CN114195860A - Anti-angiogenesis and anti-tumor active peptide from codonopsis pilosula thunb, and preparation method and application thereof - Google Patents
Anti-angiogenesis and anti-tumor active peptide from codonopsis pilosula thunb, and preparation method and application thereof Download PDFInfo
- Publication number
- CN114195860A CN114195860A CN202111544949.4A CN202111544949A CN114195860A CN 114195860 A CN114195860 A CN 114195860A CN 202111544949 A CN202111544949 A CN 202111544949A CN 114195860 A CN114195860 A CN 114195860A
- Authority
- CN
- China
- Prior art keywords
- codonopsis lanceolata
- active peptide
- angiogenesis
- codonopsis
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of plant small molecule active peptides, and particularly relates to an anti-angiogenesis and anti-tumor active peptide from Codonopsis lanceolata, and a preparation method and application thereof. The invention firstly adopts high performance liquid chromatography-tandem mass spectrometry to analyze and identify the codonopsis pilosula oligopeptide products and efficiently screen the activity to obtain high-activity small molecular peptides with potential anti-angiogenesis and anti-tumor functions, and the small molecular peptides are obtained by SephadexLH-20 sephadex column chromatography and high performance liquid chromatography separation and purification and are subjected to activity verification. The result shows that the small molecular active peptide has stronger anti-angiogenesis activity and the effect of inhibiting the growth of cervical cancer tumor, belongs to food-borne active peptide, can be used for preventing and treating cervical cancer related diseases, can be used for developing foods, health care products and biological medicine products, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of plant small molecule active peptides, and particularly relates to an anti-angiogenesis and anti-tumor active peptide from campanumoea lanceolata, and a preparation method and application thereof.
Background
Cervical cancer is a common malignancy that occurs in the cervico-vaginal region or the transitional zone, second only to breast cancer, as the second major gynecological malignancy in the world. The existing means for treating the cervical cancer mainly comprise surgery, radiotherapy and chemotherapy, wherein the chemotherapy has poor curative effect on the cervical cancer at the middle and late stages and recurrence and metastasis and is easy to generate serious adverse drug reactions. 30-40% of cervical cancer patients still have treatment failure after comprehensive treatment, and molecular targeted therapy provides a new treatment method for the part of patients, and anti-angiogenesis therapy is one of the patients. Since its introduction in 1971 by the new tumor angiogenesis, tumor angiogenesis has been identified as an important target for the treatment of malignancies. The vascular abnormality is very important for the growth and the metastasis of solid tumors, and the clinical application proves that the curative effect of the anti-vascular targeting treatment on the cervical cancer is considerable, but the price is high, and the popularization and the application are limited. Therefore, the development of the medicament which has good anti-angiogenesis and anti-tumor effects and is cheap and easy to obtain has important significance.
Codonopsis lanceolata is a perennial herb of Codonopsis genus of Campanulaceae family, has white milk, also called goat milk, Codonopsis lanceolata, Daucus carota, etc., and Codonopsis lanceolata contains triterpenoid saponin, sterol, alkaloid, lignan, etc., and protein, dietary fiber, mineral, vitamin, etc., and is a plant for both food and medicine. Codonopsis lanceolata is widely eaten as wild vegetable and is also used as traditional Chinese medicinal material, and has the effects of nourishing, invigorating qi, promoting lactation, expelling pus, eliminating phlegm, diminishing inflammation and the like. Modern researches show that Codonopsis lanceolata has biological activities of resisting tumor, resisting inflammation, resisting oxidation, protecting liver, reducing blood lipid, reducing blood sugar, resisting thrombosis, protecting nerve, etc. In recent years, the artificial planting area of Codonopsis lanceolata in northeast China is continuously increased, and the method has great development value and wide market prospect.
