CN108659111A - Scorpion activated protein - Google Patents
Scorpion activated protein Download PDFInfo
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- CN108659111A CN108659111A CN201810473979.2A CN201810473979A CN108659111A CN 108659111 A CN108659111 A CN 108659111A CN 201810473979 A CN201810473979 A CN 201810473979A CN 108659111 A CN108659111 A CN 108659111A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/646—Arachnids, e.g. spiders, scorpions, ticks or mites
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/06—Antimigraine agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses scorpion activated protein, the extracting method of the activated protein is：Add water, shikimic acid and p-Coumaric Acid into dry scorpio coarse powder, impregnate, is centrifuged after soybean dietary fiber, supernatant is extracting solution；The pH of extracting solution is adjusted, then ultrafiltration, trapped fluid washing is clean, and concentration is spare；Saturated ammonium sulfate solution is added into concentrate, static, the sodium phosphate buffer of centrifugation equivalent fully dissolves, and dialysis, dialyzate is up to crude protein solution；Crude protein solution is purified with cellulose column, dextran gel filtration successively, is freeze-dried to get scorpion activated protein.It has the beneficial effect that：Activity of active protein of the present invention and purity are high, and containing more protein species, gain effect is played between each activated protein, improve the pharmacological action of activated protein, and the activated protein is not easy body immunogenicity and allergic reaction occur, reduces side effect, has preferable development prospect.
Description
Technical field
The present invention relates to bioactive ingredients technical fields, more particularly to scorpion activated protein.
Background technology
Scorpion belongs to the Arthropoda of the animal kingdom, Arachnoidea, Scorpionida.Be oneself know most ancient terrestrial arthropod it
One.Currently, oneself knows that whole world scorpion type has 6 sections, 70 belong to, more than 650 kinds.In China, scorpion is there are about 15 kinds, such as buthus martensii Karscs, mountain
Scorpion hides scorpion, spot scorpion, the Liao Dynasty Ke Er scorpions, ten leg scorpions etc., and distribution is also very wide, be mainly distributed on North China between the forbidden word 32-42 degree of north,
Wide geographic area on the south northeast and the Changjiang river, and the less Northwest inland of moisture be then distributed it is less.Buthus martensii Karscs are also known as Buthus martesiiKarsch,
In China, distribution is most wide, is mainly distributed on Henan, Hebei, Shandong, the ground such as Fujian, Taiwan are also distributed.It is also China's tradition name
Your Chinese medicine, it is most well-known with the produced scorpion in ground such as Nanyang, henan, the Laohekou in Hubei, Qingzhou in Shandong province.Scorpio is buthus martensii Karscs
Hirudo leech, nature and flavor are pungent, flat, toxic, return liver warp, have effects that ceases wind antispastic, dispersing pathogen accumulation, remove obstruction in channels to relieve pain.Clinical application is more
Enter ball, powder, next enters water decoction.Contain scorpion toxin, trimethylamine, Taurine, glycine betaine, cholesterol, lecithin, sweet in scorpio
A variety of chemical compositions such as grease, palmitic acid, stearic acid, ammonium salt, saturated fatty acid, main active are scorpion venom.In scorpion venom
Active function it is predominantly protein-based, there are the multiple pharmacological effects such as antitumor, analgesia, anti-epileptic, antithrombotic.Protein exists
It is typically all to exist with complicated mixed form in tissue and cell, various types of cells all contain thousands of kinds of different eggs
White matter.Obviously, to be a difficult task from mixing, obtaining target protein in numerous protein.Scorpion protein at present
Extracting method grind make internal disorder or usurp it is main include water decoct, alcohol extracting method and enzymatic isolation method etc., the application of enzymatic isolation method is the most universal, it may have
Universal pharmacodynamics effect, but extraction obtains the active not satisfactory with purity of scorpion activated protein, and recovery rate is relatively low.
