EA043096B1 - COMPOSITION INCLUDING A STRUCTURED AQUEOUS PHASE AND SERICIN - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to compositions that include a structured aqueous phase and 0.1-10 wt.% sericin relative to the mass of water, and the specified sericin is characterized by a proportion of beta-sheet in the secondary structure of more than 80%.

Prerequisites for the creation of the invention

In cosmetics, pharmaceuticals, detergents, tissue engineering and water-based food products, it is necessary to control the rheological properties of the formulation in order to give the formulation the desired feel, appearance, viscosity, etc. A structurant (water structuring component) is used in the aqueous phase of such formulations to adjust the rheological properties. Depending on the nature of the structure-forming component for water, the composition of the aqueous solution, and the concentration of the structure-forming component used, these rheological properties can vary over a wide range.

Sericin, a component of silk, is a protein that can act as a structurant for water. Silk is a natural protein fiber derived from the silkworm Bombyx mori and is usually composed of the two proteins fibroin (70-80%) and sericin (20-30%), the remainder being a mixture of wax, carbohydrates and inorganic substances. Fibroin is a fibrous glycoprotein present as a thin double strand coated with layers of sericin which promote cocoon formation. To produce silk from dried silkworm cocoons, fibroin is separated from sericin using the so-called degumming process, and sericin is sent to wastewater. Degumming under the influence of heat or heat under pressure has the advantage that it results in fewer impurities. Many industrial removal methods include extraction with soaps and detergents, enzymatic hydrolysis, acid or alkaline hydrolysis.

Until recently, sericin was considered a waste product of silk production. Sericin exists in a wide range of molecular weights from 10 to over 400 kDa. Sericin is found mainly in an amorphous random helix structure and to a lesser extent in a beta sheet structure (Padamwar et al., Silk sericin and its applications: A review, Journal of Scientific & Industrial Research, 2004, 63, pp. 323-329).

US 2009/176965 discloses that the molecular structure of isolated sericin can be modified by irradiating a solution of sericin to produce high molecular weight sericin with improved radical scavenging and tyrosinase inhibiting properties. A maximum of 50% of the beta sheet structure is formed (Fig. 2, irradiation dose 200 kGy). Sericin having a modified molecular structure can be used in the manufacture of various products to improve the antioxidant and tyrosinase inhibitory capacity, which include foods, cosmetics and/or pharmaceutical products and drugs.

JP 2006111667 discloses a method for producing sericin hydrogel. A silk extract with a molecular weight of more than 50 kDa is proposed, to which alcohol is added and the mixture is kept. Sericin in aqueous solution is characterized by absorption at 1641 cm ^-1 that refers to the statistical structure in the absorption band of the amide. On the other hand, sericin hydrogel absorbs at 1620 cm ^-1 , which corresponds to the beta sheet structure in the absorption band of amide I. The addition of ethanol to promote hydrogen bonding between the sericin molecules and the resulting three-dimensional structure forms the sericin hydrogel.

US 2016/0136241 discloses a sericin hydrogel. Bombyx mori fibroin-deficient mutant silkworm cocoons are used as raw materials. The sericin solution is obtained by extraction and purification of the starting material, and then cross-linked with a cross-linking agent (aldehydes and geniposide) to obtain a sericin hydrogel. The cross-linked sericin hydrogel retains the natural conformation of sericin.

Brief description of the essence of the invention

The inventors have developed a method which allows sericin to be isolated from silk, such that such sericin has an extremely high proportion (>80%) of the beta sheet in the secondary structure. It has also been found that such sericin is an excellent structurant for water and can accordingly be used to structurize the aqueous phase of a wide range of products such as cosmetics, pharmaceuticals, food and detergent compositions.

Thus, the present invention relates to a composition comprising 5-100 wt.% of a structured aqueous phase, and the specified composition contains 0.1-10 wt.% sericin relative to the mass of water, and the specified sericin is characterized by a beta-sheet fraction in the secondary structure of more than 80 %.

