CN104212861A - Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer - Google Patents
Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer Download PDFInfo
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Abstract
The invention relates to ruditapes philippinarum oligopeptide, the amino acid sequence of which is Asp-Trp-Pro-His. The invention discloses a preparation method of the oligopeptide. According to the preparation method, a target oligopeptide enzymatic hydrolysate is obtained through enzymatic hydrolysis of trypsin; and through ultrafiltration, anion exchange and reversed-phase high-performance liquid chromatography, separation and purification are further carried out to obtain the target oligopeptide. Meanwhile, the invention also discloses an application of the oligopeptide in resisting prostate cancer. Specifically, the oligopeptide can effectively inhibit cell growth of prostate cancer PC-3 and DU-145 cultured in vitro, induce apoptosis and change cell cycle. In comparison with the prior art, the preparation method of the ruditapes philippinarum oligopeptide has advantages of simple technological process, few impurity and high efficiency. Meanwhile, based on the prior art, the invention discloses effectiveness of the oligopeptide in resisting prostate cancer.
Description
Technical field
The present invention relates to a kind of philippine clam whelp oligopeptides, particularly relate to the preparation method of this philippine clam whelp oligopeptides and the application in anti-prostate cancer field thereof.
Background technology
Philippine clam whelp (Ruditapes philippinarum) belongs to Mollusca Bivalvia curtain clam order Veneridae, a kind of eurythermal small-sized bivalve shellfish, south is commonly called as colored clam, the large cultivated shellfish of China four is listed as with oyster, razor clam of hanging, blood clam class, it is one of the main shellfish in offshore beach and shallow sea, its delicious flavour, nutritious, protein content is high.
Research shows, philippine clam whelp extract has a lot of physiological function, as antitumor, anti-oxidant, antibacterial and strengthening immunity etc., remarkable progress is had in recent years to the antitumor research of philippine clam whelp extract, as (foodstuffs industry science and technology such as Fan Xiuping, 2003) raw product of philippine clam whelp glycosaminoglycan (CRG) is utilized to act on the HL-60 cell of vitro culture, find that CRG has remarkable restraining effect to HL-60 cell under 1.0mg/ml concentration, 59.8%, 67.7% and 96.2% can be reached respectively at 24h, 48h, 72h tumour inhibiting rate.(the Oceanologia et Limnologia Sinica such as Zhang Li, 2007) philippine clam whelp meat is utilized to extract two kinds of proteoglycan PG1 and PG2, wherein PG1 reaches 73.30% when 200ug/ml concentration to the growth inhibition ratio of human liver cancer cell SMMC7721, and does not affect growth and the function of Human normal hepatocyte HL-7002.Prostate cancer is one of modal malignant tumour of the male sex, and the death toll caused by prostate cancer in American-European countries accounts for the second that malignant tumour causes death toll, is only second to lung cancer.In recent years along with the prolongation of people's mean lifetime and the raising of diagnostic techniques, the sickness rate of China's prostate cancer is in the trend significantly increased, quality of life and the life-span of China more than 50 years old male sex are drastically influence, become the research topic that Urological Field one is more and more important, but, and less about the application anti-prostate cancer of philippine clam whelp bioactive peptide on less about the report of philippine clam whelp activity extract to the effect of prostate cancer inhibition of cancer cell.
At present, the preparation of biologically active peptides mainly contains three kinds of modes: one is from natural organism, extract itself intrinsic various natural radioactivity peptide matters; Two is obtain the biologically active substance with various physiological function by the approach of protein degradation; Three is utilize biosynthetic method to prepare biologically active peptides.Wherein, in first kind of way, the content of nature biotechnology body weight bioactive peptide material is lower, and in leaching process, productive rate is lower, and needs more raw material individual, makes cost higher; Biosynthetic process complicated difficult control in the third mode, intermediate product is more, is not suitable for extensive generation; Comprehensive, due to enzymolysis specificity and the high efficiency of proteolytic enzyme, make enzymolysis process produce the method security of biologically active peptides high, productive rate is high.
Summary of the invention
Technical problem to be solved by this invention provides a kind of protease hydrolyzed legal system that utilizes for the method for philippine clam whelp oligopeptides.
