Patent Application
20240109942
The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBZYGJ171 Sequence_Listing.xml, created on 11/23/2023, and is 5,222 bytes in size.
The present invention belongs to the field of bioactive peptides, and particularly relates to a cholecystokinin (CCK) secretion-promoting peptide targeting the calcium-sensing receptor, and a preparation method and use thereof.
Obesity has been emerged as a global public health issue. A wide range of chronic diseases such as cardiovascular diseases, type II diabetes, atherosclerosis, fatty liver and gout are all associated with obesity. Imbalance between energy intake and energy expenditure directly leads to individual overweight and obesity. In view of this, it is optimal to limit excessive energy intake, i.e. limit food intake, in order to prevent obesity. The hypothalamus and digestive tract constitute the key parts of body appetite control, in which a wide range of brain-gut peptides associated with regulation of food intake are distributed. Cholecystokinin (CCK) is a member of a classic brain-gut peptides, which can regulate the body to produce a satiety signal, and reduce food intake of the body, thereby achieving the effect of suppressing appetite. It is of great significance to increase secretion of the intestinal CCK to prevent and alleviate obesity.
Researches have confirmed that the secretion of the CCK is regulated by dietary factors. As a result, dietary regulation of secretion of the intestinal CCK can improve or alleviate obesity and related chronic diseases. Food-derived bioactive peptides are common dietary factors featuring easy digestion and absorption by human body and high food safety. Many countries have made significant effects to promote the development of food-derived bioactive peptide industry, and clearly pointed out that it is necessary to accelerate development of functional food, support development of functional foods such as the bioactive peptide, and carry out related application. At present, the food-derived bioactive peptides are mainly derived from animal and plant proteins. Increasing attentions have been paid to the plant proteins because of environmental, economic, and sustainable considerations. Avena sativa L, a widely cultivated crop worldwide, has important edibleness. Its protein content is higher than that of other crops such as wheat, corn, and rice. At present, oat polysaccharides and oils are widely developed and utilized, but there is a lack of development and utilization of oat proteins. The development of the bioactive peptides by using proteins deriving from Avena sativa L as a raw material has important application value and development prospect.
An objective of the present invention is to provide a cholecystokinin (CCK) secretion-promoting peptide targeting calcium-sensing receptor (CaSR), and a preparation method and use thereof.
The objective of the present invention can be implemented by the following technical solution:
In a first aspect of the present invention, a CCK secretion-promoting peptide targeting a CaSR is provided. The CCK secretion-promoting peptide targeting a CaSR is an active peptide of QGDVVALPA, and has an amino acid sequence as follows: Gln-Gly-Asp-Val-Val-Ala-Leu-Pro-Ala shown as SEQ ID NO: 1.
An oat protein is mainly globulin, where avena 12S seed storage globulin 1 has an amino acid sequence shown as SEQ ID NO: 2. It may be found that the active peptide of QGDVVALPA exists in the oat protein. The active peptide of QGDVVALPA may be prepared from the oat protein.
The active peptide of QGDVVALPA according to the present invention is derived from Avena sativa L, and can target the CaSR to activate a Gq signal pathway, thereby further increasing an intracellular calcium ion concentration to significantly promote secretion of CCK in the enteroendocrine cells; and moreover, the active peptide has the advantages of safety, no toxic side effect, tolerance to digestive enzyme hydrolysis of a gastrointestinal tract, easy absorption, etc.
In a second aspect of the present invention, a polynucleotide encoding the CCK secretion-promoting peptide targeting a CaSR is provided.
In a third aspect of the present invention, a preparation method of the CCK secretion-promoting peptide targeting a CaSR is provided. The CCK secretion-promoting peptide targeting a CaSR is artificially synthesized through a genetic engineering method, or is directly obtained from oat protein through a separation and purification method, or is directly prepared through chemical synthesis.
Artificial synthesis of the CCK secretion-promoting peptide targeting a CaSR through the genetic engineering method is a technical solution achievable by those skilled in the art. For example, sequence synthesis of polypeptide can be controlled by means of an appropriate deoxyribonucleic acid (DNA) template on the basis of a recombinant DNA technology.