Invention content
The purpose of the present invention is to provide a kind of activity and purity height, containing more protein species, between each activated protein
Gain effect is played, pharmacological action is high, the high scorpion activated protein of yield.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
Scorpion activated protein.The activity of active protein and purity are high, have a variety of medicines such as antitumor, analgesia, anti-epileptic, antithrombotic
Reason acts on, while containing more protein species in the activated protein, and gain effect is played between each activated protein, improves active egg
White pharmacological action, and the activated protein is not easy body immunogenicity and allergic reaction occur reduces side effect, have compared with
Good development prospect.
The extracting method of scorpion activated protein, specifically includes following steps：
Step 1：It is 1 by solid-to-liquid ratio:10-15 adds water into dry scorpio coarse powder, adds shikimic acid and p-Coumaric Acid, soaks
25-35min is steeped, then soybean dietary fiber 10-20min centrifuges 8-15min, supernatant under conditions of 5000-10000r/min
Liquid is that extracting solution is spare, and it is excellent that the dissolution of the step activated protein has that diffusional resistance is small, dissolution path is short, dissolution area is big
Point makes crushing be completed with one step of extraction, greatly improves the efficiency of extraction, while removing heating from, to effectively be avoided that albumen knot
Structure is destroyed, and improves the activity of activated protein；
Step 2：Regulating step 1 obtains the pH to 6.5-7.5 of extracting solution, then through 5- under the conditions of pressure is 0.25-0.35Mpa
6KDa molecular weight hollow-fibre membrane ultrafiltration 20-30min, trapped fluid are rinsed well repeatedly with distilled water, and being concentrated into relative concentration is
0.9-1.2g/mL, spare, the step condition design is reasonable so that protein-based to increase with water affinity, membrane flux reaches most
Greatly, soybean dietary fiber extracting solution small molecular nucleic acid object can be effectively removed under the premise of not destroying activated protein
Matter is removed by ultrafiltration；
Step 3：Saturated ammonium sulfate solution is added into step 2 concentrate to its a concentration of 65-75%, was stood under cryogenic conditions
At night, then centrifuge 8-15min under conditions of 5000-10000r/min, precipitation with equivalent concentration be 0.008-0.012mol/L,
PH is that the sodium phosphate buffer of 7.0-8.0 fully dissolves, and is dialysed, and dialyzate is crude protein solution, is added in the step a large amount of
Ammonium sulfate, so that the solubility of protein is reduced and Precipitation, it is continuous that this method advantage is that ammonium sulfate concentrations variation has
Property, effect of saltouing is good, saves energy consumption；
Step 4：Crude protein solution is purified with cellulose column, dextran gel filtration successively, freeze-drying is to get scorpion activity
Albumen, the activity of active protein and purity are high, have the multiple pharmacological effects such as antitumor, analgesia, anti-epileptic, antithrombotic, simultaneously
Contain more protein species in the activated protein, gain effect is played between each activated protein, the pharmacology for improving activated protein is made
With, and the activated protein is not easy body immunogenicity and allergic reaction occur, reduces side effect, before there is preferable development
Scape.
Preferably, in step 1 shikimic acid a concentration of 0.02-0.03mg/mL, p-Coumaric Acid it is a concentration of
0.005-0.01mg/mL.On the one hand the presence of shikimic acid and p-Coumaric Acid can change the osmotic pressure of cell, assist wet method
Ultramicro grinding rapid damage cell membrane material, improves the dissolution rate of activated protein, substantially increases the efficiency of extraction, another party
Face can act on the hydrophobic side of scorpion protein, improve the solubility of protein in water in scorpio, make gained activated protein
In contain more protein species, play gain effect between each activated protein, improve the pharmacological action of activated protein.
