WO2021052450A1 - Reversed phase chromatography isolation of active component in amniotic fluid of non-human animal - Google Patents

Reversed phase chromatography isolation of active component in amniotic fluid of non-human animal Download PDF

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WO2021052450A1
WO2021052450A1 PCT/CN2020/116079 CN2020116079W WO2021052450A1 WO 2021052450 A1 WO2021052450 A1 WO 2021052450A1 CN 2020116079 W CN2020116079 W CN 2020116079W WO 2021052450 A1 WO2021052450 A1 WO 2021052450A1
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amniotic fluid
eggs
reversed
phase chromatography
phase
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PCT/CN2020/116079
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French (fr)
Chinese (zh)
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张可中
张利红
高翔
骆严
钱进
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浙江楚沅生物科技有限公司
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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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features

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  • the present invention belongs to the field of animal extracts, and specifically relates to the use of reversed-phase chromatography to separate the active ingredient extraction from non-human animal amniotic fluid and its effect on cell proliferation activity.
  • Animal amniotic fluid contains a wide variety of amino acids, organic acids, sugars, steroids, nucleotides, lipids, their derivatives, trace elements and other substances. According to the differences in physical and chemical properties of different substances, including molecular weight, solubility, isoelectric point, and hydrophobicity, the techniques for purifying amniotic fluid usually include precipitation techniques, chromatography techniques, and two-liquid phase extraction techniques. However, these technologies currently have high costs, complex processes, and low recovery rates, and often cause the biological activity of the target compound to be reduced or even lost during the separation and purification process.
  • Reversed phase chromatography refers to an elution chromatography that uses a non-polar reverse phase medium as the stationary phase and an aqueous solution of a polar organic solvent as the mobile phase to separate and purify the solute according to the polarity (hydrophobicity) of the solute.
  • High performance liquid chromatography and ultra high performance liquid chromatography can effectively separate, identify, and semi-quantitate target compounds in crude products and are widely used. Therefore, RPC is very suitable for separating substances or composite substances with a relative molecular mass of less than 2000 Daltons, which are neutral and have good stability.
  • the invention uses C18 and its derivatives as a reverse phase medium, and combines the AKTA system and the HPLC system to separate the active substances in the amniotic fluid of non-human animals.
  • the extract of the present invention can promote cell proliferation.
  • the present invention provides an extract of non-human animal amniotic fluid.
  • the octanol/water partition coefficient Log P of the active ingredients contained in the extract is in the range of 0.05-1.897.
  • the octanol/water partition coefficient Log P of the active ingredient in the extract is between 0.3-1.5.
  • the extract does not bind to the ion exchange column at a pH between 5.8 and 8.0.
  • the amniotic fluid is amniotic fluid derived from poultry eggs.
  • the poultry eggs are eggs with embryo age of 5-12 days, preferably eggs with embryo age of 6-11 days, more preferably eggs with embryo age of 7-9 days, more preferably embryos Eggs with an age of 7-8 days, or eggs of other poultry other than chickens whose developmental period corresponds to the developmental period of the embryo-aged eggs.
  • the poultry eggs are poultry eggs, such as eggs, duck eggs, ostrich eggs, goose eggs, and the like.
  • the amniotic fluid is non-human mammal amniotic fluid; preferably, the amniotic fluid is derived from rodents; preferably, the amniotic fluid is derived from embryos of rodents with a gestational age of 8-20 days , Or embryos from non-human mammals whose developmental period corresponds to that of rodents with a gestational age of 8-20 days.
  • the extract is separated by reverse phase chromatography.
  • the present invention also provides a method for preparing a non-human animal amniotic fluid extract, the method comprising using reversed-phase chromatography to separate the octanol/water partition coefficient Log P between 0.05-1.897 and having cell proliferation activity from the amniotic fluid And/or a fraction that promotes the recovery of cardiac function after myocardial infarction.
  • the method includes performing two reverse phase chromatographic separations, wherein the resolution of the reverse phase chromatography column used in the second reverse phase chromatographic separation is higher than that of the first reverse phase chromatographic separation. The resolution of the reversed-phase column used.
  • the method includes:
  • step (2) The fraction obtained in step (1) is separated again by reversed-phase chromatography, and the fraction with the octanol/water partition coefficient Log P in the range of 0.05-1.897 is obtained.
  • the method includes two reversed-phase chromatography, wherein, in the first reversed-phase chromatography, the elution volume of the fraction between 51-731 ml, preferably the elution volume is 250-504
  • the fraction between milliliters, more preferably the fraction with an elution volume between 278-353 milliliters and/or between 354-429 milliliters and/or between 430-504 milliliters, or other similar columns in the same proportion
  • the obtained similar fractions are used in the second reverse phase chromatography; in the second reverse phase chromatography, take the fraction with the elution time between 11-12.5 minutes and the fraction between 13-14 minutes, or use other similar chromatography
  • the column obtains similar fractions in the same proportions.
  • the stationary phase of the reversed-phase chromatography column used in reversed-phase chromatography uses silica gel as a carrier, and the non-polar groups bonded on the surface are selected from phenyl, C8 or C18 alkyl groups and their derivatives.
  • organic solvents such as methanol, acetonitrile, ethanol and water as the mobile phase.
  • the mobile phase used in step (1) is acetonitrile and ddH 2 O.
  • step (1) adopts gradient elution, and the mobile phase is acetonitrile and ddH 2 O, wherein the concentration gradient of acetonitrile is 5%-12%, and the concentration gradient of ddH 2 O is 95%- 88%.
  • the mobile phase of step (2) is acetonitrile and pure water.
  • step (2) adopts gradient elution, the mobile phase is acetonitrile and pure water, the concentration gradient of acetonitrile is 0%-7%, and the concentration gradient of pure water is 100%-93%.
  • a C18 reverse phase chromatography column is used for reverse phase separation.
  • the method further includes, after the first reverse phase chromatography separation, testing the cell proliferation activity of the separated fractions, picking the fraction with the strongest cell proliferation activity and performing the second reverse phase Chromatographic separation.
  • the present invention also provides active ingredients derived from non-human animal amniotic fluid obtained by the method described herein.
  • the present invention also provides a pharmaceutical composition containing the extract or active ingredient described herein, and optionally a pharmaceutically acceptable carrier.
  • the invention also provides the application of the reverse phase chromatography column in separating the components with cell proliferation activity in the amniotic fluid of non-human animals.
  • the present invention also provides the application of the active ingredients described herein in the preparation of products that promote cell proliferation and/or promote cardiac function recovery after myocardial infarction.
  • Figure 1 HPLC chart of egg amniotic fluid.
  • Figure 2 HPLC chart of duck egg amniotic fluid.
  • Figure 3 Cell proliferation test results of egg and duck egg amniotic fluid extract.
  • Figure 4 Chromatogram of fresh egg amniotic fluid separated by UniSil 10-100 C18.
  • Figure 5 HPLC chart of peaks P6, P7 and P8 of the amniotic fluid extract.
  • Figure 6 Cell viability detection found that the P6, P7, and P8 peaks in Figure 4 have biological activity.
  • FIG. 10-11 P6 and P8 peaks with cellular activity help to promote the repair of heart function in myocardial infarction.
  • the present invention provides a method for purifying and extracting active ingredients in the amniotic fluid of non-human animal embryos, and obtains a group of growth factors that can promote cell proliferation and recovery of cardiac function after myocardial infarction.
  • the method herein includes the step of separating the fraction with the octanol/water partition coefficient Log P between 0.05 and 1.897 from the amniotic fluid of non-human animal embryos, preferably between 0.1 and 1.897, more preferably between 0.5 and 1.897 or Between 0.5-1.5.
  • amniotic fluid can come from poultry eggs and non-human mammals.
  • Poultry eggs refer to poultry eggs.
  • the preferred birds are poultry, such as chickens, ducks, ostrich eggs, and geese.
  • the present invention uses poultry eggs whose embryonic age is 5-20 days, preferably 6-15 days. It should be understood that the appropriate embryo age may not be the same for different eggs.
  • eggs with embryo age of 5-12 days are preferably used, eggs with embryo age of 6-11 days are more preferably used, eggs with embryo age of 7-9 days are more preferably used, and embryo age is more preferably used.
