163 results about "Androstenedione" patented technology

A process of fermenting a phytosterol composition to produce androstenedione (androst-4-ene-3,17-dione) and subsequently androstadienedione (and rosta-1,4-diene,3,17-dione) comprises propagating a microbial culture of the genus Mycobacterium in a first nutrient medium, placing the microbial culture and the phytosterol composition in a bioreactor for a sufficient time to transform the composition substantially to androstenedione (the "AD solution"), propagating a fungal culture of the genus Fusarium in a second nutrient medium, and then injecting one or more of 1) Fusarium sp, or 2) the fungal culture medium into the bioreactor for a sufficient time to transform the AD solution to androstadienedione.

Methods are provided for detecting the amount of one or more CAH panel analytes (i.e., pregnenolone, 17-OH pregnenolone, progesterone, 17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione, testosterone, deoxycorticosterone, 11-deoxycortisol, and cortisol) in a sample by mass spectrometry. The methods generally involve ionizing one or more CAH panel analytes in a sample and quantifying the generated ions to determine the amount of one or more CAH panel analytes in the sample. In methods where amounts of multiple CAH panel analytes are detected, the amounts of multiple analytes are detected in the same sample injection.

Methods for treating or reducing the likelihood of acquiring estrogen-related (e.g. estrogen-exacerbated) diseases including endometriosis include administering to a patient a selective estrogen receptor modulator (SERM), in combination with inhibiting ovarian secretions, e.g., by administering an LHRH agonist or antagonist. In some embodiments, a precursor of sex steroids, said precursor being selected from the group consisting of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), androst-5-ene-3β,17β-diol (5-diol), and androstenedione or a compound transformed into one of these, is also administered

The invention discloses a microbial strain for preparing androstenedione. The collection number of the microbial strain is CCTCC NO:M2012522. The preparation of androstenedione by the utilization of the strain comprises the following steps: A, primary seeding tank fermentation: inoculation amount of the strain is 0.5-1.5%, and cultivation is carried out at 160-200rpm and at 0.03-0.07MPa for 30-42h; B, secondary seeding tank fermentation: inoculation amount of the strain is 8-12%, and cultivation is carried out under the same conditions for 20-30h; C, fermentation tank fermentation for conversion to generate androstenedione: inoculation amount of the strain is 10-14%, and cultivation is carried out under the conditions of 27-35 DEG C and 0.03-0.07MPa; and D, termination of conversion: sterol is lower than 0.5%, pH is 8.9-9.0, discharging, heating to 90-100 DEG C and keeping for 30-50min, cooling to 30-50 DEG C, stopping stirring and standing for 4-6h. The invention provides a high-efficiency fermentation strain of mycobacterium. Meanwhile, a surfactant, soya-bean oil and a broth are used to form a two-way system. The fermentation technology is improved, and feeding amount of raw materials and conversion rate of microorganisms are greatly raised.

The invention belongs to the technical field of bio-catalysis, and in particular relates to a method for improving regeneration of mycobacterium coenzyme and for simultaneously promoting production ofandrostenedione. According to the method, sterol C27 site monooxygenase Cyp125 in a phytosterol metabolic pathway is introduced into steroid transformation microorganism, so as to improve androstenedione conversion efficiency of recombinant microorganism in a fermentation system; and therefore, a novel method for solving a problem of slow metabolism of mycelial phytosterol is offered. Through over-expression of the Cyp125, specific enzyme activity of the mycobacterium sterol C27 site monooxygenase is improved by 22.2%; regeneration of intracellular NAD+ is improved, and an intracellular NAD+ / NADH ratio is improved by 131%; and meanwhile, it is unexpectedly discovered that a bacterium biomass amount can be improved by 18.7% through the over-expression of the enzyme, and finally, effects ofshortening a conversion cycle by 24h and improving an AD(D) yield by 10.0% can be achieved.

