CN113912688A

CN113912688A - Method for extracting and purifying pancreatic ANP in chickpea

Info

Publication number: CN113912688A
Application number: CN202111336767.8A
Authority: CN
Inventors: 王家欢; 张金花; 陈安琪; 刘宇
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-01-11

Abstract

The invention belongs to the technical field of protein purification, in particular to a method for extracting and purifying pancreatin from chickpeas. The method comprises the following steps: adding alginic acid to the mixed solution of hydrochloric acid and ethanol, filtering the filter residues with hydrochloric acid, distilled water and acetic acid in sequence to obtain the treated alginic acid, which is refrigerated for later use; the chickpeas are soaked and germinated, and the germinated The obtained chickpeas are homogenized in an acetic acid solution containing mercaptoethanol, and a supernatant is obtained after extraction; alginic acid is added to the obtained supernatant, stirred and adsorbed, and the protein-adsorbed alginic acid is collected with a Buchner funnel, After washing, the adsorbed protein was fully eluted with hydrochloric acid, the eluate was adjusted to be acidic, sodium chloride was added for salting out, and the salting-out product was collected. The process for extracting pancreatin adopted in the present invention is simple in operation, mild in conditions and few in restrictions, easy to realize large-scale production, and has great commercial value.

Description

Method for extracting and purifying pancreatic ANP in chickpea

Technical Field

The invention belongs to the technical field of protein purification, and particularly relates to a method for extracting and purifying pancreatic ANP from chickpeas.

Background

Pancreatic Ampeptide is a novel bioactive polypeptide with 37 amino acid residues extracted from the seeds of legumes. As one of plant polypeptides, the tryptanthrin is related to glycolipid metabolism, has a structure similar to insulin, can also effectively reduce the blood sugar level, and is one of effective medicaments for clinically treating diabetes. The research at present shows that the efficacy of the pancreatic anmin for reducing blood sugar is superior to that of insulin, the side effect is low, the curative effect is high, and the pancreatic anmin meets the standards of raw material medicines, most importantly, the insulin can only achieve the purpose of reducing blood sugar through injection, and the pancreatic anmin can play the curative effect through oral administration, so that the convenience of taking medicines for diabetics can be effectively improved, and the pain is relieved. The high-grade structure of the tryptophane cystine can resist the enzymolysis of pepsin, trypsinase and the like, and no toxic or side effect is observed in experimental animals, so that the tryptophane cystine can be expected to be developed into a new oral hypoglycemic medicament.

Chickpeas, a plant of the genus Cicer of the family Leguminosae, have a high protein content and are one of the major protein sources of humans. The chickpea is rich in nutrition, contains various amino acids and mineral substances, and nutrient components such as carbohydrate, nicotinic acid, vitamins, dietary fiber, fat and the like, thereby having higher edible and medicinal values. The current research on the pancreatic anmin is mainly focused on peas and soybeans, and the research on the pancreatic anmin in the chickpeas is less, and the content of the pancreatic anmin in the chickpeas is reported to be considerable.

Until now, no experiment for extracting and purifying the tryptophanin in the chickpeas exists.

Disclosure of Invention

The technical means such as an infrared spectrophotometer, an ultraviolet spectrophotometer and a high performance liquid chromatography are used in the technical project, the pancreatic ANP is successfully extracted and purified from the chickpea, and important technical support can be provided for research and development of the pancreatic ANP oral preparation.

Compared with the prior art, the invention has the following technical effects:

1. the extraction process adopted by the invention can practically extract a certain amount of protein from the chickpeas and prove that the protein is the pancreatic anmin.

2. The pancreatic ANP freeze-dried powder prepared by the invention has great exploration value for researching the separation and purification process of pancreatic ANP in chickpeas and searching for new diabetes treatment medicines and means.

3. The technology for extracting the pancreatic Ampelopsin is simple to operate, mild in condition, few in limitation, easy to realize large-scale production and high in commercial value.

Drawings

FIG. 1 is an Infrared (IR) spectrum of a chick pea protein polypeptide extracted with methanol;

FIG. 2 is an Infrared (IR) spectrum of a chick pea protein polypeptide extracted with acetone;

FIG. 3 is a graph of the Ultraviolet (UV) spectrum of a protein solution diluted by an appropriate factor extracted with methanol;

FIG. 4 is a graph of the Ultraviolet (UV) spectrum of a protein solution extracted with acetone diluted by an appropriate factor;

FIG. 5 is a liquid chromatogram of a protein solution extracted with methanol;

fig. 6 is a liquid chromatogram of a protein solution extracted with acetone.