  • Eggs for 7-8 days When using eggs of other avians, eggs whose developmental stage corresponds to the developmental stage of the eggs of the embryonic age mentioned above can be used.
  • duck eggs duck eggs with embryo age of 8-10 days, especially 8-9 days are the best.
  • amniotic fluid from poultry eggs. For example, you can knock the blunt end of an egg of the corresponding embryonic age to break the eggshell, and then peel the eggshell to form a hole with a diameter of about 2 cm. Then use tweezers to carefully tear open the shell membrane and vitelline membrane, taking care not to damage the amniotic membrane. Pour the amniotic membrane and connected tissues covering the embryo from the shell to a petri dish, and pierce the amniotic membrane with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.
  • amniotic fluid can also be derived from non-human mammals, especially rodents, such as from mice.
  • the amniotic fluid is derived from embryos of rodents with a gestational age of 8-20 days, or from non-humans whose developmental period corresponds to that of rodents with a gestational age of 8-20 days. The embryo of a mammal. Conventional methods can be used to obtain amniotic fluid.
  • the amniotic fluid is derived from rodent embryos with a gestational age of 8-14 days or 11-16 days, more preferably 13-14 days, or from a developmental period and a gestational age of 8-14 days or 11-16 days, more preferably The embryos of non-human mammals corresponding to the developmental stage of rodents of 13-14 days are preferred.
  • amniotic fluid can be centrifuged to separate possible impurities, such as egg yolk, etc., to obtain as pure amniotic fluid as possible.
  • the amniotic fluid can be pretreated to remove the fat-soluble phase and obtain the water phase. For example, adding an appropriate amount of hexane, centrifuging to obtain an aqueous phase, and filtering with a membrane filter can obtain a sample for the next step of separation.
  • Reversed phase chromatography can be performed on amniotic fluid.
  • the stationary phase of reversed-phase chromatographic columns usually uses silica gel as a carrier, and a layer of non-polar molecules is bonded on the surface.
  • the bonded non-polar group can be selected from C18 alkyl, C8 alkyl, phenyl, C4 alkyl, etc. and their derivatives.
  • the present invention preferably uses a C18 reverse phase chromatography column, that is, a reverse phase chromatography column bonded with a C18 alkyl group.
  • the present invention can be implemented using reversed-phase chromatography columns known in the art.
  • reversed-phase chromatography columns can be obtained from commercially available sources, including UniSil 10-100 C18, LaChrom-C18, Inertsil ODS, Zorbax ODS, ACE C18, SunFire C18, Symmetry C18, Hypersil GOLD C18, Luna C18, Hypersil BDS C18, Hypersil ODS C18, SyncronisaQ C18 and Syncronis C18, etc.
  • the mobile phase of reversed-phase chromatography is a certain proportion of water and an organic solvent that is miscible with water.
  • the organic solvent can be selected from methanol, acetonitrile, ethanol, tetrahydrofuran, isopropanol, dioxane, acetone, etc., preferably methanol and acetonitrile are used.
  • the organic solvent used is a chromatographic grade organic solvent, and the water is 100% ultrapure water.
  • the mobile phase Before performing reversed-phase chromatography, the mobile phase can be used to equilibrate the reversed-phase chromatography column. After the absorption curve stabilizes and returns to the baseline, the balance can be stopped.
  • the sample can be loaded in a conventional manner, and the flow rate of the sample can be determined according to the actual production situation, such as the material, specifications and flow of the column used.
  • gradient elution can be performed.
  • the concentration of the organic solvent in the mobile phase may vary slightly depending on the type of organic solvent, which can be easily determined by those skilled in the art.
  • the percentage gradient of organic solvent in the mobile phase of the present invention can be from 5% to 12% (volume percentage), and the gradient of water can be from 95% to 88% (volume percentage).
  • the flow rate of the mobile phase can also be determined according to the actual production situation. Choose a fraction with an elution volume between 51-731 ml, preferably a fraction with an elution volume between 250-504 ml, more preferably an elution volume between 278-353 ml and/or between 354-429 ml And/or the fraction between 430-504 ml, or the similar fraction obtained by other similar chromatographic columns in the same proportion, is the extract described herein.
  • the elution volume is determined as follows:
  • Reverse phase separation column C18 reverse phase separation column
  • Elution method gradient elution, 0-10CV, acetonitrile (A) from 5% to 12%, ddH 2 O (B) from 95% to 88%;
  • the elution volume can be selected according to the above method; in other words, when other chromatographic columns are used, the selected elution volume should correspond to the elution volume determined by the above method.
  • two reverse phase chromatographic separations can be performed.
  • the resolution of the first reversed phase chromatographic separation may be lower than that of the second reversed phase chromatographic separation.
  • the percentage gradient of the polar organic solvent can vary from 5% to 12% (volume percentage), and the percentage gradient of water can vary from 95% to 88% (volume percentage).
  • the first reversed-phase chromatographic separation when performing the first reversed-phase chromatographic separation, take the fraction with an elution volume between 51-731 ml, preferably the fraction with an elution volume between 250-504 ml, and more preferably with an elution volume of 278-353 ml between and/or between 354-429 milliliters and/or between 430-504 milliliters, or other similar chromatographic columns with similar fractions obtained in the same proportion, the second reverse phase chromatographic separation is performed.
  • the elution volume will be different due to the different loading sample volume and/or the different column volume of the reversed-phase separation column.
  • the gradient of organic solvent can be from 0% to 7% (volume percentage), and the gradient of water can be from 100% to 93% (volume percentage).
  • the organic solvent when performing gradient elution in the second reverse phase chromatographic separation, in the first 0-3 minutes, the organic solvent changes from 0% to 5.5%, and the ultrapure water changes from 100% to 94.5%; In 3-50 minutes, the organic solvent changes gradually from 5.5% to 7%, and the ultrapure water changes gradually from 94.5% to 93%.
  • the eluent with an elution time between 11-12.5 minutes and the eluent with an elution time between 13-14 minutes are taken. The elution time is determined as follows:
  • Reverse phase separation column C18 reverse phase separation column
  • Elution method gradient elution, 0-3 minutes, acetonitrile from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile from 5.5% to 7%, The ultrapure water changes gradually from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%;
  • Loading volume 20 microliters.
  • the fractions when performing the first reverse phase chromatography separation, are collected, and then the activity of each fraction to promote the proliferation of cells (such as the human cardiomyocyte cell line AC16) is tested using conventional techniques in the art. Afterwards, the fractions with the activity of promoting cell proliferation were further separated by reversed-phase chromatography. After the second reverse phase chromatography separation, the collected fractions can be tested for cell proliferation activity to obtain fractions with cell proliferation activity.
  • the fraction can be a mixture of different components.
  • the present invention provides a method for isolating a component having cell proliferation activity from the amniotic fluid of a non-human animal embryo, the method comprising:
  • step (3) Perform a second reverse phase chromatographic separation on the fraction with cell proliferation activity obtained in step (2);
  • the reversed-phase chromatographic separation and cell proliferation activity test described in this method can be as described in any of the embodiments herein.
  • the present invention particularly provides a method for isolating a component having cell proliferation activity from egg amniotic fluid, the method comprising:
  • the reversed-phase chromatography columns used in steps (1) and (2) are all C18 reversed-phase chromatographic columns.
  • the mobile phases used in the two steps are both acetonitrile and pure water; wherein, in the reverse phase chromatographic separation of step (1), the percentage gradient of acetonitrile can vary from 5 to 12% (volume percentage), water The percentage gradient change of acetonitrile can be from 95% to 88% (volume percentage), and the sixth peak, the seventh peak and the eighth peak are obtained separately; in step (2), the gradient change of acetonitrile can be from 0% to 7% (volume percentage) Percentage), the gradient of water can vary from 100% to 93% (volume percentage).
  • step (2) in the first 0-3 minutes, acetonitrile changes from 0% to 5.5%, and water changes from 100% to 94.5%; from 3 to 50 minutes, acetonitrile changes from 5.5% Change to 7%, and the water changes gradually from 94.5% to 93%.
  • the extract of the present invention is an extract obtained by using the method described in any of the embodiments herein.
  • this document also provides a composition for promoting cell proliferation, promoting wound healing, and repairing organ damage.
  • the composition is a pharmaceutical composition containing the extract or active ingredient described in any of the embodiments herein, and optionally a pharmaceutically acceptable carrier.
