Recombinant production of a collagen peptide preparation and use thereof
11673940 · 2023-06-13
Assignee
Inventors
- Stephan Hausmanns (Heidelberg, DE)
- Hans-Ulrich FRECH (Weinheim, DE)
- Steffen OESSER (Glücksburg, DE)
- Martin Hahn (Gronau, DE)
Cpc classification
C07K14/78
CHEMISTRY; METALLURGY
A61K8/65
HUMAN NECESSITIES
A61K38/39
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
International classification
C07K14/78
CHEMISTRY; METALLURGY
A61K8/65
HUMAN NECESSITIES
Abstract
The present invention relates to a method for producing collagen peptide preparations comprising recombinant collagen peptides, to collagen peptide preparations produced by means of said methods, to products containing the collagen peptide preparations and to uses of the aforementioned preparations and products.
Claims
1. A method for producing a collagen peptide preparation, comprising: a) expressing, from a coding nucleotide sequence, a collagen peptide having a molecular weight in a range from 8 to 100 kDa, and b) enzymatically hydrolyzing the expressed collagen peptide under conditions to produce a collagen peptide preparation comprising hydrolyzed collagen peptides having an average molecular weight of 1 to 5 kDa, wherein no more than 5.5 wt.-% of the hydrolyzed collagen peptides have a molecular weight less than 500 Da, no more than 2.8 wt.-% of the hydrolyzed collagen peptides have a molecular weight between 7500 Da and 13500 Da, and from 35 to 60 wt.-% of the hydrolyzed collagen peptides have a molecular weight between 1500 Da and 3500 Da.
2. The method according to claim 1, wherein the collagen peptide is expressed in a host cell selected from the group consisting of bacterial cell, yeast cell, fungal cell, mammalian cell, insect cell and plant cell.
3. The method according to claim 2, wherein the host cell is capable of hydroxylating proline residues, lysine residues or proline residues and lysine residues of the expressed collagen peptide.
4. The method according to claim 3, wherein the host cell comprises a polynucleotide sequence that encodes a prolyl 4-hydroxylase.
5. The method according to claim 3, wherein the host cell comprises a polynucleotide sequence that encodes a lysyl hydroxylase.
6. The method according to claim 2, wherein the host cell is incapable of causing hydroxylation of proline residues, lysine residues or proline and lysine residues of the expressed collagen peptide.
7. The method according to claim 6, wherein the expressed collagen peptide is hydroxylated before the hydrolysis.
8. The method according to claim 6, wherein the expressed collagen peptide is further hydroxylated after the hydrolysis.
9. The method according to claim 1, wherein the collagen peptide is a collagen peptide of a vertebrate, a mammal, a bird, a fish, an amphibian, a reptile, or an invertebrate animal.
10. The method according to claim 1, wherein the hydrolyzed collagen peptides have a molecular weight in a range 0.1 to 13.5 kDa.
11. A method for improving a skin, for accelerating the growth or reducing the fragility of nails, for improving hair, for increasing the number or activity of mitochondria, or for improving endurance or mental performance of a subject, comprising administering the subject an effective amount of hydrolyzed collagen peptides producible by the method of claim 1.
12. A food supplement comprising hydrolyzed collagen peptides producible according to claim 1 and at least one food-acceptable additive.
13. A cosmetic product comprising hydrolyzed collagen peptides producible according to claim 1 and at least one skin-compatible additive.
Description
(1) The invention is described below without restricting the general inventive concept with reference to figures, tables and the associated exemplary embodiments.
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EXAMPLES
Example 1—Production of Collagen Peptides
(14) 1.1 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Bovine Origin with a Size of 45 kDa, Produced Recombinantly in Pichia pastoris, with Neutral Protease
(15) A 0.789% collagen solution was initially heated to 50° C. in a 50 ml bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on the dry substance (TS) of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 6.2 with a 10% NaOH solution. In a next step, 0.8% Sumizyme BNP-L (based on TS of the collagen) was added to the collagen solution.