The invention discloses a method for preparing progesterone by taking 1,4-androstenedione as a raw material, which comprises the following steps: 1) dissolving 1,4-androstenedione into an organic solvent, adding the acid of trimethyl orthoformate or triethyl orthoformate, and introducing nitrogen to protect the 1,4-androstenedione to synthesize the enol ether of 1,4-androstenedione, namely 3-methoxy-androstane 3,5-diene-20-ketone; and 2) dispersing (1-methoxy ethyl)-triphenylphosphine salt in a reaction medium, an organic solvent, adding alkali at low temperature, performing a Wittig reaction of the 3-methoxy-androstane 3,5-diene-20-ketone synthesized in the step 1), and purifying and crystallizing to obtain progesterone. By adopting the 1,4-androstenedione as the raw material, the method solves the problem that of lack in raw materials for synthesizing steroid drugs such as progesterone, and improves the utilization rate of 1,4-androstenedione and the yield of progesterone; the preparation process is simple.

The invention relates to a method for simultaneously detecting five kinds of steroid hormones in serum. The five kinds of steroid hormones are respectively testosterone, androstenedione, 11-deoxycortisol, cortisol and cortisone. The method for simultaneously detecting the five kinds of steroid hormones in the serum comprises the following steps: sample pretreatment: adding an acetonitrile solution of an internal standard substance in a serum sample for carrying out protein precipitation, then adding tert-butyl methyl ether for extracting, blow-drying supernatant, adding a combination solution, and taking the supernatant, thus obtaining a to-be-detected sample; enrichment, separation and detection: carrying out enrichment, separation and detection on the to-be-detected sample by adopting a two-dimensional liquid chromatography-tandem quadrupole mass spectrometer. According to the method for simultaneously detecting the five kinds of steroid hormones in the serum, disclosed by the invention, the method is adopted for simultaneously detecting five kinds of compounds of which the concentration ranges are inconsistent in the serum, and the time for simultaneously detecting the five kinds of the compounds is about 8.5 to 12.0 minutes; the method for simultaneously detecting the five kinds of steroid hormones in the serum is low in cost, high in flux, high in precision degree and strong in specificity.

The invention relates to a high-yield simple preparation method of 17alpha-hydroxy progesterone. With 4-androstenedione as an initial raw material, the method comprises the following steps: performing a cyanogen alcoholization addition reaction between the 17-site carbonyl of 4-androstenedione and acetone hydrogen alcohol to obtain 17-alpha hydroxyl-17-beta cyanoandrostane-4-ene-3-one; performing a ketal protection reaction of the C3-site carbonyl to obtain a ketal product; and performing a direct methylation reaction between the ketal product and zinc chloride methane, and hydrolyzing to obtain 17alpha-hydroxy progesterone. The method provided by the invention has the advantages of short process, high yield, high product purity, mild reaction conditions, low cost and low energy consumption and is particularly suitable for industrial production.

The invention provides a method for preparing 9a-hydroxy androstendione, which comprises a step of performing fermentation culture on phytosterol by use of mycobacterium fortuitum ATCC 35855 so as to convert the phytosterol into 9a-hydroxy androstendione. The method is used for preparing 9a-hydroxy androstendione by use of mycobacterium fortuitum ATCC 35855 and corresponding fermentation liquid and fermentation technology; the cheap phytosterol can be adopted as a substrate; and moreover, the yield of the prepared 9a-hydroxy androstendione is high, a few byproducts are produced, the fermentation time is short, and a good way is provided for high-efficiency industrial production of 9a-hydroxy androstendione.

The invention provides a method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation. Recombinant Mycobacterium smegmatis is fermented to convert phytosterin to produce the 9-alpha-hydroxyandrostenedione. The method comprises the following steps: constructing a recombinant expression vector pMV261-ksh; constructing a recombinant strain; and carrying out culture propagation and fermentation conversion. Gene engineering means are utilized to obtain the gene engineering strain for overexpressing 3-sterone-9-alpha-hydroxylase gene (ksh); after the strain is fermented for 120 hours, the conversion rate of the phytosterin is up to 95% above; and the method has the characteristics of high yield, fewer byproducts, short fermentation time simple extraction, small pollution, low pressure for environmental protection and the like, and has huge advantages in industrial production.