The sericin of the present invention, that is, the sericin having more than 80% beta-sheet structure, can be isolated from silkworm cocoons by a method which includes the following steps:

cutting cocoons into pieces approximately 5x5 mm;

first washing of cocoons with demineralized water (demin water) at about 25°C;

second washing of cocoons with demineralized water at about 70°C;

second washing of cocoons with ethanol at about 25°C;

- 1 043096 removal of ethanol from washed cocoons by evaporation at temperatures below 30°C;

mixing 1 wt.h. dry washed cocoons with about 20 wt.h. hot water (~95°C) to form an aqueous suspension;

keeping the aqueous suspension at about 120° C. for about 5-20 minutes;

centrifugation of the heated suspension at 4000 g for 20 min, followed by decantation to extract the sericin protein extract.

In the process described above, preferably the aqueous suspension is maintained at a temperature of about 120° C. for up to 15 minutes, preferably 10 minutes and most preferably up to 5 minutes. The present inventors have determined that when the aqueous suspension is held at a temperature of about 120° C. for up to about 20 minutes, the sericin thus obtained has a betalyst content in the secondary structure of more than 80%. The amount of sericin in beta-sheet form decreases if the indicated temperature is maintained longer.

In contrast to the prior art method described in US 2009/0176965, beta-sheet high sericin can be easily used without the use of gamma, electron or x-ray irradiation.

It has been found that the rheological properties of the sericin-structured aqueous phase in the composition of the invention can be controlled by changing the pH and/or by adjusting the concentration of multivalent cations (eg Zn ²⁺ , Cu ²⁺ , Ca ²⁺ ).

Also proposed is a method for obtaining a composition of the present invention, including obtaining an aqueous component containing sericin, characterized by a beta-sheet share in the secondary structure of more than 80%; and mixing 100 wt.h. the specified water component with at least 5 wt.h. one or more other components.

Detailed description of the invention

As used herein, the term comprising encompasses the terms consisting essentially of and consisting of. When the term including is used, the listed steps or options are not necessarily exhaustive.

Unless otherwise noted, numeric ranges expressed in the format x to y are treated as including x and y.

When specifying any range of values or amounts, any specific higher value or amount can be combined with any specific lower value or amount.

Except for examples and comparative experiments or where expressly stated otherwise, all numbers are to be understood as modified by the word about.

All percentages and ratios contained in this document are by weight unless otherwise noted.

As used herein, the indefinite article a or an and its corresponding definite article the mean at least one or one or more, unless otherwise indicated.

The various features of the present invention mentioned in the individual sections above apply, where appropriate, to other sections mutatis mutandis (ie, with necessary modifications). Therefore, features listed in one section may be combined with features listed in other sections where appropriate. Any section headings are added for convenience only and are not intended to limit the present disclosure in any way.

It should be understood that, when the features indicated in the claims are accompanied by reference numbers, such numbers are included solely for the purpose of improving the clarity of the claims and in no way limit the scope of the claims.

The present invention relates to a composition comprising 5-100 wt.% structured aqueous phase, and the composition contains 0.1-10 wt.% sericin relative to the mass of water, and the specified sericin is characterized by a beta-sheet share in the secondary structure of more than 80%.

Used in the description, the term structured aqueous phase, unless otherwise indicated, refers to the aqueous phase, which is characterized by non-Newtonian behavior due to the presence of one or more hydrocolloids.

As used herein, the term sericin, unless otherwise indicated, refers to a sericin polypeptide derived from the Bombyx mori silkworm, said polypeptide having a molecular weight of at least 2 kDa. Sericin from Bombyx mori is identified as P07856 in the UniProtKB database. The term sericin also encompasses partially hydrolysed forms of naturally occurring sericin.

As used herein, the term secondary structure, unless otherwise indicated, refers to the spatial arrangement of local segments of sericin polypeptides, referred to as alpha helix, beta helix, or random helix.

As used herein, the term alpha helix, unless otherwise indicated, refers to

- 2 043096 parts of a molecular structure, which is a rod-like structure, in which a tightly twisted frame forms the inside of the rod, and the side chains are directed outward in the form of a helical structure. The alpha helix is stabilized by hydrogen bonds between the NH and CO groups of the backbone. In particular, the CO group of each amino acid forms a hydrogen bond with the NH group of the amino acid, which is located four residues forward (i+4) in the main chain (Berg JM, Tymoczko JL, Stryer L., Biochemistry 5th Ed. New York, WH Freeman, 2002, 3.3.1).