  • a suitable carrier may be a carrier well-known in the art, for example, a carrier suitable for preparing the active ingredient into tablets, injections, freeze-dried preparations or ointments.
  • the composition is a cosmetic, and contains the extract described herein and a carrier commonly used in cosmetics.
  • purification and extraction and separation are used interchangeably, meaning the separation or extraction of active ingredients from amniotic fluid.
  • Example 1 Composition analysis of eggs and duck eggs
  • the separation column is LaChrom-C18 AQ.
  • the elution solvent is degassed chromatography grade acetonitrile and ddH 2 O.
  • the mobile phase is acetonitrile (ACN) and ultrapure water.
  • ACN acetonitrile
  • the flow rate of the mobile phase was 0.8 ml/min
  • the column temperature was 25.0°C
  • the injection volume was 20 ⁇ l.
  • the detection wavelength of the DAD detector is 250 nm. Collect fractions in equal volume, 3 ml/tube for cell viability test.
  • FIG 3 shows that egg amniotic fluid extract (CEE) and duck egg amniotic fluid extract (DEE) can promote cell proliferation.
  • CEE egg amniotic fluid extract
  • DEE duck egg amniotic fluid extract
  • phase separation column is UniSil 10-100 C18;
  • the elution solvent is degassed chromatography grade acetonitrile and ddH 2 O.
  • Sample treatment 400ml fresh egg amniotic fluid, add appropriate amount of hexane, centrifuge at 2500rpm, 4°C for 20 minutes to obtain the water phase, filter with 0.22 ⁇ m filter membrane.
  • the first step using acetonitrile (A) and ddH 2 O (B) as mobile phases, UniSil 10-100 C18 reversed-phase separation column;
  • Equilibrate the reversed-phase column equilibrate the reversed-phase column with mobile phase 5% acetonitrile (A) at a flow rate of 10ml/min until the 280nm UV absorption curve is stable and returns to the baseline;
  • Sample loading flow rate 1ml/min, sample loading volume is 50ml;
  • the second step Hitachi C18 reversed phase separation column separation
  • the 8th peak of the primary active fraction separated by AKTA was further separated and purified by HPLC (Hitachi).
  • the mobile phase is acetonitrile and ultrapure water.
  • Use gradient elution, the specific parameters are as follows: 0-3 minutes, acetonitrile changes from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile changes from 5.5% to 7 %, the ultrapure water changes from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%.
  • the flow rate of the mobile phase was 0.8mL/min, the column temperature was 25.0°C, and the injection volume was 20 ⁇ l.
  • the detection wavelength of the DAD detector is 250 nm, and the detection time is 0-20 minutes.
  • Figure 8 shows that the hydrophobicity of P8 is between L-dopa and VB12. Since the octanol/water partition coefficient Log P of L-dopa is 0.05, and the octanol/water partition coefficient Log P of VB12 is 1.897, the octanol/water partition coefficient Log P of P8 is between 0.05 and 1.897. It is preferably between 0.1 and 1.897, more preferably between 0.5 and 1.897 or between 0.5 and 1.5.
  • Example 3 Effect of active ingredients in eggs on repairing cardiac function after myocardial infarction
  • mice were anesthetized with 5% isoflurane in the induction box, fixed with medical tape, straightened the neck, and then quickly connected to the ventilator with a 20g indwelling needle tracheal intubation through the oral cavity. Perform mechanical ventilation and switch the air anesthesia channel to the ventilator. The amount of anesthetic is adjusted to 2.5% to maintain anesthesia. The frequency of the ventilator is 115 breaths/minute, the breathing ratio is 1:1, and the tidal volume is 1.5 ml. Fix the mouse on the operating table, maintain the temperature of the hot plate at 37°C, connect the mouse limbs to the signal acquisition and processing system, and monitor the changes in the electrogram of the operating center.
  • the left chest is depilated and skinned, disinfected with iodophor and covered with autoclaved gauze. Open the chest through the fourth intercostal space on the left anterolateral side, exposing the heart. Open the pericardium and penetrate 2mm from the lower edge of the left atrial appendage. Use 7-0 prolene thread to ligate through the interventricular sulcus, and ligate the left anterior descending coronary artery through the interventricular sulcus. The apex will turn white and the ST segment of the ECG will be elevated. . Suture the intercostal space, muscles and skin. disinfect. Turn off the anesthetic and continue to ventilate until the mouse wakes up.
  • Example 2 Inject the P6 and P8 obtained in Example 2 with control 5% glucose and glucose dissolved in the tail vein, 100 microliters each, and injected every other day.

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Abstract

An extract of the amniotic fluid of a non-human animal, the octanol/water partition coefficient Log P of the active component contained in the extract being within the range of 0.05-1.897. The active component obtained by isolation has cell proliferation activity and/or promotes heart function recovery after a myocardial infarction.

Description

反相色谱分离非人动物羊水中的活性成分Separation of Active Components in Amniotic Fluid of Non-human Animals by Reversed Phase Chromatography 技术领域Technical field
本发明属动物提取物领域，具体涉及使用反相色谱分离非人动物羊水中的活性成分提取及在细胞增殖活性中的作用。The present invention belongs to the field of animal extracts, and specifically relates to the use of reversed-phase chromatography to separate the active ingredient extraction from non-human animal amniotic fluid and its effect on cell proliferation activity.
背景技术Background technique
动物羊水中含有种类繁多的氨基酸、有机酸、糖类、类固醇、核苷酸、脂质、及其衍生物、微量元素等物质。根据不同物质之间物理和化学性质的差异，包括分子量、溶解度、等电点以及疏水性等性质，纯化羊水的技术通常有沉淀技术、层析技术、双液相萃取技术等。然而目前这些技术成本较高，过程复杂，回收率低，并且往往在分离纯化过程中造成目标化合物的生物活性降低甚至丢失。Animal amniotic fluid contains a wide variety of amino acids, organic acids, sugars, steroids, nucleotides, lipids, their derivatives, trace elements and other substances. According to the differences in physical and chemical properties of different substances, including molecular weight, solubility, isoelectric point, and hydrophobicity, the techniques for purifying amniotic fluid usually include precipitation techniques, chromatography techniques, and two-liquid phase extraction techniques. However, these technologies currently have high costs, complex processes, and low recovery rates, and often cause the biological activity of the target compound to be reduced or even lost during the separation and purification process.
作为化学分离的重要手段，根据流动相和固定相极性的相对不同，液相色谱分为正相色谱和反相色谱。反相色谱(RPC)是指利用非极性的反相介质为固定相，极性有机溶剂的水溶液为流动相，根据溶质极性(疏水性)的不同进行溶质分离与纯化的洗脱色谱法。高效液相色谱和超高效液相色谱，能有效地分离、鉴定、半定量粗品中的目标化合物而被广泛地运用。因此，RPC非常适合于分离相对分子质量小于2000道尔顿、中性、稳定性好的物质或复合物质。As an important means of chemical separation, liquid chromatography is divided into normal phase chromatography and reverse phase chromatography according to the relative difference of the polarity of mobile phase and stationary phase. Reversed phase chromatography (RPC) refers to an elution chromatography that uses a non-polar reverse phase medium as the stationary phase and an aqueous solution of a polar organic solvent as the mobile phase to separate and purify the solute according to the polarity (hydrophobicity) of the solute. . High performance liquid chromatography and ultra high performance liquid chromatography can effectively separate, identify, and semi-quantitate target compounds in crude products and are widely used. Therefore, RPC is very suitable for separating substances or composite substances with a relative molecular mass of less than 2000 Daltons, which are neutral and have good stability.
本发明采用C18及其衍生物作为反相介质，结合AKTA***和HPLC***两步分离非人动物羊水中的活性物质。本发明的提取物能促进细胞增殖。The invention uses C18 and its derivatives as a reverse phase medium, and combines the AKTA system and the HPLC system to separate the active substances in the amniotic fluid of non-human animals. The extract of the present invention can promote cell proliferation.
发明内容Summary of the invention
本发明提供一种非人动物羊水的提取物，所述提取物所含活性成分的辛醇/水分配系数Log P在0.05-1.897的范围内。The present invention provides an extract of non-human animal amniotic fluid. The octanol/water partition coefficient Log P of the active ingredients contained in the extract is in the range of 0.05-1.897.
