CN106560518A - Preparing method of authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides - Google Patents
Preparing method of authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides Download PDFInfo
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Abstract
The invention discloses a preparing method of authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides. The preparing method comprises the steps of preprocessing of a sample, enzymolysis, ultrafiltration, anion exchange chromatography, Sephade*G25 gel chromatography, separation and purification of reversed-phase high-performance liquid chromatography, and the final obtaining of the authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides. According to the authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides, the safety is good, the reaction condition is mild, process control is easy, the purity of the obtained authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides is high, the authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides have obvious anti-proliferative effects on prostate cancer cell DU-145, and the authopleura midori uchida muramatsu resisting prostatic cancer oligopeptides have good medical application value.
Description
Technical field
The present invention relates to marine active substance extractive technique field, more particularly to a kind of green Anthopleura anti-prostate cancer widow
The preparation method of peptide.
Background technology
Sea anemone (Sea anemone) belongs to primitive ocean biology, and a mesh of Zoantharia has 37 kinds of six section,
It is widely distributed, it is common in the torrid zone and temperate zone marine rock and silt.At present, various countries scientist is to actinocongestin potassium-channel
Substantial amounts of research has been done with sodium-ion channel activity；In addition, the research of its blood pressure lowering, antifungal and antineoplastic also has a large amount of reports
Road.But as actinocongestin content is few, it is difficult to extract, seriously restrict the research in terms of its physiology and pathology.With reference to
The progress discovery enzymatic isolation method of oceanic biological active peptides prepares biologically active peptide and has become new study hotspot, extracts
To bioactive peptide can supplement body nutritional labeling, again have antioxidation, anticoagulation, blood pressure lowering, antitumor isoreactivity.
Carcinoma of prostate (Prostate cancer PCa) be a kind of early stage difficulty be found, high risk sexual disease, contrast Europe
Man of the U.S., current China's prostate-cancer incidence are much lower, but in recent years as people's diet is unbalanced and environmental pollution,
Its sickness rate also has obvious ascendant trend, becomes the key factor for threatening China's men's health.The generation development of carcinoma of prostate
Regulation and control are lost to tumor with host immune system relevant.If timely and effectively cannot treat, will to develop into androgen non-
Dependency carcinoma of prostate, carcinoma of prostate late period will bring greatly difficulty to treatment plus Bone tumour.Therefore, searching can be improved
Human body immunity again can the active matter of effectively cancer cell specific induction of apoptosis just seem and be even more important.
The content of the invention
It is an object of the invention to provide a kind of preparation method of green Anthopleura anti-prostate cancer oligopeptide, safety is good,
Reaction condition is gentle, and easily, the green Anthopleura anti-prostate cancer oligopeptide purity of gained is high, to prostate gland cancer cell for process control
DU-145 has obvious inhibited proliferation, is worth with preferable medical application.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of preparation method of green Anthopleura anti-prostate cancer oligopeptide, step are as follows：
(1) sample pretreatment：The green Anthopleura of fresh wild obtains sea anemone meat slurry after pretreatment；
(2) digest：According to every gram of sea anemone meat slurry plus the proportioning of 4-5mL pure water, sea anemone meat slurry and pure water are mixed, is adjusted
Section pH=10-11, adds alkaline protease, 35-45 DEG C of enzymolysis 4-6 according to the enzyme concentration of every gram of sea anemone meat slurry plus 2000-2500U
Hour obtains enzymolysis solution；
(3) ultrafiltration：Enzymolysis solution is inactivated, centrifugation, takes supernatant, and Jing ultrafiltration obtains active component of the molecular weight less than 8kd；
(4) anion-exchange chromatography：Molecular weight is cloudy less than the activearm lease making DEAE-Sepharose Fast Flow of 8kd
Ion-exchange chromatography, eluting detects absorbance in 280nm, and merges eluting peak according to absorbance curve, and what collection was obtained is each
Eluting peak product lyophilization, mtt assay are screened to prostate gland cancer cell DU-145 proliferation inhibition rate highest anion exchange layers
Division thing；
(5) SephadexG25 gel chromatographies：Prostate gland cancer cell DU-145 proliferation inhibition rate highests anion will be handed over
Change chromatographed product to continue to isolate and purify using G-25 gel chromatographies, ultrapure water elution, absorbance is detected in 280nm, and according to suction
Luminosity curve merges eluting peak, and each eluting peak product lyophilization obtained to collection, mtt assay are screened to prostate gland cancer cell
DU-145 proliferation inhibition rate highest Sephadex G25 gel chromatography products；
(6) reversed-phase high-performance liquid chromatography is isolated and purified：To prostate gland cancer cell DU-145 proliferation inhibition rate highests
Sephadex G25 gel chromatography products are isolated and purified with reversed-phase high-performance liquid chromatography Jing after membrane filtration, finally obtain green side flower
Sea anemone anti-prostate cancer oligopeptide.
