CN114213506A - Ginseng-derived anti-angiogenesis and anti-tumor active peptide and preparation method and application thereof - Google Patents
Ginseng-derived anti-angiogenesis and anti-tumor active peptide and preparation method and application thereof Download PDFInfo
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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 derived from ginseng, and a preparation method and application thereof. The invention firstly adopts high performance liquid chromatography-tandem mass spectrometry to analyze and identify ginseng oligopeptide products and efficiently screen activity to obtain high-activity small-molecular peptides with potential anti-angiogenesis and anti-tumor effects, and 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 good anti-angiogenesis activity and the effect of inhibiting the growth of lung cancer tumor, belongs to food-borne active peptide, can be used for preventing and treating lung cancer and other related diseases, and has wide application prospect in the fields of food, health care products and medicine.
Description
Technical Field
The invention belongs to the field of plant small molecule active peptides, and particularly relates to a ginseng-derived small molecule anti-angiogenesis and anti-tumor active peptide, and a preparation method and application thereof.
Background
The distribution of the bioactive peptide is very wide, and the bioactive peptide has important physiological action on the life activity of organisms. Many bioactive peptides are usually present in the long chain of proteins in an inactive state, and can be hydrolyzed by an appropriate protease to release their molecular fragments and activities. Compared with protein, the bioactive peptide has simpler structure, faster and better digestion and absorption, higher stability, lower immunogenicity or even no immunogenicity, and simultaneously has various biological functions such as antioxidation, immunoregulation, blood pressure reduction, anti-inflammation, anticancer and the like. Active polypeptides have been applied to the fields of medicine, nutritional therapy, health care, food and the like, and more polypeptide drugs are developed and applied to clinical application. The prior polypeptide medicine is mainly derived from some natural polypeptides or endogenous polypeptides, has the problems of low content in organisms, difficult extraction, difficult acquisition and the like, and with the rise and continuous progress of biological enzymolysis technology, the active polypeptides from enzymolysis become an effective way for obtaining the active polypeptides, so that the application prospect is wide.
China has abundant resources, and a plurality of raw materials for researching and developing enzymolysis active polypeptides are available, from plants to animals, and from land to sea. At present, it has been reported that active peptides with antioxidant and hypoglycemic functions, such as soybean, balsam pear, ossein, marine fish, oyster, giant salamander, etc. are separated from proteolytic hydrolysates from different sources.
Ginseng is a perennial herb of the genus Panax of the family Araliaceae, and has a long medicinal history in China, and has a name of 'Ginseng crown baicao' from ancient times. The earliest existing pharmaceutical monograph in China is recorded in Shen nong Ben Cao Jing: ginseng is sweet in taste and slightly cold in nature, and has the effects of tonifying five internal organs, calming soul, stopping palpitation, eliminating pathogenic factors, improving eyesight, and improving intelligence. It can be taken for a long time, and has effects of reducing weight and prolonging life. Modern pharmacological research shows that ginseng has the effects of resisting oxidation, resisting fatigue, improving brain function, resisting tumor, enhancing immunity, reducing blood sugar, protecting liver, regulating blood pressure, resisting aging and the like. The ginseng protein is subjected to enzymolysis by different proteases to obtain various active peptide fragments. Currently, there are reports on the antioxidant and hypoglycemic functions of active peptides in ginseng, for example, patent CN201910600912.5 reports an antioxidant food composition containing ginseng oligopeptide; patent CN201810646091.4 reports an auxiliary hypoglycemic product composition added with ginseng peptide; patent CN201510357188.X reports antioxidant effect of Jilin ginseng oligopeptide, but the ginseng peptide components in the Jilin ginseng oligopeptide are mixed peptides prepared by enzymolysis technology, and are not separated and purified, so that the types of the peptides are complex, the amino acid sequence of the peptide segment is not clear, and the specific peptide has unclear function and insignificant efficacy. At present, no report exists on active peptides which are derived from ginseng, have definite amino acid compositions obtained by separation and purification and have the effects of resisting angiogenesis and inhibiting the growth of lung cancer tumors.
At present, in China, the incidence rate of lung cancer ranks the first of all malignant tumors, and is one of the biggest malignant tumors threatening the life and health of human beings. The traditional treatment means mainly combines operations with radiotherapy and chemotherapy, the period is long, toxic and side effects are large, and many lung cancer patients are found to be in an advanced stage, so the treatment effect is not ideal. 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 formation of new blood vessels is very important for the growth and the metastasis of solid tumors, the new blood vessels can bring rich nutrients for tumor cells, and the tumor cells can also be transferred and spread outwards through the new blood vessels. Moreover, the treatment cost of lung cancer is high, and serious economic burden is brought to the society and families. Therefore, the development of the medicament which has good anti-angiogenesis and anti-tumor effects and is cheap and easy to obtain has very important significance.