As used herein, the term beta sheet, unless otherwise indicated, refers to the part of the molecular structure formed by hydrogen bonding two or more beta strands. The beta strand is a stretch of the polypeptide chain, typically 3-10 amino acids long along the main chain in a stretched conformation. Adjacent strands in a beta sheet can run in opposite directions (anti-parallel β-sheet) or in the same direction (parallel β-sheet). In the antiparallel arrangement, the NH group and the CO group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of the partner in the adjacent strand, in the parallel arrangement, the hydrogen bonding scheme is more complex. For each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid of the adjacent chain, while the CO group is hydrogen bonded to the NH group of the amino acid two residues further down the chain (Berg JM, Tymoczko) JL, Stryer L., Biochemistry 5 ^th Ed. New York, W.H. Freeman, 2002, 3.3.2).

As used herein, the term random helix, unless otherwise indicated, refers to a portion of a molecular structure that does not have a defined pattern in hydrogen bonding, such as an alpha helix or beta sheet.

The secondary structure of sericin can be determined using circular dichroism spectroscopy in the far-UV spectral region (190-250 nm). At these wavelengths, the chromophore is a peptide bond and the signal occurs when it is arranged in a normal fold. The structure of the beta sheet is characterized by maximum ellipticity per residue (theta) at 195 nm and minimum ellipticity at 220 nm. The alpha helix is characterized by a maximum ellipticity per remainder (theta) at about 190 nm and double minima at about 205 nm and 225 nm. The structure of the statistical helix is characterized by a minimum ellipticity of about 200 nm and a wide maximum between 210 and 220 nm.

According to a most preferred embodiment, the sericin in the composition of the present invention has a beta-sheet fraction in the secondary structure of greater than 80% or even greater than 85% and most preferably greater than 90%.

In one preferred embodiment, sericin has a molecular weight in the range of 2-400 kDa, preferably 25-300 kDa, more preferably 40-250 kDa, even more preferably in the range of 60-200 kDa.

In a preferred embodiment of the invention, the composition contains 0.2-4 wt.% sericin relative to the mass of water. Preferably the composition contains 0.3-3 wt.% sericin relative to the mass of water, more preferably 0.4-2 wt.% sericin relative to the mass of water.

Preferably, at least 80% by weight of the sericin contained in the composition is present in the structured aqueous phase. More preferably at least 90% by weight of the sericin, most preferably at least 95% by weight of the sericin contained in the composition is present in the structured aqueous phase.

The inventors have found that the ability of sericin to structure water is optimal when the pH of the structured aqueous phase is in the range of 2.0-4.0 or 5.5-10.5. Preferably the structured aqueous phase has a pH in the range of 5.8-9.5, even more preferably in the range of 6-8.5.

The composition of the present invention may be in the form of a single phase aqueous composition or in the form of a composition that includes one or more additional separate phases. Examples of such multi-phase compositions are emulsions and suspensions. According to a most preferred embodiment, the composition comprises 1-80% by weight, more preferably 5-70% by weight, most preferably 10-50% by weight of one or more non-aqueous phases.

According to a particularly preferred embodiment, the structured aqueous phase is a continuous aqueous phase. Compositions comprising such a continuous structured aqueous phase preferably have a storage modulus (G') at 25°C in the range of 50-3000 Pa, more preferably in the range of 100-2000 Pa, and most preferably 200-800 Pa. The loss modulus (G) of this composition is preferably in the range of 10-300 Pa, more preferably in the range of 20-200 Pa, and most preferably in the range of 50100 Pa.

Using oscillatory methods for determining rheological properties, it is possible to quantify both the viscous-like and elastic-like properties of a material on different time scales. The basic principle of an oscillating rheometer is to induce a sinusoidal shear strain in the sample and measure the resulting stress; the measured time scale is determined by the oscillation frequency ω of the shear strain. The sample is placed between two plates. While the top plate remains stationary, the motor rotates the bottom plate, thereby creating a time-dependent strain γ(t)=γsin(ωt) on the specimen. The simultaneously time-dependent stress σ(t) is quantified by measuring the torque that the specimen transmits to the top plate.

Both the storage modulus (G') and the loss modulus (G) of the liquid product are determined at 25° C. using an AR1000 rheometer (TA Instruments) using a conical plate geometry, with a cone diameter of 40 mm, a cone angle of 2° and a gap of 58 µm.