Contain 0.13-0.16mM sodium cyclohexylsulfamates preferably, being saturated in ammonium sulfate in step 3.Sulfuric acid
Often contain a small amount of heavy metal ion in ammonium, have a sensitization to protein sulfhydryl, and sodium cyclohexylsulfamate there are energy
The structure of enough protected proteins is not destroyed by heavy metal ion, ensures the activity of gained scorpion activated protein, while can increase albumen
Solubility of the matter in sodium phosphate buffer improves separating effect, the final yield for improving scorpion activated protein.
Compared with the prior art, the advantages of the present invention are as follows：Activity of active protein of the present invention and purity are high, containing more a variety of
Proteinoid plays gain effect between each activated protein, improves the pharmacological action of activated protein, and the activated protein to body not
It is susceptible to immunogenicity and allergic reaction, reduces side effect, there is preferable development prospect.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
Scorpion activated protein.The activity of active protein and purity are high, have a variety of medicines such as antitumor, analgesia, anti-epileptic, antithrombotic
Reason acts on, while containing more protein species in the activated protein, and gain effect is played between each activated protein, improves active egg
White pharmacological action, and the activated protein is not easy body immunogenicity and allergic reaction occur reduces side effect, have compared with
Good development prospect.
The extracting method of scorpion activated protein, specifically includes following steps：
Step 1：It is 1 by solid-to-liquid ratio:15 add water into dry scorpio coarse powder, add shikimic acid and p-Coumaric Acid, impregnate
25min, soybean dietary fiber 20min, then centrifuge 15min under conditions of 5000r/min, and supernatant is that extracting solution is standby
With the dissolution of the step activated protein has the advantages that diffusional resistance is small, dissolution path is short, dissolution area is big, makes crushing and carries
It takes a step to complete, greatly improves the efficiency of extraction, while removing heating from, to effectively be avoided that protein structure is destroyed, improve
The activity of activated protein；
Step 2：Regulating step 1 obtains the pH to 6.5 of extracting solution, then through in 5KDa molecular weight under the conditions of pressure is 0.35Mpa
Empty fiber membrane ultrafiltration 30min, trapped fluid are rinsed well repeatedly with distilled water, and it is 0.9g/mL to be concentrated into relative concentration, spare, should
Step condition design is reasonable so that protein-based to increase with water affinity, membrane flux reaches maximum, can not destroy active egg
Under the premise of white, soybean dietary fiber extracting solution small molecular nucleic acid material can be effectively removed and removed by ultrafiltration；
Step 3：Be added into step 2 concentrate saturated ammonium sulfate solution to its a concentration of 75%, stand overnight under cryogenic conditions,
15min is then centrifuged under conditions of 5000r/min, precipitation is that the sodium phosphate that 0.008mol/L, pH are 8.0 is slow with equivalent concentration
Fliud flushing fully dissolves, and dialysis, dialyzate is crude protein solution, and a large amount of ammonium sulfate is added in the step, makes the molten of protein
Xie Du is reduced and Precipitation, and this method advantage is that ammonium sulfate concentrations variation has continuity, and effect of saltouing is good, saves energy
Consumption；
Step 4：Crude protein solution is splined on DEAE-52 cellulose columns（2.5×20cm）, dense with the sodium chloride containing 0-1mol/L
Degree is that the Tris-HCl buffer solutions that 0.05mol/L, pH are 8.0 carry out linear gradient elution, and fraction collector is collected, detection
A280nm light absorption values collect eluting peak, merge Peak Activity and respectively manage, concentrate, concentrate loading to Sephacryl S-100 glucans
It in gel column, is eluted with a concentration of 0.05mol/L, pH Tris-HCl buffer solutions for being 8.0, fraction collector is collected, inspection
A280nm light absorption values are surveyed, eluting peak is collected, merges Peak Activity and respectively manages, concentrate, be freeze-dried to get scorpion activated protein, the work
Property protein active and purity it is high, there is the multiple pharmacological effects such as antitumor, analgesia, anti-epileptic, antithrombotic, while the activated protein
In contain more protein species, play gain effect between each activated protein, improve the pharmacological action of activated protein, and the activity
Albumen is not easy body immunogenicity and allergic reaction occur, reduces side effect, has preferable development prospect.