The invention provides compositions and methods for producing androstenedione (4-androstenedione), of improved purity and for modulating its production, for example by deletion or inactivation of ksdA, cxgA, cxgB, cxgC, or cxgD. The invention also provides methods and compositions, including nucleic acids that encode enzymes, for producing 1,4-androstadiene-3,17-dione (ADD) and related pathway compounds, including 20-(hydroxymethyl) pregna-4-en-3-one and 20-(hydroxymethyl)pregna-1,4-dien-3-one. The compositions of the invention include nucleic acids, probes, vectors, cells, transgenic plants and seeds, transgenic animals, kits and arrays.

The invention discloses a method of extracting 4-androstenedione from plant sterol fermentation liquor. Particularly, 4-androstenedione crude products (I) and unreacted plant sterol are extracted from mycelium and slag charges which are separated from the fermentation liquor and soaked by acetone; the 4-androstenedione crude products (I) are dissolved and crystallized by acetone aqueous solution with a concentration of 50% so that crude products (II) are obtained; and the crude products (II) are recrystallized by alcohol so that pure 4-androstenedione is obtained, wherein the ratio of the alcohol to water is 1:10, and the unreacted plant sterol is recrystallized by the alcohol so that recovered plant sterol is obtained, wherein the ratio of the alcohol to water is 1:10. The method of extracting the 4-androstenedione from the plant sterol fermentation liquor is good in extraction effect, high in product purity, easy to operate, few in number of types of solvents used, capable of reducing environmental pollution, low in cost, short in process route and suitable for industrialized production. The unconverted plant sterol can be recovered and recycled.

The invention provides a method for preparing androstenedione using a double liquid phase fermentation system by fermenting Mycobacterium sp.DE6823, and aseptic distilled water is selected as a fermentation medium water phase, and rice oil, sunflower oil, soybean oil and peanut oil can be selected as an oil phase, after fermentation conversion, a solution containing androstenedione is obtained, and after condensation, decolouring, filtering, condensation, recrystallization and vacuum drying and other operations, and androstenedione whose purity is above 99% is obtained. Different oil phases can be selected, and the conversion rate of the plant sterol is in the range of 59%-85%. The invention solves the problems that the raw materials of the present androstenedione production technology in our country are limited by season and region, thereby effectively reducing destroy to ecological environment; the invention has the characteristics of simple operation, small environmental pollution, high conversion rate, low production cost, etc., and is suitable for industrialized production.

The invention provides a method for screening a I type 17 beta-hydroxysteroid dehydrogenase inhibitor through an immobilized enzyme, and belongs to the technical field of enzymology and enzyme engineering. I type 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1) has the important function in treating hormone-dependent diseases. At present, a substrate radiolabelling method is mainly adopted in studying the activity of 17 beta-HSD1, due to the fact that the free enzyme of 17 beta-HSD1 is likely to be inactivated, 17 beta-HSD1 is hard to prepare, a fresh placenta is needed for preparing an enzyme source in a new medicine screening experiment of every time, raw materials are hard to obtain, the price is high, and difficulty is brought to the screening work of new medicine. According to the method, an amino-modified silicon ball is adopted as a carrier, a glutaraldehyde crosslinking method is utilized, 17 beta-HSD1 extracted from the placenta is fixed, an external immobilization 17 beta-HSD1 enzyme model is built, androstenedione is adopted as a substrate, a high performance liquid chromatography method is used for detecting products, and the potential 17 beta-HSD1 inhibitor is screened. According to the method, instability of the free enzyme is overcome, operation is easy, the manufacturing cost is low, and repeated using is achieved.

