CN116444606A - Donkey skin oligopeptide and application thereof - Google Patents

Donkey skin oligopeptide and application thereof Download PDF

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CN116444606A
CN116444606A CN202310401192.6A CN202310401192A CN116444606A CN 116444606 A CN116444606 A CN 116444606A CN 202310401192 A CN202310401192 A CN 202310401192A CN 116444606 A CN116444606 A CN 116444606A
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oligopeptide
donkey skin
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刘思如
包国良
贾建萍
李青青
陈智涛
郑安博
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Hangzhou Medical College
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    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
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    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • A23L33/18—Peptides; Protein hydrolysates
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention discloses donkey skin oligopeptide, the amino acid sequence of which is selected from Glu-Thr-Trp-Arg or Ala-Tyr-Phe-Pro-Lys. The invention also discloses application of the novel compound. The donkey skin oligopeptide disclosed by the invention has the alpha-glucosidase and alpha-amylase inhibiting activities, can assist in reducing blood sugar, has very important significance for developing foods, health-care products and medicines with the function of reducing blood sugar, and can be used for preparing alpha-glucosidase, alpha-amylase inhibitor and anti-type II diabetes medicines.

Description

Donkey skin oligopeptide and application thereof
Technical Field
The invention relates to the technical field of oligopeptides, in particular to donkey skin oligopeptides and application thereof.
Background
Donkey hide is the epidermis of donkey (Equus asinus L.) of equine, is rich in high-quality collagen, donkey hide is the traditional Chinese medicine decoction piece processed by decocting, concentrating and other processes of donkey hide (dried skin or fresh skin), has the functions of replenishing blood, nourishing yin, moistening dryness, stopping bleeding and the like, can be used for mainly treating blood deficiency sallow, dizziness, palpitation and the like, and is recorded in the pharmacopoeia of the people's republic of China (one part), the dictionary of Chinese medicine and the like, so that the amino acid composition and the amino acid sequence of donkey hide collagen may be different from those of other animal sources.
The donkey-hide collagen peptide is a mixture of proteins, polypeptides, oligopeptides and amino acids (the content of the oligopeptides is mainly) prepared from donkey-hide by an enzyme method. The oligopeptide is a small peptide with the number of amino acid residues of 2-10, and the oligopeptide with the molecular weight of less than 1000Da and some protein molecules in the donkey-hide gelatin are reported to be the main substance basis for the donkey-hide gelatin to exert the pharmacological action.
The Chinese patent document with the application number of CN202111417049.3 discloses a preparation method and a product of donkey-hide gelatin peptide with low pro-inflammatory reaction, wherein the preparation method comprises the following steps: (1) crushing donkey-hide gelatin blocks; (2) The crushed donkey-hide gelatin is subjected to enzymolysis for 3-5 hours under the action of nonspecific enzyme chain protease E; (3) Adding protease into the sugar enzymolysis liquid for enzymolysis, so that the molecular weight of an enzymolysis product is between 1000 and 3000Da, and inactivating enzyme at high temperature; (4) And (5) freeze-drying the enzymatic hydrolysate to obtain the donkey-hide gelatin peptide for pro-inflammatory reaction.
The Chinese patent document with the application number of CN202011410008.7 discloses a preparation method of donkey-hide collagen, which comprises the following steps: (1) donkey hide pretreatment; (2) performing enzymolysis membrane circulation separation; (3) decoloring and dearomatizing; (4) Freeze drying to obtain collagen powder with molecular weight of 2-5kD, which has no damaged triple helix structure and retains original biological activity.
Further, for example, chinese patent document No. CN202110143314.7 discloses a preparation method of homoglutamic acid donkey collagen peptide, and (1) the compound enzymolysis technology (2) is adopted to separate the homoglutamic acid collagen active peptide from donkey collagen by using an affinity chromatography method, which has strong chelating activity for ferrous ion, calcium ion and zinc ion.
