CN106632633B

CN106632633B - Abelmoschus moschatus antitumor oligopeptide and application thereof

Info

Publication number: CN106632633B
Application number: CN201611045737.0A
Authority: CN
Inventors: 丁国芳; 吴宗泽; 杨最素; 贾盈露; 郑媛媛; 陈锐; 余方苗
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2016-11-24
Filing date: 2016-11-24
Publication date: 2020-03-20
Anticipated expiration: 2036-11-24
Also published as: CN106632633A

Abstract

The invention discloses an actinia viridis anti-tumor oligopeptide and application thereof, wherein the amino acid sequence of the actinia viridis anti-tumor oligopeptide is as follows: Tyr-Val-Pro-Gly-Pro. The invention can improve the human body immunity, has obvious proliferation inhibition effect on the prostate cancer cell DU-145, and has better medical application value.

Description

Abelmoschus moschatus antitumor oligopeptide and application thereof

Technical Field

The invention relates to a marine active substance, in particular to an actinia viridis anti-tumor oligopeptide and application thereof.

Background

Sea anemone (Sea anerone) belongs to the original marine organism, an order of the subclass of the sikaempferia, has thirty-seven species in six families, is widely distributed and is commonly found in tropical and temperate submarine rocks and sediments. At present, scientists in various countries make a great deal of research on the activity of potassium ion channels and sodium ion channels of actinia okra toxin; in addition, many studies on blood pressure reduction, antifungal and antitumor properties have been reported. However, the research on the physiology and pathology of the actinocongestin is severely restricted due to the extremely low content and difficult extraction of the actinocongestin. The research development of the marine bioactive peptide is referred to, the preparation of the bioactive peptide by an enzymolysis method is found to be a new research hotspot, and the extracted bioactive peptide not only can supplement nutrient components of organisms, but also has activities of oxidation resistance, anticoagulation, blood pressure reduction, tumor resistance and the like.

Prostate cancer (Prostate cancer PCa) is a disease which is difficult to be found in early stage and is at high risk, and compared with European and American countries, the incidence rate of Prostate cancer in China is much lower, but in recent years, due to unbalanced diet and environmental pollution, the incidence rate of Prostate cancer also has a remarkable rising trend, and becomes an important factor threatening the health of men in China. The development of prostate cancer is associated with the loss of regulation of the host immune system against the tumor. If timely and effective treatment is not available, androgen-independent prostate cancer will develop, and advanced prostate cancer plus bone metastasis will cause great difficulty in treatment. Therefore, it is important to find active substances that can enhance the immunity of human body and effectively induce cancer cell apoptosis.

Disclosure of Invention

The invention aims to provide an actinia viridis anti-tumor oligopeptide, which can improve the human body immunity, has an obvious proliferation inhibition effect on prostate cancer cells DU-145, and has a good medical application value.

The invention also provides application of the malva virginiana anti-tumor oligopeptide.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an actinia viridis antitumor oligopeptide, the amino acid sequence of which is as follows: Tyr-Val-Pro-Gly-Pro. The invention extracts active peptide from malva virginiana meat, and carries out separation and purification, and reports that the extracted oligopeptide has the effect of resisting prostate cancer for the first time.

Tyr: tyrosine (Y), Val: valine (V), Pro: proline (P), Gly: glycine (G). An application of an actinia viridis antitumor oligopeptide, an application of the actinia viridis antitumor oligopeptide as a raw material for preparing an antitumor drug.

Preferably, the sea anemone viridis anti-tumor oligopeptide is used as a raw material for preparing the anti-prostate cancer medicine. Application of an actinia viridis anti-tumor oligopeptide to preparation of health food for resisting prostate cancer.

The invention has the beneficial effects that: not only can improve the immunocompetence of a human body, but also has obvious proliferation inhibition effect on prostate cancer cells DU-145, and has better medical application value.

Drawings

FIG. 1 shows the inhibition effect of four different enzymatic products on cell proliferation of DU 145.