At present, researches on Codonopsis lanceolata focus on polysaccharide, saponin, flavone and other components, related patents relate to the cultivation and planting of Codonopsis lanceolata and the application of a composition which only contains Codonopsis lanceolata, and the related patents are small in quantity, for example, ZL201810473358.4 discloses a method for improving the germination rate of Codonopsis lanceolata seeds, ZL201810748981.6 discloses a traditional Chinese medicine composition preparation which contains the Codonopsis lanceolata component and is used for treating respiratory system diseases, and ZL201310334283.9 discloses health care bread containing the Codonopsis lanceolata. The protein content of the Codonopsis lanceolata dry product is about 11-15%, the research on the protein component of Codonopsis lanceolata is very little, and no related patent and literature is available for researching the preparation process of Codonopsis lanceolata oligopeptide and the anti-angiogenesis and anti-tumor functions of the active peptide thereof.
Disclosure of Invention
Aiming at the problems, the current situation and the development prospect, the invention provides the anti-angiogenesis and anti-tumor active peptide from the codonopsis pilosula in campanumoea, which has the effects of anti-angiogenesis activity and inhibition of cervical cancer tumor growth, has small molecular weight, high activity, easy separation and purification and simple preparation process, can be used for health care and treatment of related diseases such as cervical cancer and the like, and has wide application prospect in the field of medical biology.
The technical scheme adopted by the invention for realizing the purpose is as follows: an anti-angiogenesis and anti-tumor active peptide derived from Codonopsis lanceolata has an amino acid sequence of Lys-Arg-Trp-Cys-Phe-Leu-Pro.
The preparation method of the active peptide comprises the following steps:
s1, preparing Codonopsis lanceolata oligopeptide;
s2, separating and purifying the Codonopsis lanceolata oligopeptide into small molecule active peptides.
As a preferred embodiment, the method for preparing codonopsis pilosula oligopeptide in step S1 comprises the following steps: coarsely crushing a Codonopsis lanceolata sample, adding water with the mass volume of 10-15 times to prepare homogenate, placing the homogenate into an enzymolysis tank, adding cellulase with the mass of 0.5-1.0% of that of Codonopsis lanceolata into the enzymolysis tank, carrying out enzymolysis for 1-2 hours at 45-50 ℃, and controlling the pH value of the enzymatic reaction to be 5.0-6.0; and adding compound protease accounting for 3-5% of the protein mass of Codonopsis lanceolata into the enzymolysis tank, wherein the mass ratio of the compound protease is as follows: the pineapple protease is neutral protease, alkaline protease (1-3), (3-5) and (2-3); carrying out enzymolysis for 3-4 hours at the temperature of 45-55 ℃, and controlling the pH value of the enzyme reaction to be 7.0-8.0; after enzymolysis, heating to 80-90 ℃ to inactivate enzyme for 10-15 minutes to obtain Codonopsis lanceolata enzymatic hydrolysate; and (3) centrifuging the Codonopsis lanceolata enzymolysis liquid at 6000-8000 rpm for 8-10 minutes, removing granular substances, separating clear liquid by adopting an ultrafiltration membrane with the molecular weight of 3000Da, and performing spray drying on the membrane passing liquid to obtain Codonopsis lanceolata oligopeptide powder.
As a preferred scheme, the method for separating and purifying codonopsis pilosula oligopeptide into small molecule active peptide in step S2 comprises the following steps: dissolving codonopsis pilosula thunb oligopeptide powder in water to prepare a solution with the concentration of 100mg/mL, separating and purifying by adopting Sephadex LH-20 column chromatography, wherein a mobile phase is 40% methanol, the flow rate is 0.3-0.6 mL/min, the absorbance of eluent is measured at 220nm, and the required peak is collected according to the absorbance value; adopting high performance liquid chromatography for purification again, collecting chromatographic peak with retention time of 24 minutes, concentrating, and freeze-drying to obtain the small molecule active peptide, wherein the chromatographic conditions are as follows: c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percentage of 0.07-0.1%, mobile phase B is acetonitrile, gradient elution conditions are as follows: 0-10 min, 5% B; 10-20 min, 5% -15% of B; 20-30 min, 15% -30% B; 30-45 min, 30-45% B; the flow rate is 0.8-1.0 mL/min, and the detection wavelength is 220 nm.
As a preferred embodiment, the preparation method of the active peptide further comprises the following steps of determining the purity and amino acid sequence of the active peptide: the collected small molecular peptides are detected to be single peaks by liquid chromatography, and the structure is determined by utilizing high performance liquid chromatography-mass spectrometry, and the amino acid sequence is as follows: Lys-Arg-Trp-Cys-Phe-Leu-Pro, with a molecular weight of 990.51 Da.