However, the donkey skin collagen peptide or donkey collagen active peptide obtained by the technical scheme belongs to an enzymolysis product obtained by hydrolyzing a collagen raw material by using active enzymes, and is mainly a mixture composed of peptides, proteins, amino acids and the like with different molecular weights, and has the characteristic of a certain molecular weight distribution range.
The peptides composed of the enzymolysis products have relatively close molecular weights, the separation is difficult to achieve by means of a single separation method, and the current separation method of the peptides mainly adopts a method combining a plurality of separation means such as membrane separation, chromatography and the like according to the differences among physical properties such as molecular weight, chargeability, hydrophobicity and the like of the peptides.
The biological activity of the active oligopeptide reported at present has the functions of resisting oxidation, reducing blood sugar, improving immunity, and the like, such as XU, and the like (XU Q Y, ZHENG L, HUANG M T, et al, expling structural features of potent dipeptidyl peptidase IV (DPP-IV) inhibitory peptides derived from tilapia (Oreochromis niloticus) skin gelatin by an integrated approach of multivariate analysis and Gly-Pro-based peptide library [ J ]) is used for hydrolyzing the fishskin of tilapia mossambica, and the oligopeptide with the inhibitory activity of several dipeptidyl peptidase-IV is obtained after separation and purification, and the peptide chain structure of the oligopeptide has-Gly-Pro-residue, so that the secretion of glucagon-like peptide and insulin can be promoted, the insulin level can be improved, and the blood sugar can be reduced.
Mudgil P et al (Mudgil P, jobe B, kamal H, et al, dipeptidyl peptidase-IV, alpha-amylase, and angiotensinIconverting enzyme inhibitory properties of novel camel skin gelatin hydrolysates [ J ]) hydrolyzed camel skin collagen by three enzymatic hydrolysis methods (alkaline protease, neutral protease, mixture of alkaline protease and neutral protease) to obtain hydrolysate with dipeptidyl peptidase-IV inhibitory activity and alpha-amylase inhibitory activity, which can promote insulin secretion, inhibit decomposition of amylase and glucosidase, and maintain normal blood glucose level in vivo.
Diabetes is a group of metabolic diseases characterized by hyperglycemia, which is caused by defective insulin secretion or impaired biological action, or both. Hypoglycemic agents are broadly classified into three types according to the mechanism of hypoglycemic action: (1) stimulating insulin secretion, such as sulfonylurea drugs; (2) Increasing the utilization of glucose by peripheral tissues, such as biguanides hypoglycemic agents; (3) Alpha-glucosidase inhibitors such as acarbose and the like commonly used in clinic.
However, it has not been reported yet that the donkey hide enzymatic hydrolysate is separated and purified to obtain collagen peptide with specific amino acid sequence and alpha-glucosidase and alpha-amylase inhibitory activities, and can be applied to type II diabetes.
Disclosure of Invention
The invention provides donkey skin oligopeptide with a novel amino acid sequence aiming at the problems in the prior art, and the donkey skin oligopeptide has alpha-glucosidase and alpha-amylase inhibition activities and can assist in reducing blood sugar through an activity test and a blood sugar reducing animal test.
The invention adopts the following technical scheme:
in a first aspect, the invention provides a donkey skin oligopeptide, the amino acid sequence of which is selected from Glu-Thr-Trp-Arg or Ala-Tyr-Phe-Pro-Lys.
The second aspect of the invention provides the application of the donkey hide oligopeptide in preparing hypoglycemic foods, health-care products and medicines
In a third aspect, the invention provides the use of the donkey hide oligopeptide above in the preparation of alpha-glucosidase and alpha-amylase inhibitors.
The fourth aspect of the invention provides application of the donkey hide oligopeptide in preparing anti-type II diabetes medicines.
The invention has the beneficial effects that:
1. the invention obtains three donkey skin oligopeptides Arg-Arg-Glu-Val-Pro, glu-Thr-Trp-Arg and Ala-Tyr-Phe-Pro-Lys with novel amino acid sequence structures, and the three donkey skin oligopeptides have alpha-glucosidase and alpha-amylase inhibition activities, can assist in reducing blood sugar, have very important significance for developing foods, health care products and medicines with blood sugar reduction, and can be used for preparing alpha-glucosidase, alpha-amylase inhibitors and anti-type II diabetes medicines.