Detailed Description

Example 1 extraction of pancreatic Ampelopsin from chickpeas

1. Treatment of alginic acid

100g of alginic acid (Sigma) was weighed and added to 1L of treatment solution (0.2L 2 mol. L)^-1Hydrochloric acid +0.8L ethanol), stirring for 30min, and collecting the filter residue with a buchner funnel. A small amount of the above treatment solution was repeatedly added to a Buchner funnel and filtered with suction until the filtrate was colorless. The collected filter residue (alginic acid) was then suspended in 2L of 0.2 mol. L^-1And (3) in the hydrochloric acid solution, pouring out supernatant after complete precipitation, and collecting the precipitated alginic acid by using a Buchner funnel. Adding a certain amount of distilled water on a Buchner funnel to wash out hydrochloric acid adsorbed on alginic acid. Alginic acid was then resuspended in 0.5 mol.L^-1The precipitate was collected again using a buchner funnel and finally stored in a refrigerator at-20 ℃ until use.

2. Seed germination and extraction

Weighing chickpea 100g, soaking at room temperatureAfter 12 hours, the water was poured off and spread on a white porcelain dish with filter paper to germinate for 60 hours at 25 ℃. Preparing 2L 0.5 mol.L of extracting solution^-1Acetic acid solution (containing mercaptoethanol with volume fraction of 0.1%) is pre-cooled to 4 ℃. Adding 400ml of the extract into germinated seeds, homogenizing, adding the rest 1.6L of the extract, removing foam with tributyl phosphate, extracting at 4 deg.C under stirring for 24 hr, and centrifuging at 7000rpm to obtain supernatant.

3. Adsorption and elution of proteins

4 mol/L for supernatant^-1Adjusting pH to 2.7 with hydrochloric acid, adding appropriate amount of alginic acid, stirring for 12 hr, collecting alginic acid adsorbed with protein with Buchner funnel, and adding distilled water to the Buchner funnel to clean alginic acid. Then 0.2 mol. L is used^-1The adsorbed protein was sufficiently eluted with hydrochloric acid, and the eluate (filtrate) was adjusted to pH3.5 with anhydrous sodium acetate, and finally, saturated sodium chloride (320g/L) was added for salting out, and the salted-out product was collected by suction filtration with a Buchner funnel.

4. Extraction of methanol and acetone

1g (wet weight) of the salted-out product was weighed out and dissolved in 10mL of ultrapure water (pre-cooled to 4 ℃ C., containing 0.1% mercaptoethanol by volume), and centrifuged at 12000rpm to obtain a supernatant. Taking two equal parts of supernatant, respectively adding methanol and acetone to ensure that the final concentration of the methanol is 60% and the final concentration of the acetone is 80%, then placing the supernatant in a refrigerator at the temperature of minus 20 ℃ for 12 hours, centrifuging the supernatant at 12000rpm, taking the supernatant, volatilizing the methanol and the acetone in the solution by the supernatant through low-temperature decompression, placing the supernatant in the refrigerator at the temperature of minus 70 ℃ for 8 hours, and freeze-drying the supernatant through a freeze-drying machine to obtain the extracted and purified pancreatic Ampelopsin sample.

Example 2 detection of pancreatic Ampelopsin in chickpeas

1. Detection by infrared-visible spectrophotometry

Weighing about 1mg of the dried and balanced extract sample in an agate mortar, fully grinding, adding about 100mg of potassium bromide powder, uniformly grinding until the particle size is less than 2 mu m, placing the mixture in a mold, pressing the mixture on an oil press by using 60Pa pressure to form transparent sheets, respectively carrying out infrared scanning, wherein the scanning wavelength is 400-4000 cm, and respectively recording an FT-IR picture.As can be seen from FIGS. 1 and 2, the wave number was 3303cm^-1A typical-OH absorption peak appears at 2935cm^-1The peak appearing is represented by-CH₂Generated by antisymmetric telescopic vibration, at 2356cm^-1The absorption peak is formed by CO₂1653cm caused by antisymmetric stretching vibration^-1The absorption peak is formed by protein amide I with characteristic group R-CO-NH₂Produced by stretching vibration of medium-C ═ O —, 1530cm^-1The absorption peak is caused by-NH in-plane bending vibration in the secondary amide group of the protein, 1390cm^-1The weaker absorption peak is generated by free COO-symmetric stretching vibration in the protein. The successful extraction of pancreatic Ampelopsin from chickpeas was demonstrated.

