TW201443075A - Extraction and purification method for enzymolysis product of Spirulina Phycocyanin - Google Patents

Abstract

The present invention discloses an extraction and purification method for enzymolysis product of Spirulina Phycocyanin, which includes the following steps: impregnating the Spirulina frond into the phosphate buffering solution under room temperature, and placing it into the refrigerator for refrigeration after the Spirulina frond is placed into the ultrasonic oscillator to be oscillated and extracted; after de-icing and thawing under normal temperature, the extraction solution is centrifuged under high speed, and the upper clear solution is separately added into the salt to carry out the protein salting-out sedimentation, wherein the precipitated protein is frozen to dry after being subject to the further centrifugation; after weighing the Phycocyanin being salting-out precipitated and dialyzed to be dissolved back in the phosphate buffering solution, adding the Proteinase K to carry out the degradation; stopping the enzymolysis reaction by reacting in the water bath after reaction is performed under room temperature; and using the gel chromatography to dialyze through the dialysis membrane to be followed by the freeze-drying to carry out the biological activity analysis to complete the method.

Description

Extraction and purification method of enzymatic hydrolysate of spirulina phycocyanin

The invention relates to a method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin, in particular to an enzymatic hydrolysate product which can significantly inhibit the growth of tumor cells in oral cells OEC-M1 and lung cancer cells (A549). Extraction and purification method of enzymatic hydrolysate of spirulina phycocyanin and preparation method thereof.

At present, studies on the biological activity and mechanism of phycocyanin have been reported, but most of them focus on phycocyanin with higher purity, and less research on phycocyanin with lower purity; and phycocyanin after enzymatic hydrolysis There are not many detailed studies on the polypeptide products of proteins against tumors. With the development and application of enzyme engineering technology, the use of protease to hydrolyze phycocyanin to obtain bioactive peptides has more research and development potential.

A phycocyanin technology, such as the method of extracting a pharmaceutical composition capable of inhibiting infection and replication of influenza virus, is disclosed in the Republic of China I272947 invention patent, which discloses a pharmaceutical composition for inhibiting influenza virus infection and replication. An effective amount of C-Phycocyanin (C-PC), Allophycocyanin (APC), Spirulina Growth Factor (SGF), or a mixture thereof. The present invention also provides a method for extracting the aforementioned pharmaceutical composition, the steps of which include a. adding organic cyanobacteria powder to a low-tension buffer, and uniformly stirring; b. standing at room temperature below room temperature overnight; c. separating and purifying the separator; Collecting the upper liquid spectrum to determine the component content; and e. freeze drying drying; characterized by using low temperature extraction to maintain the biological activity and nutrient composition of the aforementioned pharmaceutical composition.

Another conventional technique is the oral dosage form of the anti-viral infection prophylactic agent disclosed in the invention patent of the Republic of China I240631, which comprises a C-phycocyanin, an isoformin or An oral dosage form of a water-soluble formulation and an enteric formulation of any of the components of cyanobacteria alone or in combination. However, the above two prior patents did not reveal their The enzymatic hydrolysate of the extracted spirulina phycocyanin has extremely significant effects on inhibiting the growth of tumor cells in oral cells OEC-M1 and lung cancer cells (A549), and is a drawback.

In order to solve the above disadvantages of the prior art, one object of the present invention is to provide a method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin, wherein the extracted spirulina phycocyanin can be used for oral cell OEC- M1 and lung cancer cells (A549) have extremely significant inhibition of tumor cell growth.

In order to solve the above disadvantages of the prior art, another object of the present invention is to provide a method for extracting and purifying an enzymatic hydrolysate of phycocyanin from spirulina and a preparation method thereof, wherein the phycocyanin need not be purified to a high phycocyanin value ( Phycocyanin, PC value) phycocyanin, crude phycocyanin can be enzymatically digested, which will greatly increase the yield.

In order to solve the above disadvantages of the prior art, another object of the present invention is to provide a method for extracting and purifying an enzymatic hydrolysate of phycocyanin from spirulina and a preparation method thereof, wherein the extracted phycocyanin can be provided as an anti-tumor The development and application of drugs.

For the above purpose, a method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin of the present invention comprises the steps of: immersing the spirulina algae in a phosphate buffer solution (PBS) at room temperature; And placed in the ultrasonic oscillator for shock extraction, and then frozen in the refrigerator; after freezing at room temperature, the extract is centrifuged at high speed, and the supernatant is added to the salt for protein salting out and sedimentation. The protein was centrifuged and dried by freeze drying. After the salting-out sedimentation and dialysis, the phycocyanin is dissolved in the phosphate buffer solution, and then the proteinase K (Proteinase K) is added for degradation; after the reaction at room temperature, the reaction is stopped in a water bath to terminate the enzymatic hydrolysis reaction. And extracting the enzymatic hydrolysate of spirulina phycocyanin by gel chromatography, dialysis by dialysis membrane, lyophilization and bioactivity analysis.

The detailed description of the drawings and the preferred embodiments are set forth in the accompanying drawings.

Step 1: The spirulina algae body is firstly immersed in phosphate buffer solution (PBS) at room temperature, placed in an ultrasonic oscillator and shaken and extracted, and then frozen in a refrigerator; Step 2: ice thawing at normal temperature After that, the extract is centrifuged at high speed, and the supernatant is added to the salt to carry out protein salting out and sedimentation, and the precipitated protein is centrifuged and dried by freeze-drying method; Step 3: Sedimentation and sedimentation by salting out After the phycocyanin is dissolved in the phosphate buffer solution, the proteinase K (Proteinase K) is added for degradation; Step 4: after the reaction at room temperature, the reaction is stopped in a water bath to terminate the enzymatic hydrolysis reaction, and step 5: Gel chromatography, dialysis by dialysis membrane, freeze-drying and then biological activity analysis to obtain an extract of the enzymatic hydrolysate of spirulina phycocyanin.

1 is a schematic view showing the flow of a method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin according to a preferred embodiment of the present invention.

2 is a schematic view showing the flow of a method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin according to a preferred embodiment of the present invention.

3 is a schematic diagram which illustrates the present invention, three kinds of Spirulina proteolysis product F1, then F2 and F3 (80,160 and 320 μ g / mL) treatment at different doses 48 hours, human lung adenocarcinoma cells (of A549) of Schematic diagram of cell viability.

