CN110819665A - Preparation method of 6, 7, 8-trihydroxy coumarin - Google Patents

Preparation method of 6, 7, 8-trihydroxy coumarin Download PDF

Info

Publication number
CN110819665A
CN110819665A CN201910998671.4A CN201910998671A CN110819665A CN 110819665 A CN110819665 A CN 110819665A CN 201910998671 A CN201910998671 A CN 201910998671A CN 110819665 A CN110819665 A CN 110819665A
Authority
CN
China
Prior art keywords
preparation
culture
solution
anaerobic
steps
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.)
Granted
Application number
CN201910998671.4A
Other languages
Chinese (zh)
Other versions
CN110819665B (en
Inventor
李笃信
赵磊
张健
朱雪雪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou University
Original Assignee
Suzhou University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Suzhou University filed Critical Suzhou University
Priority to CN201910998671.4A priority Critical patent/CN110819665B/en
Publication of CN110819665A publication Critical patent/CN110819665A/en
Application granted granted Critical
Publication of CN110819665B publication Critical patent/CN110819665B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a preparation method of 6, 7, 8-trihydroxy coumarin, which comprises the following steps: (1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution; (2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant; (3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution; (4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure; (5) and (3) purifying a product: purification was performed by preparative liquid chromatography. The invention has the advantages of high conversion rate of over 98.0 percent, simple steps, high speed, environmental protection and suitability for industrial production.

Description

Preparation method of 6, 7, 8-trihydroxy coumarin
Technical Field
The invention belongs to the technical field of microbial transformation, and particularly relates to a preparation method of 6, 7, 8-trihydroxy coumarin.
Background
Fraxins, fraxinin and coumarins are effective components of cortex Fraxini, and fraxins are hydrolysate of fraxins. Bruno M.Bizzarri et al react with aesculin as a substrate and 2-iodoxybenzoic acid as an oxidant to produce 6, 7, 8-trihydroxy coumarin, which has stronger oxidation resistance and antibacterial activity than aesculin. However, the synthesis method has the defects of more byproducts, difficult control of reaction end point and difficult separation of products. Therefore, there is a need to research a novel preparation method of 6, 7, 8-trihydroxy coumarin.
Microbial transformation studies began in the 20 th century and aimed at the co-cultivation of bacteria, fungi or other microorganisms with exogenous substrates to yield valuable products, among which the enzymes produced by the microorganisms play a major role. The traditional Chinese medicine contains various complex active ingredients, each kind of ingredient plays an important role, but the research of the traditional Chinese medicine can not meet the increasing demand of people for medication, so the traditional Chinese medicine is combined with microbial transformation, and the active ingredients in the traditional Chinese medicine are favorably and better extracted and transformed.
Disclosure of Invention
The invention aims to provide a preparation method of 6, 7, 8-trihydroxy coumarin, which solves the problems.
The technical scheme of the invention is as follows: a preparation method of 6, 7, 8-trihydroxy coumarin comprises the following steps: (1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution;
(2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant;
(3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution;
(4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure;
(5) and (3) purifying a product: purification was performed by preparative liquid chromatography.
Further, the first solution in step (1) is physiological saline, phosphate buffer, carbonate buffer, Tris buffer or water.
Further, the feces in the step (2) is feces of a rat, a mouse, a rabbit, a guinea pig or a human, and is preferably rat feces.
Further, the components of the nutrient broth in the step (3) are peptone and beef extract powder, and the components of the nutrient broth can be any one or the combination of two components.
Further, the anaerobic condition in the step (3) is CO2, N2, Ar or He atmosphere, and the culture time is 2-10 d.
Further, the anaerobic culture solution in the step (3) comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, calcium chloride, magnesium sulfate, resazurin, L-cysteine and L-ascorbic acid; the amount of said anaerobic culture fluid added is 0.2-5 times the volume of said nutrient broth.
Further, the solvent for extraction in the step (4) is ethyl acetate, n-butanol, chloroform, dichloromethane or n-hexane, and the amount of the extracting agent is 0.5-2 times, and the times are 1-3.
Further, the separation in the step (5) is carried out by using any one of silica gel, a C8 column and a C18 column.
The invention provides a preparation method of 6, 7, 8-trihydroxy coumarin, which has the advantages that: the method solves the problems of complex production process and low efficiency of the existing 6, 7, 8 trihydroxy coumarin, and has the characteristics of simple operation steps, high conversion efficiency, high conversion rate of more than 98.0 percent, high speed, environmental friendliness and suitability for industrial production.
Drawings
The invention is further described with reference to the following figures and examples:
FIG. 1 is a schematic diagram of the microbial transformation of fraxin and fraxin in an embodiment of the present invention, wherein (A) is fraxin, (B) is fraxin, and (C) is 6, 7, 8-trihydroxycoumarin;
FIG. 2 is a schematic diagram showing the effect of time on the conversion of 6, 7, 8-trihydroxy coumarin, a product obtained in example 2, wherein 1 is aesculin and 2 is 6, 7, 8-trihydroxy coumarin;
FIG. 3 is a schematic diagram showing the effect of time on the conversion rate of 6, 7, 8-trihydroxy coumarin, which is a product obtained in example 3, wherein 1 is aesculin, 2 is aesculin, and 3 is 6, 7, 8-trihydroxy coumarin.
Detailed Description
The invention provides a preparation method of 6, 7, 8-trihydroxy coumarin, which comprises the following steps:
(1) the aesculin and/or the fraxinin are used as starting materials: adding physiological saline or other solution to dissolve;
(2) animal dung bacteria are used as carriers: collecting fresh feces of animals under aseptic condition, wherein the feces can be feces of rat, mouse, human or other animals, preferably rat feces, adding normal saline to homogenate, centrifuging, and collecting supernatant, wherein the centrifugation speed is 2000-;
(3) anaerobic culture: adding the supernatant to a nutrient broth, CO2、N2Or culturing at 36.0-38.0 deg.C for 12-36 hr under anaerobic condition, adding raw materials, adding 0.5-1.0 times volume of anaerobic culture solution, and performing anaerobic constant temperature culture;
(4) and (3) extraction of a product: extracting the culture solution with ethyl acetate, n-butanol or other water-immiscible organic solvent, preferably ethyl acetate, for 1-3 times, mixing extractive solutions, and recovering under reduced pressure;
(5) and (3) purifying a product: the separation is carried out with silica gel, ODS or other fillers, preferably silica gel or ODS.
The microbial transformation process is shown in figure 1.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
The preparation method of 6, 7, 8-trihydroxy coumarin is shown in the embodiment by the following steps
1. Raw material preparation
Accurately weighing 50.0mg of aesculin and 0.83mL of DMSO for assisting dissolution, and dissolving in 4.17mL of physiological saline.
2. Preparation of nutrient broth and anaerobic culture solution
Taking 18.0g of nutrient broth and 9.5g of anaerobic culture solution, respectively adding 1000mL of deionized water, dissolving, sealing, and sterilizing with 120 deg.C high pressure steam for 15 min.
3. Collection and in vitro culture of different faeces
Collecting fresh feces of 6 SD male rats on clean bench (5.0 g), adding 25.0mL sterile physiological saline, homogenizing, collecting supernatant (25 mL), adding into sterilized 100mL nutrient broth, mixing, and placing into 37 deg.C constant temperature incubator CO2Anaerobic culture is carried out for 24 hours to ensure that the bacteria fully grow. Collecting feces of mouse, human, guinea pig and rabbit by the same method, and culturing in vitro
4. Fecal bacteria transformation of fraxins
5mL of the starting material and 50mL of the anaerobic culture medium were added to 100mL of the fecal suspension cultured for 24 hours, and the mixture was anaerobically cultured at 37 ℃ for 7 days, and the reaction was monitored by HPLC.
Figure BDA0002240579710000041
TABLE 1
Referring to table 1, table 1 shows the effect of feces of different species in example 1 of the present invention on the conversion rate of 6, 7, 8-trihydroxy coumarin; as shown in table 1, the effect of feces of each species on the conversion of 6, 7, 8-trihydroxy coumarin was: mouse > human > rat > guinea pig > rabbit.
Example 2
The implementation case shows a preparation method of 6, 7, 8-trihydroxy coumarin according to the following steps:
1. raw material preparation
Accurately weighing 50.0mg of aesculin and 0.83mL of DMSO for assisting dissolution, and dissolving in 4.17mL of physiological saline.
2. Preparation of nutrient broth and anaerobic culture solution
Taking 18.0g of nutrient broth and 9.5g of anaerobic culture solution, respectively adding 1000mL of deionized water, dissolving, sealing, and sterilizing with 120 deg.C high pressure steam for 15 min.
3. In vitro culture of rat fecal bacteria
Collecting fresh feces of 6 SD male rats on clean bench (5.0 g), adding 25.0mL sterile physiological saline, homogenizing, collecting supernatant (25 mL), adding into sterilized 100mL nutrient broth, mixing, and placing into 37 deg.C constant temperature incubator CO2Anaerobic culture is carried out for 24 hours to ensure that the bacteria fully grow.
4. Fecal bacteria transformation of fraxins
5mL of the starting material and 50mL of the anaerobic culture medium were added to 100mL of the fecal suspension cultured for 24 hours, and the mixture was anaerobically cultured at 37 ℃ for 7 days, and the reaction was monitored by HPLC.
As shown in fig. 2, metabolites were produced from day two, with a conversion of 17.21% on day three, 42.79% on day five, and 100% on day seven.
5. Isolated preparation of metabolites
Extracting the above culture solution with 1 volume of ethyl acetate, repeating for three times, mixing the extractive solutions, recovering under reduced pressure, dissolving in 5mL of 50% methanol, filtering with 0.22 μm filter membrane, and separating liquid phase.
6. Identification of metabolites
After 15mg of the metabolite was dissolved in DMSO-d6, it was analyzed by 1H-NMR (600MHz) and 13C-NMR (151 MHz). Metabolite 1mg was dissolved in 2mL 50% chromatographic methanol and analyzed by LC-MS.
The conversion product is yellow powdery solid, is easily soluble in ethyl acetate and dimethyl sulfoxide, is slightly soluble in methanol, and has the maximum absorption of 334 nm. 1H-NMR (600MHz, dmso), δ:7.82(1H, d, J ═ 9.42, 3H),6.55(1H, s,5H),6.14(1H, d, J ═ 9.42, 4H); 13C-NMR (151MHz, dmso), delta: 160.69(C, C-2),145.02(C, C-4),143.01(C, C-6),139.18(C, C-7),138.10 (C, C-8a),132.92(C, C-8),111.45(C, C-3),110.31(C, C-4a),103.16(C, C-5); ESI-MS, m/z: 193.9. The conversion product can be determined to be 6, 7, 8-trihydroxy coumarin by the information.
Example 3
The implementation case shows a preparation method of 6, 7, 8-trihydroxy coumarin according to the following steps:
1. raw material preparation
Accurately weighing 50.0mg of aesculin and 0.83mL of DMSO for assisting dissolution, and dissolving in 4.17mL of physiological saline.
2. Preparation of nutrient broth and anaerobic culture solution
Taking 18.0g of nutrient broth and 9.5g of anaerobic culture solution, respectively adding 1000mL of deionized water, dissolving, sealing, and sterilizing with 120 deg.C high pressure steam for 15 min.
3. In vitro culture of rat fecal bacteria
Collecting fresh feces of 6 SD male rats 5.0g on clean bench, adding 25.0mL sterile physiological saline, homogenizing, collecting supernatant 25mL, adding into sterilized nutrient broth 100mL, mixing, and placing into constant temperature incubator at 37 deg.C with CO2Anaerobic culture is carried out for 24 hours to ensure that the bacteria fully grow.
4. Fecal bacteria transformation of aesculin
5mL of the starting material and 50mL of the anaerobic culture medium were added to 100mL of fecal suspension cultured for 24 hours, and the mixture was anaerobically cultured at 37 ℃ for 10 days, and the reaction was monitored by HPLC.
As shown in FIG. 3, the next day, the conversion of fraxin to fraxin was complete, and the metabolites were produced from the next day, with 100% conversion at the tenth day.
5. Isolated preparation of metabolites
Extracting the culture solution by using n-butanol with the volume of 1 time, repeating the extraction for three times, combining the extraction solutions, and then decompressing and recovering, wherein the petroleum ether: ethyl acetate ═ 1: 1 silica gel column separation.
In conclusion, the preparation method of 6, 7, 8-trihydroxy coumarin disclosed by the invention is simple to operate, high in yield, more environment-friendly and suitable for industrial production.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (8)

1. A preparation method of 6, 7, 8-trihydroxy coumarin is characterized by comprising the following steps:
(1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution;
(2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant;
(3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution;
(4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure;
(5) and (3) purifying a product: purification was performed by preparative liquid chromatography.
2. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the first solution in the step (1) is physiological saline, phosphate buffer, carbonate buffer, Tris buffer or water.
3. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the feces in the step (2) are feces of rats, mice, rabbits, guinea pigs or humans, and are preferably rat feces.
4. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the nutrient broth in the step (3) is composed of peptone and beef extract powder, and can be any one or the combination of two components.
5. According toThe process for preparing 6, 7, 8-trihydroxy coumarin as claimed in claim 1, wherein: the anaerobic condition in the step (3) is CO2、N2Ar or He atmosphere, and the culture time is 2-10 d.
6. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the anaerobic culture solution in the step (3) comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, calcium chloride, magnesium sulfate, resazurin, L-cysteine and L-ascorbic acid; the amount of said anaerobic culture fluid added is 0.2-5 times the volume of said nutrient broth.
7. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the solvent for extraction in the step (4) is ethyl acetate, n-butanol, chloroform, dichloromethane or n-hexane, the amount of the extracting agent is 0.5-2 times, and the times are 1-3.
8. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the separation in the step (5) is carried out by using any one of silica gel, a C8 column and a C18 column.
CN201910998671.4A 2019-10-21 2019-10-21 Preparation method of 6,7,8-trihydroxy coumarin Active CN110819665B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910998671.4A CN110819665B (en) 2019-10-21 2019-10-21 Preparation method of 6,7,8-trihydroxy coumarin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910998671.4A CN110819665B (en) 2019-10-21 2019-10-21 Preparation method of 6,7,8-trihydroxy coumarin

Publications (2)

Publication Number Publication Date
CN110819665A true CN110819665A (en) 2020-02-21
CN110819665B CN110819665B (en) 2023-02-24

Family

ID=69549766

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910998671.4A Active CN110819665B (en) 2019-10-21 2019-10-21 Preparation method of 6,7,8-trihydroxy coumarin

Country Status (1)

Country Link
CN (1) CN110819665B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115074313A (en) * 2022-06-29 2022-09-20 五邑大学 Application of wedelolactone in preparation of product for promoting embryonic development
CN115433151A (en) * 2022-09-02 2022-12-06 苏州大学 Preparation method of 6,7,8-trihydroxy coumarin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1398861A (en) * 2002-08-29 2003-02-26 成都迪康药物研究所 Prepn and application in preparing medicine of Fraxinus general coumarin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1398861A (en) * 2002-08-29 2003-02-26 成都迪康药物研究所 Prepn and application in preparing medicine of Fraxinus general coumarin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIZZARRI B M ET AL.: "Regioselective IBX-mediated synthesis of coumarin derivatives with antioxidant and anti-influenza activities", 《JOURNAL OF NATURAL PRODUCTS》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115074313A (en) * 2022-06-29 2022-09-20 五邑大学 Application of wedelolactone in preparation of product for promoting embryonic development
CN115074313B (en) * 2022-06-29 2023-10-17 五邑大学 Application of wedelolactone in preparation of embryo development promoting product
CN115433151A (en) * 2022-09-02 2022-12-06 苏州大学 Preparation method of 6,7,8-trihydroxy coumarin

Also Published As

Publication number Publication date
CN110819665B (en) 2023-02-24

Similar Documents

Publication Publication Date Title
CN110819665B (en) Preparation method of 6,7,8-trihydroxy coumarin
CN111321095B (en) Butenolide dimer with alpha-glucosidase inhibition effect and application thereof
CN102766184B (en) Protopanoxadiol peroxide derivatives as well as preparation method and application thereof
CN106010980B (en) A kind of endogenetic fungus Brazil class shell roundlet spore bacterial strain and its application
CN105218447B (en) Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent
CN101358173A (en) Aspergillus niger ZJUT712 and application thereof in arctium fruit preparation by solid-state fermentation
JPS5874651A (en) Macbecin derivative and its preparation
CN105861363B (en) Like that grignard bacterium, S-equol generate engineering bacteria and its construction method and application
D’Antona et al. Synthesis of novel cyano-cyclitols and their stereoselective biotransformation catalyzed by Rhodococcus erythropolis A4
CN114381478B (en) Method for preparing daidzein by solid fermentation of soybean meal
CN108727169B (en) Preparation method of marine fungus-derived diphenyl ether compound and application of compound as antibacterial agent
CN103554005A (en) Novel simple synthesis method of L-5-hydroxytryptophan
CN107686492A (en) A kind of method of rhodioside in extraction purification zymotic fluid using macroporous absorbent resin
CN109370919B (en) Bacterial strain for producing penicillic acid and application thereof
CN102329829B (en) Method for converting daidzein into 8-hydroxydaidzein by utilizing penicillium
CN111534553A (en) Method for biotransformation of dihydrodeoxyartemisinin B by artemisia annua cells and application of dihydrodeoxyartemisinin B
CN101619055B (en) Oxidated genistein and biotransformation preparation and application method thereof
CN101585809B (en) Novel compound with antibacterial and antitumor activities
KR101915847B1 (en) A method for producing bioconversion derivatives using microorganisms
CN113396214A (en) Method for producing nigericin from streptomyces sp.mcc-0151
CN110004084B (en) Paenibacillus polymyxa, application of paenibacillus polymyxa in preparation of genistein and method for preparing genistein
CN1172914C (en) Glutarimide compound S632A3 and its prepn and application in preparing medicine for treating viral infection and tumor
CN108929857B (en) Salicornia europaea-derived marine fungus and application thereof in preparation of topoisomerase I inhibitor
JP4749652B2 (en) Novel FT-0554A substance and production method thereof
CN106591155A (en) Fusarium oxysporum strain and application thereof in preparation of 3beta, 7alpha, 15alpha-trihydroxy androstane-5-ene-17-ketone

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