2. Ultraviolet-visible spectrophotometry

Weighing a certain mass of tryptanthrin sample, dissolving the tryptanthrin sample in deionized water, diluting the solution to a proper amount to prepare a protein solution with a certain mass concentration, and then performing scanning measurement in a wavelength range of 200-400 nm by using an ultraviolet-visible spectrophotometer. As shown in FIGS. 3 and 4, when the protein solution is scanned by the ultraviolet-visible spectrum within the wavelength range of 200-400 nm, the maximum absorption peak appears at the wavelength of 200nm, the curves at other wavelengths are relatively flat, no obvious miscellaneous peak is seen, and the maximum absorption peak of the ultraviolet-visible spectrum of the protein solution is near the wavelength of 214 nm. Therefore, the extracted chickpea protein has high purity, and the result of the purity data measured by combining the spectrophotometry is clear, which shows that the method for extracting and preparing the chickpea pancreatic Ampeptide is reliable.

Example 3 purification of pancreatic Ampelopsin from chickpeas

The apparatus was an Agilent analysis type high performance liquid chromatograph using ODS-C18(4.6 mm. times.150 mm,5u) column. Solution A was ultrapure water (Millipore) containing 0.1% TFA (TEDIA); solution B was acetonitrile (Fisher) with 0.1% TFA; the sample was dissolved in solution A, centrifuged at 15000rpm and injected. The detection wavelength was set at 214nm and the flow rate was set at 1.0 ml/min. The gradient was set to 60 min with the B solution rising from 10% to 70%. As can be seen from FIGS. 5 and 6, the pancreatic Ampelopsin extracted from Cicer arietinum was subjected to high performance liquid chromatography to detect a sharp peak, which proves that a large amount of pancreatic Ampelopsin was successfully extracted and the components were single. The pancreatic Ampelopsin with uniform distribution and high purity is finally obtained in the experiment, and technical support is provided for the structural research and activity research of protein polypeptide in chickpeas.

Claims

1. an extraction and purification method of pancreatin in chickpeas, is characterized in that, comprises the following steps:

1) in the mixed solution of hydrochloric acid and ethanol, add alginic acid, filter residue with hydrochloric acid, distilled water, acetic acid to carry out suction filtration successively after filtration, obtain the alginic acid after treatment, refrigerate for subsequent use;

2) the chickpeas are soaked and germinated, the germinated chickpeas are put into an acetic acid solution containing mercaptoethanol and homogenized, and a supernatant is obtained after the extraction;

3) Add the alginic acid obtained in step 1) to the supernatant obtained in step 2), stir and adsorb, collect the alginic acid that has adsorbed the protein with a Buchner funnel, and fully elute the adsorbed protein with hydrochloric acid after cleaning. The deliquoring was adjusted to acidity, sodium chloride was added for salting out, and the salting-out product was collected.

2. the extraction and purification method of pancreatin in a kind of chickpea according to claim 1, is characterized in that, in step ¹ ), the mixed solution of described hydrochloric acid, ethanol is 0.2L 2mol L A mixed solution of hydrochloric acid and 0.8L ethanol.

3. the extraction and purification method of pancreatin in a kind of chickpea according to claim 2, is characterized in that, in step 1), by solid-liquid ratio, alginic acid: the mixed solution of hydrochloric acid, ethanol=100g: 1L.

4 . The method for extracting and purifying pancreatin from chickpeas according to claim 3 , wherein in step 2), the soaking time is 12 hours, and the germination condition is 25° C. for 60 hours. 5 .

5 . The method for extracting and purifying pancreatin in a kind of chickpea according to claim 4 , wherein in step 2), the concentration of the acetic acid solution is 0.5 mol·L ⁻¹ . 6 .

6 . The method for extracting and purifying pancreatin in a chickpea according to claim 5 , wherein in step 2), the extraction temperature is 4° C. and the extraction time is 24 hours. 7 .

7. the extraction and purification method of pancreatin in a kind of chickpea according to claim 6, is characterized in that, in step 3), the pH of described supernatant is 2.7, described will be washed out The pH of the eluate was adjusted to 3.5 for acid adjustment.

8. the extraction and purification method of pancreatin in a kind of chickpea according to claim 7, is characterized in that, in step 3), in, the salted out substance is extracted with methanol and acetone, freeze-dried, through high performance liquid phase The solid pancreatin was obtained after chromatographic purification.