As a preferable scheme, the raw material of the Codonopsis lanceolata is Codonopsis lanceolata in Changbai mountain areas.
Preferably, the Sephadex LH-20 column in the step S2 has an inner diameter of 3.0cm and a length of 100 cm; the inner diameter of the C18 chromatographic column is 4.6mm, the length of the column is 250mm, and the particle size is 5 mu m.
An application of the anti-angiogenesis and anti-tumor active peptide from the Codonopsis lanceolata in the anti-angiogenesis activity or the inhibition of the tumor growth of cervical cancer.
An application of the anti-angiogenesis and anti-tumor active peptide from the Codonopsis lanceolata in anti-cervical cancer drugs.
The invention has the beneficial effects that:
(1) the invention fills the blank of research on preparing the small molecule active peptide from the protein component of the Codonopsis lanceolata and develops new preparation, separation and purification processes and efficacy research of the Codonopsis lanceolata oligopeptide.
(2) Performing high performance liquid chromatography-mass spectrometry identification on Codonopsis lanceolata oligopeptide in the early stage, and performing activity screening by using an online database to obtain high-activity small molecular peptide, separating and purifying the active peptide by Sephadex LH-20 gel column chromatography, high performance liquid chromatography and other separation technologies on the basis, verifying the activity of the active peptide, and finally obtaining the high-activity anti-angiogenesis and anti-cervical cancer peptide, wherein the amino acid sequence of the peptide is as follows: Lys-Arg-Trp-Cys-Phe-Leu-Pro, which is a novel small molecule active peptide by the search of BIOPEP and EROP-Moscow of an online database.
(3) The active peptide has the advantages of small molecular weight, high activity, simple separation and purification steps, easy obtainment, belongs to food-derived active peptide, has the effects of anti-angiogenesis activity and cervical cancer tumor growth inhibition, can be applied to the health care and treatment of cervical cancer and other related diseases and the development of related products, and has wide application prospects in the field of medicines and the like.
(4) On the basis, computer-aided drug design is carried out on the anti-angiogenesis active peptide, a plurality of small molecular peptides with potential anti-angiogenesis activity are found, and subsequently, activity verification and related product research and development can be further carried out.
Drawings
FIG. 1 is a graph of the rate of inhibition of angiogenesis of small molecule active peptides of the invention.
FIG. 2 is a diagram of the cancer suppression rate of the small molecule active peptide of the invention on cervical cancer-bearing Kunming mice.
FIG. 3 is a graph showing the rate of inhibition of angiogenesis of small molecule active peptides in example 3.
Detailed Description
The invention will be further described with reference to the following drawings and examples, but the invention is not limited to the specific examples.
Example 1
An anti-angiogenesis and anti-tumor active peptide from Codonopsis lanceolata (Codonopsis lanceolata) has an amino acid sequence of Lys-Arg-Trp-Cys-Phe-Leu-Pro.
Example 2
The preparation method of the Codonopsis lanceolata-derived anti-angiogenesis and anti-tumor active peptide in the embodiment 1 comprises the following steps:
s1 preparation of Codonopsis lanceolata oligopeptide
Selecting 1000g of Codonopsis lanceolata sample from Changbai mountain area, coarsely pulverizing, adding 10 times of water by mass volume to prepare homogenate, placing into an enzymolysis tank, adding cellulase accounting for 0.6% of Codonopsis lanceolata by mass (based on dry weight), and performing enzymolysis at 50 deg.C for 1 hr, wherein the pH value of the enzyme reaction is controlled at 5.5; adding compound protease with the protein mass of 4% of that of Codonopsis lanceolata, performing enzymolysis at 50 ℃ for 4 hours, controlling the pH value of the enzyme reaction at 7.8, heating to 90 ℃ after the enzymolysis is finished, deactivating enzyme for 10 minutes to obtain Codonopsis lanceolata enzymolysis liquid, centrifuging the enzymolysis liquid at 8000 rpm for 8 minutes, removing granular substances, separating clear liquid by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and performing spray drying on the membrane passing liquid to obtain Codonopsis lanceolata oligopeptide powder. The mass ratio of the compound protease is as follows: bromelain neutral protease alcalase 2:4: 3.
