CN114588179A - Method for separating and purifying conjugated estrogens from pregnant mare urine by using preparative chromatography - Google Patents
Method for separating and purifying conjugated estrogens from pregnant mare urine by using preparative chromatography Download PDFInfo
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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
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
- A61P5/30—Oestrogens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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Abstract
The invention provides a method for separating and purifying natural pregnant mare conjugated estrogen from pregnant mare urine by using a preparative chromatography, which is used for preparing a natural pregnant mare conjugated estrogen product without pungent smell by using pregnant mare urine as a raw material, a preparative chromatography separation and purification means, a polar group modified silica gel filler and an organic solvent as an eluent (60 target objects meet the content requirement by mass spectrometry). The method has the characteristics of simple process flow, good repeatability, high recovery rate, low solvent consumption, simple and controllable operation and the like, and is suitable for separation and purification in production.
Description
Technical Field
The method relates to the technical field of separation and purification of animal medicinal components, in particular to a method for separating and purifying natural conjugated estrogens from pregnant mare urine by using a preparative chromatography.
Background
Conjugated Estrogens (CE) are a collective term for the mixture of water-soluble conjugated estrogens of various natural sterols that were first extracted from pregnant mare urine (pregnant mare urine). The traditional Chinese medicine composition is mainly used for hormone replacement therapy in clinic to relieve any clinical symptoms caused by estrogen deficiency. It has unique curative effect on climacteric syndrome and ovariectomy common diseases, and can be used for preventing and treating heart disease and senile osteoporosis, activating body function, reducing circulating blood lipid level, removing wrinkle and delaying aging.
With the progress of society and the aging of population, more and more women pursue high-quality, safe and effective estrogen products. More than 50% of climacteric women in europe and more than 30% in the usa use hormone replacement therapy, in combination with estrogens, for nearly 80% of the global estrogen supply market. The product has a history of medication in the world for nearly 70 years, and is named as Premarin in English and Beimeili in Chinese.
The prosperity and conspiracy have been long since the first time they were marketed in the united states in 1942. Despite the numerous synthetic estrogen drugs, natural conjugated estrogens of pregnant mares are not a replacement in hormone replacement therapy.
The average content of the estrogen in the urine of each pregnant mare is 70-130 mg, and each pregnant mare can form 5-100 g of estrogen in the pregnancy period. Pregnant mare urine contains a large amount of soluble impurities, such as inorganic salts, urea, saccharides, flavones, isoflavones, phenols and polyphenols, which are metabolites of pregnant mares. Also contains insoluble impurities, such as insoluble salts in urine, specifically the generated precipitate and the precipitate generated in the storage process: calcium carbonate such as calcium carbonate, calcium bicarbonate, calcium phosphate, and calcium hydrogen phosphate, oxalate, and the like. Interference of these impurities can affect the purification and isolation of estrogen.
At present, most processes adopt an extraction method and a precipitation method, and polar macroporous adsorption resin or ion exchange resin is used for separating and purifying combined estrogen, and a plurality of related patent publication resin purification methods exist, but the defects of the prior art are that the macroporous resin has low mechanical strength, is easy to break, has less recovery times, is unstable in process, has low recovery rate, large water consumption and serious pollution. With the development of new technologies and materials, the separation and purification of pregnant mare urine needs to be continuously optimized and adjusted to meet the market demand.
The present invention is proposed in view of the above technical background and development prospects.
Disclosure of Invention
The invention aims to provide a method for separating and purifying conjugated estrogens from pregnant mare urine, which is based on the principle that components in a mixture with similar structure are in a fixed phase and have different migration speeds along with the movement of a mobile phase by virtue of the surface adsorption effect of the fixed phase, so that the components flow out of the fixed phase according to a certain sequence, and a target fraction is picked up according to a chromatogram map to obtain a high-purity product.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention relates to a method for separating and purifying pregnant mare urine by using a preparative chromatography, which adopts a chromatographic separation mechanism, uses an organic solvent mixed solution to elute conjugated estrogens, and collects conjugated estrogens target analytical solution.