(16) The average molecular weight of the collagen peptides after a hydrolysis time of 180 minutes was determined to be 5.04 kDa.
(17) 1.2 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Bovine Origin with a Size of 45 kDa, Produced Recombinantly in Pichia pastoris, with Alkaline Protease
(18) A 0.792% collagen solution was initially heated to 63° C. in a 50 ml bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 7.75 with 10% NaOH solution. In a next step, 0.3% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(19) The average molecular weight of the collagen peptides after a hydrolysis time of 180 minutes was determined to be 3.01 kDa.
(20) 1.3 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Human Origin with a Size of 25 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(21) A 2.85% collagen solution was initially heated to 63° C. in a 50 ml bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the tempered collagen solution and the pH value of the solution was adjusted to 7.6 with 10% NaOH solution. In a next step, 0.3% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(22) The average molecular weight of the collagen peptides after a hydrolysis time of 45 minutes was 1.8 kDa.
(23) 1.4 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Human Origin with a Size of 100 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(24) First, a 2.85% collagen solution in a 50 ml bottle was heated to 63° C. in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 7.6 with 10% NaOH solution. Then 0.4% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(25) After a hydrolysis time of 45 minutes, the solution included collagen peptides having an average molecular weight of 2.4 kDa. The resulting collagen peptide preparation according to the invention was used as sample 2 in Example 6.
(26) 1.5 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Human Origin with a Size of 100 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(27) A 1.5% collagen solution was heated to 63° C. in a 50 ml bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 7.6 with 10% NaOH solution. Finally, 0.3% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(28) The average molecular weight of the collagen peptides after a hydrolysis time of 60 minutes was 1.8 kDa. The resulting collagen peptide preparation according to the invention was used as sample 1 in Example 6.
(29) 1.6 Hydrolysis of a Non-Hydroxylated Collagen Peptide of Human Origin with a Size of 25 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(30) A 2.85% collagen solution was initially heated to 63° C. in a 50 ml bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the tempered collagen solution and the pH value of the solution was adjusted to 7.6 with 10% NaOH solution. In a next step, 0.1% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(31) The average molecular weight of the collagen peptides after a hydrolysis time of 60 minutes was 3.4 kDa. The resulting collagen peptide preparation according to the invention was used as sample 3 in Example 6.
(32) 1.7 Hydrolysis of a Hydroxylated Collagen Peptide of Bovine Origin with a Size of 45 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(33) A 5.53% collagen solution was initially heated to 55° C. in a 250 ml glass bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 7.59 with 2% NaOH solution. In a next step, 0.2% Alcalase 2.4 L (based on TS of the collagen) was added to the collagen solution.
(34) The average molecular weight of the collagen peptides after a hydrolysis time of 150 min was determined to be 7 kDa. The resulting collagen peptide preparation according to the invention was used as sample 4 in Example 7.
(35) 1.8 Hydrolysis of a Hydroxylated Collagen Peptide of Bovine Origin with a Size of 45 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(36) A 5.00% collagen solution was initially heated to 55° C. in a 100 ml glass bottle in the cryostat. Then 200 ppm CaCl.sub.2×2H.sub.2O (based on TS of the collagen) were added to the temperature-controlled collagen solution and the pH value of the solution was adjusted to 7.60 with 2% NaOH solution. Then 0.25% NZ37071 (based on TS of the collagen) was added to the collagen solution.
(37) The average molecular weight of the collagen peptides after a hydrolysis time of 240 minutes was 5.6 kDa. The resulting collagen peptide preparation according to the invention was used in Example 7 as sample 5.
(38) 1.9 Hydrolysis of a Hydroxylated Collagen Peptide of Bovine Origin with a Size of 45 kDa Produced Recombinantly in Pichia pastoris with Alkaline Protease
(39) Starting from the collagen peptide hydrolyzate obtained according to Example 1.7, the higher molecular weight components of the hydrolyzate were removed by means of a concentrator (e.g. Vivaspin 20) with a size exclusion membrane of 5000 Da.
(40) An average molecular weight of 1.3 kDa was ascertained for the collagen peptides thus obtained. The collagen peptide preparation according to the invention was used as sample 6 in example 7.