S2 separation and purification of small molecule active peptide
Dissolving Codonopsis lanceolata oligopeptide powder in S1 in water to obtain a solution with a concentration of 100mg/mL, separating and purifying by Sephadex LH-20 column chromatography (3.0 × 100cm), eluting with 40% methanol at a flow rate of 0.5 mL/min to obtain an eluate with an absorbance of 220nm, and collecting the desired peak according to the absorbance value;
further purifying by high performance liquid chromatography under the following conditions: c18 chromatographic column (4.6mm × 250mm, 5 μm), mobile phase A is trifluoroacetic acid water containing 0.1% by volume percentage, mobile phase B is acetonitrile, and the gradient elution conditions are as follows: 0-10 min, 5% B; 10-20 min, 5% -15% of B; 20-30 min, 15% -30% B; 30-45 min, 30-45% B; the flow rate is 1.0mL/min, the detection wavelength is 220nm, the chromatographic peak with the retention time of 24 minutes is collected, and the micromolecular active peptide is obtained by freeze-drying after concentration.
S3, purity and amino acid sequence determination
The collected small molecular peptide is detected to be a single peak by liquid chromatography, and the structure is determined by utilizing high performance liquid chromatography-mass spectrometry, and the amino acid sequence of the small molecular peptide is as follows: Lys-Arg-Trp-Cys-Phe-Leu-Pro, with a molecular weight of 990.51 Da.
Determination of anti-angiogenic activity:
healthy chick embryos were incubated in an incubator at 37 ℃ and 60% relative humidity for 7 days, after which the chick embryo allantoic membrane (CAM) was exposed in a clean bench. The embryos were randomly divided into 4 groups (control and low, medium and high dose experimental groups) of 15 embryos each, and the CAM was covered with glass fiber filter paper loaded with 20. mu.L of physiological saline and 0.2mg/mL, 0.5mg/mL, 1.0mg/mL solutions of small molecule active peptides and incubated for a further 72 hours. The glass fiber filter paper was removed, the allantoic membrane was removed, fixed with 10% formaldehyde, and then angiogenesis was observed under a microscope to calculate the angiogenesis inhibition rate.
The angiogenesis inhibition rate is calculated by the formula:
angiogenesis inhibition (%) is (number of branch points of blood vessel in control group-number of branch points of blood vessel in experimental group)/number of branch points of angiogenesis in control group × 100%
The experimental result is shown in figure 1, the low, medium and high dose of small molecule active peptide solution has the inhibiting effect on the CAM blood vessels, the inhibiting rate is increased along with the increase of the concentration, wherein the angiogenesis inhibiting rate of the high dose group reaches 86.35% +/-5.34%, which shows that the small molecule active peptide has stronger anti-angiogenesis activity.
And (3) determination of anti-tumor activity:
constructing a Kunming mouse model of cervical carcinoma: kunming mice are fasted and water is not forbidden 12 hours before model building. Sucking U14 tumor cells for passage of 7-10 days under aseptic condition, and adjusting the cell density to 5 × 10 with physiological saline6cell/mL, 0.2mL was inoculated subcutaneously in the left forelimb axilla of the mouse.
Cervical cancer-bearing Kunming mice were randomly divided into 4 groups (model group and low, medium, and high dose experimental groups) 24 hours after inoculation, with 15 mice per group. The mice in the low, medium and high dose experimental groups are respectively gavaged with 200 mg/(kg.d), 500 mg/(kg.d) and 1000 mg/(kg.d) micromolecule active peptide, and the mice in the model group are gavaged with physiological saline with the same amount, and the continuous intervention is carried out for 12 days. After 12 days, the weight of the mice is averagely weighed by using electronic analysis, then the mice are killed by removing the vertebrae, the whole tumor tissues are picked up, the tumor tissues of the mice are weighed by using an electronic analysis balance, and finally, the tumor inhibition rate is calculated.