The invention relates to a method for separating and purifying pregnant mare urine by using preparative chromatography, which is characterized by comprising the following steps: the filler used in the chromatographic separation mode is HC series pure water resistant silica gel filler modified by polar groups, and a polar copolymerization bonding technology is adopted, wherein the length of an alkyl chain connected with silica gel is adjustable and ranges from C4 to C18, and the bonding amount is 0.8 to 5.0 mu mol/m2Has good enriching effect on the combined estrogen, the grain diameter is 10 to 100 mu m, and the pore diameter is
The invention relates to a method for separating and purifying pregnant mare urine by using preparative chromatography, which is characterized by comprising the following steps: as the preferable preparation chromatographic condition, the sample loading volume of the pregnant mare urine is 10-200 times of the column volume.
The preferable preparation chromatographic condition is that the separation temperature is 15-50 ℃.
The invention relates to a method for separating and purifying pregnant mare urine by using preparative chromatography, which is characterized by comprising the following steps: and collecting target analysis liquid according to the chromatogram.
The conjugated estrogen product is obtained after concentration and drying, no pungent smell exists, and the content of 60 target components of the standard Beimeili product meets the requirement by mass spectrum detection.
The invention relates to a method for separating and purifying pregnant mare urine by using a preparative chromatogram, which comprises the following steps:
1) directly adopting pregnant mare urine as a sample solution;
2) separating by adopting a column system, wherein the inner diameter of a chromatographic column is 4.6-1200 mm; a chromatographic separation and purification system is adopted, and the used filler is silica gel modified by polar groups. The particle diameter is 5-100 mu m, and the aperture is
The specific surface area is 50-800 m2(ii)/g, the shape is irregular or spherical; wherein the organic solvent is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile, acetone, ethyl acetate and the like, and accounts for 1-90% of the total volume of the mobile phase; the flow rate is 0.02-1.0 times of the column volume/min; the loading amount is 1-200 times of the column volume. And collecting target analysis solution according to the chromatogram, and obtaining the conjugated estrogen product without pungent smell by the method (60 target objects meet the content requirement by mass spectrometry).
The invention has the following advantages:
1. the method utilizes a preparative chromatographic separation and purification means, adopts the silica gel filler modified by polar groups to purify and prepare the pregnant mare urine to obtain the conjugated estrogen, and has the advantages of rapidness and high efficiency compared with other separation processes such as traditional silica gel and resin;
2. the preparation process is adopted for separation and purification, the enrichment effect is good, the product quality is high, and no pungent smell exists;
3. the preparation process is adopted for separation and purification, so that the sample loading amount is large, the recovery rate is high, and the industrial production is easier to realize;
4. the preparation process is adopted for separation and purification, the solvent consumption is low, the flow is simple, the repeatability is good, the operation is simple and controllable, the automation is easy to realize, and the process is stable.
Drawings
FIG. 1 is a spectrum of a prepared pregnant mare's urine of example 1;
FIG. 2 is a liquid phase analysis spectrum before and after purification of example 1.
Detailed Description
The present invention will now be further described with reference to examples. It should be noted that, for those skilled in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention. The examples are given solely for the purpose of illustrating the invention and are not to be construed as limiting the invention thereto.
Example 1
Pregnant mare urine (6L) was used as a sample solution, and a C8HC silica gel filler (preparation column size: 50X 250mm, particle diameter 30 μm, pore diameter)
300g mass, Wawsonia Innovative science and technology Limited) and the bonding amount is 2.216 mu mol/m2Column temperature 30 ℃, column temperature with ethanol: eluting with water at a flow rate of 50mL/min at a rate of 30:70, collecting 180-200 min fractions according to a peak shape curve after a target peak is obtained, concentrating and drying the fractions of the target resolution solution to obtain a conjugated estrogen product with a yield of 96.1%, and calibrating with the double meili product through mass spectrometry detection, wherein 60 target substances meet the content requirement.
Example 2
Pregnant mare urine 0.5L is used as sample loading liquid, C18HC silica gel filler (preparation column specification: 20)250mm, particle size 10 μm, pore diameter
50g mass, Hua Shi Xin Tech Co., Ltd.) and the bonding amount is 2.151 μmol/m2Column temperature 25 ℃, column temperature with methanol: eluting with water at a flow rate of 10mL/min at a ratio of 20:80, collecting 190-210 min fractions according to a peak shape curve after a target peak is obtained, concentrating and drying the fractions of the target resolution solution to obtain a conjugated estrogen product with a yield of 93.9%, and aligning 60 target substances according to the content requirement by mass spectrometry and the double Meili product.