Disclosure of Invention
Aiming at the problems, the current situation and the development prospect, the invention obtains the anti-angiogenesis and anti-tumor active peptide by separating and purifying the ginseng oligopeptide, the active peptide has small molecular weight, strong activity, simple preparation process and high purity, is easy to separate and purify, can be used for health care and treatment of lung cancer and other related diseases, and has wide application prospect in the fields of food, health care products, medical biology and the like.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention has the beneficial effects that:
(1) the invention carries out high performance liquid chromatography-mass spectrometry identification on the ginseng oligopeptide in the early stage and utilizes an online database to carry out activity screening to obtain the high-activity small molecular peptide, and on the basis, the active peptide is separated and purified by separation technologies such as Sephadex LH-20 gel column chromatography, high performance liquid chromatography and the like to verify the activity of the active peptide, and finally a high anti-angiogenesis and anti-tumor active peptide is obtained, wherein the amino acid sequence of the active peptide is as follows: Leu-Asp-Asp-Pro-Val-Phe-Arg-Pro, which is a novel small molecule active peptide by on-line database BIOPEP and EROP-Moscow search.
(2) The active peptide has small molecular weight, high activity, simple separation and purification steps, easy obtainment and wide application prospect in the field of medicines and the like, belongs to food-derived active peptide, has the effects of anti-angiogenesis activity and inhibiting the growth of lung cancer tumors, can be applied to the health care and treatment of lung cancer and other related diseases and the development of related products.
(3) 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 Lewis lung cancer mice.
FIG. 3 is a graph showing the angiogenesis inhibition rate 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 derived from ginseng (Panax ginseng C.A. Meyer), the amino acid sequence of the active peptide is Leu-Asp-Asp-Pro-Val-Phe-Arg-Pro.
Example 2
The preparation method of the ginseng-derived anti-angiogenesis and anti-tumor active peptide in the embodiment 1 comprises the following steps:
s1, preparation of ginseng oligopeptide:
1000g of ginseng is crushed by a crusher, water is added for homogenate to prepare homogenate liquid, the homogenate liquid is placed in an enzymolysis tank, and the mass volume ratio of the ginseng to the water is 1: 20, adding cellulase accounting for 0.6 percent of the mass (dry weight basis) of the ginseng, carrying out enzymolysis for 2 hours at 50 ℃, controlling the pH value of the enzyme reaction to be 5.5, and then adding compound protease accounting for 4 percent of the mass of the ginseng protein, wherein the protease mixture ratio is as follows: alkaline protease, bromelain and flavourzyme are subjected to enzymolysis at 52 ℃ for 4 hours, the pH value of the enzyme reaction is controlled to be 9.0, the temperature is raised to 85 ℃ after the enzymolysis is finished, enzyme deactivation is carried out for 10 minutes to obtain ginseng enzymolysis liquid, the ginseng enzymolysis liquid is centrifuged for 10 minutes at 8000 rpm to remove granular substances, then the ginseng oligopeptide powder is obtained by adopting a membrane separation technology for separation, the molecular weight cutoff is 3000Da, and the membrane passing liquid is subjected to spray drying.
S2, separation and purification of small molecule active peptide:
dissolving the ginseng oligopeptide powder in S1 with water, preparing into 100mg/mL, separating and purifying by Sephadex LH-20 column chromatography (3.0 × 100cm), wherein the mobile phase is 35% methanol, the flow rate is 0.5mL/min, the absorbance of the eluent is measured at 280nm, and the required peak is collected according to the absorbance value.
Further purifying by high performance liquid chromatography under the following conditions: elette C18 column (4.6mm × 250mm, 5 μm), mobile phase a 0.07% trifluoroacetic acid (v/v), mobile phase B acetonitrile, gradient elution conditions: 0-15 min, 5% B; 15-20 min, 5% -15% of B; 20-30 min, 15% -25% of B; 30-40 min, 25% B-40% B; the flow rate is 0.8mL/min, the detection wavelength is 280nm, a chromatographic peak with the retention time of 17 minutes is collected, and the micromolecular active peptide is obtained by freeze drying after concentration.
S3, purity and structure 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: Leu-Asp-Asp-Pro-Val-Phe-Arg-Pro with a molecular weight of 958 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 6 groups (control and experimental groups 1, 2, 3, 4, 5) of 10 embryos each, and 20. mu.L of physiological saline and 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL solutions of small molecule active peptides adsorbed onto glass fiber filters were coated onto the CAM 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 small molecule active peptide solutions with different concentrations have the inhibition effect on the CAM blood vessels, wherein the angiogenesis inhibition effect of the small molecule active peptide solution with the concentration of 0.6mg/mL is the best, and the inhibition rate reaches 87.52% +/-1.33%, which indicates that the small molecule active peptide has good anti-angiogenesis activity.