The present invention is extracted from green Anthopleura meat and obtains bioactive peptide, and isolated and purified, and reported first is carried
There is the oligopeptide for taking anti-prostate cancer to act on.
Preferably, step (1) sample pretreatment is specially：Take the green Anthopleura of fresh wild and be placed in the clean sea water of room temperature
Middle to raise one day, cleaning removes impurity, is cut open from centre with shears, is placed in beaker, and the covering that adds water, and is placed in -20 DEG C of refrigerators
Middle freezing 12h, then be placed in room temperature naturally to thaw, repeatedly for three times, extrudes tentacle venom, is then rinsed to obtaining white with tap water
Sea anemone meat, white sea anemone meat are homogenized, and homogenate is soaked in degreasing in isopropanol, are changed once per 4h, are continuously changed 3 times, go
Isopropanol is rinsed well after terminating by fat with pure water, air-dries to without water droplet and obtain sea anemone meat slurry in being subsequently placed in fume hood wind.
Preferably, step (4) is respectively with pure water, 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.8mol/L
NaCl solution gradient elution, the elution speed of each gradient is 1mL/min.
Preferably, the elution speed of the ultra-pure water of step (5) is 1mL/min.
The invention has the beneficial effects as follows：Safety is good, and reaction condition is gentle, and process control is easy, the green Anthopleura of gained
Anti-prostate cancer oligopeptide purity is high, has obvious inhibited proliferation to prostate gland cancer cell DU-145, with preferable medical science
Using value.
Description of the drawings
The different enzymatic hydrolysate of tetra- kinds of Fig. 1 are to DU145 cell inhibitory effect effects.
Fig. 2 different molecular weights product is to DU-145 cell inhibitory effect.
The quick anion-exchange chromatography curve charts of DEAE-Sepharose Fast Flow of Fig. 3 AAP-I.
After Fig. 4 anion-exchange column eluting, three peak products are to DU-145 cell inhibitory effect.
- 25 column chromatography diagram of Fig. 5 AAP-I-1 Jing sephadex Gs.
The each eluting peak product DU-145 cell-proliferation activity schematic diagrams of Fig. 6 G-25.
The C18-HPLC figures of Fig. 7 AAP-I-1-2.
The C18-HPLC figures of Fig. 8 AAP-H.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, the raw material for being adopted and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments, if no special instructions, is the conventional method of this area.
Embodiment：
1st, material
1.1 raw material
Green Anthopleura：The wild sea anemone of collection Zhoushan sea area, Jing Zhejiang Oceans university professor Zhao Shenglong are accredited as green side flower
Sea anemone (Anthopleura anjunae).
1.2 reagents and instrument
Alkaline protease, trypsin, neutral protease, pepsin：Asia-Pacific Heng Xin bio tech ltd；First
Alcohol, acetonitrile：Chromatographically pure, Merck KGaA company；Polybrene：Shanghai Bo Pu Bioisystech Co., Ltd；F12 culture medium：The U.S.