The tumor inhibition rate is calculated by the formula:
tumor inhibition rate (%) (average tumor mass in model group-average tumor mass in experimental group)/average tumor mass in model group × 100%;
the experimental result is shown in figure 2, the tumor inhibition rate is increased along with the increase of the dosage of the small molecule peptide, wherein the tumor inhibition rate of a high dosage group reaches 78.64%, which indicates that the small molecule active peptide has obvious effect of inhibiting the growth of cervical cancer tumor.
Example 3
With the active peptide sequence as the core, any corresponding modifications or modifications are made. For example, the application of the modified or modified amino acid sequence includes the further anti-angiogenesis drug design of the active peptide: the first amino acid Lys can be replaced by any other amino acid, and the replaced small molecule peptides have potential anti-angiogenesis activity.
The first amino acid Lys of the active peptide in the example 1 is replaced by Pro to obtain a new active peptide segment, and the amino acid sequence is Pro-Arg-Trp-Cys-Phe-Leu-Pro.
The anti-angiogenic activity of the compound is verified by adopting a chick embryo allantoic membrane experiment.
The specific experimental process is as follows: healthy chick embryos were incubated in an incubator at 37 ℃ and 60% relative humidity for 7 days, after which the chick embryo allantoic membrane (CAM) was exposed in a clean bench. The embryos were randomly divided into 4 groups (control and low, medium and high dose experimental groups) of 15 embryos each, and the CAM was covered with a glass fiber filter paper loaded with 20. mu.L of physiological saline and 0.2mg/mL, 0.5mg/mL, 1.0mg/mL solutions of small molecule active peptides and incubated for a further 72 hours. The glass fiber filter paper was removed, the allantoic membrane was removed, fixed with 10% formaldehyde, and then angiogenesis was observed under a microscope to calculate the angiogenesis inhibition rate.
The angiogenesis inhibition rate is calculated by the formula:
angiogenesis inhibition (%) is (number of branch points of blood vessel in control group-number of branch points of blood vessel in experimental group)/number of branch points of angiogenesis in control group × 100%
The experimental result is shown in fig. 3, and the result shows that the small molecule active peptide obtained after replacement still has strong anti-angiogenic activity, and each dose has inhibitory effect on CAM blood vessels, wherein the small molecule active peptide in the high dose group (1.0mg/mL) has the best inhibitory effect on angiogenesis, and the inhibitory rate reaches 80.33% ± 2.68%.
Example 4
The preparation method of the Codonopsis lanceolata-derived anti-angiogenesis and anti-tumor active peptide in the embodiment has the same steps as the embodiment 2, and the difference is that:
(1) step S1, after 1000g of Codonopsis lanceolata sample in Changbai mountain area is taken and coarsely crushed, 13 times of water is added to prepare homogenate liquid which is placed in an enzymolysis tank, and then 1.0% of cellulase in mass (based on dry weight) of Codonopsis lanceolata is added to carry out enzymolysis for 1.5 hours at 45 ℃, and the pH value of the enzyme reaction is controlled at 5.0; and adding composite protease accounting for 3% of the protein mass of the Codonopsis lanceolata, wherein the mass ratio of the composite protease is as follows: bromelain, neutral protease and alkaline protease are 1:5:2, enzymolysis is carried out at 45 ℃ for 3.5 hours, the pH value of the enzyme reaction is controlled at 7.5, after the enzymolysis is finished, the temperature is raised to 85 ℃ to inactivate the enzyme for 15 minutes to obtain Codonopsis lanceolata enzymolysis liquid, the Codonopsis lanceolata enzymolysis liquid is centrifuged at 6000 rpm for 10 minutes to remove granular substances, clear liquid is separated by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and the ultrafiltration membrane liquid is spray-dried to obtain Codonopsis lanceolata oligopeptide powder.
(2) The mobile phase in step S2 was 40% methanol at a flow rate of 0.4 mL/min.
(3) In step S2, the mobile phase A is trifluoroacetic acid water containing 0.08 volume percent, and the flow rate is 0.8 mL/min.