2. Aiming at the characteristics of small molecular weight, concentrated molecular weight and small dispersivity of the donkey skin collagen peptide mixed peptide prepared by an enzymolysis method, the donkey skin collagen peptide is coarsely separated by adopting a chromatographic separation method using a Sephadex G-15 as a filler, and is further separated by a reversed-phase high performance liquid chromatography technology, and finally three novel donkey skin oligopeptides are obtained by LC-TOF-MS/MS peptide structure analysis and identification, and the three donkey skin oligopeptides have alpha-glucosidase and alpha-amylase inhibition activities and can assist in reducing blood sugar through an activity test and a blood sugar reducing animal test.
Drawings
FIG. 1 is an elution curve of a separated component obtained by separation with a Sephadex G-15 Sephadex column as a packing, the abscissa represents an elution volume (mL), and the ordinate represents a light absorption value at a wavelength of 254 nm.
FIG. 2 is an elution curve of a separated component obtained by separation with a Sephadex G-10 Sephadex column as a packing, the abscissa represents an elution volume (mL), and the ordinate represents a absorbance at a wavelength of 254 nm.
FIG. 3 is an elution profile of a separated component obtained by separation using a Sephadex G-25 Sephadex column as a packing, with the abscissa representing the elution volume (mL) and the ordinate representing the absorbance at 254 nm.
FIG. 4 is a Re-HPLC chromatogram of component C with retention time (min) on the abscissa and response values on the ordinate.
FIG. 5 shows mass spectra of LC-TOF-MS/MS peptide structure analysis and identification, arg-Arg-Glu-Val-Pro, glu-Thr-Trp-Arg, ala-Tyr-Phe-Pro-Lys, respectively, from top to bottom.
Detailed Description
The invention will be further explained with reference to examples and figures. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
The following method relates to the measurement of the alpha-glucosidase inhibitory activity: 4-nitrobenzene-alpha-glucopyranoside (PNPG) is taken as a substrate, and the reaction system is as follows: 2mL of 0.1mol/L phosphate buffer (pH 6.8), 0.5mL of an aqueous solution of a sample to be measured, 50. Mu.L of 1mg/mL reduced glutathione and 10. Mu.L of alpha-glucosidase (Type I, from Saccharomyces cerevisiae, 11.4U/mL), shaking, incubating at 37℃for 10min, then adding 200. Mu.L of the incubated substrate PNPG (20 mmol/L), incubating at 37℃for 20min, and adding 10mL of 0.1mol/L Na 2 CO 3 The reaction was terminated and absorbance was measured at a wavelength of 400 nm.
Alpha-glucosidase inhibition rate (%) = (1-a) Sample /A Empty space )×100％，A Empty space Represents absorbance of a blank reagent (no sample added, replaced by deionized water), A Sample The absorbance of the sample is shown.
The following relates to the determination of the alpha-amylase inhibitory activity by the following method: 1mL of the aqueous solution of the sample to be tested, 1mL of starch azurin (prepared as a 2mg/mL suspension in 0.05mol/L, pH6.9Tris-HCl buffer, boiled for 5 min) and 50. Mu.L of alpha-amylase (prepared as Type VI-B, from porcine pancrcase,4.71U/mL,0.05mol/L, pH6.9Tris-HCl buffer) were mixed, incubated at 38℃for 10min, the reaction was stopped by adding 0.2mL of 50% v/v glacial acetic acid aqueous solution, centrifuged at 6000rpm for 5min, and the absorbance of the supernatant was measured at 595 nm.
Alpha-amylase inhibition (%) = (1-a) Sample /A Empty space )×100％，A Empty space Represents absorbance of a blank reagent (no sample added, replaced by deionized water), A Sample The absorbance of the sample is shown.