Gibco companies；Without mycoplasma hyclone (FBS)：Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological product company limited.Human Prostate Cancer Cells DU-
145 are purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences.
CF16RN type High speed refrigerated centrifuges, L8900 automatic amino acid analyzers：Instrument Ltd. of Hitachi；
Agilent-1260 type high performance liquid chromatographs：Agilent company of the U.S.；ALPHA1-4/LDplus ultrafiltration systems：Merck KGaA
Millipore Corp.；3111 types CO of Forma2Incubator：Thermo companies of the U.S.；Inverted microscope：Japanese OLYMPUS companies；
WRO-70 type ultrapure water machines：Ten thousand clean water treatment facilities company of Hangzhou；Microplate reader：BioRad scientific ＆ technical corporation of the U.S..
2nd, method
2.1 pretreatment
Take the green Anthopleura of fresh wild and be placed in raising one day in the clean sea water of room temperature, cleaning removes impurity.With shears from
Centre is cut open, is placed in large beaker, and the covering that adds water, and is placed in -20 DEG C of refrigerators and freezes 12h, then be placed in room temperature naturally to thaw, instead
Multiple three times, tentacle venom is extruded, then with originally rinsing to obtaining white sea anemone meat.The homogenate of high-speed homogenization machine (10000r/min,
5min), homogenate is soaked in into degreasing in isopropanol, changes once per 4h, continuously change 3 times, degreasing terminate after with pure water by isopropyl
Alcohol is rinsed well, the homogenate of sea anemone meat is placed in fume hood wind and is air-dried to without water droplet, and subpackage is placed in -20 DEG C and saves backup.
2.2 green Anthopleura indexs of correlation are determined
2.2.1 basic ingredient is determined
Moisture is determined with reference to GB 5009.3-2010；Ash is surveyed with reference to GB/T5009.4-2010；With reference to GB50095-2010
Survey crude protein content；The crude fat content of sea anemone meat is determined with reference to GB/T5009.6-2003.
2.2.2 the aminoacid of green Anthopleura protein is constituted and content analysis
With reference to GB/T5009.124-2003, sea anemone meat homogenised sample is weighed Jing after the hydrolysis of 6mol/L hydrochloric acid, use Hitachi
L8900 automatic amino acid analyzers determine the composition and content of aminoacid.
The screening of 2.3 enzymes
2.3.1 enzymolysis
Sea anemone meat homogenate plus 50mL pure water of the 10g through pretreatment are taken respectively, and enzyme concentration is 1000u/g according to various enzymes
Optimal pH and temperature on test kit is adjusted, and such as 1 condition of table is digested.Before enzymolysis with the sodium hydroxide of 0.5mol/L and
The hydrochloric acid of 0.5mol/L is adjusted to required pH, sample is placed in thermostat water bath and is digested, uniform speed slow stirring during enzymolysis.Enzymolysis
Inactivation 15min being boiled in water-bath after end, then removing thick impurity with three layers of filtered through gauze, filtrate in 4 DEG C of rotating speeds is
10min is centrifuged under the conditions of 12000rpm, continuous centrifugal is twice.Collect 0.45 μm of pin type membrane filtration of supernatant, adjust pH=
7th, concentrated by rotary evaporation, lyophilization, that product subpackage is placed in -20 DEG C of Refrigerator stores is standby.
1 enzymatic hydrolysis condition of table
2.3.2 mtt assay detection product is to DU-145 cell inhibitory effect activity
Cell culture：F12 culture fluid of the prostate gland cancer cell DU-145 containing 10% too Ox blood serum is in 37 DEG C, 5%CO2Carefully
Cultivate in born of the same parents' incubator, passed on when cell attachment grows to 70%～80% degree of converging or tested.
DU-145 cells are inoculated in 96 orifice plates.Matched group and medicine group are set, and per hole, addition concentration is medicine group
The culture fluid containing enzymatic hydrolysate of 5mg/mL, sets 3 multiple holes per group, adds the PBS that 200uL contains 10%MTT to delay after cultivating 24h
Liquid is rushed, and is incubated 4h.MTT culture fluid is abandoned in suction, adds DMSO concussion reactions, microplate reader detection absorbance A value to calculate suppression ratio
(IR), computing formula is：IR=[(control group A value-medicine group A value)/control group A value] × 100%.