A preparation method for dehydroepiandrosterone, comprising: in a protective atmosphere, adding potassium tert-butoxide to tert-butanol, stirring evenly, adding 4-androstenedione to obtain a mixture,and adding the mixture dropwise to a sodium ascorbate-containing acetic acid solution for a reaction to obtain 5-androstenedione; dissolving the 5-androstenedione in an organic solvent, adding a ketone reductase, a glucose dehydrogenase, glucose and a redox coenzyme to obtain a mixture, controlling the pH of the mixture to be 6.0-6.3, stirring and reacting for 1-6 hours at 22-26 DEG C to obtain areaction solution, and performing separation and purification on the reaction solution to obtain dehydroepiandrosterone, the ketone reductase and the glucose dehydrogenase being coexpressed by a microbial strain and added in the form of a crude enzyme solution. The synthesis process of the preparation method has few steps, simple operations, high yields and low costs, and may be widely applied toindustrial scale production. Also provided is an enzyme for preparation.

The invention provides a qualitative and quantitative detection method of steroid hormones in saliva and particularly relates to a liquid chromatography tandem mass spectrometry detection method of eight steroid hormones in saliva; the steroid hormones include 17-hydroxyprogesterone, progesterone, androstenedione, testosterone, dehydroepiandrosterone, cortisol, corticosterone and aldosterone. Thedetection method herein includes: freezing a human saliva sample, allowing protein precipitation, ultrasonically extracting, filtering, mixing the filtrate with an internal standard liquid, carrying out reversed-phase SPE (solid phase extraction) column purification, and chemically deriving the target products. Full dissolution of the target hormones in saliva is guaranteed; disturbance of impurities in the saliva is eliminated; accuracy and stability are ensured for detection results. The eight hormones are synchronously, qualitatively and quantitatively analyzed by liquid chromatographic separation and mass spectrometric ion-pairing detection. The detection method herein is suitable for synchronous batch detection of hormone molecules having similar structure and approximate chemi-physical properties, has the advantages of high sensitivity, high precision, good repeatability, high detection speed and the like, fully meets the needs of clinical detection, and has high clinical application value.

The invention belongs to the technical field of biological catalyst, and particularly relates to a mycobacteria genetically engineered bacterium with cofactor regeneration function and its applicationin fermentation of two liquid phases. Through a NADH oxidizing system in the cell, the genetically engineered bacterium for transforming plant sterol biology is structured and transformed for the NADH oxidizing system in the cell, thus the problem of low transforming efficiency of androstenedione in the oil water two-liquid-phase fermenting system is solved; the genetically engineered bacterium also provides possibility for the wide application of the oil water two-liquid-phase fermenting system in industrial production of the androstenedione. The genetically engineered bacterium MNR M3N2 structured by the invention can improve the AD (D) output by 1.4-2.9 times during the preparation process of androstenedione by degrading the phytosterol through the oil water two-liquid-phase fermentingsystem fermenting method.

The invention discloses a method for preparing 19-demthyl-4-androstenedione, which comprises the steps of: based on estrone as a raw material, sequentially carrying out etherealization reaction, ketalation, Birch reducing reaction and hydrolysis reaction to obtain19-demethyl-4-androstenedione, wherein the reaction formula is shown in the specification. According to the method provided by the invention, the traditional phenolic group methyl etherealization process is improved, methyl carbonate replaces traditional high-toxicity carcinogenic etherealization reagents dimethyl sulfate and iodomethane, and inexpensive potassium carbonate is used as a base, so that the production cost is lowered, the method is environment-friendly, the yield is high, and the method is suitable for scaled industrial production.

The invention relates to a method recombining Yarrowia lipolytica to efficiently and biologically convert androstenedione so as to produce testosterone and belongs to the field of genetic engineering. The method is characterized in that 17beta-hydroxyl steroid dehydrogenase from human and glucose dehydrogenase from saccharomyces cerevisiae are respectively cloned and excessively co-expressed in Y.lipolytica Polh, and the androstenedione is efficiently converted in the Yarrowia lipolytica to produce the testosterone for the first time at home and abroad by building an NADPH coenzyme circulation system. The enzyme activity determination and intracellular coenzyme level researches of the built genetic-engineering strains show that the recombinant strains can continuously and effectively convert the androstenedione to produce the testosterone. When the conversion temperature is 37 DEG C, conversion pH is 7.5, substrate cosolvent is methylated-beta-cyclodextrin, biomass is 200g / L and substrate concentration is 5g / L, 15g / L androstenedione is converted into 14.3g / L testosterone after three material-supplementing whole-cell conversions, the currently-reported highest level of testosterone production using microorganism conversion is achieved, and a foundation is provided for industrial testosterone production using microorganisms.