Example 1
(1) And (3) raw material treatment: taking fresh healthy donkey skin, removing residual meat, soaking in clear water for 3-5 days, changing water 1-2 times per day, removing donkey hair, cleaning impurities, cutting into small pieces with the length of about 10cm multiplied by 10cm, boiling in boiling water for 10min, and taking out when the donkey skin is rolled.
Decocting and concentrating: adding clear water according to the mass ratio of the feed liquid of 1:5, decocting for 36h, adding enough evaporated water at any time, stirring for 1 time every 2-3h, and preparing 500 g of donkey hide raw material per 300 g of original donkey hide by heating, evaporating and concentrating until the donkey hide is burned to soften and smashed into paste by a tissue smashing machine.
Enzymolysis: 100g of the donkey hide raw material, 0.35g of pancreatin (4000U/g of Nanning Pang Bo biological engineering Co., ltd.) and 300mL of water are placed in a 1000mL glass beaker, the mixture is uniformly mixed, the glass beaker is placed in a constant temperature water bath at 55 ℃, the pH value of an enzymolysis reaction system is regulated to 6.0 by 1mol/LNaOH or 1mol/LHCl, a stirrer is used for stirring the reaction liquid, the rotating speed is controlled to 300rpm, the reaction is carried out for 8 hours, the temperature is raised to 90 ℃ for inactivating enzyme for 20 minutes, the reaction liquid is taken out, cooling and filtering are carried out, and the obtained filtrate is the donkey hide enzymolysis product (donkey hide collagen peptide mixed peptide), and the yield of the donkey hide collagen peptide mixed peptide reaches 70.0%. The activity test shows that the donkey-hide collagen peptide mixed peptide has the inhibition activity IC to alpha-glucosidase 50 The alpha-amylase inhibitory activity was 55.56mg/mL at 38.13 mg/mL.
According to tests, the donkey skin collagen peptide mixed peptide has the molecular mass of 180-1000Da accounting for 73%, so that the obtained donkey skin collagen peptide mixed peptide is mainly an oligopeptide with 2-10 amino acid residues.
(2) Ultrafiltering the filtrate with hollow fiber polysulfone ultrafiltration membrane with molecular weight cutoff of 3000Da, collecting filtrate, concentrating by reduced pressure distillation, and lyophilizing to obtain donkey-hide collagen peptide with molecular weight of <3000 Da;
(3) 2G of donkey-hide collagen peptide is weighed, 2mL of water is added for dissolution, 1mL of donkey-hide collagen peptide aqueous solution is taken and put on a Sephadex chromatographic column, the specification of the Sephadex chromatographic column is 2.6cm multiplied by 88cm, the filler is Sephadex G-15, the mobile phase is deionized water, the column flow rate is 1.0mL/min, and separated components A, B, C and D are obtained after elution (the elution curve of four separated components is shown in figure 1, and the wavelength is 254 nm).
Activity test shows that the separated components A, B, C and D have inhibitory activity IC on alpha-glucosidase 50 40.15mg/mL, 38.30mg/mL,22.8mg/mL, 60, respectively59mg/mL, inhibitory Activity against alpha-amylase IC 50 62.37mg/mL,48.26mg/mL,40.09mg/mL, respectively, are inactive.
The packing was replaced with Sephadex G-10, and the elution profile (fig. 2) was observed to show that the separation process did not allow efficient separation.
The packing was replaced with Sephadex G-25, and the elution profile (fig. 3) was observed to show that the separation process also failed to achieve efficient separation.
(4) And (3) further separating the component C with the strongest alpha-glucosidase inhibitory activity obtained in the step (3) by utilizing a reverse phase high performance liquid chromatography (Re-HPLC) technology, wherein the separation conditions are as follows: chromatographic column: angilent Eclipse XDB-C 18 Column (250×4.6mm,5 μm), mobile phase: and (3) solution A: 0.1% v/v aqueous trifluoroacetic acid (TFA), solution B: aqueous 80% v/v acetonitrile, linear gradient elution: 0-5min,100% -80% A;5-50min,80% -0% A,50-60min,0% A,60-70min,100% A, flow rate: 1.0mL/min, column temperature: 30 ℃, detection wavelength: 340nm. Re-HPLC spectra are shown in FIG. 4.
