilution and loss of elasticity occurs [2]. In order to form cartilage
matrix type II collagen is required as well as collagen of type I, III, V, VI to form the matrix of other
tissues in the joints. Missing collagen concentrations in connective tissues can be replenished with
exogenous substances contained in nutraceuticals. Bioavailability of collagen for digestion by human
body is high in hydrolyzates of tissues, which proteins are split into amino acids and low molecular
weight peptides. Biocatalytic method allows for obtaining given peptide profile of proteins retaining
native structure of amino acids. Supercoiled structure of collagen fibrils that is formed of spiralized
collagen molecules of long length limits the influence of enzymes. Difficulties of hydrolysis of
biopolymers in raw materials of animal origin containing collagen determine low yield of products
from 3 to 10%. Enzymatic hydrolysis with subsequent chemical hydrolysis increases the yield of final
product up to 15% [4]. However, chemical hydrolysis changes the structure of amino acids.
1
To regenerate cartilage tissue the complex of collagen peptides of molecular weights from 1 000 to 12
000 Da (dietary supplement "Sigumir", Russia) from hyaline cartilages of calves was isolated [5]. In
the United States enzymatic hydrolyzates of hyaline cartilages of chickens contained peptides of type
II collagen of molecular weights in the range 1 500 – 2 500 Da (dietary supplement BioCell Collagen
II) [14]. The stimulating effect on the biosynthesis of collagen type II peptides (molecular weights
from 500 to 13 500 Da) by chondrocytes in cell culture of type II collagen was determined [13]. In the
paper [8] it is shown that peptides of collagen type I, which are tissue-specific for skin matrix,
stimulate the reproduction of skin cells, i.e. fibroblasts. This effect of collagen peptides, like an
increase in cell proliferation, can be used to accelerate the process of tissue repair. We suppose that
type II collagen peptides can participate both in the formation of the matrix of cartilage tissue and in
the regulation of the activity of connective tissue cells of joints and in the regeneration of these tissues.
The aim of this work is to obtain low-molecular type II collagen peptides to form the matrix of
cartilage tissue, as well as to increase their yield in the result of extraction from cartilage tissue of farm
animals.
METHODS OF RESEARCH
In this paper we obtained enzymatic hydrolyzates from hyaline cartilages of cattle trachea. One of the
physical factors influencing the hydrolysis is the degree of grinding of the tissue, and, consequently,
the availability of enzyme molecules for the collagen molecules packed into fibrils. The preparation of
biological material for hydrolysis involves the homogenization of cartilage having temperature and
pressure values of denaturation. Homogenization is fulfilled on special equipment under high pressure
conditions, regulated by temperature and process of time. The cartilage, purified from films, was first
manually milled to particles of 3-4 mm in size, then dispersed in aqueous suspension to a size of 3-4
μm in the mixer for home use (Braun MQ 520). Then, smaller particle sizes (~700-800 nm) were
obtained in the Donor-3 high-pressure homogenizer at the pressure of 20-60 MPa (~ 200-600 kgf /
cm2). Even smaller particle sizes (~ 300 - 400 nm) were obtained with further homogenization of the
suspension of cartilage particles under conditions of sequential increase of pressure from 40 to 80
MPa, temperatures from 30°C to 70°C during from 30 to 65 minutes (process of "fine
homogenization"). At the stage of the final homogenization, hydrothermal hydrolysis of cartilage
biopolymers was also carried out.
As a rule, not single proteases but enzyme complexes are used for the hydrolysis of proteins contained
in connective tissues [6]. These enzymes have specific action of breaking certain bonds in proteins.
From many enzymes we studied, caripazim (OOO MedFlorina, Obolensk, Moscow Region) and
himopsin (Samson-Med LLC, St. Petersburg) were chosen. Caripazim consists of three proteolytic
enzymes: papain, himopapain, proteinase, containing in their active centers sulfhydryl groups.
Himopsin contains the mixture of α-himotrypsin and trypsin. Hydrolysis was carried out in 33,4 mM
2
K-Na phosphate buffer for himopsin at pH 8.0, temperature 42°C and for caripazim at pH 6.0,
temperature 55°C. Control samples we represented by homogenates of cartilage tissue.