FIG. 2 inhibition of cell proliferation of DU-145 by different molecular weight products.

FIG. 3 is a DEAE-Sepharose Fast Flow rapid anion exchange chromatography graph of AAP-I.

FIG. 4 inhibition of DU-145 cell proliferation by three peak products after anion exchange column elution.

FIG. 5 AAP-I-1 is a graph obtained by chromatography on Sephadex G-25 column.

FIGS. 6G-25 are graphs showing the cell proliferation activity of DU-145, which is the product of each elution peak.

FIG. 7C 18-HPLC chart of AAP-I-1-2.

FIG. 8C 18-HPLC picture of AAP-H.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Example (b):

1. material

1.1 starting materials

Malva virginiana: wild sea anemone in Zhoushan sea area was collected and identified as a green-sided flower sea anemone (Anthopleura anjunae) by professor Zhaoshenglong, university of oceanic, Zhejiang.

1.2 reagents and instruments

Alkaline protease, trypsin, neutral protease, pepsin: Asia-Pacific Biotechnology Ltd; methanol, acetonitrile: chromatographically pure, merck, germany; polybrene: shanghai Bopu Biotechnology, Inc.; f12 medium: gibco, USA; mycoplasma-free Fetal Bovine Serum (FBS): hangzhou Sijiqing biological products Co. Human prostate cancer cell DU-145 was purchased from Shanghai institute of cell biology, Chinese academy of sciences.

CF16RN model high speed refrigerated centrifuge, L8900 amino acid autoanalyzer: hitachi instruments, Inc.; agilent-1260 type high performance liquid chromatograph: agilent, USA; ALPHA1-4/LDplus ultrafiltration system: merckmix, germany; forma 3111 type CO₂An incubator: thermo corporation, usa; and (3) inverting the microscope: OlyMPUS, Japan; model WRO-70 ultrapure water machine: hangzhou Wan Jieji Water treatment facilities Corp; an enzyme-labeling instrument: BioRad technologies, USA.

2. Method of producing a composite material

2.1 pretreatment of the Material

Fresh wild malva virginiana is taken and placed in clean seawater at room temperature for feeding for one day, and is cleaned to remove impurities. Cutting open with scissors from the middle, placing in a big beaker, covering with water, freezing in a refrigerator at-20 deg.C for 12h, naturally thawing at room temperature, repeating for three times, squeezing out venom, and washing with tap water to obtain white sea anemone meat. Homogenizing with a high speed homogenizer (10000r/min, 5min), degreasing by soaking in isopropanol, changing every 4h for 3 times, washing with pure water after degreasing, air drying in a fume hood until no water drops, packaging, and storing at-20 deg.C.

2.2 measurement of related indexes of Hibiscus moschatus

2.2.1 base ingredient determination

Measuring moisture with reference to GB 5009.3-2010; measuring ash content by reference to GB/T5009.4-2010; measuring the content of crude protein by referring to GB 50095-2010; the crude fat content of sea anemone meat is determined by reference to GB/T5009.6-2003.

2.2.2 analysis of amino acid composition and content of Hibiscus Lobatanus protein

With reference to GB/T5009.124-2003, sea anemone meat homogenate samples are weighed, hydrolyzed by 6mol/L hydrochloric acid, and then the composition and the content of amino acid are measured by a Hitachi L8900 amino acid automatic analyzer.

2.3 screening of enzyme species

2.3.1 enzymatic hydrolysis

Respectively taking 10g of pretreated sea anemone meat homogenate, adding 50mL of purified water, and adjusting the enzyme addition amount to 1000u/g according to the optimal pH and temperature on various enzyme kits, and carrying out enzymolysis under the conditions shown in Table 1. Before enzymolysis, 0.5mol/L sodium hydroxide and 0.5mol/L hydrochloric acid are used for adjusting to the required pH value, a sample is placed in a constant-temperature water bath for enzymolysis, and the mixture is slowly stirred at a constant speed during enzymolysis. Boiling and inactivating in a water bath for 15min after enzymolysis is finished, filtering with three layers of gauze to remove coarse impurities, centrifuging the filtrate for 10min at 4 ℃ and 12000rpm, and continuously centrifuging twice. Collecting supernatant, filtering with 0.45 μm needle filter membrane, adjusting pH to 7, rotary steaming, concentrating, freeze drying, and packaging at-20 deg.C.