FIG 4 is a schematic diagram which illustrates the present invention, three kinds of Spirulina proteolysis product F1, then F2 and F3 (10,20,40,80,160 and 320 μ g / mL) treatment at six different doses 48 hours of Schematic diagram of cell viability of two cancer cells, OEC-M1 and A549.

FIG 5 is a schematic diagram which illustrates OEC-M1 both cancer cell A549 and the case of Spirulina after proteolysis product F3 4 doses (0-160 μ g / mL) were cultured for 48 hours, cells were sub-G1 Schematic representation of the performance.

FIG 6 is a schematic diagram which illustrates the different time points in the case of cyanobacteria F3 hydrolyzate at a dose of 80 μ g / mL, the culture OEC-M1 and both cancer cell A549, at four different time points analysis Schematic representation of its sub-G1.

7 is a schematic diagram which illustrates two OECM-1 and A54 case of DNA breaks cancer schematic expression After the case of 80 μ g / ml phycocyanin Hydrolysates F3 0,16,24 and 48 hours were treated .

8 is a schematic diagram which illustrates the case of 80 μ g / ml phycocyanin proteolysis product of F3, after 0-48 h of treatment OEC-M1 schematic and experimental results mitochondrial membrane potential after the two kinds of cancer cells A549 .

9 is a schematic diagram which illustrates the OEC-M1 both cancer cell A549, the case of phycocyanin F2 of proteolysis products, in 4 doses (0-160 μ g / ml) 48 hours after treatment - related apoptosis Schematic representation of the protein.

FIG 10 is a schematic diagram which illustrates OEC-M1 as cancer, F3 hydrolyzate case of blue algae by proteases to 80 μ g / ml treated schematic ROS its performance between 0-120 minutes.

11 is a schematic diagram which illustrates a cancer cell is A549, F3 hydrolyzate case of blue algae by proteases to 80 μ g / ml treated schematic ROS its performance between 0-120 minutes.

Please refer to FIG. 1 , which illustrates a spirulina algae egg according to a preferred embodiment of the present invention. Schematic diagram of the extraction and purification methods of the white enzymatic hydrolysate. As shown in the figure, the method for extracting and purifying the enzymatic hydrolysate of spirulina phycocyanin in the present invention comprises the following steps: immersing the spirulina algae in a phosphate buffer solution (PBS) at room temperature, and juxtaposition After being oscillated and extracted into the ultrasonic oscillator, it is then frozen in the refrigerator (step 1); after the ice is thawed at normal temperature, the extract is centrifuged at high speed, and the supernatant is added to the salt for protein salting out and sedimentation. The precipitated protein is further centrifuged and dried by freeze-drying method (step 2); the phycocyanin precipitated by salting out and dialyzed is dissolved in phosphate buffer solution, and then proteinase K (Proteinase K) is added for degradation ( Step 3); after the reaction at room temperature, the reaction is stopped in a water bath to terminate the enzymatic hydrolysis reaction (step 4); and the gel is chromatographed, dialyzed against a dialysis membrane, freeze-dried, and then subjected to biological activity analysis to obtain a spiral An extract of the enzymatic hydrolysate of algal phycocyanin (step 5).

In step 1, the spirulina algae body is firstly immersed in phosphate buffer solution (PBS) at room temperature, placed in an ultrasonic oscillator, and then vortexed and then frozen in a refrigerator; wherein, the ultrasonic wave is used in the ultrasonic wave; The oscillating extraction time in the oscillator is, for example but not limited to, 1-5 hours, and the temperature in the refrigerator is, for example but not limited to, -50 to -90 ° C, and the freezing time in the refrigerator is, for example but not limited to, 12 to 36 hours.

In step 2, after the ice is thawed at normal temperature, the extract is centrifuged at a high speed, and the supernatant is added to the salt to carry out protein salting out and sedimentation, and the precipitated protein is further centrifuged and dried by lyophilization; wherein The speed of the high speed centrifugation is, for example but not limited to, 12,000 rpm, such as, but not limited to, 30 minutes, and the temperature thereof is, for example but not limited to, 1-10 °C.

In step 3, after the salting-out sedimentation and the dialyzed phycocyanin are dissolved in the phosphate buffer solution, the proteinase K (Proteinase K) is added for degradation; wherein the weight of the phycocyanin is, for example but not limited to, 220 mg, time is for example but not limited to 30 minutes, and the weight of the proteinase K is, for example but not limited to, 16 mg.

In the step 4, after the reaction at room temperature, the reaction is stopped in a water bath to terminate the enzymatic hydrolysis reaction; wherein the room temperature reaction time is, for example but not limited to, 100 minutes, and the temperature of the water bath is, for example but not limited to, 70 ° C. The reaction time in the water bath is, for example but not limited to, 40 minutes.

In step 5, an extract of the enzymatic hydrolysate of spirulina phycocyanin is obtained by gel chromatography, dialysis by dialysis membrane, freeze-drying, and then biological activity analysis; wherein the gel chromatography system utilizes, for example, It is not limited to a column for molecular weight separation, and the extract is, for example but not limited to, phosphorus. The buffer solution of the acid salt, the flow rate of the extract is, for example but not limited to, 2.8 mL per minute, such as but not limited to collecting one tube every 3 minutes, and for example, but not limited to, separately collecting for the 5-stage degradation section, and the dialysis membrane is The molecular weight dialysis level is, for example but not limited to, 1000 Dalton cut.

The enzymatic hydrolysate F3 (Fraction 3, the third division) of spirulina phycocyanin in this case is an anti-tumor organism for two kinds of cancer cells, human oral epithelial cancer cells (OEC-M1) and human lung adenocarcinoma cells (A549). The activity analysis showed that F3 had significant inhibitory effect on human oral epithelial cancer cells (OEC-M1) and human lung adenocarcinoma cells (A549). The experimental results showed that the enzymatic hydrolysate F3 of spirulina phycocyanin in this case These two cells have the phenomenon of inducing apoptosis, but for human hepatoma cells (J5), the phenomenon of cell death is more likely to be necrosis.

