CN109303789B - Cornu Cervi Pantotrichum active component with prolyl endonuclease inhibiting activity, and its preparation method and application - Google Patents
Cornu Cervi Pantotrichum active component with prolyl endonuclease inhibiting activity, and its preparation method and application Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a pilose antler active component with prolyl endonuclease inhibition activity. The invention takes the pilose antler as the raw material, establishes the preparation method of the pilose antler active component with prolyl endonuclease inhibition activity, and the active component can be applied to the prevention and treatment of cognitive disorder diseases such as Alzheimer disease and the like, and has wide application prospect.
Description
Technical Field
The invention relates to a preparation method and application of a pilose antler active component with prolyl endonuclease inhibitory activity, wherein the pilose antler active component with the prolyl endonuclease inhibitory activity can be used for treating or preventing neurodegenerative diseases such as cognitive impairment diseases such as Alzheimer's disease, Parkinson's disease and the like.
Background
Cornu Cervi Pantotrichum is unrossified young horn of male deer dense villus of Cervidae Cervus Nippon Temminck or Cervus Elaphus L, which has been recorded in Shen nong Ben Cao Jing of Han Dynasty for over 2000 years, and has been accepted in the herbal medicine of all the generations. The compendium of materia medica records that pilose antler can 'produce essence and supplement marrow, nourish blood and benefit yang, strengthen tendons and bones, treat all deficiency, deafness, dim eyesight, vertigo and deficiency dysentery' (document 1: Jijing, Qianjing, Huangfengjie, etc.. research on active substances and pharmacological actions of pilose antler, Chinese Journal of Biochemical pharmaceuticals, 2009,30(2): 141-143).
Prolyl Endopeptidase (PEP), also known as Prolyl oligopeptidase, is a high retention serine protease. PEP plays an important role in the metabolism of proline-containing neuropeptides, and the activity of PEP is related to the induction of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). Research shows that the PEP inhibitor can block the metabolism of PEP to achieve the aim of improving memory. Therefore, the PEP inhibitor can be used as a potential drug for restoring the memory of the Alzheimer disease patient to be researched.
Disclosure of Invention
The invention aims to provide a pilose antler aqueous extract with PEP inhibitory activity and application thereof. Enzymolysis products of the pilose antler aqueous extract after the action of pepsin and pancreatin have PEP inhibition activity, and can be used as a potential source for research and development of medicaments for improving cognitive impairment diseases such as Alzheimer's disease and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) cutting fresh pilose antler into pieces, adding deionized water with the mass 5-30 times of that of the pilose antler, heating to 90-100 ℃ (the optimal range is 95-100 ℃), stirring and extracting for 1-5 (the optimal range is 2-4) hours, cooling to room temperature after extraction is finished, filtering with a filter screen with 170-270 (the optimal range is 200-230) meshes, and obtaining the extract as filtrate.
(2) Adjusting the pH value of the extract to 1.0-5.0 (the optimal range is 1.5-2.0) by using 1-6M HCl, uniformly stirring, adding protease A with the mass of 0.1-5.0% (w/w) (the optimal range is 2.0-3.0%) of the antler, and carrying out enzymolysis for 0.5-24 hours (the optimal range is 3-10 hours) at the temperature of 20-80 ℃ (the optimal range is 30-50 ℃); adjusting the pH value to 6.0-8.0 (the optimal range is 6.5-7.5) by using 1-6M NaOH after the enzymolysis is finished, adding protease B with the mass of 0.1-5.0% (w/w) (the optimal range is 2.0-3.0%) of the antler, carrying out enzymolysis for 0.5-24 hours (the optimal range is 3-10 hours) at the temperature of 20-80 ℃ (the optimal range is 30-50 ℃), increasing the temperature to 90-100 ℃ (the optimal range is 95-100 ℃) after the reaction is finished, preserving the temperature for 10-30 minutes, and then reducing the temperature to the room temperature.
(3) Centrifuging the enzymolysis liquid at the speed of 15000 Xg-25000 Xg for 20-30 minutes at the temperature of 4-8 ℃, collecting supernatant, performing ultrafiltration for 20-30 minutes at the speed of 10000 Xg-16000 Xg by using an ultrafiltration membrane with the molecular weight cutoff of 5K-20K daltons, collecting filtrate, and freeze-drying to obtain the component with the prolyl endonuclease inhibition activity.