TABLE 1 conditions of the enzymatic hydrolysis

2.3.2 MTT assay of products for inhibition of DU-145 cell proliferation

Cell culture: prostate cancer cell DU-145 was cultured in F12 medium containing 10% bovine serum, 5% CO at 37 deg.C₂Culturing in a cell culture box, and carrying out passage or experiment when the cells grow to 70-80% confluence degree by adherence.

DU-145 cells were seeded in 96-well plates. And setting a control group and a drug group, adding culture solution containing an enzymatic hydrolysate with the concentration of 5mg/mL into each well of the drug group, setting 3 multiple wells in each group, culturing for 24 hours, adding 200uL PBS buffer solution containing 10% MTT, and incubating for 4 hours. Absorbing MTT culture solution, adding DMSO for oscillation reaction, detecting an absorbance A value by an enzyme-labeling instrument, and calculating an Inhibition Rate (IR), wherein the calculation formula is as follows: IR ═ [ (control a value-drug a value)/control a value ] × 100%.

2.4 orthogonal experiment optimized preparation process of anti-prostate cancer oligopeptide from malva virginiana

After determination of the optimal protease by 2.3, as L₁₆(4⁵) Orthogonal experiment optimizes preparation process of anti-prostate cancer oligopeptide from Malabar virginiana to determine optimal temperature, time, pH, enzyme addition amount and feed-liquid ratio, and the product is concentrated, freeze-dried and detected by MTT methodThe factors and levels of the orthogonal experiment for DU-145 proliferation inhibitory activity are shown in Table 2.

TABLE 2 factors and levels of enzymatic conditions in orthogonal experiments

2.5 enzymatic hydrolysate Ultrafiltration

Performing enzymolysis according to the optimal enzymolysis condition determined by 2.4 to prepare the anemonia viridis antitumor Peptide (Anthopleura anjunae Anti-tumor Peptide- - -AAP), classifying with ALPHA1-4/LDplus ultrafiltration system 8KD and 20KD filter membrane, and naming AAP-I (MW < 8KD), AAP-II (8KD < MW < 20KD) and AAP-III (MW > 20KD) according to the molecular weight from small to large. MTT method 2.3 screening the most active fraction.

2.6 DEAE-Sepharose Fast Flow anion exchange chromatography

Activating and balancing a exchange column (2cm multiplied by 35cm) in advance, carrying out ultrasonic treatment for 5min on a solution with the sample concentration of 30mg/mL, then filtering the solution by using a 0.45 mu m needle type filter membrane, loading 3mL of the solution each time, carrying out gradient elution by using purified water, 0.1mol/L, 0.3mol/L, 0.5mol/L and 0.8mol/L NaCl solutions respectively, eluting the gradients respectively for 30min at the flow rate of 1mL/min, collecting one tube every 4min by using a DBS-100-LCD computer full-automatic partial collector, detecting the absorbance at 280nm, merging elution peaks according to an absorbance curve, and finding three peaks. The product was concentrated, freeze-dried, and named AAP-I-1, AAP-I-2, AAP-I-3. Subpackaging and storing in a refrigerator at-20 deg.C for use. MTT method 2.3 screening the most active fraction.