Another technical problem to be solved by this invention is to provide this kind of application of philippine clam whelp oligopeptides in anti-prostate cancer.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of preparation method of philippine clam whelp oligopeptides, is characterized in that comprising the following steps:
1. philippine clam whelp is cleaned, add ultrapure water after getting meat and stir, then decon homogenate, obtained homogenised sample;
2. appropriate described homogenised sample is got, add the ultrapure water of appropriate volume, now the ratio of raw material and ultrapure water is 1:1 ~ 1:4, add proteolytic enzyme and carry out enzyme digestion reaction in water bath, wherein enzyme concentration is 300ug ~ 1500ug, the pH value of enzyme digestion reaction is 8 ~ 9, and enzymolysis time is 4h ~ 12h, and hydrolysis temperature is 40 DEG C ~ 60 DEG C;
3. enzyme digestion reaction terminates rear deactivation, centrifugal, to get supernatant liquor for subsequent use;
4. added in ultra-filtration membrane by described supernatant liquor and carry out ultrafiltration, collecting enzymolysis material that molecular weight is less than 3KDa, to carry out lyophilize for subsequent use;
5. utilize DEAE SepharoseFF anionresin, adopt phosphoric acid buffer and NaCl solution to carry out stepwise elution to gained enzymolysis solution, store for subsequent use after the highest peptide peak lyophilize is collected at 280nm wavelength place;
6. utilize reversed-phased high performace liquid chromatographic to be further purified at the highest peptide peak that DEAE SepharoseFF anionresin is collected, at 220nm wavelength there is unique peak in place, collects this peak, and lyophilize obtains described philippine clam whelp oligopeptides.
As preferably, the ratio of described raw material and ultrapure water is 1:2, and raw material can be made so to be fully separated in aqueous, under the prerequisite of diluted protein enzyme concn within reason, add the contact area of proteolytic enzyme and enzymolysis object, improve enzymolysis efficiency, also can ensure the biological activity of raw material simultaneously.
Described proteolytic enzyme can have multiple choices, and as preferably, described proteolytic enzyme is trypsinase, and the optimum enzymolysis condition of proteolytic enzyme is combined as: enzyme concentration is 1200ug, and enzymolysis pH value is 8.0, and enzymolysis time is 8h, and hydrolysis temperature is 50 DEG C.
As preferably, described step 3. in be deactivation 15min in boiling water bath, the centrifugal 15min of 10000r/min at 4 DEG C, boiling water bath that is 100 DEG C can make the abundant deactivation of proteolytic enzyme, and at 4 DEG C, high speed centrifugation can ensure the biological activity of related substances in enzymolysis solution.
Described step 5. in the concentration of NaCl solution be 0.1mol/L, 0.3mol/L, 0.5mol/L tri-kinds, abundant wash-out can be carried out stage by stage to enzymolysis solution.
The chromatographic condition of described high performance liquid chromatography is: chromatographic column is C
18reversed-phase column, filler is ODS, and moving phase is acetonitrile and water (15:85), and flow velocity is 0.8ml.min
-1, loading volume is 20ul, and determined wavelength is 220nm, and elution requirement is 15% acetonitrile, 20min.
Be Asp-Trp-Pro-His by the aminoacid sequence of the philippine clam whelp oligopeptides of aforesaid method gained, this kind of application of philippine clam whelp oligopeptides in anti-prostate cancer, especially the growth of vitro culture prostate cancer PC-3, DU-145 cell can be suppressed, after effect 24h, the morning of PC-3, DU-145 cell, non-viable apoptotic cell just start to increase, and along with the increase of liquor strength, apoptotic cell increases gradually.
Compared with prior art, the invention has the advantages that: utilize trypsin digestion to obtain target oligopeptides enzymolysis solution, target oligopeptides is obtained again through the further separation and purification of ultrafiltration, anionresin and RPLC, the simple efficiency of technological process is high, and the target oligopeptides obtained has anti-prostate cancer activity, effectively can suppress vitro culture prostate cancer PC-3, DU-145 Growth of Cells, apoptosis-induced, change the cell cycle.
Accompanying drawing explanation
Fig. 1 be in embodiment 1 temperature to ANN content in trypsin digestion liquid and fishy smell value interact relation schematic diagram;
Fig. 2 be in embodiment 1 time to ANN content in trypsin digestion liquid and fishy smell value interact relation schematic diagram;
Fig. 3 be in embodiment 1 pH to ANN content in trypsin digestion liquid and fishy smell value interact relation schematic diagram;