The results show the case for F3 case of two kinds of inhibition of cancer cells presenting the relationship between the concentration-dependent and time-dependent; in a dose of 80 μ g / mL, dried after 72 hours of incubation, oral cancer (OEC-M1) ( Oral squamous cell carcinoma cell line) is the best in the early stage of sub-deoxyribonucleic acid (DNA) synthesis (hereinafter referred to as sub-G1). Followed by lung cancer cells (A549). A similar situation exists for the breakdown of cancer cells by DNA.

In terms of 80 μ g / mL of both cancer cell F3 treatment, able to cause loss of membrane potential of mitochondria thereof. From the above 40 μ g / mL F3 on both cancer cell of caspase -8 precursor (pro caspase-8) and precursor caspase--9 (pro The expression of caspase-9) has a significant effect, especially for oral cancer cells (OEC-M1). F3 in (80 μ g / mL) of the above dose oral cancer (OEC-M1) and PARP lung cancer cells (of A549) has a significant impact of the expression. At the same time, the cleaved polyadenylate ribose polymerase (Cleavaged PARP, PARP) oral cancer cells (OEC-M1) was the most obvious, while the lung cancer cells (A549) were not significant, while the four doses of F3 were two. The expression of the B-cell lymphoma gene 2-related X protein bax (Bcl-2 associated X) of cancer cells seems to have no effect.

In addition, F3 caused an increase in ROS in both cancer cells during the 120-minute experiment.

First, cell culture

The cell culture system of this case is the cancer cell human oral epithelial carcinoma cell (O1 cell line (OEC-M1)) and human lung adenocarcinoma obtained by the Food Industry Development Research Institute and the National Army Hualien Hospital. Epithelial cell line, A549) was cultured in a culture medium at 37 ° C, 5% CO ₂ with RPMI 1640 ( Royal Park Memorial Institute ). The culture broth contained 10% heated fetal bovine serum (HyClone, Logan, UT) and 2 mM glutamic acid (L-glutamine, Life Technologies, Inc., MD). The proteolytic products were subjected to cell test on human oral epithelial carcinoma (M1 cell line, OEC-M1) and human lung adenocarcinoma epithelial cell line (A549), respectively.

Second, cell survival analysis

The cancer cells measured during the experiment were treated with spirulina proteolytic products, and the enzymatic hydrolysate (three concentrations in addition to the blank group) was selected to determine the IC ₅₀ of cell viability. The cell viability of the spirulina protein hydrolysate was analyzed using methyl thiazolyl tetrazolium (MTT). The cancer cells were separately implanted into 96-well culture dishes, and the cell survival was observed after 24 hours (hr) of different doses of spirulina protein hydrolysate. After the cells were first washed twice with phosphate buffered solution (PBS), was added at a concentration of 1 mg / mL of MTT 100 μ L at 37 ℃, and incubation continued for 2 hr, then was added 100 μ L of cell lysis buffer (Lysis Buffer) dissolves cells. After 30 minutes (min) of incubation, the absorbance at wavelengths of 550 and 690 nm was measured spectrophotometrically.

Third, cell cycle analysis

Propidium Iodide (PI) is a fluorescent dye of double-stranded DNA. The combination of propidium iodide and double-stranded DNA produces fluorescence, and the fluorescence intensity is directly proportional to the amount of double-stranded DNA. After the intracellular DNA is stained with propidium iodide, the DNA content of the cells can be measured by flow cytometry, and then the cell cycle and apoptosis analysis can be performed according to the distribution of the DNA content. The cancer cells determined by this plan were analyzed at four fixed time points (three concentrations) of spirulina proteolytic products, and four time points (in addition to the starting point, there were three different time points) to flow cytometry. Perform cell cycle and apoptosis analysis.

1, cells to be detected to adjust the concentration of ¹⁰⁶ / mL, and take 200 μ L, was centrifuged at 4 ℃ 1000 rpm × 5 min.

2. Rinse twice with 1 mL of pre-cooled PBS solution (phosphate buffer solution) and centrifuge at 1000 rpm for 5 minutes at 4 °C.

3, cells were suspended in 100 μ L binding buffer (binding buffer), was added 2 μ L of Annexin -V- fluorescein isothiocyanate (Annexin-V-FITC, 20 μ g / mL), gently Mix well and leave it on ice for 15 minutes.

4, go to the flow sensing tube, was added 400 μ L PBS, was added 1 μ L PI (50 μ g / ml) of each sample before the temporary machine quickly detected after 2 minutes.

5. At the same time, a tube without Annexin V-FITC and PI was used as a negative control, and the propidium iodide staining solution was prepared (currently used).

6. Statistical analysis: Mean ± standard deviation of data statistics; ANOVA analysis was used to compare the mean between groups.

Fourth, cell cycle analysis

(A) TUNEL apoptosis analysis

The apoptosis assay was performed by selecting the optimal concentration by cell viability analysis and cycle analysis.

Terminal deoxyribonucleic acid transferase-mediated in situ nick end labeling (TUNEL) apoptosis detection is a highly sensitive, rapid and simple method for detecting apoptosis. For cells and tissues that have been fixed and washed, apoptotic cells that exhibit green fluorescence can be detected by flow cytometry after one-step staining reaction.

When cells undergo apoptosis, they initiate some endonuclease enzymes that cleave genomic DNA between nucleosomes. When DNA is extracted for electrophoresis, a DNA ladder of 180-200 bp can be found. When genomic DNA is cleaved, the exposed 3'-OH can be catalyzed by terminal deoxynucleotidyltransferase (TdT) and added with green fluorescent probe luciferin (FITC, fluorescein isothiocyanate). The labeled pyrophosphatase (fluorescein-dUTP) can be detected by flow cytometry.

Preparation of reagents

1, buffer deoxy deoxynucleotidyl transferase (TdT buffer) to draw BM (Boehringer Mannheim, BM Boehringer Mannheim) of 5-fold concentrated buffer solution 200 μ L, CoCl ₂ (cobalt chloride) 1000 μ L, TdT 8 μ L, Biotin-dUTP 10 μ L of distilled water and 682 mL. Avidin green fluorescent probe fluorescein biostain (FITC-avidin staining) buffer solution.