在一个或多个实施方案中，所述提取物中活性成分的辛醇/水分配系数Log  P在0.3-1.5之间。In one or more embodiments, the octanol/water partition coefficient Log P of the active ingredient in the extract is between 0.3-1.5.
在一个或多个实施方案中，所述提取物在pH 5.8-8.0之间不和离子交换柱结合。In one or more embodiments, the extract does not bind to the ion exchange column at a pH between 5.8 and 8.0.
在一个或多个实施方案中，所述羊水为来自禽蛋的羊水。In one or more embodiments, the amniotic fluid is amniotic fluid derived from poultry eggs.
在一个或多个实施方案中，所述禽蛋为胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋、更优选胚龄为7-8天的鸡蛋，或者为发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋。In one or more embodiments, the poultry eggs are eggs with embryo age of 5-12 days, preferably eggs with embryo age of 6-11 days, more preferably eggs with embryo age of 7-9 days, more preferably embryos Eggs with an age of 7-8 days, or eggs of other poultry other than chickens whose developmental period corresponds to the developmental period of the embryo-aged eggs.
在一个或多个实施方案中，所述禽蛋为家禽的蛋，如鸡蛋、鸭蛋、鸵鸟蛋和鹅蛋等。In one or more embodiments, the poultry eggs are poultry eggs, such as eggs, duck eggs, ostrich eggs, goose eggs, and the like.
在一个或多个实施方案中，所述羊水为非人哺乳动物羊水；优选地，所述羊水来自啮齿类动物；优选地，所述羊水来自胎龄为8-20天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天的啮齿类动物的发育时期相对应的非人哺乳动物的胚胎。In one or more embodiments, the amniotic fluid is non-human mammal amniotic fluid; preferably, the amniotic fluid is derived from rodents; preferably, the amniotic fluid is derived from embryos of rodents with a gestational age of 8-20 days , Or embryos from non-human mammals whose developmental period corresponds to that of rodents with a gestational age of 8-20 days.
在一个或多个实施方案中，所述提取物采用反相色谱分离得到。In one or more embodiments, the extract is separated by reverse phase chromatography.
本发明还提供一种非人动物羊水提取物的制备方法，所述方法包括采用反相色谱从所述羊水中分离出辛醇/水分配系数Log P在0.05-1.897之间的具有细胞增殖活性和/或促进心肌梗死后心功能恢复的馏分的步骤。The present invention also provides a method for preparing a non-human animal amniotic fluid extract, the method comprising using reversed-phase chromatography to separate the octanol/water partition coefficient Log P between 0.05-1.897 and having cell proliferation activity from the amniotic fluid And/or a fraction that promotes the recovery of cardiac function after myocardial infarction.
在一个或多个实施方案中，所述方法包括进行两次反相色谱分离，其中，第二次反相色谱分离所使用的反相色谱柱的分辨率高于第一次反相色谱分离所使用的反相色谱柱的分辨率。In one or more embodiments, the method includes performing two reverse phase chromatographic separations, wherein the resolution of the reverse phase chromatography column used in the second reverse phase chromatographic separation is higher than that of the first reverse phase chromatographic separation. The resolution of the reversed-phase column used.
在一个或多个实施方案中，所述方法包括：In one or more embodiments, the method includes:
(1)对非人动物胚胎羊水进行首次反相色谱分离，分离得到辛醇/水分配系数Log P在0.05-1.897范围内的馏分；(1) The first reverse phase chromatography was performed on the amniotic fluid of non-human animal embryos, and the fractions with the octanol/water partition coefficient Log P in the range of 0.05-1.897 were obtained;
(2)对步骤(1)获得的馏分再次进行反相色谱分离，分离得到辛醇/水分配系数Log P在0.05-1.897范围内的馏分。(2) The fraction obtained in step (1) is separated again by reversed-phase chromatography, and the fraction with the octanol/water partition coefficient Log P in the range of 0.05-1.897 is obtained.
在一个或多个实施方案中，所述方法包括两次反相色谱，其中，第一次反相色谱中，洗脱体积在51-731毫升之间的馏分，优选洗脱体积在250-504毫升 之间的馏分，更优选洗脱体积在278-353毫升之间和/或在354-429毫升之间和/或在430-504毫升之间的馏分，或以其它相似色谱柱以同比例所获相似馏分，用于第二次反相色谱；第二次反相色谱中，取洗脱时间在11-12.5分钟之间的馏分和13-14分钟之间的馏分，或以其它相似色谱柱以同比例所获相似馏分。In one or more embodiments, the method includes two reversed-phase chromatography, wherein, in the first reversed-phase chromatography, the elution volume of the fraction between 51-731 ml, preferably the elution volume is 250-504 The fraction between milliliters, more preferably the fraction with an elution volume between 278-353 milliliters and/or between 354-429 milliliters and/or between 430-504 milliliters, or other similar columns in the same proportion The obtained similar fractions are used in the second reverse phase chromatography; in the second reverse phase chromatography, take the fraction with the elution time between 11-12.5 minutes and the fraction between 13-14 minutes, or use other similar chromatography The column obtains similar fractions in the same proportions.
在一个或多个实施方案中，所述反相色谱所用的反相色谱柱的固定相以硅胶为载体，表面键合的非极性基团选自苯基、C8或C18烷基及其衍生物；使用一定比例的有机溶剂如甲醇、乙腈、乙醇和水作为流动相。In one or more embodiments, the stationary phase of the reversed-phase chromatography column used in reversed-phase chromatography uses silica gel as a carrier, and the non-polar groups bonded on the surface are selected from phenyl, C8 or C18 alkyl groups and their derivatives. Use a certain proportion of organic solvents such as methanol, acetonitrile, ethanol and water as the mobile phase.
在一个或多个实施方案中，步骤(1)所用的流动相为乙腈和ddH 2O。 In one or more embodiments, the mobile phase used in step (1) is acetonitrile and ddH 2 O.
在一个或多个实施方案中，步骤(1)采用梯度方式洗脱，流动相为乙腈和ddH 2O，其中乙腈的浓度梯度为5％-12％，ddH 2O的浓度梯度为95％-88％。 In one or more embodiments, step (1) adopts gradient elution, and the mobile phase is acetonitrile and ddH 2 O, wherein the concentration gradient of acetonitrile is 5%-12%, and the concentration gradient of ddH 2 O is 95%- 88%.
在一个或多个实施方案中，步骤(2)的流动相为乙腈和纯水。In one or more embodiments, the mobile phase of step (2) is acetonitrile and pure water.
在一个或多个实施方案中，步骤(2)采用梯度方式洗脱，流动相为乙腈和纯水，乙腈浓度梯度为0％-7％，纯水浓度梯度为100％-93％。In one or more embodiments, step (2) adopts gradient elution, the mobile phase is acetonitrile and pure water, the concentration gradient of acetonitrile is 0%-7%, and the concentration gradient of pure water is 100%-93%.
在一个或多个实施方案中，采用C18反相色谱柱进行反相分离。In one or more embodiments, a C18 reverse phase chromatography column is used for reverse phase separation.
在一个或多个实施方案中，所述方法还包括，第一次反相色谱分离后，测试分离得到的各馏分的细胞增殖活性，挑取细胞增殖活性最强的馏分实施第二次反相色谱分离。In one or more embodiments, the method further includes, after the first reverse phase chromatography separation, testing the cell proliferation activity of the separated fractions, picking the fraction with the strongest cell proliferation activity and performing the second reverse phase Chromatographic separation.
本发明还提供采用本文所述的方法分离得到的来自非人动物羊水的活性成分。The present invention also provides active ingredients derived from non-human animal amniotic fluid obtained by the method described herein.
本发明还提供一种药物组合物，所述药物组合物含有本文所述的提取物或活性成分，和任选的药学上可接受的载体。The present invention also provides a pharmaceutical composition containing the extract or active ingredient described herein, and optionally a pharmaceutically acceptable carrier.
本发明还提供反相色谱柱在分离非人动物的羊水中具有细胞增殖活性的成分中的应用。The invention also provides the application of the reverse phase chromatography column in separating the components with cell proliferation activity in the amniotic fluid of non-human animals.
本发明还提供本文所述的活性成分在制备促进细胞增殖和/或促进心肌梗死后心功能恢复的产品中的应用。The present invention also provides the application of the active ingredients described herein in the preparation of products that promote cell proliferation and/or promote cardiac function recovery after myocardial infarction.