Fig. 4 be in embodiment 1 enzyme concentration to ANN content in trypsin digestion liquid and fishy smell value interact relation schematic diagram;
Fig. 5 is DEAE-sepharoseFF elution peak schematic diagram in embodiment 2;
Fig. 6 is peak I in Fig. 5
1through the eluting result schematic diagram of RP-HPLC chromatographic column;
Fig. 7 is target oligopeptides amino acid molecular structure schematic diagram;
Fig. 8 be in embodiment 3 PRO to the inhibited proliferation histogram of PC-3 cell;
Fig. 9 be in embodiment 3 DU-145 to the inhibited proliferation histogram of PC-3 cell;
Figure 10 is control group PC-3 cell AO/EB coloration result schematic diagram in embodiment 3;
Figure 11 is PC-3 cell AO/EB coloration result schematic diagram after 10mg/mL PRO effect 24h in embodiment 3;
Figure 12 is PC-3 cell AO/EB coloration result schematic diagram after 20mg/mL PRO effect 24h in embodiment 3;
Figure 13 is PC-3 cell AO/EB coloration result schematic diagram after 30mg/mL PRO effect 24h in embodiment 3;
Figure 14 is control group DU-145 cell AO/EB coloration result schematic diagram in embodiment 3;
Figure 15 is DU-145 cell AO/EB coloration result schematic diagram after 10mg/mL PRO effect 24h in embodiment 3;
Figure 16 is DU-145 cell AO/EB coloration result schematic diagram after 20mg/mL PRO effect 24h in embodiment 3;
Figure 17 is DU-145 cell AO/EB coloration result schematic diagram after 30mg/mL PRO effect 24h in embodiment 3;
Figure 18 is PC-3 apoptosis rate schematic diagram in control group in embodiment 3;
Figure 19 is PC-3 apoptosis rate schematic diagram in 10mg/mLPRO group in embodiment 3;
Figure 20 is PC-3 apoptosis rate schematic diagram in 20mg/mLPRO group in embodiment 3;
Figure 21 is PC-3 apoptosis rate schematic diagram in 30mg/mLPRO group in embodiment 3;
Figure 22 is DU-145PRO apoptosis rate schematic diagram in control group in embodiment 3;
Figure 23 is DU-145PRO apoptosis rate schematic diagram in 10mg/mLPRO group in embodiment 3;
Figure 24 is DU-145PRO apoptosis rate schematic diagram in 20mg/mLPRO group in embodiment 3;
Figure 25 is DU-145PRO apoptosis rate schematic diagram in 30mg/mLPRO group in embodiment 3;
Figure 26 be in embodiment 3 RPO to PC-3 Cell cycle influences schematic diagram;
Figure 27 be in embodiment 3 RPO to DU-145 Cell cycle influences schematic diagram;
Figure 28 is control group PC-3 cell SubG1 peak apoptosis schematic diagram in embodiment 3;
Figure 29 is 10mg/mLPRO group PC-3SubG1 peak apoptosis schematic diagram in embodiment 3;
Figure 30 is 30mg/mLPRO group PC-3SubG1 peak apoptosis schematic diagram in embodiment 3;
Figure 31 is control group DU-145 cell SubG1 peak apoptosis schematic diagram in embodiment 3;
Figure 32 is 10mg/mLPRO group DU-145 cell SubG1 peak apoptosis schematic diagram in embodiment 3;
Figure 33 is 30mg/mLPRO group DU-145 cell SubG1 peak apoptosis schematic diagram in embodiment 3.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment 1: the optimization of enzymatic hydrolysis condition
The screening of proteolytic enzyme: shell after being cleaned by fresh philippine clam whelp and get meat, then puts into refiner by young for clam meat, pours appropriate ultrapure water homogenate into.Take appropriate homogenised sample, add the ultrapure water of 2 times of weight, select trypsinase, papoid, Sumizyme MP, neutral protease is done for selecting enzyme, adopt good fortune face-phenol method measure enzyme activity (see Yang Yongfang. mussel enzymolysis polypeptide preparation technology and anti-prostate cancer activity research [D]. Zhoushan: Oceanography Institute Of Zhejiang, 2011), according to the Substrate concentration separately of proteolytic enzyme shown in table 1, add the proteolytic enzyme of 1000u/g, enzyme digestion reaction 6h, in boiling water bath deactivation 15min after having reacted, in 4 DEG C after cooling, the centrifuge 15min of 10000r/min precooling, get supernatant liquor for subsequent use.
After prepared by sample enzymolysis solution, by ANN (amino nitrogen) content free in enzymolysis solution and the enzymolysis solution fishy smell value screening index as enzyme, ANN content is higher, fishy smell value lower proteolytic enzyme effect is better.Wherein to adopt formaldehyde potentiometric titration measure (see Yang Yongfang. mussel enzymolysis polypeptide preparation technology and anti-prostate cancer activity research [D]. Zhoushan: Oceanography Institute Of Zhejiang, 2011.) ANN content in enzymolysis solution, concrete grammar is as follows: in the distilled water of 60ml, add 5ml hydrolyzed solution, detects pH value.First use the NaOH solution titration pH8.2 of 0.1mol/L, add the formaldehyde solution 20ml neutralized, the volume of the 0.05mol/LNaOH solution consumed when record is titrated to pH9.2, calculates its nitrogen content.