2, suction aqueous sodium citrate (saline sodium citrate, referred to as SSC) 20-fold concentrated solution 20 mL, polyethylene glycol octylphenyl ether X-100 (Triton X-100 ) 100 μ L, skim milk powder 5g, distilled water 80 mL, mg / mL, FITC -avidin 5.0 μ L / mL.

fixed

1. Cells (2x10 ⁶ ) treated with spirulina proteolytic products were fixed on ice for 1 minute by adding 1% formalin (deformadehyde).

2. The cells were washed with PBS.

3. The cells are suspended in 70% ethanol at -20 ° C and stored for 1-3 days.

TdT reaction

1. The fixed cells were washed with PBS.

2, cells were suspended in 50 μ L deoxy terminal deoxynucleotidyl transferase (of TdT) buffer (containing 5U (units) of TdT calibration and 0.5 nmol of biotin (biotin labeled) dUTP.

3. Place at 37 ° C for 60 minutes.

4. Wash the cells with PBS.

5, cells were suspended in 100 μ L of avidin biotin green fluorescent dye fluorescent probe (FITC-avidin staining) buffer solution.

6. Leave at room temperature for 30 minutes.

7. Wet with PBS (containing 1% Triton X-100, polyethylene glycol octyl phenyl ether).

8, cells were resuspended in 1 mL PBS containing PI (5 μ g / mL) and RNase (RNA hydrolases 100 μ g / mL).

9. Place on ice for 30 minutes.

10. Upflow cytometry analysis.

The cancer cells measured in this case were treated with a spirulina proteolytic product at a fixed concentration, and analyzed at four time points (in addition to the starting point, there were three different time points), and the cell cycle and cells were performed by flow cytometry. Apoptosis analysis.

(B) Granulocyte apoptosis-related protein expression

Many B-cell lymphoma-2 (Bcl-2) gene family proteins bind to the membrane of the mitochondria and form many pores on the surface of the mitochondria, and the formation of these pores causes mitochondrial interstitial release. Cytochrome-c and apoptosis-inducing factor (AIF) deactivate caspase and cause a series of apoptosis. Therefore, the mitochondria are called an apoptosis performer. During the respiratory oxidation process, the mitochondria store the generated energy in the inner membrane of the mitochondria (about 250 mV/5~10 nm), which is called the mitochondrial membrane potential. And this position can be used to carry out the electron transport chain, and finally produce adenosine triphosphate (ATP) for cell use. This potential difference is formed in the inner membrane of the mitochondria, which is in the form of an outer positive and negative. Many lipophilic cationic compounds bind to the inner membrane of the mitochondria and emit fluorescence under laser excitation. Now we can easily observe the morphology and movement of the mitochondria through flow cytometry.

The cancer cells measured in this case were analyzed at four time points after fixing a concentration of spirulina proteolytic products (in addition to the blank group, there were three different time points), and the granulocyte apoptosis was performed by western blot test. Related protein expression.

(C) mitochondrial membrane potential analysis

Normally viable cells have a certain physiological membrane potential (Δm), and when protons flow back from the mitochondrial membrane space back into the matrix, another protein complex molecule produces ATP. All of these processes must be carried out in an orderly manner. If cytochrome-c (Cyt-c) is absent or its dysfunction, it will lead to dysfunction of the mitochondrial respiratory chain, resulting in ATP deficiency and cell death. The release of Cyt-c cytochrome-c from mitochondria to the cytosol is a critical step in triggering apoptosis in a variety of cell death models.

The cancer cells measured in this case were analyzed at four time points after fixing a concentration of spirulina proteolytic products (in addition to the blank group, there were three different time points), and the mitochondrial membrane potential analysis was performed by flow cytometry. .

1. DiOC ₆ cell membrane fluorescent probe (3,3-Dihexyloxacarbocyanine iodide; 3,3'-dihexyloxycarbocyanine iodide) (2.5 mg/ml dissolved in methanol, molecular probe).

2. Wash the cells (1x10 ⁶ ) with PBS (phosphate buffer solution).

3, cells were suspended in PBS (phosphate buffered solution) was added 4 μ L DiOC _6.

4. Keep away from light for 30 minutes at 37 °C.

5. Upflow cytometry analysis.

V. Western ink test

The cancer cells measured in this case were analyzed by Spirulina proteolytic products at four concentrations, and the following antibodies were analyzed including Bcl-2, Bax, Bak, cytochrome c, Caspase 3, Caspase 8, Caspase 9 and PARP. The content of the act, as well as the actin content of the cells.

(A) Cellular protein collection:

The cancer cells were cultured at 37 ° C in a culture medium, and a specific spirulina proteolytic product was added to treat the sample. After different experimental conditions, the cells were added to a phosphate-buffer saline containing salt-containing phosphate buffer solution (5 mM). Na ₃ VO ₄ .), terminates its reaction. The cells were immediately collected and placed at 4 ° C, and the cells were lysed with 300 μl of lysis buffer (the formulation of the cell lysate was as follows: 20 mM Tris pH 7.4, 150 mM NaCl (sodium chloride), 1 mM ethylenediaminetetraacetic acid (Ethylene) Diamine Tetraacetic Acid (EDTA), 1% Triton, 205 mM pyrophosphate, 1 mM glycophosphate, 1 mM Na ₃ VO ₄ , 2 μg/ml leupeptin Leupeptin) and 1 mM Phenylmethanesulfonyl fluoride (PMSF) were dissolved and placed on ice for 10-15 minutes. The insoluble material was separated by centrifugation at 1,2000 x g for 15 minutes at 4 °C. The protein concentration is quantified by protein electrophoresis chromatography.

Sodium-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): The collected protein samples were separated by 10% SDS-PAGE. After electrophoresis, the protein was converted to a PVDF filter (Millipore Inc, Bedford, MA), and the converted filter was placed in 5% fat-free milk-PBS for 1 hour at room temperature (containing 0.1% polysorbate). Ester-20 (tween 20)).

