CN106632599B - clam antineoplastic enzymolysis oligopeptides - Google Patents
clam antineoplastic enzymolysis oligopeptides Download PDFInfo
- Publication number
- CN106632599B CN106632599B CN201610326513.0A CN201610326513A CN106632599B CN 106632599 B CN106632599 B CN 106632599B CN 201610326513 A CN201610326513 A CN 201610326513A CN 106632599 B CN106632599 B CN 106632599B
- Authority
- CN
- China
- Prior art keywords
- enzymolysis
- clam
- oligopeptides
- oligopeptide
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 25
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 25
- 230000000118 anti-neoplastic effect Effects 0.000 title description 2
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 235000020639 clam Nutrition 0.000 abstract description 17
- 238000000108 ultra-filtration Methods 0.000 abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 3
- 241000237519 Bivalvia Species 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 230000002929 anti-fatigue Effects 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 235000013376 functional food Nutrition 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 206010060862 Prostate cancer Diseases 0.000 description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000004365 Protease Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000012757 fluorescence staining Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 241000514743 Cyclina sinensis Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- PWUMCBLVWPCKNO-MGHWNKPDSA-N Ile-Leu-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PWUMCBLVWPCKNO-MGHWNKPDSA-N 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000000315 cryotherapy Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 208000029584 urinary system neoplasm Diseases 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses clam anti-tumor enzymolysis oligopeptides, which are prepared by taking clams as raw materials through ultrafiltration and HPLC purification, wherein the amino acid sequence of the oligopeptide is Ile-Leu-Tyr-Met-Pro, the molecular weight of the oligopeptide is 635.82Da, and the oligopeptide has anti-tumor activity and can be developed as anti-fatigue functional food or health-care food.
Description
Technical Field
The invention relates to marine bioactive oligopeptides, in particular to clam antitumor enzymatic hydrolysis oligopeptides.
Background
The incidence of prostatic cancer is in the first place of male malignant tumor and is better to be found in elderly men, the incidence of prostatic cancer is on the trend of rising year by year in China, the prostatic cancer occupies the first place of urinary system tumor and seriously threatens the health of elderly men, the effects of traditional treatment methods such as operation, radiotherapy and cryotherapy are not ideal, the recurrence rate is higher, when recurrent prostatic cancer is converted into non-androgen dependence, the treatment is more difficult, after chemotherapy drugs generate drug resistance, the curative effect is worse and the toxic and side effects are obvious, therefore, the search for drugs for resisting prostatic cancer becomes a hotspot of researchers, oceans are rich resource treasury for finding novel anti-cancer drugs, various marine oligopeptides, glycosaminoglycans and the like have physiological activities of resisting tumor, resisting virus, resisting fungi and the like, clam oligopeptides Mere15 extracted from bigeminus, philippine clam cells have proliferation inhibition effects on human chronic myelogenous leukemia K562 cells and prostatic cancer cells, and lead to concentration-dependent apoptosis, the research of the intestinal tract oligopeptide, the intestinal tract protein has the function of resisting tumor, the intestinal tract, the liver, the intestinal tract, the liver, the spleen, the liver.
Disclosure of Invention
The invention aims to solve the technical problem of providing clam anti-tumor enzymolysis oligopeptides with anti-tumor efficacy.
The technical scheme adopted by the invention for solving the technical problems is as follows: the amino acid sequence of the oligopeptide is Ile-Leu-Tyr-Met-Pro (ILYMP), and the molecular weight is 635.82 Da.
The peptide was prepared as follows:
1) taking a clam homogenate, adding 200-12000U/g protease into active enzyme under the conditions of optimal enzymolysis temperature and pH value, and preserving heat for 4-10 h for enzymolysis; then inactivating, centrifuging at the temperature of 0-4 ℃ under the condition of 12000r/min, and taking the supernatant;
2) taking the best enzyme species and the supernatant fluid for enzymolysis under the best enzymolysis condition, and carrying out ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 3 ku;
3) taking the ultrafiltration component for agarose gel chromatographic separation, wherein the concentration is as follows: 0.01-0.5 g/mL, centrifuging, taking the supernatant, filtering the supernatant through a 0.22 mu m filter membrane, and eluting; collecting the eluted product, and freeze-drying;
4) and (4) separating the components by using HPLC (high performance liquid chromatography) in step to obtain the clam anti-tumor enzymolysis oligopeptide.
Compared with the prior art, the clam anti-tumor enzymolysis oligopeptides have the advantages that the process is scientific and reasonable, the operation is simple, the proliferation inhibition rate of DU-145 cells is obviously increased along with the increase of the concentration of the clam anti-tumor enzymolysis oligopeptides (CSOP) and the prolonging of the action time, the morphological change of apoptosis of the cells is shown by observation under an inverted microscope, AO/EB fluorescence staining, Hoechst 33258 fluorescence staining and transmission electron microscope technical experiments, the flow cytometry results show that the percentage of the early apoptosis rate and the mitochondrial membrane potential reduction of the cells is increased, the early apoptosis rate is obviously increased along with the increase of the CSOP concentration, the membrane potential reduction is more obvious, namely, the mitochondrial apoptosis signal path is predicted to play an important role in the CSOP-induced DU-145 cell apoptosis process.