Example 5
The preparation method of the Codonopsis lanceolata-derived anti-angiogenesis and anti-tumor active peptide in the embodiment has the same steps as the embodiment 2, and the difference is that:
(1) step S1, after 1000g of Codonopsis lanceolata sample in Changbai mountain area is taken and coarsely crushed, 15 times of water is added to prepare homogenate liquid which is placed in an enzymolysis tank, then 0.8 percent of cellulase in mass (based on dry weight) of Codonopsis lanceolata is added, enzymolysis is carried out for 2 hours at 48 ℃, and the pH value of enzyme reaction is controlled at 6.0; and adding composite protease accounting for 5% of the protein mass of the Codonopsis lanceolata, wherein the mass ratio of the composite protease is as follows: bromelain, namely neutral protease, namely alkaline protease, namely 2:3:3, performing enzymolysis at 55 ℃ for 3 hours, controlling the pH value of the enzyme reaction to be 8.0, heating to 90 ℃ after the enzymolysis is finished, deactivating the enzyme for 12 minutes to obtain Codonopsis lanceolata enzymolysis liquid, centrifuging the enzymolysis liquid at 7000 rpm for 10 minutes, removing granular substances, separating clear liquid by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and performing spray drying on the membrane passing liquid to obtain Codonopsis lanceolata oligopeptide powder.
(2) In the step S2, the mobile phase is 40% methanol, and the flow rate is 0.6 mL/min;
(3) in step S2, mobile phase A is trifluoroacetic acid water with volume percentage of 0.07%, and the flow rate is 0.9 mL/min.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention are equivalent to or changed within the technical scope of the present invention.
Sequence listing
<110> Jilin national medicines GmbH
DALIAN BLUESCITECH TECHNOLOGY DEVELOPMENT Co.,Ltd.
<120> anti-angiogenesis and anti-tumor active peptide from campanumoea lanceolata, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 7
<212> PRT
<213> Codonopsis lanceolata (Codonopsis lancelata)
<400> 2
Lys Arg Trp Cys Phe Leu Pro
1 5
<210> 2
<211> 7
<212> PRT
<213> Codonopsis lanceolata (Codonopsis lancelata)
<400> 2
Pro Arg Trp Cys Phe Leu Pro
1 5
Claims (9)
1. An anti-angiogenesis and anti-tumor active peptide derived from Codonopsis lanceolata is characterized in that the amino acid sequence of the active peptide is Lys-Arg-Trp-Cys-Phe-Leu-Pro.
2. A process for the preparation of an active peptide according to claim 1, comprising the steps of:
s1, preparing Codonopsis lanceolata oligopeptide;
s2, separating and purifying the Codonopsis lanceolata oligopeptide into small molecule active peptides.
3. The method for preparing active peptides of claim 2, wherein the method for preparing Codonopsis lanceolata oligopeptide in step S1 comprises: coarsely crushing a Codonopsis lanceolata sample, adding water with the mass volume of 10-15 times to prepare homogenate, placing the homogenate into an enzymolysis tank, adding cellulase with the mass of 0.5-1.0% of that of Codonopsis lanceolata into the enzymolysis tank, carrying out enzymolysis for 1-2 hours at 45-50 ℃, and controlling the pH value of the enzymatic reaction to be 5.0-6.0; and adding compound protease with the protein mass of codonopsis lanceolata accounting for 3-5% of that of the codonopsis lanceolata into the enzymolysis tank, wherein the compound protease comprises the following components in parts by mass: bromelain, alkaline protease (1-3): 3-5): 2-3); carrying out enzymolysis for 3-4 hours at the temperature of 45-55 ℃, and controlling the pH value of the enzyme reaction to be 7.0-8.0; after the enzymolysis is finished, heating to 80-90 ℃ to inactivate the enzyme for 10-15 minutes to obtain Codonopsis lanceolata enzymolysis liquid; and (3) centrifuging the Codonopsis lanceolata enzymolysis liquid at 6000-8000 rpm for 8-10 minutes, removing granular substances, separating clear liquid by adopting an ultrafiltration membrane with the molecular weight cutoff of 3000Da, and performing spray drying on the membrane passing liquid to obtain Codonopsis lanceolata oligopeptide powder.