As shown in FIG. 5, three donkey skin oligopeptides were isolated in this example, which had amino acid sequences Arg-Arg-Glu-Val-Pro (RREVP), glu-Thr-Trp-Arg (ETWR), ala-Tyr-Phe-Pro-Lys (AYFPK), respectively, as identified by LC-TOF-MS/MS peptide structure analysis.
The activity test shows that the alpha-glucosidase inhibitory activity IC of the three components 50 6.8mg/mL, 9.3mg/mL, 12.5mg/mL, respectively, alpha-amylase inhibitory activity IC 50 11.4mg/mL, 20.1mg/mL, 27.7mg/mL, respectively.
Therefore, all three donkey skin oligopeptides can be applied to the preparation of alpha-glucosidase and alpha-amylase inhibitors.
The action mechanism of the alpha-glucosidase inhibitor is as follows: the competitive inhibition of various alpha-glucosidases located in the small intestine slows down the rate of decomposition of starch into glucose, thereby slowing down the absorption of glucose in the intestinal tract and reducing postprandial hyperglycemia. Type ii diabetes mellitus is due to the fact that postprandial hyperglycemia glucose toxicity aggravates insulin resistance and insulin secretion defects, and when islet beta cell function is only about 50%, fasting blood glucose elevation occurs and glucose tolerance is impaired.
The mechanism of action of the alpha-amylase inhibitor is as follows: blocking the site of action of alpha-amylase inhibits its activity, preventing starch from being digested; inhibit amylase synthesis and reduce its activity in the intestinal tract; inhibiting amylase aggregation prevents activation and inhibits blood glucose elevation in vivo. The in-vivo amylase has the function of catalyzing the compounds of the starch class to be glucuronic acid, the glucuronic acid is decomposed into glucose under the action of the in-vivo uronic acid enzyme, the glucuronic acid and the glucose are sugar substances which can be directly utilized by the body, and the enhancement of the amylase activity directly influences the blood sugar level in the human body.
Based on the research, the three donkey skin oligopeptides disclosed by the invention can be further used for preparing anti-type II diabetes medicines.
The following three isolated donkey skin oligopeptides Arg-Arg-Glu-Val-Pro, glu-Thr-Trp-Arg, ala-Tyr-Phe-Pro-Lys were each tested for hypoglycemic animals: arg-Arg-Glu-Val-Pro is provided with a donkey hide oligopeptide group with a dosage of 0.5g/kg, a donkey hide oligopeptide group with a dosage of 0.2g/kg, glu-Thr-Trp-Arg is provided with a donkey hide oligopeptide group with a dosage of 0.5g/kg, a donkey hide oligopeptide group with a dosage of 0.2g/kg, and Ala-Tyr-Phe-Pro-Lys is provided with a donkey hide oligopeptide group with a dosage of 1.0g/kg and a donkey hide oligopeptide group with a dosage of 0.5 g/kg; arg-Arg-Glu-Val-Pro, glu-Thr-Trp-Arg, ala-Tyr-Phe-Pro-Lys were set as the normal control group and the model control group, and 0.01g/kg of the bail off group was set as the positive control. Animals used in each donkey skin oligopeptide group, model control group and bailing plane group are tetraoxypyrimidine type diabetes model mice (type II diabetes), and animals used in each normal control group are ICR mice. Oligopeptides for use in hypoglycemic animal tests were obtained by solid phase synthesis and were submitted to Jil Biochemical (Shanghai) Inc.
Each animal is fasted for 3-5h, blood sugar value before starch administration (0 h) is measured, each donkey skin oligopeptide group is given a sample (aqueous solution) with corresponding concentration in the same volume, the bailing group is given bailing with 0.01g/kg dose, the normal control group and the model control group are given sterile water in the same volume, each group is orally given starch for 3-5g/kg BW after 15-20min, blood sugar value after starch administration is measured for 0.5, 1, 2 and 3h, and change of blood sugar value and area under a curve at each time point after starch administration is observed in the model control group and each donkey skin oligopeptide group, and bailing is taken as a positive control.