Drawings
FIG. 1 is a schematic diagram of the molecular structure of an embodiment of the present invention;
FIG. 2 is a schematic diagram of an elution peak of an agarose gel column according to an embodiment of the invention;
FIG. 3 is a high performance liquid chromatogram of oligopeptide components at 280nm according to the example of the invention;
FIG. 4 is a graph comparing the proliferation inhibition effect of oligopeptides of the present invention on DU-145 cells;
FIG. 5 is a schematic diagram of the expression of the nm23H1 protein of DU-145 cells in the example of the present invention.
Detailed Description
The present invention is further described in with reference to the following examples.
Example (b):
the preparation process of clam anti-tumor enzymolysis oligopeptides is as follows:
1) preparation of samples
Cleaning Cyclina sinensis, removing shell, collecting viscera, soaking in 0.1mol/L NaOH solution to remove fat, removing impurities in distilled water under stirring, and adding times volume of pure water to homogenate.
2) Enzymolysis of the green clams: taking 10.0g of homogenate samples respectively, taking 5 enzymes of alkaline protease, trypsin, papain, neutral protease and pepsin as test enzyme species, adding 1800U/g of protease under the conditions of respective optimal enzymolysis temperature and pH value (shown in table 1), and preserving heat for 1-6h for enzymolysis. Inactivating for 15min after the end, centrifuging at 12000r/min for 10min at 4 ℃, and taking the supernatant.
TABLE 1 optimum temperature and pH for the different proteases
Preferably, the most preferred enzyme species is papain. The ratio of feed to liquid is 1:4, the pH value is 7.0, the enzyme adding amount is 1500U/g, the temperature is 45.0 ℃, and the enzymolysis time is 4 h.
3) And (3) separating and purifying the clam anti-tumor enzymolysis oligopeptide: and (4) carrying out next purification on the enzymolysis liquid of the molecular segment with the supernatant fluid less than 3 ku. The specific process is as follows:
taking a freeze-dried sample of the molecular segment less than 3ku to perform agarose gel chromatography, generating three peaks at 280nm, namely a peak 1, a peak 2 and a peak 3, as shown in figure 2, collecting components of each peak, and performing freeze drying. MTT method shows that peak 1 and peak 3 have better inhibitory activity to DU-145 cells. Peak 1 was collected and freeze dried. The peak 1 is the clam enzymolysis oligopeptide, the retention time of the peak is about 12min, the peak area is 16.32, the peak height is 1999.79, and the amino acid sequence of the peptide is obtained by N-terminal sequencing: Ile-Leu-Tyr-Met-Pro, molecular weight 635.82 Da.
The results of MTT after CSOP on DU-145 cells are shown in FIG. 4. The concentration of clam enzymolysis oligopeptide (CSOP) is increased, the action time is prolonged, and the increment inhibition rate IR is obviously increased. When the CSOP concentration is 2mg/mL, the action time is 24h, and the IR is 9.89 +/-0.49%; when the concentration is 15mg/mL and the acting time is 72 hours, the IR reaches 84.17 +/-4.21 percent and the IC is505.36 + -0.27 mg/mL, which is statistically significant compared to the control group (P < 0.05).
Positive expression of nm23H1 protein showed the appearance of brown structures in the cytoplasm (see FIG. 5). A normal DU-145 cells, B2mg/mL CSOP group DU-145 cells, C8 mg/mL CSOP group DU-145 cells, D12 mg/mL CSOP group DU-145 cells. The majority of the cells in the control group appeared light brown in the cytoplasm, and individual cells were negative (see A). With the increase of the CSOP concentration, the number of cells in the visual field is obviously reduced, the color of the positive part in the cytoplasm is gradually deepened, megakaryocytes appear, and part of the cell nucleus is in a solid state.
Finally, it is also noted that the above list is only specific embodiments of the present invention, obviously, the present invention is not limited to the above embodiments, but also many variations are possible.