A concentration of 0.02mg/mL of shikimic acid in step 1, a concentration of 0.01mg/mL of p-Coumaric Acid.Shikimic acid and
On the one hand the presence of p-Coumaric Acid can change the osmotic pressure of cell, assist soybean dietary fiber rapid damage cell membrane object
Matter improves the dissolution rate of activated protein, substantially increases the efficiency of extraction, on the other hand can act on scorpion protein
The solubility of protein in water in scorpio is improved in hydrophobic side, makes to contain more protein species, each work in gained activated protein
Gain effect is played between property albumen, improves the pharmacological action of activated protein.
It is saturated in ammonium sulfate in step 3 and contains 0.13mM sodium cyclohexylsulfamates.Often contain in ammonium sulfate a small amount of
Heavy metal ion, have sensitization to protein sulfhydryl, and the knot of protected protein is capable of in the presence of sodium cyclohexylsulfamate
Structure is not destroyed by heavy metal ion, ensures the activity of gained scorpion activated protein, while can increase protein in sodium phosphate buffer
Solubility in liquid improves separating effect, the final yield for improving scorpion activated protein.
Embodiment 2：
The extracting method of scorpion activated protein, specifically includes following steps：
Step 1：It is 1 by solid-to-liquid ratio:12 add water into dry scorpio coarse powder, add shikimic acid and p-Coumaric Acid, impregnate
30min, soybean dietary fiber 15min, then centrifuge 10min under conditions of 8000r/min, and supernatant is that extracting solution is standby
With；
Step 2：Regulating step 1 obtains the pH to 7.0 of extracting solution, then through in 5.5KDa molecular weight under the conditions of pressure is 0.3Mpa
Empty fiber membrane ultrafiltration 25min, trapped fluid are rinsed well repeatedly with distilled water, and it is 1g/mL to be concentrated into relative concentration, spare；
Step 3：Be added into step 2 concentrate saturated ammonium sulfate solution to its a concentration of 70%, stand overnight under cryogenic conditions,
10min is then centrifuged under conditions of 8000r/min, precipitation is that the sodium phosphate that 0.01mol/L, pH are 7.5 is slow with equivalent concentration
Fliud flushing fully dissolves, and dialysis, dialyzate is crude protein solution；
Step 4：Crude protein solution is purified with cellulose column, dextran gel filtration successively, freeze-drying is to get scorpion activity
Albumen.
A concentration of 0.025mg/mL of shikimic acid in step 1, a concentration of 0.008mg/mL of p-Coumaric Acid.
It is saturated in ammonium sulfate in step 3 and contains 0.15mM sodium cyclohexylsulfamates.
Embodiment 3：
The extracting method of scorpion activated protein, specifically includes following steps：
Step 1：It is 1 by solid-to-liquid ratio:10 add water into dry scorpio coarse powder, add shikimic acid and p-Coumaric Acid, impregnate
35min, soybean dietary fiber 10min, then centrifuge 8min under conditions of 10000r/min, and supernatant is that extracting solution is standby
With；
Step 2：Regulating step 1 obtains the pH to 7.5 of extracting solution, then through in 6KDa molecular weight under the conditions of pressure is 0.25Mpa
Empty fiber membrane ultrafiltration 20min, trapped fluid are rinsed well repeatedly with distilled water, and it is 1.2g/mL to be concentrated into relative concentration, spare；
Step 3：Be added into step 2 concentrate saturated ammonium sulfate solution to its a concentration of 65%, stand overnight under cryogenic conditions,
8min is then centrifuged under conditions of 10000r/min, precipitation is that the sodium phosphate that 0.012mol/L, pH are 7.0 is slow with equivalent concentration
Fliud flushing fully dissolves, and dialysis, dialyzate is crude protein solution；
Step 4：Crude protein solution is purified with cellulose column, dextran gel filtration successively, freeze-drying is to get scorpion activity
Albumen.