2.7 SephadexG25 gel chromatography separation and purification

Continuously separating and purifying AAP-I-1 with the highest DU-145 proliferation inhibition rate by G-25 gel chromatography, preparing a sample into a 30mg/mL solution, carrying out ultrasonic treatment for 5min, then filtering by using a 0.45 mu m needle filter membrane, adding 3mL of the solution into a treated Sephadex G-25 chromatographic column (2cm multiplied by 50cm) each time, eluting by using ultrapure water at the flow rate of 1.0mL/min, collecting one tube by using a DBS-100-LCD computer full-automatic partial collector every 4min, detecting the absorbance at 280nm, and merging elution peaks according to an absorbance curve to find three peaks. The product was concentrated, freeze-dried and named AAP-I-1-1, AAP-I-1-2, AAP-I-1-3. MTT method 2.3 screening the most active fraction.

2.8 reversed-phase high performance liquid chromatography separation and purification

AAP-I-1-2 with the highest inhibition rate on DU-145 proliferation is prepared into 5mg/mL solution, and the solution is separated and purified by reversed phase high performance liquid chromatography after being filtered by a 0.22 mu m needle type filter membrane. Before the sample introduction, Agilent 1260 ZORBAX SB-C18(9.4mm multiplied by 250mm) column is balanced, and the column temperature is 30 ℃; in a laboratory, samples are injected into a sample by 100 mu L at each time according to 300 mu g/mL, eluent is eluted by acetonitrile ultrapure water mixed liquid according to 1mL/min, and acetonitrile (20 percent) is eluted for 0min to 15min to (50 percent), and the sample injection time is 15min to 30 min: acetonitrile (50%); the detection wavelength was 280 nm. The pure product adopts a PPSQ-31A protein sequencer to determine the amino acid sequence of the peptide chain.

2.9 statistical analysis

The experimental results were analyzed using SPASS19.0 statistical software and

and (4) showing.

3 results and analysis

3.1 measurement results of basic Components of Hibiscus Loranthus

The basic ingredients of the malva virginiana are shown in table 3, and as can be seen from table 3, the malva virginiana meat is rich in crude protein accounting for 19.76%, and low in crude fat content of only 0.89%, indicating that the malva virginiana is a high-protein low-fat marine product.

TABLE 3 Lonicera viridissima basis (g/100g, wet weight)

The amino acid composition and content of malva virginiana are shown in table 4. As can be seen from Table 4, the amino acid types of the malva verticillata are relatively complete, and the necessary amino acid content accounts for 24.31 percent of the total amino acid content; the content of umami amino acids (aspartic acid, glutamic acid, glycine and alanine) in the total amino acids is up to 50.58%, which is one of the reasons for the delicious taste of the malva virgata. According to Table 5, the ratio of the essential amino acids of malva virginiana is not very satisfactory for the amino acid balance of the human body, according to the FAO/WHO criteria for amino acid evaluation. However, it has been found that hydrophilic polypeptides (containing hydrophilic amino acids such as Asp, Thr, Ser, Glu, Arg, Lys, His, and gin) can specifically act on tumor cells by electrostatic attraction, leading to rapid rupture of their cell membranes and leakage of cell contents, which ultimately leads to death of the tumor cells. The content of hydrophilic amino acid in the enzymolysis product of the malva virginiana meat accounts for 49.3% of the total amino acid content, and the high proportion of the hydrophilic amino acid is probably related to the function of the enzymolysis product. In conclusion, the sea anemone enzymolysis product can be used for developing anti-tumor related medicines, foods or food additives.

TABLE 4 amino acid composition and content of malva virginiana (g/100g, dry weight)

Amino acid	Content	Amino acid	Content
				Aspartic acid Asp^＊	7.067	Tyrosine Tyr	1.004
Thr threonine^#	2.700	Phenylalanine Phe^#	1.643
				Serine Ser	3.198	Lysine Lys^#	3.099
Glutamic acid Glu^＊	8.988	Histidine His	0.675
				Glycine Gly^＊	13.084	Arginine Arg	6.693
Alanine Ala^＊	4.111	Proline Pro	4.649
				Cystine Cys	0.263	Total amino acids TAA	65.74
Val of valine^#	2.572	Umami amino acid FTAA	33.25
				Methionine Met^#	1.176	Essential amino acid EAA	16.01
Ile of isoleucine^#	1.868	F/T(％)	50.58％
				Leucine Leu^#	2.951	E/T(％)	24.35％

Note that "＊" indicates umami amino acids and "#" indicates essential amino acids.