2.4 orthogonal experiments optimize the preparation technology of green Anthopleura anti-prostate cancer oligopeptide
After determining optimal protease by 2.3, with L16(45) the green Anthopleura anti-prostate cancer widow of orthogonal experiment optimization
The preparation technology of peptide, to determine optimal temperature, time, pH, enzyme concentration and solid-liquid ratio, the concentrated lyophilization of product is simultaneously adopted
Mtt assay is detected to DU-145 proliferation inhibition activities, the factor and level such as table 2 of orthogonal experiment.
The factor and level of enzymatic hydrolysis condition in 2 orthogonal experiment of table
2.5 enzymolysis solution ultrafiltration
Digested according to 2.4 optimum enzymolysis conditions for determining, prepared green Anthopleura natineoplaston (Anthopleura
Anjunae Anti-tumor Peptide---AAP), with the filter membrane point of ALPHA1-4/LDplus ultrafiltration system 8KD and 20KD
Level, and by molecular weight it is ascending be named as AAP-I (MW ＜ 8KD), AAP-II (8KD≤MW ＜ 20KD), AAP-III (MW >=
20KD).Mtt assay is with 2.3 screening activity preferably component.
2.6 DEAE-Sepharose Fast Flow anion-exchange chromatographies
In advance activation, balanced exchange post (2cm × 35cm), sample concentration be 30mg/mL solution, ultrasonic 5min, Ran Houyong
0.45 μm of pin type membrane filtration, each loading 3mL, respectively with pure water, 0.1mol/L, 0.3mol/L, 0.5mol/L,
The NaCl solution gradient elution of 0.8mol/L, each gradient distinguish eluting 30min, and flow velocity is 1mL/min, electric with DBS-100-LCD
The full-automatic fraction collector of brain is collected one and is managed per 4min, detects absorbance in 280nm, and merges eluting according to absorbance curve
There are three peaks at peak, discovery.Product concentrated frozen is dried, and is named as AAP-I-1, AAP-I-2, AAP-I-3.Subpackage is stored in-
It is standby in 20 DEG C of refrigerators.Mtt assay is with 2.3 screening activity preferably component.
2.7 SephadexG25 gel chromatographies are isolated and purified
To DU-145 proliferation inhibition rates highest AAP-I-1 be continued to isolate and purify using G-25 gel chromatographies, by sample
It is configured to 30mg/mL solution, ultrasonic 5min, then with 0.45 μm of pin type membrane filtration, every time plus 3mL is to handling well
Sephadex G-25 chromatographic columns (2cm × 50cm), uses ultrapure water elution, and flow velocity is 1.0mL/min, uses DBS-100-LCD computers
Full-automatic fraction collector is collected one and is managed per 4min, detects absorbance in 280nm, and merges eluting peak according to absorbance curve and sends out
Existing three peaks.Product concentrated frozen is dried, and is named as AAP-I-1-1, AAP-I-1-2, AAP-I-1-3.Mtt assay same 2.3
Screening activity preferably component.
2.8 reversed-phase high-performance liquid chromatography are isolated and purified
0.22 μm of pin type filter membrane mistake of 5mg/mL solution Jing will be configured to DU-145 proliferation inhibition rate highests AAP-I-1-2
Isolated and purified with reversed-phase high-performance liquid chromatography after filter.1260 ZORBAX SB-C18 of sample introduction forward horizontal stand Agilent (9.4mm ×
250mm) post, 30 DEG C of column temperature；During laboratory, sample presses each 100 μ L of sample introduction of 300 μ g/mL, and eluent is mixed with acetonitrile ultra-pure water
Liquid presses 1mL/min eluting, 0min～15min acetonitriles (20%)～(50%), 15min～30min：Acetonitrile (50%)；Detection ripple
A length of 280nm.Sterling determines chain amino acids sequence using PPSQ-31A protein sequencers.