SEQUENCE LISTING
<110> Zhejiang ocean academy
<120> clam anti-tumor enzymolysis oligopeptides
<130>zjou-yzs-01
<160>1
<170>PatentIn version 3.5
<210>1
<211>5
<212>PRT
<213> Artificial Synthesis
<400>1
Ile Leu Tyr Met Pro
1 5
Claims (1)
- The clam antitumor enzymolysis oligopeptide is characterized in that the amino acid sequence of the oligopeptide is Ile-Leu-Tyr-Met-Pro, and the molecular weight is 635.82 Da.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610326513.0A CN106632599B (en) | 2016-05-17 | 2016-05-17 | clam antineoplastic enzymolysis oligopeptides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610326513.0A CN106632599B (en) | 2016-05-17 | 2016-05-17 | clam antineoplastic enzymolysis oligopeptides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106632599A CN106632599A (en) | 2017-05-10 |
| CN106632599B true CN106632599B (en) | 2020-01-31 |
Family
ID=58848693
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610326513.0A Active CN106632599B (en) | 2016-05-17 | 2016-05-17 | clam antineoplastic enzymolysis oligopeptides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106632599B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212861A (en) * | 2013-05-29 | 2014-12-17 | 浙江海洋学院 | Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer |
-
2016
- 2016-05-17 CN CN201610326513.0A patent/CN106632599B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212861A (en) * | 2013-05-29 | 2014-12-17 | 浙江海洋学院 | Preparation method of ruditapes philippinarum oligopeptide and application in resisting prostate cancer |
Non-Patent Citations (1)
| Title |
|---|
| 抗肿瘤活性青蛤多肽的提取工艺研究;闫海强;《安徽农业科学》;20141231;第42卷(第12期);第3576-3577页及第3579页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106632599A (en) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Geng et al. | A Tricholoma matsutake peptide with angiotensin converting enzyme inhibitory and antioxidative activities and antihypertensive effects in spontaneously hypertensive rats | |
| CN103980347B (en) | Antihypertensive peptide of swim bladder of large yellow croaker and preparation method and use thereof | |
| Kim et al. | Purification and characterization of a novel anticancer peptide derived from Ruditapes philippinarum | |
| Liu et al. | Hepatoprotective effects of Antrodia cinnamomea: The modulation of oxidative stress signaling in a mouse model of alcohol‐induced acute liver injury | |
| CN104232718B (en) | The preparation method and application of dendrobium candidum antineoplastic polypeptide | |
| CN110563808B (en) | Euphausia superba antioxidant oligopeptide and preparation method thereof | |
| CN109320588B (en) | Apostichopus japonicus-derived ACE (angiotensin converting enzyme) inhibitory active peptide | |
| CN103230587A (en) | Composition of crocodile hemoglobin peptide and preparation method of composition | |
| CN105175500A (en) | Active polypeptide prepared by high performance liquid chromatography and application thereof | |
| CN101343651A (en) | Mushroom ferment pure protein with antineoplastic activity, extracting method and formulation | |
| CN111253466A (en) | Anti-inflammatory tetrapeptide, extraction and separation method thereof and application of anti-inflammatory tetrapeptide in preparation of memory improving medicines | |
| CN106632599B (en) | clam antineoplastic enzymolysis oligopeptides | |
| CN106632598B (en) | Application of clam enzymolysis oligopeptide | |
| CN110105430B (en) | Ginkgo nut protein peptide with angiotensin converting enzyme inhibitory activity and preparation method thereof | |
| CN110194786B (en) | Ginkgo nut protein peptide with ACE inhibitory activity and preparation method thereof | |
| CN107245094B (en) | Antioxidant peptide and separation preparation method and application thereof | |
| CN116813711B (en) | Jerusalem artichoke peptide capable of reducing blood sugar and resisting oxidization, preparation method and application thereof | |
| CN110183516B (en) | Preparation method of blood pressure lowering ginkgo nut protein peptide | |
| CN104928339A (en) | Preparation method for oat protein peptide with effect of inhibiting intestinal inflammatory activity | |
| JP2009191009A (en) | Low molecular substance derived from grifola frondosa having immunostimulatory activity and antitumor activity | |
| CN105646656B (en) | Griseus shark cartilage protein antioxidant peptide and application thereof | |
| CN111825754B (en) | A polypeptide obtained from sesame protein and having blood pressure lowering and blood sugar lowering activities | |
| CN104131060A (en) | Corbicula fluminea anti-oxidative peptide and preparation method thereof | |
| CN102851345B (en) | Preparation method for chlorella antitumor polypeptide | |
| KR20120079977A (en) | Anticancer composition comprising enzymatic hydrolysates of ruditapes philippinarum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee after: Zhejiang Ocean University Address before: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee before: ZHEJIANG OCEAN University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231027 Address after: 516200 Shop No. 08, 1st Floor, Unit A, Building 2, Runjing Xinyuan, Danshui Yunxin Avenue, Huiyang District, Huizhou City, Guangdong Province Patentee after: Guangdong Hairuikang Traditional Chinese Medicine Research Institute (L.P.) Address before: Room 101, Unit 1, Building 11, No. 88 Qingxi Road, Licang District, Qingdao City, Shandong Province, 266000 Patentee before: Qingdao Rongzhi Intellectual Property Agency Ltd. Effective date of registration: 20231027 Address after: Room 101, Unit 1, Building 11, No. 88 Qingxi Road, Licang District, Qingdao City, Shandong Province, 266000 Patentee after: Qingdao Rongzhi Intellectual Property Agency Ltd. Address before: 316022 No. 1, Haida South Road, Changzhi Island, Lincheng street, Zhoushan, Zhejiang. Patentee before: Zhejiang Ocean University |