附图说明Description of the drawings
图1：鸡蛋羊水的HPLC图。Figure 1: HPLC chart of egg amniotic fluid.
图2：鸭蛋羊水的HPLC图。Figure 2: HPLC chart of duck egg amniotic fluid.
图3：鸡蛋和鸭蛋羊水提取物的细胞增殖检测结果。Figure 3: Cell proliferation test results of egg and duck egg amniotic fluid extract.
图4：新鲜鸡蛋羊水经UniSil 10-100 C18分离的色谱图。Figure 4: Chromatogram of fresh egg amniotic fluid separated by UniSil 10-100 C18.
图5：羊水提取物组分P6、P7、P8峰的HPLC图。Figure 5: HPLC chart of peaks P6, P7 and P8 of the amniotic fluid extract.
图6：细胞活力检测发现图4中的P6、P7、P8峰具有生物活性。Figure 6: Cell viability detection found that the P6, P7, and P8 peaks in Figure 4 have biological activity.
图7：P8峰进一步在日立HPLC***上经C18 AQ分离，细胞活力检测发现较高纯度的P8-2具有细胞活性。Figure 7: The P8 peak was further separated by C18 AQ on the Hitachi HPLC system, and the cell viability test found that the higher purity P8-2 had cell viability.
图8：较高纯度的P8、L-多巴(Log P＝0.05)和维生素B12(Log P＝1.897)的HPLC图。Figure 8: HPLC chart of higher purity P8, L-dopa (Log P = 0.05) and Vitamin B12 (Log P = 1.897).
图9：具有细胞活性的P6、P8峰有助于降低由于心肌梗死引起的小鼠死亡率。Figure 9: P6 and P8 peaks with cellular activity help reduce the mortality of mice caused by myocardial infarction.
图10-11：具有细胞活性的P6、P8峰有助于促进心肌梗塞的心脏功能修复。Figure 10-11: P6 and P8 peaks with cellular activity help to promote the repair of heart function in myocardial infarction.
具体实施方式detailed description
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，各技术之间结合顺序可以互相调整，从而构成优选的技术方案。It should be understood that, within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in the following (such as the embodiments) can be combined with each other, and the combination sequence between the technologies can be adjusted to each other to form The preferred technical solution.
本发明提供了一种针对非人动物胚胎羊水中活性成分的纯化提取方法，得到了具有促进细胞增殖和心肌梗死后心功能恢复的生长因子群。本文的方法包括从非人动物胚胎的羊水中分离出辛醇/水分配系数Log P在0.05-1.897之间的馏分的步骤，优选在0.1-1.897之间，更优选在0.5-1.897之间或在0.5-1.5之间。The present invention provides a method for purifying and extracting active ingredients in the amniotic fluid of non-human animal embryos, and obtains a group of growth factors that can promote cell proliferation and recovery of cardiac function after myocardial infarction. The method herein includes the step of separating the fraction with the octanol/water partition coefficient Log P between 0.05 and 1.897 from the amniotic fluid of non-human animal embryos, preferably between 0.1 and 1.897, more preferably between 0.5 and 1.897 or Between 0.5-1.5.
本文中，羊水可来自禽蛋和非人哺乳动物。禽蛋指禽类的蛋。优选的禽类为家禽，如鸡、鸭、鸵鸟蛋和鹅等。优选的是，本发明使用胚龄在5-20天、优选6-15天的禽蛋。应理解为，不同禽蛋，合适的胚龄未必相同。例如，当使用鸡蛋时，优选使用胚龄为5-12天的鸡蛋，更优选使用胚龄为6-11天的鸡蛋，更优选使用胚龄为7-9天的鸡蛋、更优选使用胚龄为7-8天的鸡蛋。当使用其它禽类的蛋时，可使用其发育时期与上述胚龄的鸡蛋所处的发育时期相对应的蛋。例如，当使用鸭蛋时，胚龄为8-10天、尤其是8-9天的鸭蛋是最好的。Here, amniotic fluid can come from poultry eggs and non-human mammals. Poultry eggs refer to poultry eggs. The preferred birds are poultry, such as chickens, ducks, ostrich eggs, and geese. Preferably, the present invention uses poultry eggs whose embryonic age is 5-20 days, preferably 6-15 days. It should be understood that the appropriate embryo age may not be the same for different eggs. For example, when using eggs, eggs with embryo age of 5-12 days are preferably used, eggs with embryo age of 6-11 days are more preferably used, eggs with embryo age of 7-9 days are more preferably used, and embryo age is more preferably used. Eggs for 7-8 days. When using eggs of other avians, eggs whose developmental stage corresponds to the developmental stage of the eggs of the embryonic age mentioned above can be used. For example, when duck eggs are used, duck eggs with embryo age of 8-10 days, especially 8-9 days are the best.
可采用常规的方法获得禽蛋羊水。例如，可敲击相应胚龄的蛋的钝端，使蛋壳碎裂，将蛋壳剥开形成一个直径约为2厘米的口子。然后用镊子小心撕开壳膜和卵黄膜，注意不要破坏羊膜。将包裹着胚胎的羊膜和相连组织从壳中倾倒至培养皿中，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。Conventional methods can be used to obtain amniotic fluid from poultry eggs. For example, you can knock the blunt end of an egg of the corresponding embryonic age to break the eggshell, and then peel the eggshell to form a hole with a diameter of about 2 cm. Then use tweezers to carefully tear open the shell membrane and vitelline membrane, taking care not to damage the amniotic membrane. Pour the amniotic membrane and connected tissues covering the embryo from the shell to a petri dish, and pierce the amniotic membrane with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thereby obtaining the amniotic fluid used in the present invention.
本文中，羊水还可来自非人哺乳动物，尤其是啮齿类动物，如来自小鼠。在某些实施方案中，羊水来自胎龄为8-20天的啮齿类动物的胚胎，或来自其发育时期与胎龄为8-20天的啮齿类动物所处的发育时期相对应的非人哺乳动物的胚胎。可采用常规的方法获得羊水。例如，用手术剪剖开怀孕8-20天的小鼠腹腔，小心取出并剪开子宫，用注射器刺入羊膜抽取羊水，直至羊膜紧贴胚胎，由此即可获得用于本发明的羊水。优选地，羊水来自胎龄为8-14天或11-16天、更优选13-14天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-14天或11-16天、更优选13-14天的啮齿类动物所处的发育时期相对应的非人哺乳动物的胚胎。Here, amniotic fluid can also be derived from non-human mammals, especially rodents, such as from mice. In certain embodiments, the amniotic fluid is derived from embryos of rodents with a gestational age of 8-20 days, or from non-humans whose developmental period corresponds to that of rodents with a gestational age of 8-20 days. The embryo of a mammal. Conventional methods can be used to obtain amniotic fluid. For example, using surgical scissors to open the abdominal cavity of a mouse 8-20 days pregnant, carefully take out and cut open the uterus, pierce the amniotic membrane with a syringe to extract amniotic fluid until the amniotic membrane is close to the embryo, thereby obtaining the amniotic fluid used in the present invention. Preferably, the amniotic fluid is derived from rodent embryos with a gestational age of 8-14 days or 11-16 days, more preferably 13-14 days, or from a developmental period and a gestational age of 8-14 days or 11-16 days, more preferably The embryos of non-human mammals corresponding to the developmental stage of rodents of 13-14 days are preferred.
应理解，必要时，可对羊水进行离心，以分离出可能含有的杂质，例如卵黄等，尽可能获得纯的羊水。It should be understood that, if necessary, the amniotic fluid can be centrifuged to separate possible impurities, such as egg yolk, etc., to obtain as pure amniotic fluid as possible.
获得羊水后，可对羊水进行预处理，以除去脂溶相，获得水相。例如，加入适量己烷，离心，获得水相，滤膜过滤，即可获得用于下一步分离的样品。After obtaining the amniotic fluid, the amniotic fluid can be pretreated to remove the fat-soluble phase and obtain the water phase. For example, adding an appropriate amount of hexane, centrifuging to obtain an aqueous phase, and filtering with a membrane filter can obtain a sample for the next step of separation.