2.9 statistical analysis
Experimental result is analyzed using SPASS19.0 statistical softwares, and is adoptedRepresent.
3 results and analysis
3.1 green Anthopleura basic ingredients testing results
The basic ingredients of green Anthopleura such as table 3, from table 3 it is observed that thick containing what is enriched in green Anthopleura meat
Albumen, accounts for 19.76%, and crude fat content is relatively low, and only 0.89%, show that green Anthopleura is a kind of high protein, it is low-fat
Marine product.
3 green Anthopleura basic ingredient (g/100g, weight in wet base) of table
The aminoacid composition of green Anthopleura and content such as table 4.From table 4, it can be seen that the aminoacid kind of green Anthopleura
Class is more complete, it is necessary to which amino acid content accounts for the 24.31% of total amino acidss content；Fresh ear field (aspartic acid, glutamic acid,
One of glycine, alanine) account for total amino acidss content and be up to 50.58%, the reason for this is also green Anthopleura delicious flavour.Root
According to table 5, according to FAO/WHO for the standard that aminoacid is evaluated, green Anthopleura ratio must less meet between aminoacid
The amino acid balance of human body is required.But there are some researches show, hydrophilic polypeptides (containing Asp, Thr, Ser, Glu, Arg, Lys, His,
The hydrophilic amino acids such as Gln) can be by electrostatic attraction mode, specific effect causes its cell membrane to break rapidly in tumor cell
Split, cellular content seepage finally causes death of neoplastic cells.The hydrophilic amino acid of green Anthopleura meat enzymatic hydrolysate is accounted for always
The 49.3% of amino acid content, hydrophilic amino acid at high proportion may be relevant with its function.In sum, sea anemone enzymatic hydrolysate
Can be used for the exploitation of the medicine related to antitumor, food or food additive.
The aminoacid composition of 4 green Anthopleura of table and content (g/100g, dry weight)
| Aminoacid Amino acid | Content Content | Aminoacid Amino acid | Content Content |
| Aspartic acid Asp* | 7.067 | L-Tyrosine Tyr | 1.004 |
| Threonine Thr# | 2.700 | Phenylalanine Phe# | 1.643 |
| Serine Ser | 3.198 | Lysine Lys# | 3.099 |
| Glutamic acid Glu* | 8.988 | Histidine | 0.675 |
| Glycine Gly* | 13.084 | Arginine Arg | 6.693 |
| Alanine Ala* | 4.111 | Proline Pro | 4.649 |
| Cystine Cys | 0.263 | Total amino acidss TAA | 65.74 |
| L-Valine Val# | 2.572 | Fresh ear field FTAA | 33.25 |
| Methionine Met# | 1.176 | Essential amino acids EAA | 16.01 |
| Isoleucine Ile# | 1.868 | F/T (%) | 50.58% |
| Leucine Leu# | 2.951 | E/T (%) | 24.35% |
Note：" * " represents Fresh ear field；" # " is represented must aminoacid.
5 green Anthopleura protein nutritional assessment of table
3.2 4 kinds of different protein hydrolysate MTT active testing results
As a result as shown in figure 1, the enzymatic hydrolysate of pepsin, trypsin, neutral protease and alkaline protease is to DU-
145 cell proliferation inhibition rates are 24.98 ± 6.04%, 28.55 ± 5.29%, 31.66 ± 4.04% and 73.7 respectively ±
4.31%, it is descending to DU-145 cell inhibitory effect activity order to be respectively：Alkaline protease, neutral protease, pancreas egg
White enzyme, pepsin, so the enzyme required for alkaline protease is.