TABLE 5 nutritional evaluation of actinia viridis protein

3.2 results of MTT Activity test on four different proteolysis products

As shown in FIG. 1, the inhibition rates of the enzymatic products of pepsin, trypsin, neutral protease and alkaline protease on the proliferation of DU-145 cells were 24.98 + -6.04%, 28.55 + -5.29%, 31.66 + -4.04% and 73.7 + -4.31%, respectively, and the inhibition activities on the proliferation of DU-145 cells were in descending order: alkaline protease, neutral protease, trypsin and pepsin, so the alkaline protease is the required enzyme.

3.3 Alcalase Quadrature Experimental results

Orthogonal experiment design is adopted, 5 factors including feed-liquid ratio, temperature, pH value, enzyme adding amount and time are used, and indexes are only referenced by IR values, so that the proliferation inhibition activity of the product of Alcalase protease on prostate cancer cell DU-145 under different conditions is examined. The results are shown in Table 6:

TABLE 6 orthogonal experimental results of alkaline protease enzymolysis method

When the results of the above table analysis by the range analysis method are obtained, it can be seen from the Rj value that A (feed-liquid ratio) > E (time) > D (temperature) > C (enzyme addition) > B (pH value). And the hydrolysis condition when the IR value is maximum is A4B4C3D1E3, namely the feed-liquid ratio is 1:5, the pH is 11, the enzyme adding amount is 2000U/g, the temperature is 35 ℃, and the time is 6 hours.

3.4 Primary isolation of anti-prostate cancer oligopeptides from Malva virgata

After the alkaline protease enzymolysis liquid is ultrafiltered and primarily separated, three components of AAP-I, AAP-II and AAP-III are subjected to MTT method activity screening, as shown in figure 2: the proliferation inhibition rates of the three components on DU-145 cells are 87.18 +/-1.41%, 74.36 +/-2.30% and 83.05 +/-2.03%, respectively. The component AAP-I is shown to have the best inhibition effect on the proliferation of DU-145, so the AAP-I is subjected to anion exchange chromatography.

3.5 DEAE-Sepharose Fast Flow anion exchange chromatography results

Subjecting the AAP-I obtained by ultrafiltration to DEAE-Sepharose Fast Flow anion exchange chromatography to obtain three peak products, which are respectively named as AAP-I-1, AAP-I-2, and AAP-I-3, as shown in FIG. 3.

The peak products were concentrated, lyophilized and examined for their proliferation inhibitory activity against DU-145 by MTT method. FIG. 4 shows that, at a concentration of 5mg/mL, the inhibition rates of the products of three peaks, AAP-I-1, AAP-I-2 and AAP-I-3, on the proliferation of DU-145, are respectively: 37.12 +/-1.70%, 34.78 +/-2.20% and 24.21 +/-1.6%, and the AAP-I-1 is further separated and purified to prove that the inhibition rate of AAP-I-1 on the cell proliferation of DU-145 is the highest.

3.6 Sephadex G-25 gel chromatography separation results

Separating AAP-I-1 separated in 2.5 by Sephadex G-25 gel chromatography to obtain three peaks shown in figure 5, respectively named as AAP-I-1-1, AAP-I-1-2 and AAP-I-1-3, collecting, freeze drying, and storing in refrigerator at-20 deg.C.

In the diagram of each peak of Sephadex G-25 gel chromatography, the proliferation activity of DU-145 is detected by three peak products by adopting an MTT method, and the result of a graph in FIG. 6 shows that the proliferation inhibition rates of the three peak products to DU-145 are respectively 5mg/mL and 24 h: 30.01 + -2.16%, 46.14 + -2.29%, 40.53 + -1.76%, indicating that SAAP-I-1-2 inhibits the proliferation of DU-145 best.