The invention discloses a method for preparing 9alpha-hydroxide-androstenedione by utilizing microbial conversion. The method comprises the steps of: 1) by taking rhodococcus erythropolis as a seed lot, inoculating rhodococcus erythropolis in a seed culture medium, and culturing to obtain seeds; 2) inoculating the seeds into a conversion culture medium, and culturing to obtain fermentation liquor; 3) adding an androstenedione solution into the fermentation liquor, continuing culturing to obtain conversion liquor, and detecting the content of 9alpha-hydroxide-androstenedione in the conversion liquor; and 4) separating and extracting the 9alpha-hydroxide-androstenedione in the conversion liquor. The method has the advantages of being high-efficiency and specific, environment-friendly, high in added materail concentration, high in conversion rate, product purity and yield, low in cost and the like, is simple in overall preparation process, and economic and practical, has wide application prospects, and has very important significance on the application for extending the steroid raw materail drug androstenedione.

The invention discloses Ketosteroid C27-monooxygenase derived from mycobacterium neoaurum and application of the ketosteroid C27-monooxygenase, and belongs to the technical field of gene engineering and enzyme engineering. By means of gene knockout and expression strengthening methods, three isoenzymes of a key enzyme SMO in the sterol side chain degradation process are screened out from the mycobacterium neoaurum; the isoenzymes are intensively expressed in mycobacterium neoaurum of high-yielding androstenedione (ADD) separately, the ADD yield is obviously increased, and the effect of SMO2 is most obvious. By overexpressing SMO2, the final yield of ADD is increased to 7.3 g / L from 5.2 g / L. The Ketosteroid C27-monooxygenase derived from the mycobacterium neoaurum and the application of the ketosteroid C27-monooxygenase provide beneficial guidance for industrialization of increasing the ADD yield through a microbiological fermentation method.

The invention discloses a method for extracting androstenedione in a bi-liquid phases system. which is characterized in that a broth is pretreated, and is extracted by methanol and is performed with vacuum concentration until no methanol is generated, and then filtered to obtain a crude product I, washing the crystal by a mixing solvent to obtain a crude product II, and the crude product II is performed through twice methanol purification and dried to obtain the androstenedione pure product. The method has the advantages of good extraction effect and high product purity, methanol is taken as a main solvent, the cost is low, and the method is suitable for industrial production.

The invention discloses preparation methods of danazol and an intermediate thereof. The preparation method of danazol is prepared by the steps of taking androstenedione as a starting raw material, and carrying out 3-site enol etherification, 17-site carbonyl ethinylation, 3-site hydrolysis, 2-site methylidynel hydroxylation and oximation to obtain danazol. The 3-site enol etherification comprises firstly carrying out a reaction of androstenedione and triethyl orthoformate for 4-10 h in the presence of absolute ethyl alcohol and p-toluenesulfonic acid and at the temperature of 30-50 DEG C, then adding triethylamine at the temperature of 0-10 DEG C, and continuing to carry out a reaction for 0.2-1 h; the 17-site carbonyl ethinylation comprises firstly carrying out a reaction of a potassium hydroxide powder for 1-2 h in an acetylene airflow and at the temperature of 5-10 DEG C, and then carrying out a reaction with the 3-site enol etherified product for 2-4 h in the presence of tetrahydrofuran and a catalyst, at the temperature of 15-30 DEG C and in the acetylene airflow. The 3-site enol etherification is mild in reaction conditions and relatively high in yield, and the 17-site carbonyl ethynylation is relatively high in reaction yield and relatively short in time.