可对羊水实施反相色谱。反相色谱柱的固定相通常以硅胶为载体，表面键合有非极性分子层。通常，键合的非极性基团可选自C18烷基、C8烷基、苯基和C4烷基等及其衍生物。本发明优选使用C18反相色谱柱，即键合有C18烷基的反相色谱柱。可使用本领域周知的反相色谱柱来实施本发明，如可从市售途径获得这类反相色谱柱，包括UniSil 10-100 C18、LaChrom-C18、Inertsil ODS、Zorbax ODS、ACE C18、SunFire C18、Symmetry C18、Hypersil GOLD C18、Luna C18、Hypersil BDS C18、Hypersil ODS C18、SyncronisaQ C18和Syncronis C18等。反相色谱的流动相为一定比例的水和可以与水混溶的有机溶剂。有机溶剂可选自甲醇、乙腈、乙醇、四氢呋喃、异丙醇、二氧六环、丙酮等，优选使用甲醇和乙腈。使用的有机溶剂为色谱级有机溶剂，水为100％超纯水。Reversed phase chromatography can be performed on amniotic fluid. The stationary phase of reversed-phase chromatographic columns usually uses silica gel as a carrier, and a layer of non-polar molecules is bonded on the surface. Generally, the bonded non-polar group can be selected from C18 alkyl, C8 alkyl, phenyl, C4 alkyl, etc. and their derivatives. The present invention preferably uses a C18 reverse phase chromatography column, that is, a reverse phase chromatography column bonded with a C18 alkyl group. The present invention can be implemented using reversed-phase chromatography columns known in the art. For example, such reversed-phase chromatography columns can be obtained from commercially available sources, including UniSil 10-100 C18, LaChrom-C18, Inertsil ODS, Zorbax ODS, ACE C18, SunFire C18, Symmetry C18, Hypersil GOLD C18, Luna C18, Hypersil BDS C18, Hypersil ODS C18, SyncronisaQ C18 and Syncronis C18, etc. The mobile phase of reversed-phase chromatography is a certain proportion of water and an organic solvent that is miscible with water. The organic solvent can be selected from methanol, acetonitrile, ethanol, tetrahydrofuran, isopropanol, dioxane, acetone, etc., preferably methanol and acetonitrile are used. The organic solvent used is a chromatographic grade organic solvent, and the water is 100% ultrapure water.
在实施反相色谱前，可用流动相平衡反相色谱柱。待吸收曲线平稳、回归基线后，可停止平衡。可采用常规的方式上样，样品的流速可根据实际生产情况，如所使用的柱的材料、规格以及流动相等确定。上样结束后，可进行梯度洗脱。流动相中有机溶剂的浓度可根据有机溶剂种类的不同而稍有差异，这可由本领域技术人员容易确定。在某些实施方案中，本发明的流动相中有机溶剂的百分比梯度变化可以是从5％到12％(体积百分比)，水的梯度变化可以从95％到88％(体积百分比)。流动相的流速也可根据实际生产情况确定。选择洗脱体积在51-731毫升之间的馏分，优选洗脱体积在250-504毫升之间的馏分，更优选洗脱体积在278-353毫升之间和/或在354-429毫升之间和/或在430-504毫升之间的馏分，或以其它相似色谱柱以同比例所获相似馏分，即为本文所述的提取物。洗脱体积如下确定：Before performing reversed-phase chromatography, the mobile phase can be used to equilibrate the reversed-phase chromatography column. After the absorption curve stabilizes and returns to the baseline, the balance can be stopped. The sample can be loaded in a conventional manner, and the flow rate of the sample can be determined according to the actual production situation, such as the material, specifications and flow of the column used. After sample loading, gradient elution can be performed. The concentration of the organic solvent in the mobile phase may vary slightly depending on the type of organic solvent, which can be easily determined by those skilled in the art. In some embodiments, the percentage gradient of organic solvent in the mobile phase of the present invention can be from 5% to 12% (volume percentage), and the gradient of water can be from 95% to 88% (volume percentage). The flow rate of the mobile phase can also be determined according to the actual production situation. Choose a fraction with an elution volume between 51-731 ml, preferably a fraction with an elution volume between 250-504 ml, more preferably an elution volume between 278-353 ml and/or between 354-429 ml And/or the fraction between 430-504 ml, or the similar fraction obtained by other similar chromatographic columns in the same proportion, is the extract described herein. The elution volume is determined as follows:
反相分离柱：C18反相分离柱；Reverse phase separation column: C18 reverse phase separation column;
流动相：乙腈和超纯水；Mobile phase: acetonitrile and ultrapure water;
洗脱方式：梯度洗脱，0-10CV，乙腈(A)从5％到12％，ddH 2O(B)从95％到88％； Elution method: gradient elution, 0-10CV, acetonitrile (A) from 5% to 12%, ddH 2 O (B) from 95% to 88%;
流动相流速：10ml/min；Flow rate of mobile phase: 10ml/min;
柱温：25.0℃；Column temperature: 25.0℃;
上样流速：1ml/min；Sample flow rate: 1ml/min;
上样量：50ml。Loading volume: 50ml.
当采用其它色谱柱时，洗脱体积的选择可参照上述方法进行；换言之，当采用其它色谱柱时，所选择的洗脱体积应与采用上述方法确定的洗脱体积相对应。When other chromatographic columns are used, the elution volume can be selected according to the above method; in other words, when other chromatographic columns are used, the selected elution volume should correspond to the elution volume determined by the above method.
在某些实施方案中，可实施两次反相色谱分离。首次反相色谱分离的分辨率可低于第二次反相色谱分离的分辨率。首次反相色谱分离进行梯度洗脱时，极性有机溶剂的百分比梯度变化可从5％-12％(体积百分比)，水的百分比梯度变化可从95％-88％(体积百分比)。如上所述，实施首次反相色谱分离时，取洗脱体积在51-731毫升之间的馏分，优选洗脱体积在250-504毫升之间的馏分，更优选洗脱体积在278-353毫升之间和/或在354-429毫升之间和/或在 430-504毫升之间的馏分，或以其它相似色谱柱以同比例所获相似馏分，进行第二次反相色谱分离。由于上样样品体积不同和/或反相分离柱柱体积不同，洗脱体积会相应不同。In certain embodiments, two reverse phase chromatographic separations can be performed. The resolution of the first reversed phase chromatographic separation may be lower than that of the second reversed phase chromatographic separation. When performing gradient elution for the first reverse phase chromatographic separation, the percentage gradient of the polar organic solvent can vary from 5% to 12% (volume percentage), and the percentage gradient of water can vary from 95% to 88% (volume percentage). As mentioned above, when performing the first reversed-phase chromatographic separation, take the fraction with an elution volume between 51-731 ml, preferably the fraction with an elution volume between 250-504 ml, and more preferably with an elution volume of 278-353 ml Between and/or between 354-429 milliliters and/or between 430-504 milliliters, or other similar chromatographic columns with similar fractions obtained in the same proportion, the second reverse phase chromatographic separation is performed. The elution volume will be different due to the different loading sample volume and/or the different column volume of the reversed-phase separation column.
第二次反相色谱分离进行梯度洗脱时，有机溶剂的梯度变化可以是从0％到7％(体积百分比)，水的梯度变化可以从100％到93％(体积百分比)。在某些实施方案中，第二次反相色谱分离进行梯度洗脱时，前0-3分钟，有机溶剂从0％梯度变化到5.5％，超纯水从100％梯度变化到94.5％；第3-50分钟，有机溶剂从5.5％梯度变化到7％，超纯水从94.5％梯度变化到93％。优选地，第二次反相色谱分离时，取洗脱时间在11-12.5分钟之间的洗脱液和13-14分钟之间的洗脱液。洗脱时间如下确定：In the second reverse phase chromatography for gradient elution, the gradient of organic solvent can be from 0% to 7% (volume percentage), and the gradient of water can be from 100% to 93% (volume percentage). In some embodiments, when performing gradient elution in the second reverse phase chromatographic separation, in the first 0-3 minutes, the organic solvent changes from 0% to 5.5%, and the ultrapure water changes from 100% to 94.5%; In 3-50 minutes, the organic solvent changes gradually from 5.5% to 7%, and the ultrapure water changes gradually from 94.5% to 93%. Preferably, in the second reverse phase chromatographic separation, the eluent with an elution time between 11-12.5 minutes and the eluent with an elution time between 13-14 minutes are taken. The elution time is determined as follows:
反相分离柱：C18反相分离柱；Reverse phase separation column: C18 reverse phase separation column;
流动相：乙腈和超纯水；Mobile phase: acetonitrile and ultrapure water;
洗脱方式：梯度洗脱，0-3分钟，乙腈从0％梯度变化到5.5％，超纯水从100％梯度变化到94.5％；3-50分钟，乙腈从5.5％梯度变化到7％，超纯水从94.5％梯度变化到93％；50-52分钟，乙腈从7％梯度变化到100％，超纯水从93％梯度变化到0％；Elution method: gradient elution, 0-3 minutes, acetonitrile from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile from 5.5% to 7%, The ultrapure water changes gradually from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%;
流动相流速：0.8ml/min；Flow rate of mobile phase: 0.8ml/min;
柱温：25.0℃；Column temperature: 25.0℃;
上样量：20微升。Loading volume: 20 microliters.