(B) Compressed antibodies:

The above filters were placed at room temperature with anti-Bcl-2, anti-Bax, anti-Bak, anti-cytochrome c, anti-Caspase 3, anti-Caspase 8, anti-Caspase 9 and anti-PARP. Antibody for 2 hours. Rinse three times (10 minutes each) with gentle shaking with PBS (containing 0.1% tween 20). Thereafter, the conjugated wasabi catalase (HRP-Conjugated) secondary antibody is directly pressed onto the primary antibody, and the procedure is the same as above. Finally, a fluorescent reaction is initiated at the beginning of enhanced chemiluminescence, and the tableting step is operated in a darkroom.

Determination of intracellular ROS by spirulina proteolytic products

In this case, the content of reactive oxygen molecules in the cells of human oral epithelial cancer cells (OEC-M1) and human lung adenocarcinoma cells (A549) was determined by spirulina proteolytic products.

Seven, the experimental results

Referring then to Figure 2, which illustrates the present invention, three kinds of Spirulina proteolysis product F1, F2 and F3 treatment at different doses (80, 160 and 320 μ g / mL) 48 hours, the human oral epithelial cancer (OEC-M1) Schematic diagram of cell viability. As shown, the case of three kinds of Spirulina blue Hydrolysates F1, F2 and after F3 (80 μ g / mL, 160 μ g / mL to 320 μ g / mL) treatment at different doses 48 hours the human oral cavity Cell viability of epithelial cancer cells (OEC-M1). The results showed that the phycocyanin proteolytic product F1 showed no toxic effect on oral cancer cells at three doses, while F2 and F3 showed better cytotoxicity to oral cancer cells.

Referring to FIG 3, which illustrates the present invention, three kinds of Spirulina proteolysis product F1, F2 and F3 after different doses in 48 hours (80, 160 and 320 μ g / mL) treatment, human lung adenocarcinoma cells (of A549) cells of Schematic diagram of survival rate. As shown in the figure, the three spirulina proteolytic products F1, F2 and F3 in this case were cultured for three hours under the three different doses. After 48 hours of co-culture for the three cancer cells, the results showed that the spirulina proteolytic product F1, for the two cancer cells There is almost no inhibition of growth. Hydrolysates phycocyanin and F3, is displayed with the best growth inhibitory effect on the human lung adenocarcinoma cells (A549), at 80 μ g / mL dose reached IC ₅₀ (only 48% cell viability); and A similar phenomenon occurs in the phycocyanin proteolytic product F5. Followed by the human oral cavity epithelial cancer (OEC-M1), when / mL dose of 80-320 μ g, cell viability between about 68-57%.

After Referring to Figure 4, which illustrates the present invention, three kinds of Spirulina proteolysis product F1, F2 and F3 at six different doses (10,20,40,80,160 and 320 μ g / mL) for 48 hours, OEC Schematic diagram of cell viability of both M1 and A549 cancer cells. As shown in the figure, the inhibitory effect of Spirulina proteolytic product F3 on the growth of the two cancer cells (OEC-M1 and A549) for 48 hours was compared. Dose, respectively (10,20,40,80,160 and 320 μ g / mL), the optimal concentration was found to inhibit both cancer cell to be 80 μ g / mL (greatest negative slope), so that subsequent phycocyanin F3 test concentration of protein hydrolysates are 80 μ g / mL.

Referring to FIG 5, which illustrates the OEC-M1 both cancer cell A549 and the case of Spirulina after proteolysis product F3 4 doses (0-160 μ g / mL) were cultured for 48 hours, the cells in sub-G1 Performance diagram. As shown, in this case in order to observe whether the cyanobacteria hydrolyzate F3 cause apoptosis and cell cycle effects on both cancer cells through F3 4 of doses (0-160 μ g / mL) were cultured for 48 hours after, F3 found that increasing the concentration of SUI, at high concentrations (160 μ g / mL) will cause oral cancer (OEC-M1) and lung cancer cells (A549), sub-G1 appears. Oral cancer (OEC-M1), in a dose of 40 μ g / mL, the sub-G1 about 2.1%, at a dose of 80 μ g / mL, the sub-G1 about 8.8%, but at a dose of 160 μ g / mL of the, sub-G1 up to 17.5%.

For lung cancer cells (of A549), at a dose of 40 μ g / mL, the sub-G1 only 1.4% at a dose of 80 μ g / mL, the sub-G1 about 2.3%, but in 160 μ At a dose of g/mL, the sub-G1 of lung cancer cells (A549) can be as high as 19.8%. The higher the proportion of sub-G1, the more obvious the appearance of apoptosis in cells.

Referring to FIG 6, which is shown at different time points in the case of cyanobacteria F3 hydrolyzate at a dose of 80 μ g / mL, the culture OEC-M1 both cancer cell A549 and analyzed in four different time points Schematic representation of the performance of sub-G1. As shown later, while the case cyanobacteria hydrolysis product F3 at different time points at a dose 80 μ g / mL of both cancer cell cultured for 48 hours at 4 different time points analyzed which both cancer cell sub -G1 ratio. The results obtained are as follows: oral cancer (OEC-M1), in a dose of 80 μ g / mL, and about 3.1% of its culture sub-G1 after 24 hours, cultured for 48 hours after which about sub-G1 It accounted for 6.6%, and its sub-G1 accounted for about 36.9% after 72 hours of culture.

After For lung cancer cells (of A549), at a dose of 80 μ g / mL, and after 24 hours, after which the culture sub-G1 about 1.6%, for 48 hours, followed by sub-G1 about 5.8%, which is 72 hours Sub-G1 accounts for about 15.4%. The experimental results show that after this case, F3 cyanobacteria hydrolyzate at a dose of 80 μ g / mL, and cultured for 72 hours, the resulting effect of the best sub-G1 oral cancer (OEC-M1). Followed by lung cancer cells (A549).

In terms of a concentration (80 μ g / mL) of the product of proteolysis phycocyanin F3 OEC-M1 cells stimulated after 0-72 hours, the results show that with prolonged culture time, a significant increase in the range of apoptosis zone (36.9 %), and the position drifts significantly, especially after 72 hours. After the occurrence of apoptosis, in this experimental mode, the DNA content of apoptotic cells is at a relatively constant value. As the apoptosis time is longer, the intracellular DNA content is lower, and the Sub-G1 peak shift occurs. . As for A549 cells, it was 15.4% at 72 hours.