(41) 1.10 Gel Chromatographic Analysis of the Collagen Peptide Hydrolysates
(42) The molecular weight distribution of the collagen peptide hydrolyzates obtained in Examples 1.3 to 1.5 and of two commercially available comparison products having an average molecular weight of 2.3 kDa and 1.7 kDa were determined by means of gel chromatography (Knauer, Germany). The statistical analysis was carried out using the WinGPC software (company PSS GmbH, Mainz, Germany). The following parameters were used for gel chromatography:
(43) TABLE-US-00001 Stationary phase: TSK 2000 SW XL (TOSOH Bioscience GmbH) Mobile phase: 0.4 mol/l sodium dihydrogen phosphate, pH 5.3 Flow rate: 0.5 ml/min Calibration standard: defined collagen type 1 fragments (FILK) Detection: UV detection at 214 nm (Knauer) Sample concentration: 1%
(44) For the different collagen peptide hydrolyzates, the percentages of individual collagen peptides shown in Table 1 and in
(45) TABLE-US-00002 TABLE 1 Evaluation of the percentages of the individual collagen peptides in defined molecular weight ranges. The analysis was carried out by means of gel chromatography using defined type 1 collagen peptide standards. Molecular Com- Com weight range parison parison of the product 1 product 2 Example Example Example individual 2.3 kDa 1.7 kDa 1.3 1.4 1.5 peptides Percentage Percentage Percentage Percentage Percentage in Da wt.-% wt-% wt.-% wt-% wt.-% <500 5.81 13.09 2.72 2.47 3.26 1500-500 43.63 50.84 45.78 37.69 47.84 3500-1500 31.87 25.52 43.67 40.82 39.29 7500-3500 15.63 8.93 7.64 16.74 9.23 13500-7500 2.88 1.41 0.19 2.28 0.38 >13500 0.2 0.21 0 0 0
(46) Chromatograms of a 1% solution each of the comparison products and the collagen peptide preparations according to Examples 1.3 to 1.5 are shown in
(47) The avoidance of the formation of such peptides is a further advantage for the production of sensory and organoleptically excellent collagen peptides classified as “neutral” tasting by the consumer. The same also applies to products having a higher average molecular weight in the range from 3 to 7 kDa.
(48) From the above-mentioned embodiments it may be deduced that the method according to the invention and thus the use of uniform recombinant collagen fragments as starting material for the hydrolysis enables the formation of preferred individual peptides within a narrow molecular weight distribution which, depending on the hydrolysis conditions selected, has characteristic peaks, usually between 4 and 6 (
(49) The formation of these individual peptides may be specifically controlled through the choice of the starting fragment as well as the hydrolysis conditions, which is almost impossible when using animal starting material due to their inhomogeneity.
Example 2—Ex Vivo Hydroxylation of Collagen Fragments
(50) For the post-translational modification (hydroxylation of proline residues) of collagen fragments ex vivo, a specific 4-OH prolyl hydroxylase (P4H) in the presence of the cofactors α-ketoglutarate, iron (II) ions and O.sub.2 was used, the use of nonspecific hydroxylases also being conceivable. For this purpose, 8 mM of the collagen fragment in the presence of 14 mM α-ketoglutarate, 0.5 mM iron (II) sulfate and 1.5 mM L-ascorbic acid in 50 mM MES buffer (pH 6.5) and of an enzyme solution in a total volume of 1 mL were incubated while shaking (300 RPM) at 37° C. on a thermomixer for 14 to 18 hours. Alternatively, the incubation may also take place in an incubator under the aforementioned conditions.
Example 3—Osteoblast Activity (Particularly Bone Health)
(51) To analyze the biological effectiveness of the collagen peptide preparation according to the invention in terms of maintaining bone health and the prophylaxis and treatment of bone diseases, its stimulating effect on the synthesis of matrix proteins and enzymes that play a role in the structure and mineralization of the matrix is examined via osteoblasts in vitro. This is done by determining the expression of the corresponding mRNA by means of real-time PCR and a semi-quantitative evaluation (based on a control without collagen hydrolyzate).