3.3 Alcalase Orthogonal experiment results
Orthogonal is adopted with solid-liquid ratio, temperature, pH value, 5 factors of enzyme concentration and time, just reference refers to IR values
Mark, investigate Alcalase protease it is most different under the conditions of product to prostate gland cancer cell DU-145 proliferation inhibition activities.Experiment knot
Fruit such as table 6：
6 alkaline protease enzymatic isolation method Orthogonal experiment results of table
Upper table is analyzed using extremum difference analysis, A (solid-liquid ratio) is can be seen that from Rj value sizes>E (time)>D
(temperature)>C (enzyme concentration)>B (pH value).And hydrolysising condition during IR values maximum is that solid-liquid ratio is 1 for A4B4C3D1E3.:5、pH
For 11, enzyme concentration be 2000U/g, temperature be 35 DEG C, the time be 6h.
3.4 green Anthopleura anti-prostate cancer oligopeptide initial gross separation results
Tri- components Jing of AAP-I, AAP-II, AAP-III that initial gross separation after alkaline protease enzymolysis solution ultrafiltration is obtained
After mtt assay screening active ingredients, as shown in Figure 2：Three components are 87.18 respectively to the proliferation inhibition rate of DU-145 cells ±
1.41%th, 74.36 ± 2.30%, 83.05 ± 2.03%.Illustrate that component AAP-I is best to DU-145 proliferation inhibiting effects, so
AAP-I is carried out into anion-exchange chromatography.
3.5 DEAE-Sepharose Fast Flow anion-exchange chromatography results
The AAP-I that ultrafiltration is obtained DEAE-Sepharose Fast Flow anion-exchange chromatographies obtain three peaks and produce
Thing, is respectively designated as AAP-I-1, AAP-I-2, AAP-I-3, such as Fig. 3.
Each peak product concentrated frozen is dried, and its proliferation inhibition activity to DU-145 is detected using mtt assay.Fig. 4 is tied
Fruit shows that, when concentration is 5mg/mL, tri- peak products of AAP-I-1, AAP-I-2, AAP-I-3 are to DU-145 proliferation inhibition rates point
It is not：37.12 ± 1.70%, 34.78 ± 2.20%, 24.21 ± 1.6%, it was demonstrated that AAP-I-1 is to DU-145 cell inhibitory effect
Rate highest, so AAP-I-1 is further isolated and purified.
3.6 Sephadex G-25 gel chromatography separating resultings
Detached AAP-I-1 in 2.5 is separated using Sephadex G-25 gel chromatographies, three such as Fig. 5 are as a result obtained
Peak, is respectively designated as AAP-I-1-1, AAP-I-1-2, AAP-I-1-3, lyophilization after collection, is sub-packed in -20 DEG C of refrigerators and protects
Deposit.
Three peak products are detected to DU-145 by each peak schematic diagram of Sephadex G-25 gel chromatographies using mtt assay
Proliferation activity, Fig. 6 results show that three peak product 5mg/mL, 24h to the increment suppression ratio of DU-145 are respectively：30.01±
2.16%th, 46.14 ± 2.29%, 40.53 ± 1.76%, illustrate that SAAP-I--1-2 is best to DU-145 Proliferation Abilities.
3.7 AAP-I-1-2 Jing efficient liquid phase separating resultings
By AAP-I-1-2 Jing HPLC, result is dried and is named as AAP- as shown in fig. 7, collecting main peak concentrated frozen after purification
H, carries out MTT active testings, as a result to DU-145 proliferation inhibition rates be 49.14 ± 2.29%, show that main peak AAP-H should be
The chief active thing of AAP-I-1-2, AAP-H Jing efficient liquid phases detect basic simple sample, and purity is more than 95%, meets aminoacid
Sequencing requires that AAP-H efficient liquid phases detect such as Fig. 8.
3.8 determined amino acid sequence results
Amino acid sequencing result is：Tyr-Val-Pro-Gly-Pro(SEQ ID No：1).