The proteolytic enzyme A is one or a combination of pepsin, trypsin, chymotrypsin, elastase, papain, bromelain and flavourzyme; the proteolytic enzyme B is one or more of trypsin, chymotrypsin, carboxypeptidase, papain, bromelain, flavourzyme and neutral protease. The proteolytic enzyme A and the protease B cannot be identical.
The active ingredient contains prolyl endonuclease inhibitory peptides such as LPQPPQE, GPAGPQGPR, LPPLTAD, PPGLPGSPGQ, etc.
The cornu Cervi Pantotrichum active component with prolyl endonuclease inhibiting activity can be used for treating or preventing cognitive disorder diseases such as Alzheimer's disease and Parkinson's disease.
The invention establishes a preparation method of the pilose antler active component with prolyl endonuclease inhibition activity, and the prepared pilose antler PEP inhibitory peptide can be applied to the prevention and treatment of cognitive impairment diseases such as Alzheimer's disease and the like, and has wide application prospect.
Compared with the prior art, the invention has the following beneficial effects:
1. the method has the advantages of high extraction rate of active components of cornu Cervi Pantotrichum, mild reaction conditions, good retention of original active components in cornu Cervi Pantotrichum, simple process, and applicability to large-scale production.
2. The basis of the active ingredients of the pilose antler is analyzed by combining the leading edge analysis technology LC-MS/MS, so that the result is more accurate and credible.
3. Has good application prospect. The invention has wide application prospect as a potential source for researching and developing cognitive disorder diseases.
Detailed Description
Example 1
The fresh sika deer antler is used as a raw material and is prepared according to the following process:
freeze drying and pulverizing fresh cornu Cervi Pantotrichum, adding 5L deionized water into 1kg of powder, heating to 100 deg.C, stirring and extracting for 5 hr, cooling to room temperature after extraction, and filtering with 200 mesh filter screen to obtain filtrate.
Adjusting pH of the extract to 2.0 with 6M HCl, stirring, adding 1g pepsin, and performing enzymolysis at 37 deg.C for 5 hr; and (3) after the enzymolysis is finished, adjusting the pH to 7.0 by using 1M NaOH, adding 1g of trypsin, carrying out enzymolysis for 5 hours at the temperature of 37 ℃, raising the temperature to 100 ℃ after the reaction is finished, preserving the temperature for 10-30 minutes, and then cooling to room temperature.
Centrifuging the above enzymolysis solution at 15000 × g speed at 4 deg.C for 30 min, collecting supernatant, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10K Dalton at 10000 × g speed for 30 min, collecting filtrate, and freeze drying to obtain component with PEP inhibiting activity.
Detecting PEP inhibitory activity of the obtained cornu Cervi Pantotrichum fraction
1. Principle of
The PEP can specifically hydrolyze small molecular weight polypeptide at the carboxyl terminal of proline, so the method adopts Z-Gly-Pro-4-nitroanalide as a substrate of the PEP, and the yellow paranitroaniline has a characteristic absorption peak at 405nm after the Z-Gly-Pro-4-nitroanalide is cut off by the PEP, and the inhibitory activity of the sample on the PEP can be calculated according to the absorbance change at 405nm before and after the sample and the substrate are added.
2. Method of producing a composite material
(1) Sample preparation: the samples were dissolved in 10mM PBS buffer (pH 7.0) and prepared as required for the experiment as solutions at concentrations of 0.5, 1.0 and 2.0mg/mL, respectively.
(2) Substrate solution: Z-Gly-Pro-4-nitroanalide (Z-Gly-Pro-pNA) solution, and Z-Gly-Pro-4-nitroanalide is prepared into 10mM solution by using 40% (v/v) dioxane.
(3) Positive drug: sodium Valproate (Sodium Valproate) was dissolved in 10mM PBS (pH 7.0) and prepared to concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0mM, respectively, as required for the experiment.
(4) Enzyme solution: PEP solution, PEP solution was prepared by diluting PEP with a protective solution (45mM Tris-HCl, pH 8.0, 124mM NaCl, 2.4mM KCl, 10% (v/v) glycerol, 225mM imidazole and 3mM DTT) to 1U/mL, storing in a freezer at-80 ℃ and diluting 8-fold with PBS (10mM PBS, 137mM NaCl and 2.7mM KCl, pH 7.0) just before use.