Referring to FIG 7, which illustrates a schematic diagram OECM-1 expression and DNA breaks the case of both cancer cell A549 After the case of 80 μ g / ml phycocyanin Hydrolysates 0,16,24 and F3 were treated for 48 hours. As shown, the increase it can be found after two OECM-1 and A549 cancer cell F3 are processed through 80 μ g / mL phycocyanin Hydrolysates 0,16,24 and 48 hours of DNA fragmentation phenomenon, we can clearly It is seen that the peak shifts to the right (representing an increase in the amount of fluorescence, meaning that intracellular DNA fragmentation increases), while J5 cells have no DNA fragmentation, and the peak shift to the left should be caused by TdT staining itself.

Referring to FIG. 8, which illustrates the case of 80 μ g / ml phycocyanin proteolysis product of F3, after 0-48 h of treatment with OEC-M1 schematic A549 particles both cancer cell lines after membrane potential results. As shown in the figure, after phycocyanin proteolytic product F3, OECM-1 has a significant membrane potential loss after 0-48 hours of treatment, and the peak shifts to the left (especially after 48 hours of treatment), while A549 has similar The result, but not as significant as OEC-M1, may be caused by F3 causing cell creases and rupture of the mitochondrial membrane, which in turn causes the membrane potential to disappear.

Referring to FIG 9, which illustrates the OEC-M1 both cancer cell A549, F2, 4 doses (0-160 μ g / ml) after 48 hours treatment of apoptosis-related proteins was phycocyanin Hydrolysates case of Schematic representation of the performance. Shown, F2 hydrolyzate of cyanobacteria in this case, from the above of 40 μ g / mL doses of oral cancer (OEC-M1) and lung cancer cells (of A549) of pro caspase-8 and pro caspase-9 in FIG. The expression has a significant effect, especially for oral cancer cells (OEC-M1).

Caspase-3 is frequently activated in different mammalian cell apoptosis processes Protease. In general, many pathways of apoptosis are initiated by receptors on the cell surface. Although there are at least 14 caspases in the human body, which are applied to different cells that are sufficient to kill, respectively, only a few caspase can detect proteolytic enzymes. However, caspase-3 precursors are heteromeric enzymes that are frequently present and activated by proteolytic hydrolysis to produce activity.

Caspase-3 is important for survival and is required for many apoptotic processes. For example, caspase-3 knockout mice have a low birth rate and die within a few weeks. These mice have a phenotypic defect in their brain. There are a large number of ectopic excess cells, which are caused by the inability to apoptosis during brain development. But this is not the case in other organs and tissues.

Caspase-3 is essential for the apoptosis of some cells, whereas the apoptosis of all cell types in the tested caspase-3 deficient mice lacks typical apoptotic markers (especially chromosome condensation and cleavage). .

In the case of cyanobacteria hydrolyzate F3 oral cancer cells (OECM) and lung cancer cells (of A549) at concentrations of 160 μ g / mL of pro caspase-3 expression of a slightly reduced. While caspase-3 cleavage of oral cancer (OEC-M1) and lung cancer cells (of A549) at 40-160 μ g / mL dose of already arisen, especially in oral cancer (OEC-M1) is obvious.

Hydrolysates phycocyanin in F3 (80 μ g / mL) of the above doses of oral cancer cells (OECM) and lung cancer cells (of A549) as much poly ADP-ribose polymerase (PARP) has a significant impact on the expression case . At the same time, the dividing PARP oral cancer cells (OECM) were most obvious, while the lung cancer cells (A549) were not significant.

In 1972, Kerr et al. first proposed the concept of apoptosis (Kerr et al., 1972). Recent studies have shown that Apoptosis has received attention as an important phenomenon in cancer biology. In 1987, Fesus et al. first studied the gene expression and regulation of transglutaminase during apoptosis by molecular biology techniques. The relationship between gene expression and apoptosis has been confirmed so far (Bowen, 1993). . A variety of genes are known to be involved in the regulation of apoptosis, and they play different roles in the regulation of apoptosis. In 2001, Chen et al. published the study on the expression of apoptosis-related genes bcl-2 and bax in oral cancer.

Bcl-2 is a 26kD protein molecule that was first discovered in non-Hodgkin lymphoma (NHL). When the chromosomal translocation is initiated, during this translocation, the bcl-2 gene is transferred from the normal chromosomal location (18q21) to the immunoglobulin. Beside the heavy chain (IgH) site (14q32), adjacent to a strong promoter sequence, the translocated bcl-2 gene is deregulated, overexpressing a large amount of bcl-2 gene mRNA, and encoding an excess Protein molecules (Hockenbery, et al., 1990).

In 1988, Vaux et al first reported that bcl-2 promoted cell survival (Liu et al., 1991). Vaux et al. injected the bcl-2 expression plasmid into nerve growth factor (NGF)-dependent sympathetic neurons. After bcl-2 expression, the removal of neurotrophic factors from the culture medium can delay the process of apoptosis. The expression of bcl-2 protein by gene transfer method can promote cell survival; on the contrary, blocking bcl-2 to decrease or disappear its expression can promote apoptosis of these cells after growth factor elimination.

It is currently believed that bcl-2 is a major regulatory gene that inhibits apoptosis. In Chen et al's experiment, it was found that bcl-2 positive cells were more common in stage I and II oral cancer, and the positive expression rate of stage I and II bcl-2 was higher than that of stage III and IV, and it was highly differentiated in pathological grade. The positive expression rate of the moderately differentiated group was also higher than that of the poorly differentiated group, but the expression of bcl-2 was not associated with the metastasis of cervical lymph nodes. The expression of bcl-2 was related to the degree of differentiation of cancer cells and the stage of carcinogenesis.

Inferred from this, overexpression of bcl-2 may be required for early canceration. Bcl-2 belongs to the class of anti-apoptotic genes. Its overexpression can reduce the apoptosis of epithelial cells, prolong the life span of cells, and increase the number of cancerous cells in tissues. However, with the accumulation of cancerous cells, the growth viability of advanced tumor cells decreased, and the expression ability of some oncogenes related to the maintenance of cell life decreased. On the other hand, after the tumor cells enter the late stage, the protein products of various oncogenes are produced and accumulated in large quantities, and the expression of the bcl-2 gene can also be inhibited.