4. The method for preparing active peptide of claim 3, wherein the step S2 is a method for separating and purifying Codonopsis lanceolata oligopeptide into small molecule active peptide: dissolving Codonopsis lanceolata oligopeptide powder in water to prepare a solution with the concentration of 100mg/mL, separating and purifying by Sephadex LH-20 column chromatography, wherein the mobile phase is 40% methanol, the flow rate is 0.3-0.6 mL/min, the absorbance of the eluent is measured at 220nm, and the required peak is collected according to the absorbance value; adopting high performance liquid chromatography for purification again, collecting chromatographic peak with retention time of 24 minutes, concentrating, and freeze-drying to obtain the small molecule active peptide, wherein the chromatographic conditions are as follows: c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percentage of 0.07-0.1%, mobile phase B is acetonitrile, gradient elution conditions are as follows: 0-10 min, 5% B; 10-20 min, 5% -15% of B; 20-30 min, 15% -30% B; 30-45 min, 30-45% B; the flow rate is 0.8-1.0 mL/min, and the detection wavelength is 220 nm.
5. The method of claim 4, further comprising determining the purity and amino acid sequence of the active peptide: the collected small molecular peptide is detected to be a single peak by liquid chromatography, and the structure is determined by utilizing high performance liquid chromatography-mass spectrometry, and the amino acid sequence of the small molecular peptide is as follows: Lys-Arg-Trp-Cys-Phe-Leu-Pro, with a molecular weight of 990.51 Da.
6. The method for preparing active peptide according to claim 3, wherein the raw material of Codonopsis lanceolata is Codonopsis lanceolata in Changbai mountain area.
7. The method for preparing active peptide according to claim 4, wherein the Sephadex LH-20 column of step S2 has an inner diameter of 3.0cm and a length of 100 cm; the inner diameter of the C18 chromatographic column is 4.6mm, the length of the column is 250mm, and the particle size is 5 mu m.
8. Use of the Codonopsis lanceolata-derived anti-angiogenic and anti-tumor active peptide of claim 1 for anti-angiogenic activity or inhibition of tumor growth in cervical cancer.
9. The use of the Codonopsis lanceolata-derived anti-angiogenic and anti-tumor active peptide of claim 1 in the preparation of a medicament for treating cervical cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111544949.4A CN114195860B (en) | 2021-12-16 | 2021-12-16 | Anti-angiogenesis and anti-tumor active peptide derived from codonopsis pilosula as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111544949.4A CN114195860B (en) | 2021-12-16 | 2021-12-16 | Anti-angiogenesis and anti-tumor active peptide derived from codonopsis pilosula as well as preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114195860A true CN114195860A (en) | 2022-03-18 |
| CN114195860B CN114195860B (en) | 2023-07-25 |
Family
ID=80654575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111544949.4A Active CN114195860B (en) | 2021-12-16 | 2021-12-16 | Anti-angiogenesis and anti-tumor active peptide derived from codonopsis pilosula as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114195860B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091117A (en) * | 2011-01-30 | 2011-06-15 | 延边大学 | Method for improving anticancer activity of Codonopsis lanceolata Benth.et Hook.F |
| CN106074661A (en) * | 2016-07-16 | 2016-11-09 | 吉林农业大学 | A kind of wheel blade Radix Codonopsis extract new application in preparation prevention injury of kidney medicine and health product |
| US20170340690A1 (en) * | 2016-05-31 | 2017-11-30 | Korea Institute Of Science And Technology | Method for improving intestinal health using extracts of codonopsis lanceolata |
| CN110590907A (en) * | 2019-10-31 | 2019-12-20 | 福州大学 | Preparation, separation and purification method of immunoregulatory peptide |
| CN110655556A (en) * | 2019-10-31 | 2020-01-07 | 福州大学 | Preparation and method of immunoregulatory peptide |
-
2021
- 2021-12-16 CN CN202111544949.4A patent/CN114195860B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091117A (en) * | 2011-01-30 | 2011-06-15 | 延边大学 | Method for improving anticancer activity of Codonopsis lanceolata Benth.et Hook.F |