The result shows that: all three donkey skin oligopeptides can reduce the absorption speed of sugar, obviously improve the sugar tolerance, and experimental results are shown in tables 1, 2 and 3.
Table 1 shows the effect of Arg-Arg-Glu-Val-Pro on starch load tolerance in mice with models of tetraoxypyrimidine-type diabetes, and the results in Table 1 show that the blood glucose levels were significantly lower than those in the model control group (P < 0.05) at time points of 0.5, 1, 2, and 3 hours after starch administration of the donkey skin oligopeptide group, and at time point of 0.5 hours after starch administration of the donkey skin oligopeptide group, and at 0.2 g/kg. Comparing the donkey skin oligopeptide group with the model control group, the blood sugar value of each dose is slowly increased, and the peak value is reduced; and the area under the curve can be reduced, and the obvious dosage relationship is shown.
TABLE 1 influence of Arg-Arg-Glu-Val-Pro on starch load tolerance in mice model of tetraoxypyrimidine diabetes
Note that:mean ± standard deviation, n represents 10 mice per group, P<0.05 indicates that the difference is significant, △△ P<0.05 compared to model control group, ×p<0.05 was compared to the normal control group.
Table 2 shows the effect of Glu-Thr-Trp-Arg on starch load tolerance in mice with models of tetraoxypyrimidine-type diabetes, and the results in Table 2 show that the blood glucose levels were significantly lower than those in the model control group (P < 0.05) at time points of 0.5, 1, 2, 3h after starch administration of the donkey skin oligopeptide group at 0.5g/kg and at time points of 0.5, 1, 2h after starch administration of the donkey skin oligopeptide group at 0.2 g/kg. Comparing the donkey skin oligopeptide group with the model control group, the blood sugar value of each dose is slowly increased, and the peak value is reduced; and the area under the curve can be reduced, and the obvious dosage relationship is shown.
TABLE 2 influence of Glu-Thr-Trp-Arg on starch load tolerance in mice model of tetraoxypyrimidine diabetes
Note that:mean ± standard deviation, n represents 10 mice per group, P<0.05 indicates that the difference is significant, △△ P<0.05 compared to model control group, ×p<0.05 was compared to the normal control group.
Table 3 shows the effect of Ala-Tyr-Phe-Pro-Lys on starch load tolerance in mice model of tetraoxypyrimidine-type diabetes, and the results in Table 3 show that the blood glucose levels were significantly lower than those of the model control group (P < 0.05) at the time points of 0.5, 1, 2, and 3 hours after starch administration of the donkey skin oligopeptide group at 1.0g/kg dose and at the time points of 0.5, 1, and 3 hours after starch administration of the donkey skin oligopeptide group at 0.5g/kg dose. Comparing the donkey skin oligopeptide group with the model control group, the blood sugar value of each dose is slowly increased, and the peak value is reduced; and the area under the curve can be reduced, and the obvious dosage relationship is shown.
TABLE 3 influence of Ala-Tyr-Phe-Pro-Lys on starch load tolerance in mice model of tetraoxypyrimidine diabetes
Note that:mean ± standard deviation, n represents 10 mice per group, P<0.05 indicates that the difference is significant, △△ P<0.05 compared to model control group, ×p<0.05 was compared to the normal control group.

Claims (4)

1. Donkey skin oligopeptide, characterized in that its amino acid sequence is selected from Glu-Thr-Trp-Arg or Ala-Tyr-Phe-Pro-Lys.
2. The use of donkey skin oligopeptide according to claim 1 in the preparation of hypoglycemic foods, health care products and medicines.
3. Use of the donkey skin oligopeptide of claim 1 for the preparation of alpha-glucosidase and alpha-amylase inhibitors.
4. Use of the donkey skin oligopeptide according to claim 1 for preparing an anti-type ii diabetes drug.
CN202310401192.6A 2023-04-12 2023-04-12 Donkey skin oligopeptide and application thereof Pending CN116444606A (en)

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