Found in the case of cyanobacteria hydrolyzate F3 4 of doses (40-160 μ g / mL) bcl -2 expression in oral cancer (OECM) of a decrease in the tendency. And lung cancer cells (of A549) at 4 doses (40-160 μ g / mL), its expression of bcl-2, there are significantly reduced.

Bax is a homologous analog of bcl-2 and is a 21 kD protein consisting of 192 amino acid residues. The bax protein has a transmembrane site with 21% homology to bcl-2 and may bind to bcl-2 to form a heterodimeric form, an inactive form of bcl-2. Although bax is structurally similar to bcl-2, its function is opposite. Bax acts against bcl-2 and promotes apoptosis (Yin, et al., 1997).

At present, we know that the expression of bax gene is related to the degree of differentiation of cancer cells. As the degree of differentiation decreases, the positive expression rate also decreases. The Bax gene is a pro-apoptotic gene. In highly differentiated tumor cells, the biological behavior of cells is similar to that of normal cells. The host may induce spontaneous tumor cell apoptosis through overexpression of bax gene. The bax expression rate decreases as the degree of differentiation decreases.

However, in the experiment of this case, the four doses of the cyanobacterial hydrolysate F3 did not seem to affect the expression of the bax protein of the three cancer cells. Bcl-2 and bax were more expressed in early differentiated tumor cells, and the expression rates of bcl-2 and bax were also decreased with the decrease of differentiation. Cyanobacteria hydrolyzate F3 while 4 of doses have little effect on three kinds of cancer cells of Cytochromec only at 160 μ g / mL for oral cancer cells, a decrease in the expression of the phenomenon is generated.

Reactive oxygen species (ROS) are hydrogen peroxide (H ₂ O ₂ ), hydroxyl radicals (OH.), and superoxide anions (O ₂ ^- ). Both glutathione reductase and catalase can scavenge reactive oxygen species. Mitochondria, membrane lipids, proteins, and nuclear genes can all be targets of reactive oxygen species attack.

Reactive oxygen species attack unsaturated fatty acids in biofilm phospholipids to form lipid peroxides; they can also interact with bases or deoxyribose in DNA molecules to cause DNA molecular chain breaks; in addition, they can interact with protein molecules to cause proteins. Deuterium destruction, protein cross-linking, inactivation of biologically active molecules. In conclusion, reactive oxygen species can induce apoptosis, and many physical and chemical factors that induce hepatocyte apoptosis can also cause oxidative stress.

Active oxygen is mainly derived from mitochondria. In the cell, 95%-98% of molecular oxygen is consumed by mitochondria to produce water, and 2% to 5% form superoxide and hydrogen peroxide through the electron transport chain (Jacobson and Duchen 2002). Any stimulus that reduces the effect of the electron transport process can increase the formation of superoxide anion and lipid peroxide.

These produced reactive oxygen species can further inhibit the respiratory chain, accelerate the production of superoxide, open the MPTP pores on the mitochondrial membrane, and simultaneously initiate the release of calcium from the endoplasmic reticulum calcium pool, leading to calcium accumulation in the mitochondria, which further increases reactive oxygen species. The generation, amplification of oxidative stress stimulation and calcium loading triggers a vicious circle.

Therefore, mitochondrial reactive oxygen species and calcium loading promote the opening of MPTP pores. In addition, some thiol oxidative mitochondria can also trigger the opening of MPTP pores, causing ATP depletion, energy production disorders, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3. In addition to mitochondria, Lysosomes and endoplasmic reticulum are also important sources of reactive oxygen species.

Mueller et al. (Mueller, et al., 2002) demonstrated that some membranous organs in the cell, such as the endoplasmic reticulum/Golgi, are capable of producing an apoptotic initiation signal, which then acts on the mitochondria to cause electron transport chains to be disturbed to produce large amounts of reactive oxygen species. And this initial signal is the active oxygen produced by the endoplasmic reticulum/Golgi matrix. The accumulation of reactive oxygen species in a single mitochondria causes mitochondrial depolarization, which increases the violent increase in reactive oxygen species (Jacobson and Duchen 2002).

If the stress stimulation of the endoplasmic reticulum persists, the unfolded protein response of the cells regulates the oxidative folding mechanism in the endoplasmic reticulum, causing the accumulation of reactive oxygen species to cause cell death. It can be seen that mitochondria, endoplasmic reticulum, lysosomes and other organelles can produce reactive oxygen species, and a small amount of active oxygen accumulation triggers a series of waterfall-like reactions, resulting in a vicious circle.

The role of oxidative stress stimulation is mitochondria and the calcium storage of the endoplasmic reticulum. Lewen et al. (Lewén, et al., 2000) demonstrated the production of reactive oxygen species in a mouse model of hypoxia-ischemia, the endoplasmic reticulum calcium pump is sensitive to oxidative damage, and the endoplasmic reticulum protein is oxidatively modified. The endoplasmic reticulum may be one of the important targets of reactive oxygen species attack.

After hepatic ischemia and hypoxia, reactive oxygen species can oxidize and retain proteins in the lumen of the endoplasmic reticulum and remain in the lumen of the endoplasmic reticulum, thereby inhibiting protein synthesis, endoplasmic reticulum calcium depletion and endoplasmic reticulum molecules. Companion production (DeGracia, et al., 2002).

There is an RNA-dependent protein kinase eIF-2a kinase/PERK on the endoplasmic reticulum membrane. Normally, the endoplasmic reticulum molecule binds to GRP78 on PERK to inhibit its phosphorylation. Under the action of reactive oxygen species, the protein in the lumen of the endoplasmic reticulum is oxidized and retained in the lumen of the endoplasmic reticulum. When the unfolded protein in the endoplasmic reticulum accumulates, GRP78 is dissociated from PERK and combined with non-folded protein. Phosphorylation occurs when PERK that loses GRP78. Phosphorylated PERK phosphorylates elf-2a, and once eIF-2a is phosphorylated, it loses its activity and inhibits protein synthesis, and phosphorylated eIF-2a activates a transcription factor that reduces Bcl-2, Caspase-12 Activation leads to apoptosis.