(52) For this purpose, human osteoblasts are first isolated from knee joints by incubating bone material under vigorous agitation at 37° C. for 1 h in Hanks' solution, supplemented with 7 mg/ml hyaluronidase type I and III-S and 5 mg/ml pronase. The digestion is then continued at 37° C. for 3-5 h in Hanks' solution supplemented with 16 mg/ml collagenase type CLS IV. The primary osteoblasts obtained are cultivated after enzymatic digestion in Ham's F12 medium, supplemented with 10% fetal calf serum, 20 U/ml penicillin-streptomycin, 50 μg/ml partricin, 0.05 mg/ml ascorbic acid and 0.15 mg/ml glutamine. Alternatively, primary osteoblasts (Article No. C-12760; 2019) may also be obtained from PromoCell GmbH, Heidelberg, Germany for investigating the biological effectiveness. The cells are then cultivated in Ham's F12 medium, supplemented with 10% fetal calf serum, 20 U/ml penicillin-streptomycin, 50 μg/ml partricin and 0.15 mg/ml glutamine.
(53) To investigate the biological effectiveness, monolayer cell cultures of the isolated human osteoblasts are incubated for a period of 24 hours in a medium that is supplemented with 0.5 mg/ml of the respective collagen peptide preparation. A control is incubated in each case in a medium without preparation. The respective mRNA expression is then determined.
Example 4—Fibroblast Activity (Particularly Skin Health)
Example 4.1—Stimulation of mRNA Synthesis
(54) The stimulation of the synthesis of collagen (type 1) and the proteoglycans biglycan and versican is investigated in vitro on human dermal fibroblasts (skin cells). For this purpose, the cells are incubated for 24 hours with 0.5 mg/ml of a low molecular weight or the collagen peptide preparation according to the invention, and the expression of collagen RNA, biglycan RNA and versican RNA is then determined by real-time PCR and semi-quantitatively (based on a control without preparation).
Example 4.2—Stimulation of the Synthesis of Connective Tissue Proteins
(55) To determine the stimulation of the synthesis of proteins of the connective tissue via the collagen peptide preparations according to the invention, primary human dermal fibroblasts after enzymatic digestion are initially cultured in HAM's F12 medium, comprising 10% FCS, 20 U/ml penicillin-streptomycin, 50 μg/ml partricin, 0.05 mg/ml ascorbic acid and 0.15 mg/ml glutamine. After reaching a confluence of 80%, the respective culture medium is replaced by a medium without collagen peptide (control) or with 0.5 mg/ml of a collagen peptide preparation to be tested, and the primary human fibroblasts are incubated in the respective medium for a period of at least 14 days, preferably 14 to 21 days, in particular 14 days. The expression of different proteins of the connective tissue may then be determined and evaluated by means of suitable assays (see, for example, Examples 6 and 7).
Example 5—Chondrocyte Activity (Particularly Cartilage Health)
(56) For the cell cultures, porcine or human chondrocytes are isolated from cartilage tissue in a known manner and sown on culture plates at a density of approximately 350,000 cells/cm.sup.2. Ham's F12 medium with 10% fetal calf serum, 10 μg/ml gentamicin and 5 μg/ml amphotericin B is used as the culture medium. As an alternative to 10 μg/ml gentamicin, 10 μg/ml penicillin-streptomycin may also be used. The cultivation took place at 37° C. in an oxygen-reduced atmosphere (5% 02, 5% CO.sub.2 and 90% N.sub.2).
(57) Determination of Collagen Biosynthesis:
(58) The quantification of the collagen synthesized by the chondrocytes (essentially type II) is carried out by radioactive labeling with .sup.14C-proline, which is incorporated into the collagen.