And (3) determination of anti-tumor activity:
construction of mouse Lewis lung cancer model: kunming mice are fasted and water is not forbidden 12 hours before model building. Under the aseptic condition, the well-grown Lewis lung cancer mouse tumor tissue is taken, added with normal saline, prepared into tumor tissue cell suspension according to the proportion of 1:5, and inoculated with 0.2mL of subcutaneous tissue of the left forelimb armpit of the mouse.
Lewis lung carcinoma Kunming mice were randomly divided into 6 groups (model and experimental groups 1, 2, 3, 4, 5) with 10 mice per group 24 hours after inoculation. The mice of the experimental groups 1, 2, 3, 4 and 5 are respectively gavaged with 200 mg/(kg.d), 400 mg/(kg.d), 600 mg/(kg.d), 800 mg/(kg.d) and 1000 mg/(kg.d) micromolecular active peptides, and the mice of the model groups are gavaged with physiological saline with the same amount for continuous intervention for 12 days. Weighing the weight of the mouse by using an electronic analytical balance 12 days later, then killing the mouse by removing the vertebra, picking up the complete tumor tissue, weighing the mass of the tumor tissue of the mouse by using the electronic analytical balance, and finally calculating the tumor inhibition rate.
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 molecular peptide, wherein the tumor inhibition rate of the 1000 mg/(kg. d) dosage group is the highest and reaches 88.05 percent, and the small molecular active peptide has obvious effect of inhibiting the growth of the lung cancer tumor.
Example 3
With the active peptide sequence as the core, any corresponding modifications or modifications are made. Such as the application of corresponding adjustment or modification, including the further anti-angiogenesis drug design of the active peptide: the amino acid Pro at the fourth position and the amino acid Pro at the eighth position can be replaced by the amino acid Leu singly or simultaneously, and the replaced small molecular peptides have potential anti-angiogenesis activity.
The eighth amino acid Pro of the active peptide sequence in the embodiment 2 is replaced by Leu to obtain a new active peptide segment, and the amino acid sequence is Leu-Asp-Asp-Pro-Val-Phe-Arg-Leu.
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 6 groups (control and experimental groups 1, 2, 3, 4, 5) of 10 embryos each, and 20. mu.L of physiological saline and 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL solutions of small molecule active peptides adsorbed onto glass fiber filters were coated onto the CAM 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 good anti-angiogenesis activity, wherein the inhibition effect of the small molecule active peptide of 0.8mg/mL on angiogenesis is the best, and the inhibition rate reaches 83.66% +/-4.01%.
Example 4
The steps of the preparation method of the ginseng-derived anti-angiogenesis and anti-tumor active peptide in the embodiment are the same as those in the embodiment 2, and the differences are as follows:
(1) in step S1, 1000g of ginseng sample is pulverized, and then 17 times the mass volume of water is added to prepare homogenate and the homogenate is placed in an enzymolysis tank, cellulase accounting for 1.0% of the mass (by dry weight) of ginseng is added, enzymolysis is carried out at 48 ℃ for 1 hour, the pH value of the enzymatic reaction is controlled at 6.0, and then composite protease accounting for 3% of the mass of ginseng protein is added, and the ratio of the protease is as follows: alkaline protease, bromelain and flavourzyme are subjected to enzymolysis at 50 ℃ for 4 hours, the pH value of the enzymatic reaction is controlled at 9.5, the temperature is increased to 80 ℃ after the enzymolysis is finished, the enzyme is inactivated for 15 minutes to obtain ginseng enzymolysis liquid, the ginseng enzymolysis liquid is centrifuged for 8 minutes at 7000 rpm to remove granular substances, then the ginseng oligopeptide powder is obtained by adopting a membrane separation technology for separation, the molecular weight cutoff is 3000Da, and the membrane passing liquid is subjected to spray drying.
(2) In the step S2, the mobile phase is 35% methanol, and the flow rate is 0.4 mL/min;
(3) in step S2, mobile phase A is trifluoroacetic acid water containing 0.05% by volume percentage, and the flow rate is 0.9 mL/min.