The invention provides a Chinese herbal compound for treating the pubertal polycystic ovary syndrome. The Chinese herbal compound is prepared from, by weight, 30-35 parts of radix astragali, 20-25 parts of American ginseng, 15-20 parts of Radix Salviae Miltiorrhizae,, 15-20 parts of hawthorn, 15-20 parts of poria cocos, 20-25 parts of bighead atractylodes rhizome and 15-20 parts of tangerine peel. The pubertal polycystic ovary syndrome (PCOS) is treated through the method of tonifying qi and spleens, reducing phlegm, activating blood and regulating constitution; compared with other traditional Chinese medicine research, the Chinese herbal compound can reduce levels of luteinizing hormone (LH), luteinizing hormone / follicle-stimulating hormone (LH / FSH), testosterone (T) and androstenedione (AND) in serum of patients and also can reduce the levels of total cholesterol (TC) and apolipoprotein B (APOB) in blood fat, and therefore the aims of regulating the menstrual cycle, improving hyperandrogenism and correcting dyslipidemia are achieved.

The invention relates to a method for detecting steroid hormones in blood. The method comprises the following steps: preparation of a standard curve dried blood spot sample: preparing red blood cells by using whole blood; by taking hormone-removed serum as a blank matrix, adding standard substances such as 17alpha-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycortisol and 11-deoxycortisol solutions to prepare a series of serum standard curve working solutions; mixing the red blood cells with the serum standard curve working solution, and preparing a standard curve dried blood spot sample by using the obtained mixed solution; pretreatment of the dried blood spot sample: taking the dried blood spot sample, adding an internal standard-containing extraction liquid, extracting, centrifuging, drying, adding a reconstitution fluid, centrifuging, and determining by adopting liquid chromatography-tandem mass spectrometry. The detection method disclosed by the invention is simple in pretreatment operation, short in time consumption, high in stability and good in reproducibility, and various steroid isomers can be efficiently separated at the same time.

The invention dsiclsoes a method of preparing androstane-4 alkene-3,17 diketone and / or androstane-1,4-diene-3,17-diketone by taking a phytosterol composition as a substrate. The method is characterized by comprising the following steps: adding the phytosterol composition into a reactor which contains nocardia or mycobacterium microorganisms and a culture medium for fermentation, regulating the PH value to be larger than 7, fermenting at 20-37 DEG C, converting the phytosterol composition into androstane-4 alkene-3,17 diketone and / or androstane-1,4-diene-3,17-diketone, wherein the conversion process is carried out in an electric field.

The invention provides a method for preparing androstenedione using a double liquid phase fermentation system by fermenting mycobacterium sp.DE6823, and aseptic distilled water is selected as a fermentation medium water phase, and rice oil, sunflower oil, soybean oil and peanut oil can be selected as an oil phase, after fermentation conversion, a solution containing androstenedione is obtained by employing a membrane separating method, and after condensation, decolouring, filtering, condensation, recrystallization and vacuum drying and other operations, androstenedione with purity of 99% or more is obtained. Different oil phases can be selected, and the conversion rate of the plant sterol is in the range of 59%-85%. The invention solves the problems that the raw materials of the present androstenedione production technology in our country are limited by season and region, thereby effectively reducing destroy to ecological environment; and the invention has the characteristics of simple operation, small environmental pollution, high conversion rate, low production cost, etc., and is suitable for industrialized production.

The invention belongs to the technical field of androstenedione production and specifically relates to a treating method for androstenedione fermented wastes and application of recovered materials. The method comprises the following steps: adding a demulsifying agent into the androstenedione fermented wastes at material liquor ratio of 1:(1-3); standing by, splitting phases and taking an upper phase under the condition at 20-60 DEG C; adding isometric demulsifying agent into a lower phase, and then standing by, splitting phases and taking the upper phase under the same temperature again; filtering the lower phase, thereby acquiring a filtrate; mixing the filtrate with the upper phase, thereby acquiring a mixed solution; crystallizing the mixed solution under the condition at 0-10 DEG C for two times, filtering and taking crystal, thereby acquiring sterol crystal; concentrating the filtrate and then precipitating the grease, thereby acquiring the grease. An organic solvent demulsification method and a low-temperature secondary crystallization technique are adopted by the invention for effectively recycling and reusing grease and sterol from the androstenedione fermented wastes, so that the production cost is saved and the pollution of the wastes is reduced.