The degree of hydrolysis was determined by the parameter DH - degree of hydrolysis: DH is
calculated as the ratio of the mass fraction of amine nitrogen to the mass fraction of total nitrogen in
the hydrolyzate. In the hydrolysates, the content and composition of amino acids characteristic of
collagen type II was determined. Next, we studied the enzymatic hydrolysis of cartilage, depending on
the concentration of enzymes and time.
Hydrolysis was carried out in Excella T-24 thermostat shaker (USA). The unique equipment of
scientific Pushchino town is the high-pressure homogenizer (GVD) Donor-3, which was developed on
the basis of the Donor-1 GVD at the Institute of Cell Biophysics of the Russian Academy of Sciences
in Pushchino town (Author V.V. Kaptsov). The analysis of hydrolyzates was carried out by
spectroscopic methods on devices: SPECORD UV VIS spectrophotometer (GDR), mass spectrometer
MALDI-TOF Autoflex speed (Bruker, Germany). The solubility of lyophilized samples in distilled
water was verified by the method developed in the pharmacopeia [1].
The statistical reliability of the obtained data was determined by the Student's test with probability of
p < 0,05 – 0,001.
RESULTS OF RESEARCH
Samples that have parameters of the "fine homogenization" were studied; the effect of himopsin and
caripazime on the hydrolysis of cartilage tissue was tested.
Table 1. Degree of hydrolysis (DH) of various homogenates of hyaline cartilages under the
influence of proteolytic enzymes
Homo-
genate
Homogenization mode,
sequence and time
Pressure,
MPa
Temperature
оС
Enzyme DH, %
1
1 time - 10 min.
2 time - 10 min.
3 time - 10 min.
40
60
80
30
45
60
Control
Himopsin
Caripazim
1,5±0,1
10,4±0,5
57,8±4,5
2
1 time - 15 min.
2 time - 15 min.
3 time - 35 min.
40
60
80
30
45
70
Control
Himopsin
Caripazim
2,8±0,2
12,1±0,8
71,7±5,6
Note. The difference between the data comparatively to the control for different enzymes is true
(p < 0,001).
Applying sequential pressure change from 40 MPa to 80 MPa, temperatures from 30°C to 60°C and
70°C, time from 10 to 65 minutes, we obtained particles of minimal size in the homogenate No. 2 (320
nm), which are close to the length of collagen molecules (300 nm). Data of the Table 1 indicate that
the homogenization under high pressure and temperature conditions activates subsequent enzymatic
3
hydrolysis. The degree of hydrolysis of homogenized samples respondent to the influence of himopsin
increases by 20% with increasing temperature and time. Analysis of the effect of caripazim on
homogenates showed increase in the degree of hydrolysis by 24% in the sample No. 2. If we compare
caripazim and himopsin, the proteolytic activity of caripazim significantly increases the degree of
hydrolysis. High degree of hydrolysis indicates high content of amino acids and peptides. When amino
acids enter the cells, the biosynthesis of collagen begins [3] and then the formation of the collagen
fibrils and the connective tissue matrix. These experiments demonstrated that during the
homogenization stage it is possible to regulate the yield of amino acids and peptides in the
hydrolyzates of cartilage tissue and to increase it.
On the basis of the obtained data homogenate No. 2 was chosen for the study of enzymatic hydrolysis.
Under the influence of himopsin, peptides of molecular weights from 1000 to 7000 Da (Figure 1) were
formed, which after cleavage to amino acids in the human body can participate in the biosynthesis of
collagen molecules and the formation of collagen fibrils. It is suggested that collagen peptides have
immunomodulatory, antioxidant and anti-inflammatory properties [10, 11]. It is also known that
peptides of the molecular weight 1 000-2 000 Da reduced blood pressure in laboratory animals.
Consequently, the peptides obtained under the influence of himopsin can have polypharmaceutical
effect, affecting both structure of the matrix of the cartilage tissue and metabolism.