X=（V1-V2）×C×0.014×100/(5×V)
Wherein: X is the content (g/100ml) of ANN in sample; V1 is the NaOH reference liquid (ml) adding formaldehyde dilution post consumption; V2 is the NaOH reference liquid volume (ml) that blank test adds formaldehyde post consumption; V is the sample liquid amount of taking (ml); C is the concentration (mol/L) of NaOH reference liquid; 0.014 is the mmole quality (g/mmoL) of nitrogen.The fishy smell evaluation of enzymolysis solution and subjective appreciation form subjective appreciation group by the academics and students of 10 Majors of Foods, carry out Comprehensive Assessment to the fishy smell of philippine clam whelp enzymolysis solution and other organoleptic features such as color, turbidity etc.Fishy smell is evaluated using distilled water as with reference to (be 0 point without fishy smell score value), very light according to fishy smell, fishy smell is light, fishy smell is general, fishy smell is heavier and fishy smell weighs 5 ranks and marks very much, full marks are 10 points, mark is larger, then show that fishy smell is heavier, when philippine clam whelp enzymolysis solution fishy smell value 4 points and following time think that it can accept.
ANN content after four kinds of protease hydrolyzed philippine clam whelp meat in enzymolysis solution and sense organ fishy taste evaluation result are in table 2, trypsin digestion best results as seen from Table 2, after 6h enzymolysis, in enzymolysis solution, ANN content is the highest, and fishy smell is lighter, within acceptable scope, and sense organ color is also best; Next is Sumizyme MP, and the hydrolysis result of papoid and neutral protease is the poorest and fishy smell is heavier, and sense organ color is also the poorest, therefore, considers in the present embodiment 1 and selects trypsinase as hydrolysising protease.
The determination of enzymatic hydrolysis condition: adopt experiment of single factor to filter out tryptic the best use of condition (temperature, time, pH, enzyme concentration), point four groups of experiments are carried out.
Experiment one: temperature single factor test: the philippine clam whelp 5 parts taking appropriate identical amount (10g) respectively, regulates pH to 8.0, adds the trypsinase of 1000u/g, and at different temperatures (40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C) enzymolysis 6h;
Experiment two: time single factor test: the philippine clam whelp 5 parts taking appropriate identical amount (10g) respectively, regulates pH to 8.0, adds the trypsinase of 1000u/g, constant temperature enzymolysis different time (4h, 6h, 8h, 10h, 12h) at 50 DEG C;
Experiment three: pH value single factor test: the philippine clam whelp 5 parts taking appropriate identical amount (10g) respectively, different pH value (7,7.5,8,8.5,9), adds the trypsinase of 1000u/g, constant temperature enzymolysis 6h at 50 DEG C;
Experiment four: enzyme concentration single factor test: the philippine clam whelp 5 parts taking appropriate identical amount (10g) respectively, regulates pH to 8.0, adds the trypsinase of different amount (300u/g, 600u/g, 900u/g, 1200u/g, 1500u/g), constant temperature enzymolysis 6h at 50 DEG C.
Tentatively determining on the basis of enzymatic hydrolysis condition, selecting enzymolysis time, temperature, pH value, enzyme concentration four factors to set up three level design orthogonal experiment L9(34) orthogonal table, determine that trypsin digestion solution philippine clam whelp prepares the optimum enzymolysis condition of oligopeptides.
Experimental result as shown in figures 1-4, as can be seen from Figure 1 free ANN content then starts to decline after then reaching a maximum value along with the rising of temperature increased before this, constantly increase when temperature is 40-50 DEG C, maximum value is reached when arriving 50 DEG C, then start to decline along with temperature raises, this also just in time to meet in enzymatic reaction temperature to the Changing Pattern of enzyme activity; The fishy smell of enzymolysis solution constantly declines along with the rising of temperature.Therefore, to sum up consider, the present embodiment selects 50 DEG C as a reference at first.
Can learn from Fig. 2, the ANN content in enzymolysis solution constantly increases along with the increase of time, and start increasing ratio comparatively slowly, between 8-10h, speedup increases suddenly, sharply increases, and between 10-12h, speedup becomes again very slow subsequently.Fishy smell value diminishes along with the increase of time equally, As time goes on starts to remain unchanged after arriving 10h.Therefore, the present embodiment chooses 10h enzymolysis time as a reference at first.
As can be seen from Figure 3, free ANN content subtracts afterwards along with the rising of pH first increases, and reaches maximum when pH is 8.0, and this is also just meeting enzyme and is living and relation between pH.The fishy smell of enzymolysis solution along with the rising of pH just in time contrary with the relation of ANN content and pH, first decline and raise afterwards, enzymolysis solution reaches minimum when pH value is 8.0, increases again and has certain peculiar smell to produce subsequently along with pH raises fishy smell.Therefore, the initial pH value of pH8.0 the present embodiment is the most chosen.
Can to learn from Fig. 4 in enzymolysis solution that free ANN content can constantly increase along with the increase of enzyme amount, and speedup is from fast to slow, the fishy smell of enzymolysis solution along with the increase of enzyme amount just started to reduce a reaching minimum value after start again to increase.Therefore, considering the present embodiment selects the enzyme amount of 1200u/g for select enzyme amount at first.