3.7 results of high performance liquid separation of AAP-I-1-2

The result of the AAP-I-1-2 purified by HPLC is shown in figure 7, the main peak is collected, concentrated, freeze-dried and named as AAP-H, MTT activity test is carried out, the result shows that the DU-145 proliferation inhibition rate is 49.14 +/-2.29%, the main peak AAP-H is the main active substance of AAP-I-1-2, the AAP-H is detected by high performance liquid phase to be basically a single sample, the purity is more than 95%, the determination requirement of amino acid sequence is met, and the AAP-H high performance liquid phase detection is shown in figure 8.

3.8 amino acid sequence determination results

The amino acid sequencing results are: Tyr-Val-Pro-Gly-Pro (SEQ ID No: 1).

4 conclusion

The alkaline protease is determined to be the best enzyme species through screening, and the best process conditions for extracting the sea anemone antitumor peptide by adopting an orthogonal experiment to further optimize an enzymolysis method are that enzymolysis is carried out for 6 hours at 35 ℃ according to the material-liquid ratio of 1:5(W/V), the pH value of 11 and the enzyme adding amount of 2000 u/g. Separating and purifying by ultrafiltration, anion exchange chromatography, G-25 gel chromatography, reverse high performance liquid chromatography and other methods, and finally purifying by taking the proliferation inhibition rate of the product on DU-145 as an index to obtain the peptide chain with the sequence Tyr-Val-Pro-Gly-Pro.

There are three more common methods currently used to prepare bioactive peptides: firstly, preparing bioactive peptide by a chemical synthesis method; secondly, directly separating and extracting organisms or tissues serving as raw materials by various separation means; and thirdly, degrading biological tissues or macromolecular products by an enzymolysis method to prepare the bioactive peptide. The protease enzymolysis method for producing bioactive peptides has the advantages of high safety, mild reaction conditions and relatively high yield, so the method becomes a research hotspot for preparing bioactive peptides in recent years. Because the growth environment of marine organisms is unique to that of terrestrial organisms, and some marine organisms even grow in the environments of extremely high pressure or extremely low temperature and the like, the marine organisms form active peptides with unique structures and novel functions in the growth process. The active peptide partially exists in a natural state, partially serves as a structural domain of protein macromolecules, a peptide chain with better activity than that of the protein can be obtained by shearing protease at a specific point position, and the bioactive peptides play important roles in regulating an immune system, resisting bacteria, thrombus and high blood pressure, regulating gastrointestinal tract movement, scavenging free radicals, resisting viruses, promoting mineral element absorption and resisting cancers. The existing research shows that: the biological activity of the peptide chain is mainly influenced by the amino acid components and the molecular weight of the peptide chain, and the active peptide has stronger biological activity than protein, polypeptide and simple amino acid. In addition, aromatic amino acids or hydrophobic amino acids such as Trp, Tyr, Met, Gly, Lys, His and Pro in the peptide chain can well enhance the biological activity of the peptide chain. The peptide chain prepared by the invention contains Tyr, Pro and Gly, and the structure is considered to be one of important reasons for the anti-tumor activity of the peptide chain. In summary, the sea anemone green-side meat is a natural resource with high protein content, and the peptide prepared by the enzymolysis method can be used for developing the drugs related to the prostate cancer resistance or being developed into health-care food.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

SEQUENCE LISTING

<110> Zhejiang ocean university

<120> Abelmoschus moschatus anti-tumor oligopeptide and application thereof

<130>2016.11

<160>1

<170>PatentIn version 3.3

<210>1

<211>5

<212>PRT

<213> Malabar virginiana

<400>1

Tyr Val Pro Gly Pro

1 5

Claims

1. The application of the anemonia viridis anti-tumor oligopeptide as a raw material in the preparation of the anti-prostate cancer medicine is characterized in that the amino acid sequence of the anemonia viridis anti-tumor oligopeptide is as follows: Tyr-Val-Pro-Gly-Pro.