| US20170340690A1 (en) * | 2016-05-31 | 2017-11-30 | Korea Institute Of Science And Technology | Method for improving intestinal health using extracts of codonopsis lanceolata |
| CN106074661A (en) * | 2016-07-16 | 2016-11-09 | 吉林农业大学 | A kind of wheel blade Radix Codonopsis extract new application in preparation prevention injury of kidney medicine and health product |
| CN110590907A (en) * | 2019-10-31 | 2019-12-20 | 福州大学 | Preparation, separation and purification method of immunoregulatory peptide |
| CN110655556A (en) * | 2019-10-31 | 2020-01-07 | 福州大学 | Preparation and method of immunoregulatory peptide |
Non-Patent Citations (2)
| Title |
|---|
| WEI LI ET AL.: ""Anti-Tumor Effect of Steamed Codonopsis lanceolata in H22 Tumor-Bearing Mice and Its Possible Mechanism"", 《NUTRIENTS》, vol. 7, pages 8294 - 8307 * |
| 姜国哲 ET AL.: ""轮叶党参的植物化学物及生物活性研究进展"", 《食品科学》, vol. 37, no. 17, pages 257 - 262 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114195860B (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109293740B (en) | Oyster-derived ACE (angiotensin converting enzyme) inhibitory and antitumor active peptide | |
| CN102847134B (en) | Preparation method for chlorella polypeptide microcapsule | |
| CN105586379B (en) | A kind of preparation method with the collagen active peptide for inhibiting cancer cell multiplication effect | |
| CN105203671A (en) | HPLC (high performance liquid chromatography) and GPC (gel permeation chromatography) based separation and purification method for marine organism sourced polypeptide | |
| CN105524963A (en) | Broccoli polypeptide extract as well as preparation method and application thereof | |
| CN114213506B (en) | Anti-angiogenesis and anti-tumor active peptide derived from ginseng as well as preparation method and application thereof | |
| CN105601707B (en) | A kind of anti-oxidant tetrapeptide of dandelion seed and preparation method thereof | |
| CN114195860B (en) | Anti-angiogenesis and anti-tumor active peptide derived from codonopsis pilosula as well as preparation method and application thereof | |
| CN113893310A (en) | Mountain plant fermentation composition and preparation method and application thereof | |
| KR102160672B1 (en) | Composition for Anti-diabetic comprising Hydrorized Extract of Glub | |
| CN114989258B (en) | Application of plant extract composition in preparing product for treating constipation and reducing weight | |
| CN106822331A (en) | To lock application of the lichee bark extract based on shape polymer polyphenol in treatment antihyperuricemic disease drug or health products are prepared | |
| CN112851764B (en) | Antioxidant peptide derived from pleurotus tuber-regium fruit body protein and application thereof | |
| KR101431882B1 (en) | A composition for anti-aging or wrinkle improvement of skin comprising enzyme-treated red ginseng extracts | |
| CN107915772B (en) | Novel sweet potato glycoprotein and sweet potato glycoprotein extract containing the same | |
| CN115124591A (en) | Spirulina platensis phycocyanin angiotensin converting enzyme inhibitory peptide and preparation method and application thereof | |
| CN114213516B (en) | Active peptide derived from radix codonopsis pilosulae for promoting lactation and resisting breast tumor, and preparation method and application thereof | |
| JP5034071B2 (en) | Kawariharatake extract that suppresses breast cancer | |
| KR101133739B1 (en) | A Method for Preparing Acid Hydrolyzed White Ginseng Extracts Having High Content of Gingenoid Rg3 | |
| KR101975018B1 (en) | Composition for lowering cholesterol comprising a mixture culture of Ginseng Berry-Auricularia auricula mycelia | |
| CN114377107B (en) | Application of sea buckthorn seed meal protein peptide in preparation of medicine for preventing and treating prostatic hyperplasia | |
| Gao et al. | ACE inhibitory, antitumor and antioxidant activities of submerged culture materials of three medicinal mushrooms | |
| KR20080044942A (en) | Ginseng preparations containing a high concentration of ginseng sapogenin using a bacillus natto and method thereof | |
| KR101454067B1 (en) | A composition for anti-aging or wrinkle improvement of skin comprising mixture of enzyme-treated red ginseng extracts and malt extracts | |
| CN116854774B (en) | Derived peptides, telomere length modulators and their use in anti-aging |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |