CN118878552B - A preparation method of rifamycin S sodium salt - Google Patents
A preparation method of rifamycin S sodium salt Download PDFInfo
- Publication number
- CN118878552B CN118878552B CN202410929459.3A CN202410929459A CN118878552B CN 118878552 B CN118878552 B CN 118878552B CN 202410929459 A CN202410929459 A CN 202410929459A CN 118878552 B CN118878552 B CN 118878552B
- Authority
- CN
- China
- Prior art keywords
- rifamycin
- extractant
- added
- preparation
- acid
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D498/08—Bridged systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本申请公开了一种利福霉素S钠盐的制备方法，对含有利福霉素SV的发酵液依次氧化、酸化、气浮后静置分层，分去酸水后加入萃取剂萃取，然后固液分离，将得到的液体静置分层，去除酸水层，向萃取剂层加入碱液，然后加碱调节pH搅拌结晶，分离干燥得到利福霉素S钠盐。本发明的方法大大减少了萃取剂的耗用量，废水产生量少，操作步骤简单，杂质含量低，适用于自动化生产。
The present application discloses a method for preparing sodium salt of rifamycin S, wherein the fermentation liquid containing rifamycin SV is oxidized, acidified, and flotated in sequence, and then allowed to stand for stratification, after the acid water is separated, an extractant is added for extraction, and then the solid-liquid separation is performed, the obtained liquid is allowed to stand for stratification, the acid water layer is removed, an alkali solution is added to the extractant layer, and then an alkali is added to adjust the pH and stir to crystallize, and the sodium salt of rifamycin S is obtained by separation and drying. The method of the present invention greatly reduces the consumption of the extractant, generates less wastewater, has simple operation steps, has a low impurity content, and is suitable for automated production.
Description
Technical Field
The invention belongs to the field of compound purification and preparation, and particularly relates to a preparation method of rifamycin S sodium salt.
Background
Rifamycin antibiotics are broad-spectrum antibiotics, and the main varieties are fosinomycin sodium, rifampin, rifadine, rifabutin, rifaximin, rifapentine and the like. They have high activity against tubercle bacilli and can be used for the treatment of tuberculosis and other diseases caused by drug-resistant mycobacteria. Rifamycins exert their antibacterial effects by inhibiting the activity of bacterial RNA polymerase, thereby preventing bacterial RNA synthesis, and thus blocking protein synthesis, ultimately leading to bacterial death. Rifamycin is commonly used in conjunction with other antitubercular drugs (e.g., isoniazid, pyrazinamide, etc.) to reduce the production of resistant strains and to enhance therapeutic effects. Rifamycin is sometimes also used to treat other infections, such as brucellosis and lymphomatosis virus infections, due to its broad-spectrum antibacterial effect.
Different components of rifamycin, including A, B, C, D, E, O, S, SV, etc. can be obtained after fermentation of different strains.
Rifamycin is a semisynthetic antibiotic, and is typically obtained by fermentation to yield the parent rifamycin S sodium salt. By taking the rifamycin as an initiator, rifamycin sodium, rifampicin, rifapentine, rifabutin and the like are obtained through synthesis.
The rifamycin is prepared by fermenting Nocardia Mediterranean, filtering fermentation liquor to remove bacterial residues, proteins and other impurities through a plate frame, and obtaining rifamycin sodium salt from filtrate through two technological processes, namely (1) adding an oxidant into the filtrate for oxidization, then extracting with butyl acetate, demulsifying, washing, concentrating, crystallizing, centrifuging and drying extract to obtain rifamycin S-Na, (2) extracting the rifamycin SV in the filtrate with butyl acetate, then oxidizing to obtain the rifamycin S, and demulsifying, washing, concentrating and crystallizing the oxidized liquor to obtain the rifamycin S-Na. The two processes differ in that the order of extraction and oxidation is different, with the other steps being substantially identical.
However, in the process of removing bacterial residues by plate and frame filtration of fermentation liquor, fermentation liquor is usually hydraulically fed into a refined receiving tank, and is stirred by adding a flocculating agent. And pressing the plate frame into the filter. This process is for several hours. Bacteria die during the process of asphyxiation, decomposition and destruction, and abnormal metabolism. When the fermentation is abnormally and severely infected, the filtrate after plate and frame filtration can be subjected to further extraction. In this case, the filter is always completely or partially discarded.
Therefore, it is necessary to provide a preparation process of rifamycin S sodium salt with good process stability, simple operation and low cost.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of rifamycin S sodium salt aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a preparation method of rifamycin S sodium salt, which comprises the steps of sequentially oxidizing, acidifying, air-floating, standing and layering fermentation liquor containing the rifamycin SV, separating to remove acid water, adding an extractant for extraction, then carrying out solid-liquid separation, standing and layering the obtained liquor, removing an acid water layer, adding alkali liquor into the extractant layer, adding alkali to adjust pH, stirring for crystallization, separating and drying to obtain the rifamycin S sodium salt.
Wherein the fermentation broth comprising rifamycin SV may be derived from a fermentation plant, comprising mycelium, a medium and rifamycin SV. In some embodiments, the rifamycin SV. is typically present in an amount of 0.3% -0.6% and is dissolved in the fermentation broth. The mycelium and the solid components of the culture medium are about 2%, the other soluble substances are about 3%, and the balance is water.
The oxidizing agent used in the oxidation is any one or a mixture of more than one of hydrogen peroxide, bleaching powder, sodium hypochlorite, sodium nitrite, trichloroisocyanuric acid, potassium ferricyanide, chlorine dioxide or peracetic acid, the use amount of the oxidizing agent is calculated by mole number, the ratio of rifamycin s to the oxidizing agent is 1-5, and the oxidation time is 2-3 hours.
The acidulant used for acidification is any one of acetic acid, hydrochloric acid, sulfuric acid or oxalic acid, and the acidulant is added to enable the pH value of the system to be 1-4.
The air floatation is to introduce compressed air and stir. Wherein, during the stirring process, vortex suction air and chemical reaction generated gas can also play the role of air floatation. The amount of air introduced by air floatation is calculated by enabling the air to be wrapped by the feed liquid, and the ventilation amount is 10-12 cubic meters of air per cubic meter of feed liquid per hour in general. During the oxidation and acidification, air is introduced, and the volume of the feed liquid is gradually increased in the process.
The extractant is any one of butyl acetate, chloroform or dichloromethane, and the dosage of the extractant is 10-20 times of the sv mass of the rifamycin.
The solid-liquid separation adopts any one of a horizontal decanter centrifuge, an automatic discharging centrifuge or a disc centrifuge.
Wherein the alkali liquid added in the extractant layer is sodium bicarbonate water solution, and the alkali for adjusting the pH is sodium hydroxide or sodium carbonate.
Preferably, alkali is added to adjust the pH to 8-12, and stirring crystallization is carried out.
Preferably, after adding alkali to adjust pH, stirring and crystallizing, cooling to below 10 ℃ and separating by a centrifuge, and then drying under vacuum at below 100 ℃.
Compared with the prior art, the application has the following advantages:
(1) By directly adding an oxidant to the fermentation broth, rapid sterilization can be achieved, avoiding the undesirable related impurities of catabolism (i.e., homologs of the product such as norrifamycin S, norrifamycin S);
(2) When the fermentation is abnormally and severely infected, the conventional process can refine the filtrate after plate and frame filtration only by the next step, and the filtration is always completely or partially scrapped. The application can be normally refined without influence because of no filtering step by using an air floatation method;
(3) Compared with the traditional production method, the application removes the filtering link of the fermentation liquor, directly oxidizes the fermentation liquor, adopts an air floatation method to separate mycelium and rifamycin S, separates the waste water, then uses an extractant such as butyl acetate and methylene dichloride to extract the solid, and only has a small amount of waste water containing organic solvent. The traditional method has the advantages of high labor intensity and low efficiency in plate and frame filtration, and the filtrate is produced by 1.5-2 cubic meters per cubic meter of fermentation liquor. The application greatly reduces the production wastewater of rifamycin, and only 20% of the wastewater of the traditional method contains organic solvent. Meanwhile, the process is suitable for automatic production, and only 10-30% of the traditional process is used by personnel;
(4) The consumption of the extractant is also greatly reduced, and the extractant is not contacted with a large amount of water and is not dissolved in the water, so that the consumption of the extractant is obviously reduced to be less than 50 percent of that of the traditional method.
Drawings
FIG. 1 is a schematic diagram of fermentation broth under air-float stirring state, and FIG. 1 is a schematic diagram of fermentation broth after air-float standing to separate acid water;
FIG. 2 is a liquid phase representation of rifamycin S prepared in example 1, showing the peaks of the main peak (i.e., rifamycin S) incompletely in order to clearly show the content of other impurities.
Detailed Description
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
In the following examples, fermentation broth was derived from a fermentation plant and contained mycelium, medium and rifamycin SV, wherein the rifamycin SV. content was 0.3% -0.6%, and was dissolved in the fermentation broth. The mycelium and the solid components of the culture medium are about 2%, the other soluble substances are about 3%, and the balance is water. It will be appreciated by those skilled in the art that fermentation broths derived from other fermentation plants containing rifamycin SV are equally suitable for use in the methods of the application, and the specific properties of the fermentation broths are not limited.
Example 1 preparation of rifamycin S sodium salt.
To 1000L of fermentation broth containing rifamycin SV, 0.5kg of bleaching powder and 0.4kg of trichloroisocyanuric acid are added per kg of rifamycin SV for oxidation, and air is introduced for stirring and air floatation, and the ventilation amount is 10 cubic meters of air per cubic meter of feed liquid per hour, as shown in the left graph of FIG. 1. Adding 30% hydrochloric acid to acidify to pH 2.5, continuously introducing air during oxidation and acidification, standing for layering, and separating acid water. The state diagram of the fermentation broth after separating out the acid water is shown in the right diagram of fig. 1. From the figure, the water content of the fermentation broth is greatly reduced after the fermentation broth is subjected to air floatation stirring treatment and layering. 20kg of butyl acetate per kg of rifamycin sv is added and stirred. Separating solid and liquid by a decanter centrifuge, and standing the obtained liquid for layering. The lower layer is an acid water layer, and the upper layer is a butyl acetate layer. Separating the deacidified water layer, adding 3% sodium bicarbonate water with equal volume of butyl acetate as water phase, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for crystallization, cooling to below 10deg.C, separating with centrifuge, and vacuum drying below 100deg.C to obtain rifamycin S sodium salt.
The content of the obtained rifamycin S is characterized and detected, and the results are shown in table 1. Wherein, the content detection adopts an external standard method, and is determined by referring to high performance liquid chromatography (general rule 0512). Wherein, a C19 stainless steel column is adopted, the flow rate is 2.0mL/min, the sample injection amount is 20 mu L, the detection wavelength is 254nm, and the column temperature is 30 ℃. The mobile phase was mixed with 0.025mol/L disodium hydrogen phosphate (dihydrate) -acetonitrile (51:49), and the pH was adjusted to about 6.9 with phosphoric acid.
Wherein, the liquid phase result diagram is shown in fig. 2.
TABLE 1
| Inspection item | Test results |
| Rifamycin SV | 1.82% |
| 25-Deacetyl-27-norrifamycin S | 0.34% |
| 16-Hydroxymethyl rifamycin S | 0.51% |
| 27-De-methylli Fucomycin S | 0.67% |
| 25Desacetyl rifamycin S (25-deacetylrifamycin S) | 0.77% |
| Sum of peaks (Total impurity free 25-deacetylrifamycin S) | 3.9% |
| Loss on drying (moisture) | 4.9% |
| Content of | 84.7% |
| Solvent impurity | 5.33% |
Wherein the total impurities comprise rifamycin SV, 25-deacetyl-27-norrifamycin S, 16-hydroxymethyl rifamycin S, 27-norrifamycin S and other impurities not listed.
Example 2
The basic procedure is as in example 1, except that 0.1kg hydrogen peroxide per kg rifamycin sv is used for oxidation.
Example 3
The basic procedure is as in example 1, except that oxidation is carried out with 0.6kg of trichloroisocyanuric acid per kg of rifamycin sv.
Example 4
The basic procedure was as in example 1, except that solid oxalic acid was used to acidify to pH 3.0, the acid water was separated by air flotation layering, the solid fraction was dewatered to below 50% water content by an automatic discharge centrifuge, butyl acetate was added and stirred, and the butyl acetate layer was separated by standing. Adding 3% sodium bicarbonate water with equal volume of butyl acetate as water phase, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for crystallization, cooling to below 10deg.C, separating with centrifuge, and vacuum drying at below 100deg.C to obtain rifamycin S sodium salt.