The assay was performed in 96-well plates and absorbance was measured at 405nm using a microplate reader. Firstly, 140 mu L of PBS, 20 mu L of sample (or buffer solution) and 20 mu L of PEP enzyme solution are sequentially added into a sample hole, incubated for 5min at 37 ℃, then 20 mu L of substrate is added, mixed uniformly and incubated for 30 min at 37 ℃, and the absorbance at 405nm is measured by a microplate reader. The total volume of the reaction was 200. mu.l. Triplicate were done per well and the sample inhibition was calculated.
TABLE 1 PEP inhibitory Activity assay Components and sample amounts
Note: "-" indicates no addition of any reagent.
Inhibition rate calculation formula:
wherein I represents the inhibition ratio, ASampleDenotes the absorbance value of the sample, ASampleblankDenotes the blank absorbance value of the sample, AControlThe absorbance values of the negative control groups, AblankIndicating blank absorbance values.
3 Positive control test results
The PEP inhibition activity of the positive control sodium valproate was determined as above when the positive control sodium valproate concentration was 0.2, 0.4, 1, 2 and 4 mM. The results are shown in table 2:
TABLE 2 Positive control sample concentration and PEP inhibition
Warp IC50IC for calculating sodium valproate by calculator50It was 1.98 mM.
When the concentration of the pilose antler sample is 2.0mg/mL, the PEP inhibition rate is 26.52 percent
Desalting the ultrafiltrate of the enzymolysis product of cornu Cervi Pantotrichum extract with C18-SPE column. After desalting, freeze-drying, redissolving in 0.1% (v/v) formic acid water, and mass spectrometry of the sample using LTQ Orbitrap XL (ion trap cyclotron resonance combination mass spectrometer). The peptide composition of the active ingredients of deer antler is shown in table 3.
TABLE 3 composition of peptides of the active ingredient of velvet antler
Example 2
Freeze drying and pulverizing fresh cornu Cervi Pantotrichum, adding 10L deionized water into 1kg of powder, heating to 90 deg.C, stirring and extracting for 3 hr, cooling to room temperature after extraction, and filtering with 230 mesh filter screen to obtain filtrate, i.e. cornu Cervi Pantotrichum extract.
Adjusting pH of the extract to 5.0 with 3M HCl, stirring, adding 1g pepsin, and performing enzymolysis at 50 deg.C for 3 hr; after the enzymolysis is finished, the pH value is adjusted to 8.0 by using 2M NaOH, 1g of chymotrypsin is added, the enzymolysis is carried out for 3 hours at the temperature of 50 ℃, the temperature is increased to 90 ℃ after the reaction is finished, the temperature is kept for 10 minutes, and then the temperature is reduced to the room temperature.
Centrifuging the above enzymolysis solution at 25000 × g speed at 6 deg.C for 20 min, collecting supernatant, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 15K Dalton at 15000 × g speed for 20 min, collecting filtrate, and freeze drying to obtain component with PEP inhibiting activity.
PEP inhibitory activity was detected and mass spectrometry was performed on the obtained velvet antler fraction according to the method in example 1.
When the concentration of the system is 1.0mg/mL, the PEP inhibition rate of the sample prepared by the method is 20.83%, and the components comprise main active peptides: LPQPPQE, GPAGPQGPR, LPPLTAD, PPGLPGSPGQ.
Example 3
Freeze drying and pulverizing fresh cornu Cervi Pantotrichum, adding 20L deionized water into 1kg of powder, heating to 100 deg.C, stirring for 1 hr, cooling to room temperature, and filtering with 170 mesh filter screen to obtain filtrate.
Adjusting pH of the extract to 4.0 with 2M HCl, stirring, adding 1g pepsin, and performing enzymolysis at 30 deg.C for 10 hr; adjusting pH to 6.0 with 5M NaOH, adding elastase with a mass of 1g of cornu Cervi Pantotrichum, performing enzymolysis at 30 deg.C for 10 hr, heating to 100 deg.C, holding for 20 min, and cooling to room temperature.
Centrifuging the above enzymolysis solution at 20000 × g at 8 deg.C for 30 min, collecting supernatant, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 20K Dalton at 12000 × g for 30 min, collecting filtrate, and freeze drying to obtain component with PEP inhibiting activity.
PEP inhibitory activity was detected and mass spectrometry was performed on the obtained velvet antler fraction according to the method in example 1.
When the concentration of the system is 3.0mg/mL, the PEP inhibition rate of the sample prepared by the method is 35.27%, and the components comprise main active peptides: LPQPPQE, GPAGPQGPR, LPPLTAD, PPGLPGSPGQ.