应理解，两次反相色谱分离时，当使用不同的色谱分离柱时，可根据不同分离柱的组成、性能和规格选用不同的流动相、梯度变化及流速，以获得最佳的洗脱效果。此外，当采用不同的反相柱、流动相和洗脱方式时，洗脱液的选取会有所不同，可参照本文所述的确定洗脱液的方法选择合适的洗脱液，作为本发明的提取物。It should be understood that when two reversed-phase chromatographic separations are used, when different chromatographic separation columns are used, different mobile phases, gradient changes and flow rates can be selected according to the composition, performance and specifications of the different separation columns to obtain the best elution effect. . In addition, when different reversed-phase columns, mobile phases and elution methods are used, the selection of the eluent will be different. You can refer to the method of determining eluent described herein to select a suitable eluent as the present invention. Extracts.
在某些实施方案中，进行首次反相色谱分离时，收集馏分，然后采用本领域常规的技术测试各馏分促进细胞(如人心肌细胞系AC16)增殖的活性。之后用对具有促进细胞增殖活性的馏分做出进一步的反相色谱分离。第二次反相色谱分离后，可测试收集到的馏分的细胞增殖活性，以获得具有细胞增殖活性 的馏分。该馏分可以是一种不同成分的混合物。In some embodiments, when performing the first reverse phase chromatography separation, the fractions are collected, and then the activity of each fraction to promote the proliferation of cells (such as the human cardiomyocyte cell line AC16) is tested using conventional techniques in the art. Afterwards, the fractions with the activity of promoting cell proliferation were further separated by reversed-phase chromatography. After the second reverse phase chromatography separation, the collected fractions can be tested for cell proliferation activity to obtain fractions with cell proliferation activity. The fraction can be a mixture of different components.
在某些实施方案中，本发明提供一种从非人动物胚胎羊水中分离具有细胞增殖活性的成分的方法，所述方法包括：In certain embodiments, the present invention provides a method for isolating a component having cell proliferation activity from the amniotic fluid of a non-human animal embryo, the method comprising:
(1)对非人动物胚胎羊水进行首次反相色谱分离；(1) The first reversed phase chromatographic separation of non-human animal embryo amniotic fluid;
(2)测试步骤(1)获得的各馏分的细胞增殖活性；(2) Test the cell proliferation activity of each fraction obtained in step (1);
(3)对步骤(2)获得的具有细胞增殖活性的馏分进行第二次反相色谱分离；和(3) Perform a second reverse phase chromatographic separation on the fraction with cell proliferation activity obtained in step (2); and
(4)测试步骤(3)获得的各馏分的细胞增殖活性；(4) Test the cell proliferation activity of each fraction obtained in step (3);
从而分离得到具有细胞增殖活性的成分。Thereby, components with cell proliferation activity can be isolated.
该方法所述的各反相色谱分离以及细胞增殖活性测试可如本文任一实施方案所述。The reversed-phase chromatographic separation and cell proliferation activity test described in this method can be as described in any of the embodiments herein.
在某些实施方案中，本发明尤其提供一种从鸡蛋羊水中分离具有细胞增殖活性的成分的方法，所述方法包括：In certain embodiments, the present invention particularly provides a method for isolating a component having cell proliferation activity from egg amniotic fluid, the method comprising:
(1)对鸡蛋羊水进行首次反相色谱分离，获取具有生物活性的第6峰、第7峰和第8峰的馏分；任选地，(1) Perform the first reverse phase chromatographic separation of the egg and amniotic fluid to obtain the biologically active fractions of the sixth peak, the seventh peak and the eighth peak; optionally,
(2)对第8峰的馏分进行第二次反相色谱分离，获取第2峰馏分；(2) Perform a second reverse phase chromatographic separation on the fraction of the 8th peak to obtain the fraction of the second peak;
从而分离得到具有细胞增殖活性的成分；其中，步骤(1)和(2)使用的反相色谱柱均为C18反相色谱柱。Thereby, components with cell proliferation activity can be separated; wherein, the reversed-phase chromatography columns used in steps (1) and (2) are all C18 reversed-phase chromatographic columns.
优选的实施方案中，两个步骤使用的流动相均为乙腈和纯水；其中，步骤(1)的反相色谱分离中，乙腈的百分比梯度变化可从5到12％(体积百分比)，水的百分比梯度变化可从95％到88％(体积百分比)，分离获得第6峰、第7峰和第8峰；步骤(2)中，乙腈的梯度变化可以是从0％到7％(体积百分比)，水的梯度变化可以从100％到93％(体积百分比)。更优选的实施方案中，步骤(2)中，前0-3分钟，乙腈从0％梯度变化到5.5％，水从100％梯度变化到94.5％；第3-50分钟，乙腈从5.5％梯度变化到7％，水从94.5％梯度变化到93％。In a preferred embodiment, the mobile phases used in the two steps are both acetonitrile and pure water; wherein, in the reverse phase chromatographic separation of step (1), the percentage gradient of acetonitrile can vary from 5 to 12% (volume percentage), water The percentage gradient change of acetonitrile can be from 95% to 88% (volume percentage), and the sixth peak, the seventh peak and the eighth peak are obtained separately; in step (2), the gradient change of acetonitrile can be from 0% to 7% (volume percentage) Percentage), the gradient of water can vary from 100% to 93% (volume percentage). In a more preferred embodiment, in step (2), in the first 0-3 minutes, acetonitrile changes from 0% to 5.5%, and water changes from 100% to 94.5%; from 3 to 50 minutes, acetonitrile changes from 5.5% Change to 7%, and the water changes gradually from 94.5% to 93%.
在某些实施方案中，本发明的提取物为采用本文任一实施方案所述的方法提取得到的提取物。In some embodiments, the extract of the present invention is an extract obtained by using the method described in any of the embodiments herein.
本文的提取物能有效加快细胞***的速度，改善老化细胞的再生功能；帮 助减少皱纹，让皮肤更红润、水嫩、有光泽，同时促进伤口愈合，修复器官损伤(如本案例中提出的修复心肌梗死后心功能)。因此，在某些实施方案中，本文还提供一种组合物，用于促进细胞增殖、促进伤口愈合、修复器官损伤。在某些实施方案中，该组合物为药物组合物，该药物组合物含有本文任一实施方案所述的提取物或活性成分，和任选的药学上可接受的载体。合适的载体可以是本领域周知的载体，例如适用于将活性成分制成片剂、注射剂、冻干剂或膏剂等的载体。在某些实施方案中，该组合物为化妆品，含有本文所述的提取物和化妆品中常用的载体。The extract of this article can effectively accelerate the speed of cell division, improve the regeneration function of aging cells; help reduce wrinkles, make the skin more rosy, supple and shiny, at the same time promote wound healing, repair organ damage (such as the repair proposed in this case Heart function after myocardial infarction). Therefore, in certain embodiments, this document also provides a composition for promoting cell proliferation, promoting wound healing, and repairing organ damage. In certain embodiments, the composition is a pharmaceutical composition containing the extract or active ingredient described in any of the embodiments herein, and optionally a pharmaceutically acceptable carrier. A suitable carrier may be a carrier well-known in the art, for example, a carrier suitable for preparing the active ingredient into tablets, injections, freeze-dried preparations or ointments. In certain embodiments, the composition is a cosmetic, and contains the extract described herein and a carrier commonly used in cosmetics.