The applicable program settings are as follows.

The voltage t is selected in the linear-viscous-elastic range of the product (LVER is determined using an amplitude sweep). The voltage is kept constant at 1 Pa.

The rate of rise of the angular frequency ω on the sample is set from low to high frequency starting from 6.28 rad/s. The final ω is 628.32 rad/s.

The setting at which the measuring points are dialed is the setting without time. In this mode, the device is in standby mode until it reaches its measuring point.

For each ten, 10 measurement points are taken.

According to another preferred embodiment, the structured aqueous phase is a gelled aqueous phase which is structured with a three-dimensional hydrocolloid structure. The gelled aqueous phase may be gelled with a three-dimensional structure that is composed solely of sericin (sericin hydrogel) or, alternatively, it may include a three-dimensional structure that also includes other gelling agents.

The structured aqueous phase usually contains at least 50% by weight of water. Preferably, the structured aqueous phase contains at least 60 wt% water, more preferably at least 70 wt% water, more preferably at least 80 wt% water, and most preferably at least 85 wt% water.

In accordance with the most preferred embodiment, the present composition is an emulsion comprising 1-80 wt.% hydrophobic phase and 20-99 wt.% structured aqueous phase. More preferably, the emulsion comprises 5-70% by weight of a hydrophobic phase and 30-95% by weight of a structured aqueous phase. Most preferably, the emulsion contains 10-50% by weight of a hydrophobic phase and 50-90% by weight of a structured aqueous phase. In addition to the hydrophobic phase and the structured aqueous phase, the emulsion composition may contain other phases, such as a dispersed phase consisting of fine particles of solid material.

The inventors have found that the ability of sericin to structure water can be enhanced by the presence of multivalent metal cations. Therefore, in yet another preferred embodiment, the composition comprises at least 0.08 mmol of a multivalent metal cation, said multivalent metal cation being selected from Zn ²⁺ , Cu ²⁺ , Ca ²⁺ and combinations thereof. More preferably, the composition comprises at least 0.1 mmol of said polyvalent metal cation, even more preferably in the range of 0.2-0.9 mmol of said polyvalent metal cation, more preferably 0.3-0.8 mmol of said polyvalent metal cation and most preferably 0.4-0.6 mmol of said multivalent metal cation.

The sericin used in accordance with the present invention is preferably high purity sericin. Accordingly, in another preferred embodiment, the present composition contains fibroin and sericin in a mass ratio of fibroin:sericin of not more than 1:5, preferably not more than 1:10.

In the most preferred embodiment, the structured aqueous phase comprises a sericin hydrogel. It was found that the sericin of the present invention forms a hydrogel without additional hydrocolloids. Typically, the sericin contained in the composition is able to form a stable gel at a sericin concentration of 1% relative to the mass of water. More preferably, said stable gel has a storage modulus (G') at 25° C. of at least 200 Pa.

Unlike the sericins described in US 2016/036241, the sericin of the present invention is not a gluteraldehyde-crosslinked sericin. More preferably, the sericin is not a cross-linked sericin.

Unlike the sericins described in US 2009/176965, the sericin of the present invention is not irradiated sericin that has been irradiated with an absorbed dose of 10-500 kGy.

The structured phase of the composition of the present invention preferably contains less than 0.1 wt.% ethanol, more preferably less than 0.01 wt.% ethanol.

In a specific embodiment, the present composition comprises at least 0.3%, preferably at least 0.4% sericin, based on the weight of water. This sericin preferably has a molecular weight of at least 10 kDa and is characterized by a beta-sheet fraction in the secondary structure of more than 80%.

In a more preferred embodiment, the composition comprises at least 0.3%, preferably at least 0.4% sericin, based on the weight of water, having a molecular weight of at least 15 kDa and having a beta-sheet fraction in the secondary structure of more than 85%.

- 4 043096

The composition of the present invention is preferably a cosmetic product, more preferably a cosmetic product selected from a lotion, cream, ointment and salve.

Another aspect of the present invention relates to a process for preparing a composition as defined in the description above, said process comprising:

obtaining an aqueous component containing sericin, characterized by a beta-sheet share in the secondary structure of more than 80%; and mixing 100 wt.h. of the specified water component with at least 5 wt.h., preferably at least 10 wt.h., more preferably at least 20 wt.h. one or more other components.