(59) .sup.14C-proline is first added to the culture medium and the chondrocytes are cultivated under these conditions until the time of the determination. In order to be able to distinguish the incorporated from non-incorporated .sup.14C-proline during the detection, the isotope-containing culture medium is then replaced by pure culture medium for a period of 3 days. The culture medium is then discarded and the adherent cell layer is mixed with distilled water in order to destroy the cell membranes through osmotic stress and to release cytosolic, unbounded .sup.14C-proline. The cell debris with the synthesized extracellular matrix is pelleted by centrifugation. The pellet is re-suspended in fresh distilled water and a xylene scintillation cocktail is added. The amount of synthesized collagen may then be quantified by detecting the 14C-Proline with a beta counter.
(60) Alternatively, the quantification may be carried out using the Sircol Collagen Assay Kit (Article No. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions (see Examples 6 and 7).
(61) Determination of Proteoglycan Biosynthesis:
(62) The proteoglycans synthesized by the chondrocytes are quantified by means of Alcian blue staining and photometric determination of the glycosaminoglycans (GAG), which are components of the proteoglycans.
(63) In order to determine the GAG content in the cell culture, the culture medium is first discarded and the adherent cell layer is rinsed with PBS buffer (pH 7). The cells are then fixed in a 10% formaldehyde solution in PBS at 4° C. for 2 hours. After removing the formaldehyde, the Alcian blue staining reagent (5% Alcian blue in 3% acetic acid) is applied to the cell lawn and incubated at 4° C. overnight. Unbound Alcian blue is discarded and washed out by carefully rinsing three to four times with PBS. By adding acidic guanidine solution (8 mol/l), the GAG complexes are released from the cell layer. The amount of glycosaminoglycans may then be quantified photometrically at a wavelength of 620 nm.
(64) Alternatively, the quantification may be carried out using the Blyscan Glycosaminoglycan Assay Kit (Article No. 054B3000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions (see Examples 6 and 7).
Example 6—Stimulation of the Synthesis of Collagen, Elastin and Proteoglycan in Primary Human Fibroblasts Via Non-Hydroxylated Collagen Peptide Preparations According to the Invention
(65) To determine the stimulation of the synthesis of collagen, elastin and proteoglycan, primary human dermal fibroblasts according to Example 4.2 were incubated for a period of at least 14 days, preferably 14 to 21 days, in particular 14 days, in a medium without collagen peptide (control 1) and in the presence of 0.5 mg/ml of a 100 kDa collagen peptide (control 2), of a collagen peptide preparation having an average molecular weight of 1.8 kDa (sample 1), of a collagen peptide preparation having an average molecular weight of 2.4 kDa (sample 2) and one of a collagen peptide preparation having an average molecular weight of 3.4 kDa (sample 3).
Example 6.1—Determination of the Stimulation of the Synthesis of Collagen
(66) The determination of collagen synthesis by primary human dermal fibroblasts was carried out using the Sircol Collagen Assay Kit (Article No. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 2 and
(67) TABLE-US-00003 TABLE 2 Determination of the optical density (OD) at a wavelength of 450 nm for ascertaining the collagen synthesis according to the Sircol Collagen Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 1 Sample 2 Sample 3 Control 2 (average (average (average (molecular molecular molecular molecular weight = weight = weight = weight = Control 1 100 kDa) 1.8 kDa) 2.4 kDa) 3.4 kDa) Average 1 1.01 1.2 1.24 1.21 (OD.sub.450) Standard 0 0.01 0.07 0.1 0.06 deviation
Example 6.2—Determination of the Stimulation of the Synthesis of Elastin
(68) The determination of elastin synthesis via primary human dermal fibroblasts was carried out using the Fastin Elastin Assay (Article No. 054F2000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 3 and