Table 3 is trypsin digestion Orthogonal experiment results, as shown in Table 3, from enzymolysis solution, free ANN content is known, temperature, time, pH, enzyme concentration four factors all can affect tryptic hydrolysis result, wherein B(temperature) > D(enzyme concentration) > A(pH) the > C(time), and A2B2C1D3 combined effect is better, the free ANN content obtained is the highest; Known from enzymolysis solution fishy smell value, the impact of four factors is yet more remarkable, B(temperature is known by extreme difference R ' is known) > D(enzyme concentration)=A(pH)=C(the time), and A2B2C1D2 or A2B2C1D3 combined effect is better, the enzymolysis solution fishy smell value obtained is minimum.Consider, show that best of breed is A2B2C1D3, namely the optimum enzymolysis condition of proteolytic enzyme is: enzymolysis pH is 8.0, and temperature is 50 DEG C, and enzymolysis time is 8h, and enzyme concentration is 1200u/g.Under this conditional combination, in the enzymolysis solution that enzymolysis obtains, free ANN content is 3.125mg/g, and fishy smell value is between 7 ~ 8.
In order to verify the reliability of this optimization experiment condition, this optimal conditions parameter is amplified several times, and each experiment all repeats 3 times, and record in enzymolysis solution ANN content average of dissociating at about 2.968mg/g, fishy smell value, between 7 ~ 8, demonstrates the reliability of orthogonal test.
Embodiment 2: the preparation of enzymolysis oligopeptide and separation and purification
Prepare philippine clam whelp oligopeptides according to the enzymatic hydrolysis condition that embodiment 1 obtains, that is: shell after fresh philippine clam whelp being cleaned and get meat, then young for clam meat is put into refiner, pour appropriate distilled water homogenate into.Take appropriate homogenised sample, add the water of 2 times of weight, add the trypsinase of 1200u/g, in enzymolysis pH8.0, enzyme digestion reaction 8h under temperature 50 C condition, in boiling water bath deactivation 15min after having reacted, in 4 DEG C after cooling, the centrifuge 15min of 10000r/min precooling, gets supernatant liquor.Be the ultra-filtration membrane of 3KDa with intercepting molecular weight, in 20 DEG C of environment, ultrafiltration 11h obtains enzymolysis solution, freeze-dried back.With DEAE SepharoseFF anionresin, column type is 2.6 × 50cm, loading volume 3mL, stepwise elution is carried out respectively by the NaCl solution of phosphoric acid buffer, 0.1mol/L, 0.3mol/L, 0.5mol/L, elution speed is 1ml/min, ultraviolet monitoring under 280nm, automatic Fraction Collector often pipe 4mL is collected.As shown in Figure 5, after DEAE SepharoseFF post wash-out, there is 2 peaks, i.e. peak 1(I in the enzymolysis solution getting below 3KDa
1) and peak 2(I
2), be respectively the elution fraction of phosphoric acid buffer and 0.1mol.L-1NaCl solution, 0.3mol.L
-1and 0.5mol.L
-1naCl solution there is no obvious elution peak.Collect I
1, obtain 1.3g dry sample after lyophilize, yield is 0.33%.
To the peak 1(I collected
1) utilize reversed-phased high performace liquid chromatographic to be further purified and detect, chromatographic condition is C18 reversed-phase column, and filler is ODS; Column type is 10 × 250mm; Using acetonitrile/water (15:85) as moving phase, flow velocity is 0.8ml.min-1, and loading volume is 20 μ L, wavelength 220nm.Elution requirement is acetonitrile concentration 15%, and elution time is 20min, repeats loading.As shown in Figure 6, when retention time is about 6.00min, occur simple spike, peak height is 1022.1, and peak area is 5191.3, collects this peak, obtains 0.37g sample after lyophilize, and yield is 0.1%.
Aminoacid sequence detects, the amino acid sequence analysis of target peptide detects and adopts N-terminal degraded detection method, measure with Protein Sequencer, obtain philippine clam whelp enzymolysis oligopeptide (Ruditapes philippinarum oligopeptides, RPO), the aminoacid sequence of this target peptide is: Asp-Trp-Pro-His, molecular weight is 607.6Da, called after philippine clam whelp enzymolysis oligopeptide (Ruditapes philippinarum oligopeptides, RPO), molecular structural formula is shown in Fig. 7.
Embodiment 3: anti-prostate cancer Activity determination
Prostate cancer PC-3, DU-145 cell cultures: preparation 1000ml F12 nutrient solution, add 1.176g NaHCO3, penicillin, the Streptomycin sulphate of each 100,000 IU, carry out aseptic suction filtration, in ultra-clean platform after packing in-20 DEG C of refrigerators cryopreservation, often add the foetal calf serum of 10ml before use in 100ml nutrient solution.Cellar culture human prostata cancer PC-3, DU-145 cell strain, is placed in 37 DEG C, cultivates in 5%CO2 incubator, within 2 ~ 3 days, goes down to posterity once.