Example 4
Freeze drying and pulverizing fresh cornu Cervi Pantotrichum, adding 1kg powder into 30L deionized water, heating to 95 deg.C, stirring and extracting for 5 hr, cooling to room temperature after extraction, and filtering with 270 mesh filter screen to obtain filtrate.
Adjusting pH of the extract to 2.0 with 3M HCl, stirring, adding 1g pepsin, and performing enzymolysis at 37 deg.C for 20 hr; after the enzymolysis is finished, the pH value is adjusted to 7.0 by 6M NaOH, 0.5g of trypsin and 0.5g of chymotrypsin are added for enzymolysis at the temperature of 37 ℃ for 20 hours, and after the reaction is finished, the temperature is raised to 95 ℃ and is kept for 30 minutes, and then the temperature is reduced to the room temperature.
Centrifuging the above enzymolysis solution at 20000 × g at 4 deg.C for 30 min, collecting supernatant, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 10K Dalton at 16000 × g for 30 min, collecting filtrate, and freeze drying to obtain component with PEP inhibiting activity.
PEP inhibitory activity was detected and mass spectrometry was performed on the obtained velvet antler fraction according to the method in example 1.
When the concentration of the system is 1.0mg/mL, the PEP inhibition rate of the sample prepared by the method is 57.45%, and the components comprise main active peptides: LPQPPQE, GPAGPQGPR, LPPLTAD, PPGLPGSPGQ.
Claims (5)
1. A preparation method of cornu Cervi Pantotrichum active component with prolyl endonuclease inhibiting activity is characterized by: extracting cornu Cervi Pantotrichum, performing enzymolysis, and purifying to obtain cornu Cervi Pantotrichum active component with prolyl endopeptidase inhibiting activity, which comprises the following steps:
freeze drying and pulverizing fresh cornu Cervi Pantotrichum, adding 1kg powder into 30L deionized water, heating to 95 deg.C, stirring and extracting for 5 hr, cooling to room temperature after extraction, and filtering with 270 mesh filter screen to obtain filtrate (cornu Cervi Pantotrichum extract);
adjusting pH of the extract to 2.0 with 3M HCl, stirring, adding 1g pepsin, and performing enzymolysis at 37 deg.C for 20 hr; adjusting the pH value to 7.0 by using 6M NaOH after the enzymolysis is finished, adding 0.5g of trypsin and 0.5g of chymotrypsin, carrying out enzymolysis for 20 hours at the temperature of 37 ℃, raising the temperature to 95 ℃ after the reaction is finished, preserving the temperature for 30 minutes, and then cooling to the room temperature;
the enzymolysis solution is prepared with 20000gCentrifuging at 4 deg.C for 30 min, collecting supernatant, and collecting supernatant with ultrafiltration membrane with cut-off molecular weight of 10K dalton in 16000gThe mixture was ultrafiltrated at the speed of (1) for 30 minutes, and the filtrate was collected and freeze-dried to obtain a fraction having prolyl endonuclease inhibitory activity.
2. The method of claim 1, wherein: the prepared active component contains prolyl endonuclease inhibiting peptide selected from LPQPPQE, GPAGPQGPR, LPPLTAD and PPGLPGSPGQ.
3. A cornu Cervi Pantotrichum active ingredient with prolyl endonuclease inhibiting activity prepared by the preparation method of any of claims 1-2.
4. A use of the active fraction of deer antler having prolyl endonuclease inhibitory activity of claim 3, wherein: the cornu cervi pantotrichum active component with prolyl endonuclease inhibiting activity is used for preparing a medicament for treating and/or preventing nervous system diseases;
the neurological disease is a neurodegenerative disease.
5. Use according to claim 4, characterized in that: the disease is Alzheimer's disease or Parkinson's disease cognitive impairment.
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| CN113499353A (en) * | 2021-08-02 | 2021-10-15 | 长春中医药大学 | Preparation method and application of ginseng and pilose antler refined component with anti-Alzheimer disease activity |
| CN113817019B (en) * | 2021-09-09 | 2023-09-12 | 集美大学 | Prolyl endopeptidase natural inhibitor, and preparation method and application thereof |
| WO2023136573A1 (en) * | 2022-01-11 | 2023-07-20 | 유니메드제약주식회사 | Composition for improving memory or cognition or preventing or treating cranial nerve diseases, comprising porcine brain hydrolysates |
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