(69) TABLE-US-00004 TABLE 3 Determination of the optical density (OD) at a wavelength of 450 nm for ascertaining the elastin synthesis according to the Fastin Elastin Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 1 Sample 2 Sample 3 Control 2 (average (average (average (molecular molecular molecular molecular Control weight = weight = weight = weight = 1 100 kDa) 1.8 kDa) 2.4 kDa) 3.4 kDa) Average 1 0.96 1.28 1.25 1.38 (OD.sub.450) Standard 0 0.22 0.13 0.02 0.3 deviation
Example 6.3—Determination of the Stimulation of the Synthesis of Glycosaminoglycan
(70) The determination of the synthesis of glycosaminoglycans via primary human dermal fibroblasts was carried out using the Blyscan glycosaminoglycan assay (Article No. 054B3000, 2019, tebu-bio, Offenbach, or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 4 and
(71) TABLE-US-00005 TABLE 4 Determination of the optical density (OD) at a wavelength of 450 nm for ascertaining the synthesis of glycosaminoglycans according to the Blyscan Glycosaminoglycan Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 1 Sample 2 Sample 3 Control 2 (average (average (average (molecular molecular molecular molecular Control weight = weight = weight = weight = 1 100 kDa) 1.8 kDa) 2.4 kDa) 3.4 kDa) Average 1 1 1.28 1.26 1.2 (OD.sub.450) Standard 0 0.09 0.08 0.08 0.06 deviation
Example 7—Stimulation of the Synthesis of Collagen, Elastin and Proteoglycan in Primary Human Fibroblasts Via Hydroxylated Collagen Peptide Preparations According to the Invention
(72) To determine the stimulation of the synthesis of collagen, elastin and proteoglycan, primary human dermal fibroblasts according to Example 4.2 were incubated for a period of at least 14 days, preferably 14 to 21 days, in particular 14 days, in a medium without collagen peptide (control 1) and in the presence of 0.5 mg/ml of a collagen peptide preparation having an average molecular weight of 7.0 kDa (sample 4), of a collagen peptide preparation having an average molecular weight of 5.6 kDa (sample 5) and of a collagen peptide preparation having an average molecular weight of 1.3 kDa (Sample 6).
Example 7.1—Determination of the Stimulation of the Synthesis of Collagen
(73) The determination of collagen synthesis via primary human dermal fibroblasts was carried out using the Sircol Collagen Assay Kit (Article No. Article No. 054S5000, 2019, tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 5 and
(74) TABLE-US-00006 TABLE 5 Determination of the optical density (OD) for ascertaining the collagen synthesis according to the Sircol Collagen Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 4 Sample 5 Sample 6 (average (average (average molecular molecular molecular weight = weight = weight = Control 1 7.0 kDa) 5.6 kDa) 1.3 kDa) Average 1 1.35 1.36 1.39 Standard 0 0.16 0.1 0.09 deviation
Example 7.2—Determination of the Stimulation of the Synthesis of Elastin
(75) The determination of elastin synthesis by primary human dermal fibroblasts was carried out using the Fastin Elastin Assay (Article No. 054F2000, 2019, tebu-bio, Offenbach, Germany or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 6 and
(76) TABLE-US-00007 TABLE 6 Determination of the optical density (OD) for ascertaining the elastin synthesis according to the Fastin Elastin Assay (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 4 Sample 5 Sample 6 (average (average (average molecular molecular molecular weight = weight = weight = Control 1 7.0 kDa) 5.6 kDa) 1.3 kDa) Average 1 1.32 1.33 1.31 Standard 0 0.11 0.07 0.04 deviation
Example 7.3—Determination of the Stimulation of the Synthesis of Glycosaminoglycan
(77) The determination of the synthesis of glycosaminoglycans via primary human dermal fibroblasts was carried out using the Blyscan glycosaminoglycan assay (Article No. 054B3000, 2019, tebu-bio, Offenbach, or Biocolor Ltd., UK) according to manufacturer instructions. The results of the experiment are shown in Table 7 and
(78) TABLE-US-00008 TABLE 7 Determination of the optical density (OD) for ascertaining the synthesis of glycosaminoglycans according to the Blyscan Glycosaminoglycan Assays (tebu-bio, Offenbach, Germany, or Biocolor Ltd., UK). Sample 4 Sample 5 Sample 6 (average (average (average molecular molecular molecular weight = weight = weight = Control 1 7.0 kDa) 5.6 kDa) 1.3 kDa) Average 1 1.37 1.41 1.40 Standard 0 0.15 0.11 0.12 deviation