The distribution of peptides according to the molecular mass after the action of caripazime on the
homogenate is in the range from 200 to 600 Da (Fig. 2). According to the collagen amino acid data,
the molecular weights correspond to the following oligopeptides: dipeptides, tripeptides, tetrapeptides,
pentapeptides, hexapeptides. It should be noted that the peptides of dietary supplement "Sigumir"
contain more than 10 amino acids, and peptides of dietary supplements "BioCell Collagen II" from
15 to 30 amino acids. The information about peptides available in the EROP-MOSKOW database,
including less than 50 amino acids, indicate the regulatory role of exogenous peptides in human body
[15]. Moreover, native collagen peptides consisting of small number of amino acids can carry out
specific regulatory functions, and, consequently, stimulate the formation of cartilage tissue of the
joints. When exogenous introduction of collagen occurs, preparations can be replaced by damaged link
in human body, both at the level of the structure, and at the level of signaling between the cells of the
matrix [7].
The development of biological material from raw materials with the properties necessary for
hydrolysis begins at the stage of homogenization of tissues. We have found conditions for the
homogenization of cartilage tissue that activate proteolytic cleavage of collagen under the influence of
caripazime in such the way that the yield of peptides increases twofold comparatively to the peptides
obtained by authors of the patent of the Russian Federation No. 2290936 [5] and increases threefold if
to compare with the yield of collagen after hydrolysis of cartilage tissues of hydrobionts, carried out
by the authors of the Russian Federation patent No. 2250047 [4]. Enzymes that catalyze the cleavage
4
of different bonds in polypeptide chains can affect the size of peptides and develop samples containing
controlled-molecular weight collagen peptides: caripazim allows to obtain low-molecular peptides
from 200 to 600 Da, and himopsin ― peptides from 1 000 to 7 000 Da. The solubility of lyophilized
samples obtained in the result of enzymatic proteolysis was different: from readily soluble after action
of karipazim to moderately soluble after the action of chymopsin. Consequently, low molecular weight
peptides from 200 to 600 Da had higher degree of dissolution in water.
The novelty of the work is to improve the technology of manufacturing of collagen nutraceuticals that
combines homogenization of the cartilages and their hydrothermal hydrolysis. The combination of
high pressure conditions and denaturing temperatures for homogenization of cartilage facilitates
subsequent enzymatic hydrolysis. We have obtained peptides that have not only quantitative but also
qualitative advantage since they contain from two to seven amino acids. The effectiveness of the
action of low-molecular collagen peptides is determined by their structure, including Gly―, Pro― and
Hyp―, as well as by high absorption rate of dipeptides and tripeptides proceeding by the mechanism
of ATP-dependent transport that exists in the gastrointestinal tract along with passive transfer of
substances. We suppose that the peptides obtained during our research work, that have lower
molecular weight (200―600 Da) than peptides of Russian and American manufacturers (molecular
weights from 1000 to 12000 Da), can more efficiently influence the biosynthesis of collagen proteins
in the tissues of joints, restore and strengthen joints regulating structure of the matrix and stimulating
functioning of the cells.
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4. Patent RF No. 2250047. Nutritional health-improving preventive product from cartilage tissue of
hydrobionts and the method of its preparation. // Newsletter No. 11. Published 20.04.2005.
5. Patent RF No. 2290936. Method of obtaining an agent for supportive therapy having tissue-specific
activit. // Newsletter No. 1. Published 10.01.2007.
6. L.Ya. Telishevskaya Protein hydrolyzates. Production, composition, use. М., 2000.
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14. United States Patent №6025327. Hydrolysed collagen type II and use thereof. 15.02.2000.
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D261–D266. doi:10.1093/nar/gkj008.
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Figures
Fig. 1. Distribution of molecular masses of peptides formed under the influence of himopsin in 33.4
mM K-Na phosphate buffer, pH 8.0 at 42 ° C, concentration 5% for 6 hours.
Fig. 2. Distribution of molecular masses of peptides formed under the influence of caripazim in 33.4
mM K-Na phosphate buffer, pH 6.0 at 55 ° C, concentration 5% for 6 hours.
6
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Patent RU No. 2250047. Food restorative prophylactic product out of cartilaginous tissue of hydrobionts and method for its obtaining
  • T N Pivnenko
  • Klychkova Gju
  • N N Kovalev
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  • Blinov Jug
Hydrolysed collagen type II and use thereof. US Patent 6025327A
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Bioactive compounds from marine processing byproducts
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  • E Mendis
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