Preferably, the sericin-containing aqueous component is liquid when mixed with one or more other components. In the event that the aqueous component contains sericin at a concentration sufficient to gel the aqueous component, the aqueous component may be liquefied prior to mixing and allowed to gel after mixing. This can be achieved by adjusting the pH of the aqueous component to a pH in the range of 4.0-5.5 before mixing, and then adjusting the pH to less than 3.9 or greater than 5.6 after said mixing.

One or more other components that are mixed with the aqueous component in the present process are preferably selected from the group consisting of fillers, colorants, pH modifiers, solvents, polymers and thickeners.

In accordance with the most preferred embodiment, the method includes adding a water-soluble salt of a multivalent metal cation selected from Zn ²⁺ , Cu ²⁺ , Ca ²⁺ and combinations thereof.

Examples

Example 1: extraction of sericin.

Sericin is extracted from Bombyx mori cocoons using the following procedure.

i) The cocoons are soaked in ultrapure and degassed water at ambient temperature for 15 minutes, shaking the contents every 5 minutes. Cocoons are separated from the water with tweezers.

ii) The pre-washed pieces are again soaked in ultrapure and degassed water at 70° C. for 30 minutes (with shaking every 5 minutes). Cocoons are separated from the water with tweezers.

iii) Cocoons are soaked in ethanol at room temperature for 15 minutes (shaking every 3 minutes). Cocoons are separated from ethanol with tweezers.

Cocoons are dried on Whatman filter paper at ambient temperature and atmospheric pressure. Dried cocoons are used as starting material for the extraction process.

Known amounts of water are introduced into the containers and heated to 95° C. (using a bottom agitated autoclave). The pre-washed cocoons (ratio of cocoons to water 1:30) are added to water and the suspensions thus obtained are subjected to various extraction regimes.

Sample 1 is kept under stirring and heating to 120°C for 5 minutes.

Sample 2 is extracted under the same conditions using an extraction time of 30 minutes.

Sample 3 is also extracted under the same conditions using an extraction time of 90 minutes.

At the end of the heating period, the heating is turned off and the pressure is immediately released (this allows the slurry to cool more quickly).

Sericin is separated from fibroin by centrifugation (6000 rpm (4000 g) for 30 min) and decanting. The content of serecin in the extracts is about 1.1 wt.%

The molecular weight of the extracted sericin from sample 1 was determined by SDS-PAGE analysis and gave values between 35 kDa and 250 kDa.

Example 2: secondary structure.

The secondary structure of the sericin protein in samples 1-3 of Example 1 was analyzed using circular dichroism (CD) measurements. Standard CD spectra of protein secondary structures are recorded using scanning in the wavelength range of 195-240 nm, and then data is obtained using K2D3 online analysis software.

The folded protein consists of α-helix secondary structures; β-sheet; loops (linker residues connecting domains of the secondary structure). The K2D3 software uses scanning in the 200-240 nm wavelength range. The mean circular dichroism residue ellipticity refers to the percentage of amino acids that contribute to the secondary structures (α-helix and β-sheet) and does not include residues in the linker region. The maximum sum of all secondary structures (46%) was taken from a computer model of the sericin protein. The structure of the sericin beta sheet was evaluated using the results obtained using the K2D3 software.

CD data are presented as Mean Residual Ellipticity (MRE) expressed in deg cm ² dmol ^-1 as a function of wavelength using the following equation:

- 5 043096 where is the calculated average ellipticity of the residue (deg cm ² dmol ^-1 ); MRW is the average mass of a residue for a peptide bond (MRW is calculated as MRW=M/N ^-1 where M is the molecular weight of the peptide chain (Da) and N is the number of amino acids in the chain); [θ]^, observed ellipticity (expressed in degrees); d optical path length (cm); c, protein concentration (chl ^-1 ). All CD spectra are corrected for buffer concentration and secondary structure calculated using the K2D3 web tool for K2D3 analysis: Kelly et al. (2005) Biochimica et Biophysica Acta 1751: 119-39. PMID: 16027053 neural network software package.

The results of measurements of CD are given in table. 1.

Table 1

Sample Extraction time/minutes Temperature/°С % beta lista 1 5 120 92% 2 thirty 71% 3 90 thirty%

Example 3: rheological studies.