4 conclusions
Determine that alkaline protease is optimal enzyme by screening, enzymatic isolation method is further optimized using orthogonal experiment and extracts sea anemone
The optimum process condition of natineoplaston is:It is 1 according to solid-liquid ratio:5 (W/V), pH=11, enzyme concentration are 2000u/g, 35 DEG C of conditions
Lower enzymolysis 6 hours.Separated by methods such as ultrafiltration, anion-exchange chromatography, G-25 gel chromatographies and reverse efficient liquid phases pure
Change, with product to DU-145 proliferation inhibition rates as index, final purification obtain sequence be Tyr-Val-Pro-Gly-Pro peptide
Chain.
Currently preparing the more commonly used method of biologically active peptide has three kinds：One is that chemical synthesis process prepares biological activity
Peptide；Two are with organism or are organized as raw material and are directly separated extraction by various separation means；Three are degraded using enzymatic isolation method
Biological tissue or macromole product prepare biologically active peptide.As protease hydrolyzed method production biologically active peptide has safety
High, reaction condition is gentle, the advantage that yield is relatively high, therefore the method has become the research for preparing bioactive peptide in recent years
Focus.As marine organism growth environment is unique to terrestrial organism, what is had is even grown in the environment such as extra-high voltage or extremely low temperature
In, cause marine organisms that unique structure, the bioactive peptide of novel functions are formed in growth course.This active peptide moiety is with natural
State is present, and partly as the domain of protein macromolecule, protease is by specific point position shearing, obtaining activity
Than the more preferable peptide chain of protein, these biologically active peptides are adjusting immune system, antibacterial, antithrombotic, resisting hypertension, are adjusting gastrointestinal
Road motion, removing free radical, antiviral, the absorption of promotion mineral element and anticancer aspect play an important role.Existing research table
It is bright：The biological activity of peptide chain is mainly affected by amino acid composition and peptide chain molecule amount size, bioactive peptide than protein, polypeptide and
Simple aminoacid has higher biological activity.The fragrance such as Trp, Tyr, Met, Gly, Lys, His and the Pro in other peptide chain
Race's aminoacid or hydrophobic amino acid can strengthen the biological activity of peptide chain well.Contain in the peptide chain that the present invention is prepared
Tyr, Pro and Gly, it is believed that this structure is probably which has one of major reason of anti-tumor activity.In sum, green side flower
Sea anemone meat is a kind of very high natural resourcess of protein content, the peptide prepared by enzymatic isolation method, can be used for anti-prostate cancer related
Drug development or be developed into health food.
Embodiment described above is one kind preferably scheme of the present invention, not the present invention is made any pro forma
Limit, also have other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Ocean university
<120>A kind of preparation method of green Anthopleura anti-prostate cancer oligopeptide
<130> 2016.11
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 5
<212> PRT
<213>Green Anthopleura
<400> 1
Tyr Val Pro Gly Pro
1 5
Claims (4)
1. a kind of preparation method of green Anthopleura anti-prostate cancer oligopeptide, it is characterised in that step is as follows：
(1) sample pretreatment：The green Anthopleura of fresh wild obtains sea anemone meat slurry after pretreatment；
(2) digest：According to every gram of sea anemone meat slurry plus the proportioning of 4-5mL pure water, sea anemone meat slurry and pure water are mixed, pH is adjusted
=10-11, adds alkaline protease, 35-45 DEG C of enzymolysis 4-6 hour according to the enzyme concentration of every gram of sea anemone meat slurry plus 2000-2500U
Obtain enzymolysis solution；
(3) ultrafiltration：Enzymolysis solution is inactivated, centrifugation, takes supernatant, and Jing ultrafiltration obtains active component of the molecular weight less than 8kd；
(4) anion-exchange chromatography：Activearm lease making DEAE-Sepharose Fast Flow anion of the molecular weight less than 8kd
Displacement chromatography, eluting detect absorbance in 280nm, and merge eluting peak according to absorbance curve, to collecting each eluting for obtaining
Peak product lyophilization, mtt assay screening are produced to prostate gland cancer cell DU-145 proliferation inhibition rate highests anion-exchange chromatography
Thing；
(5) SephadexG25 gel chromatographies：Will be to prostate gland cancer cell DU-145 proliferation inhibition rate highest anion exchange layers
Division thing continues to isolate and purify using G-25 gel chromatographies, ultrapure water elution, detects absorbance in 280nm, and according to absorbance
Curve merges eluting peak, and each eluting peak product lyophilization obtained to collection, mtt assay are screened to prostate gland cancer cell DU-145
Proliferation inhibition rate highest Sephadex G25 gel chromatography products；
(6) reversed-phase high-performance liquid chromatography is isolated and purified：To prostate gland cancer cell DU-145 proliferation inhibition rate highests Sephadex
G25 gel chromatography products are isolated and purified with reversed-phase high-performance liquid chromatography Jing after membrane filtration, finally obtain green Anthopleura it is anti-before
Row adenocarcinoma oligopeptide.