The manner of directly obtaining the CCK secretion-promoting peptide targeting a CaSR from the oat protein through the separation and purification method may be as follows: on the basis of a given amino acid sequence of the CCK secretion-promoting peptide targeting a CaSR, the CCK secretion-promoting peptide targeting a CaSR is obtained from Avena sativa L through conventional enzymatic hydrolysis, separation and purification methods using a biological technology.
The preparation method through chemical synthesis synthesizes the oligopeptide described above through a traditional solid phase synthesis method.
In a fourth aspect of the present invention, a preparation method of an enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR is provided. The preparation method enzymatically hydrolyses oat protein by means of a two-step enzymatic hydrolysis method, and includes: sequentially enzymatically hydrolyzing the oat protein through pepsin and trypsin to obtain an oat protein hydrolysate, i.e. enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR.
In an embodiment of the present invention, the sequentially enzymatically hydrolysing the oat protein through pepsin and trypsin includes:
In an embodiment of the present invention, the extracting the oat protein from Avena sativa L includes:
In an embodiment of the present invention, a mass ratio of the pepsin or the trypsin to the oat protein is 1:10-100.
In an embodiment of the present invention, an enzymatic hydrolysis condition of the pepsin or the trypsin is enzymatic hydrolysis for 1 h-4 h at 37° C.
In an embodiment of the present invention, step 3) of the obtaining an oat protein hydrolysate further includes:
In a fifth aspect of the present invention, an enzymatic hydrolysate of the CCK secretion-promoting peptide targeting a CaSR prepared on the basis of the preparation method described above is provided.
In a sixth aspect of the present invention, a use of a CCK secretion-promoting peptide targeting a CaSR and an enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR in preparation of a product having at least one function of 1)-4) as follows:
The CCK secretion-promoting peptide targeting a CaSR and the enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR have the function of promoting secretion of CCK, and can slow down gastric emptying, suppress appetite to reduce food intake, and prevent or adjunctively treat obesity.
In a seventh aspect of the present invention, a product is provided. The product includes the CCK secretion-promoting peptide targeting a CaSR and the enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR, and has at least one function of 1)-4) as follows:
The product includes food, functional food/health food and medicine.
In an eighth aspect of the present invention, a use of the CCK secretion-promoting peptide targeting a CaSR and the enzymatic hydrolysate containing the CCK secretion-promoting peptide targeting a CaSR in preparation of a kit. The kit is configured to activate the CaSR or a downstream Gq signal pathway of the CaSR.
Compared with the prior art, the present invention has the advantages and beneficial effects as follows:
The present invention will be described in detail below with reference to the accompanying drawings and particular examples.
I. Synthesis of the Active Peptide of QGDVVALPA
An active peptide of Gln-Gly-Asp-Val-Val-Ala-Leu-Pro-Ala(QGDVVALPA) was synthesized by “Zhejiang Hongtuo Technology Co., Ltd.” through a peptide solid phase synthesis method. Purity of the synthesized peptide was verified to be greater than 95% by means of a high-performance liquid chromatography and a mass spectrometry technology. A liquid phase diagram and a mass spectrum diagram of QGDVVALPA were shown in
II. Effects of QGDVVALPA on Viability and Secretion of CCK of SCT-1 Cell
(1) STC-1 Cell Culture
An STC-1 cell was cultured in a dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 100 U/mL penicillin and 0.1 mg/mL streptomycin. The cell was incubated in a cell incubator containing 5% CO2 at 37° C. and was subcultured by means of trypsin digestion when reaching 80%-90% density.