Cell growth inhibition assay: adopt mtt assay to detect vitro culture prostate cancer PC-3, DU-145 cell survival rate.Select Growth of Cells to more than 80% during experiment and form is better, be in PC-3, DU-145 cell suspension of logarithmic phase, be seeded to 96 orifice plates with 1 × 104/mL, every hole 200 μ l, inhale after cultivating 24h and abandon supernatant liquor.If control group and 10mg/mL, 15mg/mL, 20mg/mL, 25mg/mL and 30mg/mL RPO dosing group, often group establishes 3 multiple holes, and abandon nutrient solution after cultivating 24h, 48h and 72h respectively, dosing group adds 200 μ l1mg/mL MTT, continues to cultivate 4h.MTT is abandoned in suction, and every hole adds the DMSO of 150 μ l, vibration 10min, put microplate reader and survey absorbancy at 490nm wavelength place, calculate cell inhibitory effect index (IR), experiment repetition 3 times, IR=[(control group A value-dosing group A value)/(control group A value-zeroing hole A value] × 100%.As shown in Figure 8 and Figure 9, after the RPO of different concns acts on PC-3, DU145 cell respectively, result shows increase along with RPO concentration and the prolongation of action time, and Proliferation Ability index obviously rises, and has statistical significance (P<0.05) compared with control group.Therefore, the display of MTT result PC-3, DU-145 cell, after RPO effect, present obvious Proliferation Ability phenomenon, and along with the increase of drug level and the prolongation of action time, inhibition index obviously increases, and is dependency with time and dosage.
AO/EB fluorescent dye: put in 6 well culture plates through processed cover glass, PC-3, DU-145 cell concn is adjusted to 1 × 105/mL, be inoculated on the cover glass in culture plate, every hole 2ml, observe after 24h and change liquid, set up control group and 10mg/mL, 20mg/mL, 30mg/mLRPO dosing group, clean 3 times with PBS after 24h, then fix 30min with 95% ethanol.Get each 1mg of AO and EB to be dissolved in respectively in the PBS damping fluid of 10ml pH7.2, shake up mixing, now with the current, keep in Dark Place.On slide glass, drip 40 μ l PBS and 10 μ l AO/EB mixed solutions before observing, 10 × 20 fluorescence microscopy Microscopic observations are also taken pictures.As shown in Figure 10 ~ 17, PC-3, DU-145 cell is AO/EB dyeing after RPO effect 24h, and at the visible four kinds of cells of fluorescence microscopy Microscopic observation, i.e. 1. viable cell: nuclear chromatin is even, in green; 2. viable apoptotic cell: cytolemma is complete, nuclear chromatin condensation, in comparatively strong green; 3. non-viable apoptotic cell: nuclear chromatin condensation, in orange or orange; 4. non-viable non-apoptotic cell: cell is homogeneous orange; Wherein Figure 10 ~ 13 are PC-3 cell AO/EB cell dyeing metamorphosis, and Figure 14 ~ 17 are DU-145 cell AO/EB cell dyeing metamorphosis.The full growth in Epithelial of cellular form in control group, nuclear morphology is normally bright green, as Figure 10 and Figure 14; There is a little viable apoptotic cell in PC-3 and the DU-145 cell of 10mg/mL group, core is in green, and chromatin pyknosis becomes lumps be positioned at nucleus side or occur the ANOMALOUS VARIATIONS such as crescent, as Figure 11 and Figure 15; PC-3 and the DU-145 early apoptosis of cells cytosis of 20mg/mL group, cell rounding, kernel reduced number, chromatin pyknosis, respectively in safran and yellow-green colour, as Figure 12 and Figure 16; Cell rounding appears that late apoptic and dead cell increase, in PC-3 and the DU-145 cell of 30mg/mL group, and obvious pyknosis appears in nuclear chromatin, and color burn is safran; After birth outwardly forms apoptotic body and part nuclear fragmentation is orange, as Figure 13 and Figure 17.
Flow cytomery apoptosis: PC-3, DU-145 cell is inoculated in 6 well culture plates according to 1 × 105/mL, changes liquid after 24h.Set up control group and 10mg/mL, 20mg/mL and 30mg/mL RPO dosing group, continue to cultivate 24h.With 0.25% tryptic digestive juice peptic cell, add PBS damping fluid, blow and beat into single cell suspension, under room temperature, after the centrifugal 5min of 1200r/min, abandon supernatant liquor, then use PBS repeated washing 1 time.Add the test kit dedicated buffering liquid of 400 μ l, blow and beat into cell suspension, in cell suspension, then add 5 μ lAnnexin V-FITC, add the PI of 5 μ l after mixing again, ambient temperatare is upper machine analysis after entering in darkroom to leave standstill 5min.