2. preparation method according to claim 1, it is characterised in that step (1) sample pretreatment is specially：Take fresh open country
The green Anthopleura of life is placed in the clean sea water of room temperature raises one day, and cleaning removes impurity, is cut open from centre with shears, is placed in beaker
In, and the covering that adds water, it is placed in -20 DEG C of refrigerators and freezes 12h, then is placed in room temperature naturally to thaw, repeatedly for three times, extrusion tentacle poison
Liquid, is then rinsed to white sea anemone meat is obtained with tap water, and white sea anemone meat is homogenized, homogenate is soaked in isopropanol
Fat, changes once per 4h, continuously changes 3 times, and isopropanol is rinsed well after terminating by degreasing with pure water, is subsequently placed in fume hood wind
Air-dry to without water droplet and obtain sea anemone meat slurry.
3. preparation method according to claim 1 and 2, it is characterised in that step (4) respectively with pure water, 0.1mol/L,
The NaCl solution gradient elution of 0.3mol/L, 0.5mol/L, 0.8mol/L, the elution speed of each gradient is 1mL/min.
4. preparation method according to claim 1 and 2, it is characterised in that the elution speed of the ultra-pure water of step (5) is
1mL/min。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111876457A (en) * | 2020-07-22 | 2020-11-03 | 海南医学院 | Preparation of sea anemone tectorial enzymolysis polypeptide and insecticidal application thereof |
| CN112458138A (en) * | 2020-11-30 | 2021-03-09 | 海南医学院 | Preparation method and application of actinia violaceus enzymatic hydrolysis polypeptide |
| CN113481271A (en) * | 2021-06-22 | 2021-10-08 | 中国科学院南海海洋研究所 | Marine biological active peptide capable of effectively relieving skin sunburn and preparation method and application thereof |
| CN113564219A (en) * | 2021-09-26 | 2021-10-29 | 渤海水产股份有限公司 | Separation and purification method of active starfish peptide, active starfish peptide and application |
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| WO2012174412A2 (en) * | 2011-06-16 | 2012-12-20 | La Jolla Institute For Allergy And Immunology | Compositions targeting pkc-theta and uses and methods of treating pkc-theta pathologies, adverse immune responses and diseases |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876457A (en) * | 2020-07-22 | 2020-11-03 | 海南医学院 | Preparation of sea anemone tectorial enzymolysis polypeptide and insecticidal application thereof |
| CN112458138A (en) * | 2020-11-30 | 2021-03-09 | 海南医学院 | Preparation method and application of actinia violaceus enzymatic hydrolysis polypeptide |
| CN112458138B (en) * | 2020-11-30 | 2023-05-05 | 海南医学院 | Preparation method and application of sea anemone enzymatic polypeptide |
| CN113481271A (en) * | 2021-06-22 | 2021-10-08 | 中国科学院南海海洋研究所 | Marine biological active peptide capable of effectively relieving skin sunburn and preparation method and application thereof |
| CN113564219A (en) * | 2021-09-26 | 2021-10-29 | 渤海水产股份有限公司 | Separation and purification method of active starfish peptide, active starfish peptide and application |
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|---|---|
| CN106560518B (en) | 2020-02-07 |
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