Example 5
The steps of the method for separating and purifying the anti-angiogenesis and anti-tumor active peptide from the ginseng in the embodiment are the same as those in the embodiment 2, and the difference is as follows:
(1) in step S1, 1000g of ginseng sample is pulverized, then 15 times the mass volume of water is added to prepare homogenate, the homogenate is placed in an enzymolysis tank, cellulase 0.8% of the ginseng mass (by dry weight) is added, enzymolysis is carried out at 45 ℃ for 1.5 hours, the enzymatic reaction pH is controlled at 5.0, and then composite protease 5% of the ginseng protein mass is added, the protease ratio is: alkaline protease, bromelain and flavourzyme are 6:4:3, enzymolysis is carried out at 50 ℃ for 3 hours, the pH value of the enzyme reaction is controlled at 9.7, the temperature is increased to 90 ℃ after the enzymolysis is finished, enzyme deactivation is carried out for 10 minutes, ginseng enzymolysis liquid is obtained, the ginseng enzymolysis liquid is centrifuged at 6000 rpm for 10 minutes, granular substances are removed, then separation is carried out by adopting a membrane separation technology, the molecular weight cutoff is 3000Da, and the ginseng oligopeptide powder is obtained by spraying and drying membrane passing liquid.
(2) In the step S2, the mobile phase is 35% methanol, and the flow rate is 0.6 mL/min;
(3) in step S2, the mobile phase A is trifluoroacetic acid water with volume percentage of 0.06%, and the flow rate is 1.0 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 able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Sequence listing
<110> Dalian deep blue peptide research & development Co Ltd
<120> anti-angiogenesis and anti-tumor active peptide derived from ginseng, and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Ginseng radix (Panax ginseng C.A. Meyer)
<400> 1
Leu Asp Asp Pro Val Phe Arg Pro
1 5
<210> 2
<211> 8
<212> PRT
<213> Ginseng radix (Panax ginseng C.A. Meyer)
<400> 2
Leu Asp Asp Pro Val Phe Arg Leu
1 5
Claims (8)
1. An anti-angiogenesis and anti-tumor active peptide derived from ginseng is characterized in that the amino acid sequence of the active peptide is Leu-Asp-Asp-Pro-Val-Phe-Arg-Pro.
2. A process for the preparation of an active peptide according to claim 1, comprising the steps of:
s1, preparing ginseng oligopeptide;
s2, separating and purifying the ginseng oligopeptide into small molecule active peptide.
3. The method for preparing active peptide according to claim 2, wherein the method for preparing ginseng oligopeptide in step S1 comprises: after the ginseng sample is subjected to coarse grinding treatment, adding 15-20 times of water by mass volume to prepare homogenate, placing the homogenate in an enzymolysis tank, adding cellulase accounting for 0.5-1.0% of the ginseng mass 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; adding compound protease with the mass of 3-5% of ginseng protein into an enzymolysis tank, carrying out enzymolysis for 3-4 hours at 50-55 ℃, controlling the pH value of the enzyme reaction to be 9.0-10.0, wherein the mass ratio of the compound protease is as follows: alkaline protease (bromelain) and flavourzyme (4-6): 2-4); and after enzymolysis, heating to 80-90 ℃ to inactivate enzyme for 10-15 minutes to obtain ginseng enzymolysis liquid, centrifuging the ginseng enzymolysis liquid for 8-10 minutes at 6000-8000 rpm, removing granular substances, separating clear liquid by adopting a membrane with the molecular weight cutoff of 3000Da, and performing spray drying on the membrane passing liquid to obtain ginseng oligopeptide powder.
4. The method for preparing active peptide according to claim 3, wherein step S2 is a method for separating and purifying ginseng oligopeptide into small molecule active peptide: dissolving ginseng 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 35% methanol, the flow rate is 0.4-0.6 mL/min, the absorbance of an eluent is measured at 280nm, and a required peak is collected according to the absorbance value; purifying by high performance liquid chromatography, collecting chromatographic peak with retention time of 17 min, concentrating, and freeze drying to obtain small molecule active peptide; the chromatographic conditions were as follows: c18 chromatographic column, mobile phase A is trifluoroacetic acid water with volume percentage of 0.05% -0.08%, mobile phase B is acetonitrile, gradient elution conditions are as follows: 0-15 min, 5% B; 15-20 min, 5% -15% of B; 20-30 min, 15% -25% of B; 30-40 min, 25% B-40% B; the flow rate is 0.8-1.0 mL/min, and the detection wavelength is 280 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: Leu-Asp-Asp-Pro-Val-Phe-Arg-Pro with a molecular weight of 958 Da.
6. 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.
7. Use of the active peptide of claim 1 for anti-angiogenesis or inhibiting the growth of a lung cancer tumor.
8. Use of the active peptide of claim 1 for the manufacture of a medicament for the treatment of angiogenesis or lung cancer.
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