Example 5
The basic procedure is as in example 1, except that after the acid water has been separated off in layers by air flotation, the solids fraction is dewatered to a water content of less than 50% by means of an automatic discharge centrifuge. 20kg of chloroform was added to 1kg of rifamycin sv and stirred, and the mixture was allowed to stand for delamination. Chloroform was under. Separating chloroform, adding 3% sodium bicarbonate water with chloroform volume as water phase, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for crystallization, cooling to below 10deg.C, separating with centrifuge, and vacuum drying below 100deg.C to obtain rifamycin S sodium salt
Example 6
The basic procedure was as in example 2, except that chloroform was added and stirred after the acid water was separated by air-float separation. 20kg of chloroform was added to 1kg of rifamycin sv and stirred, and the mixture was allowed to stand for delamination. Chloroform was under. Separating chloroform, adding 3% sodium bicarbonate water with chloroform volume as water phase, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for crystallization, cooling to below 10deg.C, separating with centrifuge, and vacuum drying below 100deg.C to obtain rifamycin S sodium salt
Example 7
The basic procedure was as in example 1, except that after the acid water was separated off by air-flotation, methylene chloride was added and stirred. And (5) standing and layering. Dichloromethane was in the lower layer. Separating dichloromethane, adding 3% sodium bicarbonate water as water phase, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for crystallization, cooling to below 10deg.C, separating with centrifuge, and vacuum drying below 100deg.C to obtain rifamycin S sodium salt
The test data for each example are shown in table 1.
TABLE 1
Comparative example
1000L of fermentation broth was added with 1.5kg of zinc sulfate and stirred for 10 minutes, and the mixture was filtered through a plate frame. After all the fermentation liquor enters the plate frame, the fermentation liquor is top-washed to 1500L by tap water. The filtrate concentration was checked. 1kg of bleaching powder and 20kg of butyl acetate are added to each kg of rifamycin sv, and air is introduced for stirring and oxidation. After the oxidation is checked, the pH is regulated to 1-4 by 30% hydrochloric acid, and water is separated after standing for a few hours. After emulsification, adding dodecyl trimethylamine chloride, stirring and standing, demulsifying, and separating water. The butyl acetate solution was washed with tap water, 1% sodium bicarbonate water, 1% hydrochloric acid water, respectively. Adding 50% butyl acetate by volume and 3% sodium bicarbonate water as crystal water, heating to above 40deg.C, adjusting pH to 9.5 with 5% sodium hydroxide, stirring for 2 hr, and cooling to below 10deg.C. After standing for several hours, the mixture was filtered by a centrifuge to separate out the solvent. The solid is dried in vacuum in a vacuum drying oven at a temperature below 100 ℃.
The content of the obtained rifamycin S is characterized and detected, and the results are shown in Table 2.
TABLE 2
In summary, the present application provides for the first time that the preparation concept of rifamycin S sodium salt, which can realize rapid sterilization and avoid unnecessary impurities of catabolism, by directly adding an oxidizing agent into a fermentation broth, and the specific implementation method of the technical scheme are numerous, the above is only a preferred embodiment of the present application, and it should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present application, and these improvements and modifications should also be regarded as the protection scope of the present application. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A preparation method of rifamycin S sodium salt is characterized in that fermentation liquor containing the rifamycin SV is sequentially oxidized and acidified, air floatation is continuously carried out in the oxidation and acidification processes, then standing and layering are carried out, extractant is added for extraction after acid removal, then solid-liquid separation is carried out, the obtained liquid is stood and layered, an acid water layer is removed, alkali liquor is added into the extractant layer, then alkali is added for regulating pH value, stirring crystallization is carried out, and separation and drying are carried out, thus obtaining the rifamycin S sodium salt.
2. The preparation method of the rifamycin compound is characterized in that the oxidizing agent used in the oxidation is any one or a mixture of more than one of hydrogen peroxide, bleaching powder, sodium hypochlorite, sodium nitrite, trichloroisocyanuric acid, potassium ferricyanide, chlorine dioxide or peracetic acid, the amount of the oxidizing agent is calculated by mole number, the ratio of rifamycin s to the oxidizing agent is 1-5, and the oxidation time is 2-3 h.
3. The preparation method of claim 1, wherein the acidulant used for acidification is any one of acetic acid, hydrochloric acid, sulfuric acid or oxalic acid, and the acidulant is added to make the pH of the system be 1-4.
4. The method according to claim 1, wherein the air floatation is performed by introducing compressed air and stirring.
5. The preparation method of claim 1, wherein the extractant is any one of butyl acetate, chloroform or dichloromethane, and the extractant dosage is 10-20 times of the rifamycin sv mass.
6. The method according to claim 1, wherein the solid-liquid separation is performed by any one of a decanter centrifuge, an automatic discharge centrifuge, and a disk centrifuge.
7. The process of claim 1, wherein the alkaline solution added to the extractant layer is aqueous sodium bicarbonate and the pH adjusting base is sodium hydroxide or sodium carbonate.
8. The preparation method according to claim 1, wherein the pH is adjusted to 8-12 by adding a base.
9. The preparation method according to claim 1, wherein after adding alkali to adjust the pH, stirring and crystallizing, cooling to below 10 ℃ and separating by a centrifuge.