(2) Cell Activity Assay
The effect of the active peptide of QGDVVALPA on the viability of the STC-1 cell was tested and evaluated by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [3-(4,5-di methyl-2-thiazolyl)-2,5-di phenyl]-2H-tetrazolium bromide, methyl thiazolyl tetrazolium (MTT)) cell proliferation and cytotoxicity. MTT was added into the STC-1 cell treated in a 96-well plate, and a metabolically active cell cleaved yellow tetrazolium salt MTT into a purple formazan crystal. Formazan formed was dissolved, absorbance was measured with a microplate reader at a measurement wavelength of 570 nm, and results were expressed as a percentage of a control group. Results were shown in
(3) Assay of Secretory Hormone Content of STC-1 Cell
The active peptide of QGDVVALPA was prepared into peptide solutions having molar concentrations of 0.2 mM, 2 mM and 5 mM respectively with a Hank's buffer solution. The STC-1 cell was inoculated in a 24-well culture plate at a density of 1.25*105 cells. When the cell reached 80%-90% confluence, the cell was washed twice with the Hank's buffer solution to remove the medium. Seven oligopeptide solutions were added into the STC-1 cell, and the cell was incubated in an incubator for 2 h at 37° C.. At the end of incubation, 1000 g of solution was centrifuged for 20 min to obtain supernate. A CCK content was assayed by a commercial CCK kit of Wuhan Cloud Clone Science and Technology Co., Ltd.
The effect of the active peptide of QGDVVALPA on secretion of CCK by the STC-1 cell was shown in
(1) Preparation of Oat Protein Hydrolysate
Avena sativa L was ground into powder through an 80 mesh, and then the Avena powder was degreased with hexane. Degreased Avena powder was soaked into a beaker through distilled water in a mass ratio of 12:1, pondus Hydrogenii (pH) was adjusted to 5.0 through 1 mol/L HCl, and a solution was treated through cellulase for 1 h at 50° C. Then, the pH was adjusted to 11.0 through 1 mol/L NaOH, and the solution acted for 2 h in a magnetic stirrer to obtain supernate. The pH of the supernate was adjusted to an isoelectric point (pH 4.5) through 1 mol/L HCl, the supernate was left to stand for 1 h, then centrifuged, washed with water, precipitated to neutrality, redissolved through a small amount of distilled water, and frozen and dried to obtain oat protein, and the oat protein was stored at 4° C. for later use.
One g of lyophilized protein powder was dissolved into 20 mL of K2HPO4—KH2PO4 phosphate buffer solution (0.1 mol/L) containing 25 mg of freshly prepared pepsin, the pH of the solution was adjusted to 2.0 through HCl (1 mol/L), and the solution was incubated for 2 h at 37° C.. At the end of incubation, the pH of the solution was adjusted to 6.8 through NaOH (1 mol/L), and 50 mg of trypsin was added to continue enzymatic hydrolysis for 2 h. After completion, enzyme was inactivated by a boiling water bath for 8 min, the solution was centrifuged to obtain supernate, and the supernate was frozen and dried to obtain oat protein hydrolysate.
(2)Activity Evaluation
The oat protein hydrolysate was prepared into solutions having mass concentrations of 3 mg/mL, 4 mg/mL and 5 mg/mL separately with a Hank's buffer solution, and the effect of the hydrolysate on secretion of CCK by an STC-1 cell was assayed through the above method. Results were shown in
Further, the effect of the oat protein hydrolysate on hormone secretion by the intestinal endocrine cell in a mouse was evaluated at an animal level. After one week of adaptation period, institute for cancer research (ICR) mice were randomly divided into 2 groups (28 mice in each group). Control group: normal saline was administered intragastrically; and oat protein hydrolysate group: oat protein hydrolysate (1.0 g/kg body weight) was administered intragastrically. After intragastric administration, eyeballs were removed at 0 min, 15 min, 30 min, 60 min, 90 min, 120 min and 150 min to collect blood, the blood was put into centrifuge tubes containing ethylene diamine tetraacetic acid (EDTA) (final concentration 1 mg/mL) and aprotinin (final concentration 0.6 TIU/mL), a solution was centrifuged to obtain supernate, and CCK hormone content in serum was assayed through an enzyme-linked immunosorbent assay (ELISA) method. A CCK level in the serum in the intragastric administration normal saline group was maintained at 10 pg/mL during the period. Results of the intragastric administration oat protein hydrolysate group was shown in