The flow cytomery cell cycle: get PC-3, DU-145 single cell suspension, cell concn is adjusted to 1 × 105/mL, be inoculated in 6 well culture plates, every hole 2ml, changes liquid after cultivating 24h, sets up the RPO dosing group of control group and 10mg/mL, 30mg/mL, cultivate 24h, collect each group of cell.Fix with 75% ethanolic soln of precooling, and blow and beat into single cell suspension, at 4 DEG C, place 30min, the centrifugal 20min of 1000r/min; PBS re-suspended cell, adding PI and RNaseA to final concentration is 50 μ g/mL, 37 DEG C of water-bath 30min and lucifuge.Examination with computer, the red fluorescence at record excitation wavelength 488nm place.Each sample utilizes sub-two times of peaks and sub-G1 phase (SubG1) method to analyze apoptotic cell, measures the apoptosis rate of cell, and data processing adopts Cell Quest software analysis detected result.Annexin V-FITC/PI double-tagging flow cytometry can by normal in experiment sample, downright bad and apoptotic cell etc. separately, and cell is divided into four districts: left upper quadrant Annexin V-FITC-PI+, representative be physical abuse cell; Left lower quadrant Annexin V-FITC-/PI-, representative be normal cell; Right upper quadrant Annexin V-FITC+/PI+ is late apoptic; Right lower quadrant Annexin V-FITC+/PI-, representative be viable apoptotic cell.Figure 18 ~ 21 are the impact of PRO on PC-3 apoptosis rate, Figure 22 ~ 25 are the impact of PRO on DU-145 apoptosis rate, by the increase along with drug level Figure 18 ~ the 25 Suo Shi, early apoptosis rate and late apoptic rate rise gradually, through statistics dosing group compared with control group, difference has statistical significance (P<0.05).
After RPO acts on PC-3, DU-145 cell as shown in Figure 26 ~ 33, occur SubG1 phase peak (apoptotic peak) before G0/G1 cell cycle, peak phase, record SubG1 peak percentage of cell apoptosis is apoptosis rate.After oligopeptides effect, G0/G1 phase cellular change is little, and the S phase reduces, G2/M phase cytosis, and dosing group compares with between control group, has statistical significance (P<0.01).Wherein flow cytometry PI mono-dye detection PC-3 cell cycle finds, in control group, SubG1 peak percentage of cell apoptosis is 0.69%, as shown in figure 28; 10mg/mL group SubG1 peak percentage of cell apoptosis is 4.23%, as shown in figure 29; 30mg/mL group SubG1 peak percentage of cell apoptosis is 5.63%, as shown in figure 30.Flow cytometry PI mono-dye detection DU-145 cell cycle finds. in control group, SubG1 peak percentage of cell apoptosis is 0.66%, as shown in figure 31; 10mg/mL group SubG1 peak percentage of cell apoptosis is 3.67%, as shown in figure 32; 30mg/mL group SubG1 peak percentage of cell apoptosis is 5.74%, as shown in figure 33.
To sum up, by the known philippine clam whelp oligopeptides obtained by trypsin digestion of embodiment 3: Asp-Trp-Pro-His,
Can suppress prostate cancer PC-3 and DU-145 Growth of Cells in vitro, apoptosis-induced, change the cell cycle, make S phase Leukopenia, G2/M phase cell increases, and cells arrest is in the G2/M phase.
Unless otherwise indicated, practice of the present invention will use the routine techniques of biotechnology, organic chemistry, inorganic chemistry etc., obviously except describing especially in above-mentioned explanation and embodiment, the present invention can also be realized by other means, other technical schemes within the scope of the present invention will be apparent for those skilled in the art in the invention with improving, according to instruction of the present invention, many changes and change are feasible, therefore all within the scope of the present invention.
The peak enzymolysis-ability temperature of the different proteolytic enzyme of table 1 and pH
Table 2 four kinds of protease hydrolyzed philippine clam whelps compare
Table 3 trypsin digestion orthogonal experiments
Note: the raw material philippine clam whelp used in embodiment 1 is purchased from food market, Zhoushan;
Human prostata cancer PC-3, DU-145 cell strain used in embodiment 3, purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences, is cultivated by applicant place laboratory passage;
In each embodiment agents useful for same and key instrument information as follows:
Papoid, Sumizyme MP, neutral protease and DEAE SepharoseFF anion-exchange column (Heng Xin bio tech ltd, Asia-Pacific, Beijing); Trypsinase, MTT and propidium iodide (PI) (SIGMA company); F12 substratum (Gibco company); Foetal calf serum (Hangzhou folium ilicis chinensis bio-engineering corporation); Penicillin, Streptomycin sulphate (Lukang Medical Co., Ltd., Shandong); Reversed Phase High Performance C18 is produced by Kromasil company; (SIGMA company); AO/EB(Hangzhou Hao Tian Bioisystech Co., Ltd); The two transfection reagent box (Jing Mei biotechnology company limited) of Annexin V-FITC/PI; All the other reagent are analytical pure.