The rheological properties of the extracts of sericin from example 1 is determined by measuring the dynamic modulus of elasticity and the loss modulus of these extracts (at 25°C). The results are shown in table. 2.

table 2

Sample Sericin concentration Temperature/°С Time extraction/minutes G' (Pa) G (Pa) 1 1.1 120 5 980 120 2 1.1 thirty 65 7 3 1.1 90 5 1

Hydrogels were prepared using various concentrations of Sample 1 from Example 1. The storage modulus and loss modulus of the hydrogels were determined (at 25°C). The results of these measurements are given in table. 3.

Table 3

Sericin concentration (% w/w) G' (Pa) G” (Pa) 0.9 104 AND 1.3 1027 97 1.6 3156 323

Example 4: rheological studies in the presence of metal ions.

Hydrogels containing Zn ²⁺ are prepared using Sample 1 from Example 1 and by adding varying amounts of ZnCl ₂ .

The dynamic modulus of elasticity and the loss modulus of hydrogels are determined. The results of these measurements are given in table. 4.

The effect of pH on the storage modulus of the sericin hydrogel was determined using Sample 1 of Example 1. The pH of a 0.9% sericin aqueous solution was adjusted using HCl (molarity 0.1) or NaOH (molarity 0.1) to obtain solutions with different pH.

The dynamic modulus of elasticity and the loss modulus of hydrogels are determined. The results of these measurements are given in table. 5.

Table 5

formulation pH G' G” 2.5 104 19 3.5 260 31 4.5 sericin precipitate sericin precipitate 6.4 126 12 7.5 149 14 9 40 4

Hydrogel formation occurs at pH<3.5 and pH between 6-9.5. Hydrogel does not form in the pH range of 4-5.5.

Claims (12)

1. Cosmetic composition comprising 5-100 wt.% of a structured aqueous phase, and the composition contains 0.1-10 wt.% sericin relative to the mass of water, where sericin is characterized by a beta-sheet share in the secondary structure of more than 90%, and where the structured aqueous the phase has a pH in the range of 2.0-4 or 5.5-10.5, where the structured aqueous phase is an aqueous phase that exhibits non-Newtonian behavior and includes a sericin hydrogel. 2. Composition according to claim 1, in which sericin has a molecular weight in the range of 2-400 kDa. 3. Composition according to claim 1 or 2, containing 0.2-4 wt.% sericin relative to the mass of water. 4. Composition according to any one of claims 1 to 3, comprising 1-80 wt.% of one or more non-aqueous phases and 20-99 wt.% of a structured aqueous phase. 5. A composition according to any one of claims 1 to 4, comprising at least 0.08 mmol of a multivalent metal cation, said multivalent metal cation being selected from Zn ²⁺ , Cu ²⁺ , Ca ²⁺ and combinations thereof. 6. A composition according to any one of claims 1 to 5, wherein the structured aqueous phase is a continuous aqueous phase. 7. A composition according to any one of claims 1 to 6, wherein the structured aqueous phase contains at least 50% by weight of water. 8. A composition according to any one of claims 1 to 7, comprising at least 0.4% sericin relative to the weight of water, where said sericin has a molecular weight of at least 10 kDa and is characterized by a beta-sheet share in the secondary structure of more than 90%. 9. The composition according to any one of claims 1 to 8, which is a cosmetic product selected from lotion, cream and ointment. 10. The method of obtaining a composition according to any one of claims 1 to 9, containing one or more other components in addition to the structured aqueous phase, and the method includes obtaining an aqueous component containing sericin, characterized by a beta-sheet share in the secondary structure of more than 90%; and mixing 100 wt.h. the specified water component with at least 5 wt.h. one or more additional components, and the pH of the aqueous component is adjusted to a pH in the range of 4.0-5.5 before mixing, and then, after mixing, the pH is adjusted to a level of less than 3.9 or more than 5.6. 11. The method of claim 10, wherein one or more other components are selected from the group consisting of fillers, colorants, pH modifiers, solvents, polymers, and thickeners. 12. The method of claim 10 or 11, comprising adding a water-soluble salt of a multivalent metal cation selected from Zn ²⁺ , Cu ²⁺ , Ca ²⁺ and combinations thereof.