Toyopearl HW-40F packing conventionally swelled and was packed (a packing buffer solution was 0.1 M NaCl dissolved into 50 mM phosphate), a column height was 10 cm, an inner diameter was 2.6 cm, 1.5 cm-2 cm of water layer was needed at a top of a column at any moment, a column may be used after about 3-4 column sizes were balanced, and oat protein hydrolysate was added when 2 mm-3 mm of liquid level was reserved. About 20 mg of oat protein hydrolysate powder was weighed and dissolved into 2 mL of distilled water. Filtration was carried out through a 0.45 μm microporous membrane, then a chromatographic column was added, an eluent was distilled water at an elution speed of 2 mL/min, and an elution peak was collected. A separation graph of the oat protein hydrolysate was shown in
The four peptide components were evaluated for activity through the method described above, and results were shown in
A peptide sequence in the component F1 was identified through a mass spectrometry technology. A sample was dissolved into distilled water to prepare a 1 mg/mL sample. Separation was carried out through a reversed phase column (150 μm i.d.*150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å), a mobile phase A was 0.1% formic acid solution, a mobile phase B was 0.1% formic acid/80% acetonitrile solution, gradient elution was carried out at 600 nL/min of flow rate, and separation gradient was as follows: 0 min-2 min, 4%-8% B; 2 min-45 min, 8%-40% B; 45 min-55 min, 40%-60% B; 55 min-56 min, 60%-95% B; and 56 min-66 min, 95% B. Mass spectrometry had an ion source type of electrospray ionization (ESI), a positive ion scanning mode, a spray voltage of 2200 V, and a capillary temperature of 270° C.. Primary mass spectrometry parameters were set as follows: scanning range of 100 m/z-2000 m/z, maximum resolution of 70000, and automatic gain parameter of 3000000. Secondary mass spectrometry parameters were set as follows: scanning range of 50 m/z-2000 m/z, maximum resolution of 17500, and automatic gain parameter of 100000.
A main ion peak in the component F1 was m/z=869.472 having one charge through mass spectrometry detection. A molecular ion peak was further analyzed by secondary mass spectrometry. A secondary mass spectrum of the molecular ion peak was shown in
An oat protein was mainly globulin, where avena 12S seed storage globulin 1 had an amino acid sequence shown as SEQ ID NO: 2. It may be found that the active peptide of QGDVVALPA existed in the oat protein. An oligopeptide of DVNNNANQLEPR, as shown in SEQ ID NO: 3, may be prepared from the oat protein.
The amino acid sequence of avena 12S seed storage globulin 1 was specifically as follows:
An STC-1 cell was inoculated in a 24-well culture plate at a density of 1.25*105 cells per well. When the cell reached 80%-90% confluence, the cell was washed twice with a Hank's buffer solution to remove a medium, and then pretreated with a calcium-sensing receptor (CaSR) specific antagonist NPS 2143(25 μM), a Gq inhibitor YM 254890 (10 μM), a free calcium ion chelator BAPTA-AM (25 μM) or a carrier (0.1% dimethylsulfoxide (DMSO)) for 10 min separately. The STC-1 cell was exposed to a buffer solution containing an active peptide of QGDVVALPA for 2 h. At the end of incubation, 1000×g of solution was centrifuged for 20 min, and the CCK content outside STC-1 cells was assayed according to operations of description of a cloud clone CCK assay kit. Results were shown in
The above description of the examples is provided to facilitate understanding and use of the invention by those of ordinary skill in the art. Obviously, those skilled in the art can easily make various modifications to these examples and apply the general principles explained herein to other examples without creative efforts. Therefore, the present invention is not limited to the above examples. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the scope of protection of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210735384.6 | Jun 2022 | CN | national |
This application is a continuation application of International Application No. PCT/CN2023/091021, filed on Apr. 27, 2023, which is based upon and claims priority to Chinese Patent Application No. 202210735384.6, filed on Jun. 27, 2022, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN23/91021 | Apr 2023 | US |
| Child | 18539344 | US |