A concentration of 0.03mg/mL of shikimic acid in step 1, a concentration of 0.005mg/mL of p-Coumaric Acid.
It is saturated in ammonium sulfate in step 3 and contains 0.16mM sodium cyclohexylsulfamates.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. the extracting method of scorpion activated protein, it is characterised in that：Include the following steps：
Step 1：Add water, shikimic acid and p-Coumaric Acid into dry scorpio coarse powder, impregnate, is centrifuged after soybean dietary fiber,
Supernatant is extracting solution；
Step 2：Regulating step 1 obtains the pH of extracting solution, and then ultrafiltration, trapped fluid washing is clean, and concentration is spare；
Step 3：Saturated ammonium sulfate solution, static, the sodium phosphate buffer of centrifugation equivalent are added into step 2 concentrate
Fully dissolving, dialysis, dialyzate is crude protein solution；
Step 4：Crude protein solution is purified with cellulose column, dextran gel filtration successively, freeze-drying is to get scorpion activity
Albumen.
2. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：Shikimic acid in the step 1
A concentration of 0.02-0.03mg/mL, a concentration of 0.005-0.01mg/mL of p-Coumaric Acid.
3. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：Scorpio is thick in the step 1
The solid-to-liquid ratio of powder and water is 1:10-15 impregnates 25-35min, crushes 10-20min.
4. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：Ultrafiltration in the step 2
PH is 6.5-7.5, and pressure 0.25-0.35Mpa, membrane molecule amount is 5-6Kda, time 20-30min.
5. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：Concentrate in the step 2
Relative concentration be 0.9-1.2g/mL.
6. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：It is saturated sulphur in the step 3
Contain 0.13-0.16mM sodium cyclohexylsulfamates in acid ammonium solution.
7. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：It is saturated sulphur in the step 3
Acid ammonium solution adds to a concentration of 65-75%.
8. the extracting method of scorpion activated protein according to claim 1, it is characterised in that：Sodium phosphate in the step 3
A concentration of 0.008-0.012mol/L, pH 7.0-8.0 of buffer solution.
9. scorpion activated protein prepared by the extracting method of the scorpion activated protein described in claim 1-8 any one.
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| CN201810473979.2A CN108659111A (en) | 2018-05-17 | 2018-05-17 | Scorpion activated protein |
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| CN201810473979.2A CN108659111A (en) | 2018-05-17 | 2018-05-17 | Scorpion activated protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116874307A (en) * | 2023-09-05 | 2023-10-13 | 莱芜市兆信新材料股份有限公司 | Ceramic water reducer and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709083A (en) * | 2009-11-27 | 2010-05-19 | 广东工业大学 | Fibrinolytic protein from scorpion tails, preparation method and application thereof |
| CN103396472A (en) * | 2013-06-24 | 2013-11-20 | 陕西步长制药有限公司 | Scorpion extract with thrombolytic activity |
-
2018
- 2018-05-17 CN CN201810473979.2A patent/CN108659111A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709083A (en) * | 2009-11-27 | 2010-05-19 | 广东工业大学 | Fibrinolytic protein from scorpion tails, preparation method and application thereof |
| CN103396472A (en) * | 2013-06-24 | 2013-11-20 | 陕西步长制药有限公司 | Scorpion extract with thrombolytic activity |
Non-Patent Citations (2)
| Title |
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| 田晓然 等: "湿法超微粉碎-超滤法快速提取浓缩全蝎蛋白可行性考察", 《中国实验方剂学杂志》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116874307A (en) * | 2023-09-05 | 2023-10-13 | 莱芜市兆信新材料股份有限公司 | Ceramic water reducer and preparation method thereof |
| CN116874307B (en) * | 2023-09-05 | 2023-11-07 | 莱芜市兆信新材料股份有限公司 | Ceramic water reducer and preparation method thereof |
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