10. The method according to claim 1, wherein the drying condition is vacuum drying at 100 ℃ or less.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410929459.3A CN118878552B (en) | 2024-07-11 | 2024-07-11 | A preparation method of rifamycin S sodium salt |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410929459.3A CN118878552B (en) | 2024-07-11 | 2024-07-11 | A preparation method of rifamycin S sodium salt |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118878552A CN118878552A (en) | 2024-11-01 |
| CN118878552B true CN118878552B (en) | 2025-07-08 |
Family
ID=93234226
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410929459.3A Active CN118878552B (en) | 2024-07-11 | 2024-07-11 | A preparation method of rifamycin S sodium salt |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118878552B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1045993A (en) * | 1989-03-28 | 1990-10-10 | 五洲药厂 | A kind of preparation method of sodium salt for rifainycin S |
| CN104357586A (en) * | 2014-11-11 | 2015-02-18 | 河北欣港药业有限公司 | Fermentation production method of rifamycin SV based on phosphate glycine betaine concentration as control parameter |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE796018A (en) * | 1973-03-14 | 1973-06-18 | Archifar Ind Chim Trentino | PROCESS FOR THE EXTRACTION AND ISOLATION OF RIFAMYCINS |
| CN105713010B (en) * | 2016-04-15 | 2017-09-29 | 沈阳化工研究院有限公司 | A kind of production technology of Rifamycin Sodium sodium salt |
| CN114031628B (en) * | 2021-11-26 | 2023-02-28 | 中国科学院大连化学物理研究所 | A kind of synthetic method of rifamycin S-Na salt |
-
2024
- 2024-07-11 CN CN202410929459.3A patent/CN118878552B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1045993A (en) * | 1989-03-28 | 1990-10-10 | 五洲药厂 | A kind of preparation method of sodium salt for rifainycin S |
| CN104357586A (en) * | 2014-11-11 | 2015-02-18 | 河北欣港药业有限公司 | Fermentation production method of rifamycin SV based on phosphate glycine betaine concentration as control parameter |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118878552A (en) | 2024-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113185485B (en) | Semi-synthesis method of dihydroquercetin | |
| CN105648022B (en) | A kind of technique that liquid 6-APA leads directly to Amoxicillin | |
| CN114957122A (en) | Preparation method of 10-methoxyiminostilbene | |
| CN118878552B (en) | A preparation method of rifamycin S sodium salt | |
| CN115011661B (en) | Synthesis method of 3 beta-ursodeoxycholic acid | |
| AP610A (en) | Process for the preparation of a salt of clavulanic acid. | |
| CN104853825A (en) | Generation of low-solids second mother liquor from terephthalic acid product filter | |
| CN113083253A (en) | Weak acid cation resin for extracting vitamin B12 and synthetic method thereof | |
| CN116178188B (en) | Method for producing glycine by one-step synthesis and step-by-step purification in a mixed solvent | |
| CN106883227B (en) | The method for preparing ergometrine by ergot fermentation waste | |
| CN106854161B (en) | The synthetic method of 3,4- dimethoxy -6- nitrobenzoic acid | |
| CN114426479B (en) | Method and device for refining long-chain dibasic acid | |
| CN102180781B (en) | Method for extracting and producing high-purity xanthohumol from carbon dioxide extraction hop residue | |
| CN115505622A (en) | Method for preparing UDCA isomer of 3 alpha, 7 beta-dihydroxy-5 alpha-H | |
| EP2471939B1 (en) | Method for producing 1-(2-hydroxyethyl) 2,5,5,8a-tetramethyldecahydronaphthalene-2-ol by microbial fermentation | |
| CN113480514B (en) | Method for extracting sesamin | |
| CN118579946B (en) | A method for treating irbesartan production wastewater | |
| CN113563416B (en) | Treatment method of dipeptide mother liquor and application of dipeptide mother liquor in dipeptide production process | |
| MD4706C1 (en) | Process for producing bisulfates of benzophenanthridine alkaloids from macleaya leaves | |
| CN103773832A (en) | Method and process for extracting 4-androstenedione from phytosterol aqueous phase fermentation liquor | |
| CN115572296B (en) | Method for reducing absorbance index of caffeine product | |
| CN106589073A (en) | Pneumocandin B0 purification method | |
| CN111116500B (en) | A kind of purification method of key intermediate of Recinade | |
| RU2238103C1 (en) | Method for preparing allapinine | |
| CN111073337B (en) | Apparatus and method for refining copper phthalocyanine |
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 |