Therefore, the first step in the endoplasmic reticulum-mediated apoptosis signal is phosphorylation of extracellular signal-regulated kinase (PERK) phosphorylation. Studies have shown that SOD overexpression in copper/zinc SOD transgenic mice and the antioxidant N-acetylcysteine can prevent this damage (Hayashi, et al., 2003). The superoxide signal is generated in the same way as the PERK phosphorylation site, indicating endoplasmic reticulum stress thorn Super-oxidation in the stimuli plays an important role.

Dissociation of glucose-regulated protein 78 (GRP78) from PERK means an increase in unfolded proteins in the lumen of the endoplasmic reticulum. SOD has an antioxidant effect, which indicates that oxidative destruction of proteins leads to misfolding of proteins. When the endoplasmic reticulum stress stimulation is not severe, the eukaryotic translation initiation factor 2-alpha (eIF-2α) phosphorylation inhibitory protein synthesis initiation factor reduces the unfolded protein. The amount, on the other hand, reduces the load on the endoplasmic reticulum and therefore has a cytoprotective effect; however, when the endoplasmic reticulum stress is severely stimulated, phosphorylation of eIF-2α activates the apoptotic program (DeGracia, et al., 2002). .

There is sufficient evidence that clearance of ROS inhibits the activation of cell death-associated proteins that inhibit eukaryotic Initiation Factor-2 alpha (eIF-2 alpha) phosphorylation and glucose regulatory proteins. -78 (Glucose regulated protein-78, GRP78) production, activation of caspase-12 to inhibit apoptosis induced by endoplasmic reticulum stress stimulation. N-acetylcysteine (NAC) is a precursor of sulfhydryl synthesis and has a direct inhibitory effect on oxygen free radical reactions. N-acetylcysteine prevents the increase of cytoplasmic calcium by inhibiting the production of reactive oxygen species, inhibits JNK (Jun N-terminal kinase), Caspase-3, -7 activation, and also inhibits DNA fragmentation factor 45 cleavage. , thereby inhibiting apoptosis.

Referring collectively to FIG. 10 and FIG. 11, FIG. 10 which shows OEC-M1 as cancer cells, in this case the phycocyanin by proteolysis product F3, to 80 μ g / ml of ROS its processing performance between 0-120 minutes a schematic diagram; FIG. 11 shows the cancer cells of A549, the case of phycocyanin Hydrolysates F3, to 80 μ g / ml treated schematic ROS its performance between 0-120 minutes. , In the case of the experiment (80 μ g / mL) dose of cyanobacteria hydrolyzate F3, 120 minutes during the experiment in expression OEC-M1 both cancer cell A549 and the ROS than the control group of FIG. A lot of improvement, while adding NAC can inhibit the production of cellular ROS, the results of this study are consistent with the results of previous studies.

The disclosure of the present invention is a preferred embodiment. Any change or modification of the present invention originating from the technical idea of the present invention and being easily inferred by those skilled in the art will not deviate from the scope of patent rights of the present invention.

In summary, this case shows its difference in terms of purpose, means and function. In the technical characteristics of Xizhi, and its first invention is practical and practical, it is also in compliance with the patent requirements of the invention. Please ask your review committee to inspect it and pray for an early patent.

Claims (7)

A method for extracting and purifying an enzymatic hydrolysate of spirulina phycocyanin, comprising the steps of: immersing spirulina algae in phosphate buffer solution (PBS) at room temperature, and placing it in an ultrasonic oscillator After extraction, it is then frozen in a refrigerator; after thawing at room temperature, the extract is centrifuged at high speed, and the supernatant is added to the salt for protein salting out and sedimentation, and the precipitated protein is centrifuged again to freeze-dry Drying and quantification; weighing and dialysis phycocyanin after salting out and dialysis is dissolved in phosphate buffer solution, and then adding proteinase K (Proteinase K) for degradation; firstly reacting at room temperature, and then stopping in the water bath The enzymatic hydrolysis reaction is carried out by gel chromatography, dialysis by dialysis membrane, freeze-drying, and bioactivity analysis to obtain an extract of the hydrolyzed product of spirulina phycocyanin. The method of extracting and purifying according to claim 1, wherein the spirulina algae is first immersed in a phosphate buffer solution at room temperature, placed in an ultrasonic oscillator, and then subjected to shock extraction, and then placed. In the step of freezing in the refrigerator, the shock extraction time in the ultrasonic oscillator is 1-5 hours, the temperature in the refrigerator is -50 to -90 ° C, and the freezing time in the refrigerator is 12-36 hours. The method for extracting and purifying according to claim 1, wherein after the ice is thawed at normal temperature, the extract is centrifuged at a high speed, and the supernatant is separately added to the salt for protein salting out and sedimentation, and the precipitated protein is precipitated. After centrifugation, the step of drying and quantifying by freeze-drying was carried out at a high speed centrifugation speed of 12,000 rpm for 30 minutes and at a temperature of 1-10 °C. The method for extracting and purifying according to the first aspect of the invention, wherein the scale is precipitated and the dialyzed phycocyanin is dissolved in a phosphate buffer solution, and then the proteinase K is added for degradation. The weight of the phycocyanin was 220 mg for 30 minutes and the weight of the proteinase K was 16 mg. The method of extracting and purifying according to the first aspect of the invention, wherein in the step of reacting at room temperature and then stopping the enzymatic hydrolysis reaction in a water bath, the room temperature reaction time is 100 minutes. The temperature of the water bath was 70 ° C, and the reaction time in the water bath was 40 minutes. The method for extracting and purifying according to claim 1, wherein the spirulina phycocyanin is obtained by gel chromatography, dialysis by dialysis membrane, freeze-drying, and then biological activity analysis. In the step of extracting the enzymatic hydrolysate, the gel chromatography system utilizes a column of molecular weight separation, the extract is a phosphate buffer solution, the flow rate of the extract is 2.8 mL per minute, and a tube is collected every 3 minutes. The 5-stage degradation sections were collected separately. The method for extracting and purifying according to claim 6, wherein the extraction of the enzymatic hydrolysate of spirulina phycocyanin is carried out by gel chromatography, dialysis by dialysis membrane, freeze-drying and then biological activity analysis. In the step of the substance, the dialysis membrane has a molecular weight dialysis degree of 1000 Dalton (1000 Dalton) cut.