CF16RX II type high speed freezing centrifuge (HIT); BSA124S electronic balance (Sai Duolisi scientific instrument company limited); PPSQ-31A Protein Sequencer (Shimadzu Corporation, Japan).U2800 type ultraviolet-visible pectrophotometer (Shanghai Mei Puda company); The automatic Fraction Collector of BSZ-40-LCD and HD-21-88 Protein Detection instrument (Shanghai Qi Te Analytical Instrument Co., Ltd); Yi Lite P1201 type high performance liquid chromatograph (Dalian Yilite Analytical Instrument Co., Ltd); MSC300 ultrafiltration cup (ultra-filtration membrane: 3Kda, rub fast science equipment company limited in Shanghai); Forma3111CO2 incubator (Thermo company of the U.S.); CKX41 inverted phase contrast microscope and BX51 fluorescent microscope (OLYMPUS company); Super clean bench (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.); Microplate reader (U.S. BIO-RAD); FACS Calbur flow cytometer (U.S. company BD).
Claims (8)
1. a preparation method for philippine clam whelp oligopeptides, is characterized in that comprising the following steps:
1. philippine clam whelp is cleaned, add ultrapure water after getting meat and stir, then decon homogenate, obtained homogenised sample;
2. appropriate described homogenised sample is got, add the ultrapure water of appropriate volume, now the ratio of raw material and ultrapure water is 1:1 ~ 1:4, add proteolytic enzyme and carry out enzyme digestion reaction in water bath, wherein enzyme concentration is 300ug ~ 1500ug, the pH value of enzyme digestion reaction is 8 ~ 9, and enzymolysis time is 4h ~ 12h, and hydrolysis temperature is 40 DEG C ~ 60 DEG C;
3. enzyme digestion reaction terminates rear deactivation, centrifugal, to get supernatant liquor for subsequent use;
4. added in ultra-filtration membrane by described supernatant liquor and carry out ultrafiltration, collecting enzymolysis material that molecular weight is less than 3KDa, to carry out lyophilize for subsequent use;
5. utilize DEAE SepharoseFF anionresin, adopt phosphoric acid buffer and NaCl solution to carry out stepwise elution to gained enzymolysis solution, store for subsequent use after the highest peptide peak lyophilize is collected at 280nm wavelength place;
6. utilize reversed-phased high performace liquid chromatographic to be further purified at the highest peptide peak that DEAE SepharoseFF anionresin is collected, at 220nm wavelength there is unique peak in place, collects this peak, and lyophilize obtains described philippine clam whelp oligopeptides.
2. the preparation method of philippine clam whelp oligopeptides according to claim 1, is characterized in that: the ratio of described step 2. Raw and ultrapure water is 1:2.
3. the preparation method of philippine clam whelp oligopeptides according to claim 1, is characterized in that: described step 2. described in proteolytic enzyme be trypsinase, described enzyme concentration is 1200ug, and enzymolysis pH value is 8.0, and enzymolysis time is 8h, and hydrolysis temperature is 50 DEG C.
4. the preparation method of philippine clam whelp oligopeptides according to claim 1, is characterized in that: described step 3. in be deactivation 15min, the centrifugal 15min of 10000r/min at 4 DEG C in boiling water bath.
5. the preparation method of philippine clam whelp oligopeptides according to claim 1, is characterized in that: described step 5. in the concentration of NaCl solution be 0.1mol/L, 0.3mol/L, 0.5mol/L tri-kinds.
6. the preparation method of philippine clam whelp oligopeptides according to claim 1, is characterized in that: described step 6. middle chromatographic column is C
18reversed-phase column, filler is ODS, and moving phase is acetonitrile and water, and the ratio of acetonitrile and water is 15:85, and flow velocity is 0.8ml.min
-1, loading volume is 20ul, and determined wavelength is 220nm, and elution requirement is 15% acetonitrile, 20min.
7. by a philippine clam whelp oligopeptides for preparation method's gained of the philippine clam whelp oligopeptides described in the arbitrary claim of claim 1 ~ 7, it is characterized in that: the aminoacid sequence of described philippine clam whelp oligopeptides is Asp-Trp-Pro-His.
8. an application for philippine clam whelp oligopeptides as claimed in claim 7, is characterized in that: the application of described philippine clam whelp oligopeptides in anti-prostate cancer.
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| CN106854671A (en) * | 2016-10-26 | 2017-06-16 | 浙江海洋大学 | A kind of preparation method of clam antineoplastic extract |
| CN106868084A (en) * | 2017-03-23 | 2017-06-20 | 福建省水产研究所 | The preparation method and application of Ruditapes philippinarum high activity natineoplaston |
| CN106868084B (en) * | 2017-03-23 | 2020-10-09 | 福建省水产研究所 | Preparation method and application of Ruditapes philippinarum high-activity antitumor peptide |
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Application publication date: 20141217 |