Example 3
500 liters of pregnant mare urine was used as a sample solution, and C4HC silica gel filler (preparation column size: 300X 250mm, particle diameter 10 μm, pore diameter)
10kg mass, Hua Shi Xin science and technology Limited) and the bonding amount is 2.882 mu mol/m2The column temperature was 30 ℃ and the flow rate was 2L/min, the column temperature was measured using ethanol: eluting with 25:75 water, washing for 20min, collecting 280-310 min fractions according to a peak shape curve after a target peak is obtained, concentrating and drying a target analysis solution by adopting a film, wherein the product yield is 91.8%, and aligning with a double Meili product through mass spectrum detection, wherein 60 target objects meet the content requirement.
Comparative example 1
The difference from the example 1 is that pure silica gel (100-200 meshes) modified by non-bonding groups is used as a separation material, the other conditions are the same as the example 1, the product yield is 56.2%, 25 of 60 target components are lost through mass spectrometry, the loss has a certain pungent smell, and the mass spectrometry cannot reach the quality standard of the Beimeili product.
Comparative example 2
The difference from the example 1 is that macroporous resin is used as a separation material, the other conditions are the same as the example 1, the product yield is 68.5%, 9 of 60 target components are lost through mass spectrometry, the product has strong pungent smell, and the mass spectrometry cannot reach the quality standard of the Beimeili product.
Comparative example 3
The difference from the example 1 is that ordinary octadecylsilane chemically bonded silica is used as a separation material, the other conditions are the same as the example 1, the product yield is 42.8%, 5 of 60 target components are lost through mass spectrometry, the product has certain pungent odor, and the mass spectrometry cannot reach the quality standard of the Beimeili product.
Claims (7)
1. A method for separating and purifying pregnant mare urine by using preparative chromatography is characterized by comprising the following steps: and (3) eluting by using an organic solvent as a mobile phase in a chromatographic separation mode, and collecting a target resolution solution to obtain a conjugated estrogen product.
2. The method of claim 1, wherein: the filler used in the chromatographic separation mode is HC series pure water resistant silica gel filler modified by polar groups, and a polar copolymerization bonding technology is adopted, wherein the length of an alkyl chain connected with silica gel is adjustable and ranges from C1 to C30, and the bonding amount is 0.5 to 8.0 mu mol/m2Has good enrichment effect on the combined estrogen, the grain diameter of the filler is 5-200 mu m, and the aperture is
The shape is irregular or spherical.
3. The method according to claim 1, characterized in that the mobile phase is a mixture of organic phase and water phase, the organic phase is one or more of common solvents such as methanol, ethanol, isopropanol, acetonitrile, acetone, ethyl acetate, etc., the organic solvent accounts for 1-99% of the total volume of the mobile phase, the water phase is one or two of formic acid, acetic acid or phosphoric acid, the proportion is 0.01-5.0% (v/v), and the type of the mobile phase used is optimally adjusted according to different bonding fillers.
4. The method of claim 1, wherein: the flow velocity of the mobile phase is 0.01 to 1.5 times of the column volume/min.
5. The method of claim 1, wherein: the separation temperature of the preparative chromatography is 4-60 ℃.
6. The method of claim 1, wherein: the sample loading volume of pregnant mare urine is 1-500 times of column volume.
7. The method of claim 1, wherein: collecting target analysis solution according to chromatogram, concentrating and drying to obtain conjugated estrogen product without pungent odor, and detecting 60 target components of the standard Beimeili product by mass spectrum.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011410563.XA CN114588179A (en) | 2020-12-03 | 2020-12-03 | Method for separating and purifying conjugated estrogens from pregnant mare urine by using preparative chromatography |
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| CN1308083A (en) * | 2001-02-19 | 2001-08-15 | 中国科学院新疆化学研究所 | Extraction method of combined mixed estrin of pregnant mare |
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| CN111458442A (en) * | 2020-06-04 | 2020-07-28 | 新疆新姿源生物制药有限责任公司 | Method for measuring content of conjugated estrogen in pregnant mare urine |
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