本文中，纯化与提取和分离可互换使用，意指从羊水中分离或提取出活性成分。In this context, purification and extraction and separation are used interchangeably, meaning the separation or extraction of active ingredients from amniotic fluid.
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和试剂，除非另有说明，否则为本领域常规的方法和试剂。Hereinafter, the present invention will be explained in the form of specific embodiments. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. Unless otherwise specified, the methods and reagents used in the examples are conventional methods and reagents in the art.
实施例1：鸡蛋和鸭蛋的成分分析Example 1: Composition analysis of eggs and duck eggs
1.材料和预处理1. Materials and pretreatment
取新鲜的胚龄为7天的鸡蛋羊水和胚龄为8天的鸭蛋羊水各400ml，分别加入适量己烷，2500rpm、4℃离心20分钟，获得水相，0.22μm滤膜过滤。获得用于分析的鸡蛋羊水样品和鸭蛋羊水样品。Take 400ml of fresh egg amniotic fluid with embryo age of 7 days and duck egg amniotic fluid with embryo age of 8 days, respectively, add appropriate amount of hexane, centrifuge at 2500 rpm, 4°C for 20 minutes to obtain the water phase, and filter with 0.22 μm filter membrane. Obtained egg amniotic fluid samples and duck egg amniotic fluid samples for analysis.
2.HPLC分析2. HPLC analysis
(1)采用日立HPLC***进行，分离柱为LaChrom-C18 AQ。洗脱溶剂为经脱气的色谱级乙腈和ddH 2O。 (1) Using Hitachi HPLC system, the separation column is LaChrom-C18 AQ. The elution solvent is degassed chromatography grade acetonitrile and ddH 2 O.
(2)洗脱方法(2) Elution method
流动相为乙腈(ACN)和超纯水。采用梯度方式洗脱，具体参数如下：0-3分钟，乙腈从0％梯度变化到5.5％，超纯水从100％梯度变化到94.5％；3-50分钟，乙腈从5.5％梯度变化到7％，超纯水从94.5％梯度变化到93％；50-52分钟，乙腈从7％梯度变化到100％，超纯水从93％梯度变化到0％。流动相流 速为0.8毫升/分钟，柱温为25.0℃，进样量为20微升。DAD检测器的检测波长为250nm。等体积收集馏分，3毫升/管供细胞活性测试。The mobile phase is acetonitrile (ACN) and ultrapure water. Use gradient elution, the specific parameters are as follows: 0-3 minutes, acetonitrile changes from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile changes from 5.5% to 7 %, the ultrapure water changes from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%. The flow rate of the mobile phase was 0.8 ml/min, the column temperature was 25.0°C, and the injection volume was 20 μl. The detection wavelength of the DAD detector is 250 nm. Collect fractions in equal volume, 3 ml/tube for cell viability test.
3.测细胞活性3. Measure cell viability
将AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成含10％FBS的DMEM、DMEM和含不同体积浓度的鸡蛋羊水馏分或鸭蛋羊水馏分的培养基。培养24小时后，每孔加入10微升CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。 After AC16 was digested, it was plated in a 96-well plate, 8000 pieces/well, each group of five replicate wells. Incubate for 2 hours in a 5% CO 2 saturated humidity 37°C incubator, and the cells adhere to the wall. After starvation culture with DMEM medium for 24 hours, it was replaced with 10% FBS-containing DMEM, DMEM, and a medium containing egg amniotic fluid fraction or duck egg amniotic fluid fraction with different volume concentrations. After incubating for 24 hours, add 10 microliters of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance was measured at 450nm in a microplate reader.
4.结果4. Results
结果如图1(鸡蛋)和图2(鸭蛋)所示。由图1和2可知，鸡蛋与鸭蛋的羊水具有相同的图型，因此其活性成分相同。The results are shown in Figure 1 (egg) and Figure 2 (duck egg). It can be seen from Figures 1 and 2 that the amniotic fluid of eggs and duck eggs have the same pattern, so their active ingredients are the same.
图3显示，鸡蛋羊水提取物(CEE)和鸭蛋羊水提取物(DEE)能促进细胞增殖。Figure 3 shows that egg amniotic fluid extract (CEE) and duck egg amniotic fluid extract (DEE) can promote cell proliferation.
实施例2：鸡蛋中活性成分的分离Example 2: Separation of active ingredients in eggs
1、实验材料1. Experimental materials
(1)纯化样品：新鲜的胚龄为7天的鸡蛋羊水，400ml。(1) Purified sample: fresh egg amniotic fluid with embryo age of 7 days, 400ml.
(2)主要实验设备及耗材(2) Main experimental equipment and consumables
GE AKTA purifier；相分离柱为UniSil 10-100 C18；GE AKTA purifier; The phase separation column is UniSil 10-100 C18;
日立HPLC***；使用LaChrom-C18 AQ分离柱；Hitachi HPLC system; using LaChrom-C18 AQ separation column;
洗脱溶剂为经脱气的色谱级乙腈和ddH 2O。 The elution solvent is degassed chromatography grade acetonitrile and ddH 2 O.
2、实验方法2. Experimental method
(1)样本处理：新鲜鸡蛋羊水400ml，加入适量己烷，2500rpm、4℃离心20分钟，获得水相，0.22μm滤膜过滤。(1) Sample treatment: 400ml fresh egg amniotic fluid, add appropriate amount of hexane, centrifuge at 2500rpm, 4℃ for 20 minutes to obtain the water phase, filter with 0.22μm filter membrane.
(2)样品纯化(2) Sample purification
第一步：以乙腈(A)和ddH 2O(B)作为流动相，UniSil 10-100 C18反相分离柱分离； The first step: using acetonitrile (A) and ddH 2 O (B) as mobile phases, UniSil 10-100 C18 reversed-phase separation column;
平衡反相柱：以流动相5％乙腈(A)平衡反相柱，流速10ml/min，直到280nm紫外吸收曲线平稳，回归基线；Equilibrate the reversed-phase column: equilibrate the reversed-phase column with mobile phase 5% acetonitrile (A) at a flow rate of 10ml/min until the 280nm UV absorption curve is stable and returns to the baseline;
上样流速：1ml/min，上样量50毫升；Sample loading flow rate: 1ml/min, sample loading volume is 50ml;
梯度洗脱：0-10CV，乙腈(A)从5％到12％，ddH 2O(B)从95％到88％。流速10毫升/分钟，等体积收集馏分，3毫升/管； Gradient elution: 0-10CV, acetonitrile (A) from 5% to 12%, ddH 2 O (B) from 95% to 88%. Flow rate 10 ml/min, collect fractions in equal volume, 3 ml/tube;
重复分离纯化20次，合并每一次实验中相同出峰时间的馏分；Repeat the separation and purification 20 times, and combine the fractions with the same peak time in each experiment;
冷冻干燥每一部分样品。Freeze dry each part of the sample.
测细胞活性：将AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成含10％FBS的DMEM、DMEM和20％(体积比)不同馏分的培养基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。细胞活力(Cell viability)％＝(实验组吸收值-空白孔吸收值)/(对照组吸收值-空白孔吸收值)×100％。 Test cell viability: After AC16 is digested, it is plated in a 96-well plate, 8000 cells/well, five replicate wells in each group. Incubate for 2 hours in a 5% CO 2 saturated humidity 37°C incubator, and the cells adhere to the wall. After starvation culture with DMEM medium for 24 hours, the medium was replaced with DMEM containing 10% FBS, DMEM and 20% (volume ratio) of different fractions. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance was measured at 450nm in a microplate reader. Cell viability%=(absorption value of experimental group-absorption value of blank hole)/(absorption value of control group-absorption value of blank hole)×100%.
第二步：日立C18反相分离柱分离The second step: Hitachi C18 reversed phase separation column separation
经AKTA分离得到的初级活性馏分第8峰，用HPLC(日立)进一步分离纯化。流动相为乙腈和超纯水。采用梯度方式洗脱，具体参数如下：0-3分钟，乙腈从0％梯度变化到5.5％，超纯水从100％梯度变化到94.5％；3-50分钟，乙腈从5.5％梯度变化到7％，超纯水从94.5％梯度变化到93％；50-52分钟，乙腈从7％梯度变化到100％，超纯水从93％梯度变化到0％。流动相流速为0.8mL/min，柱温为25.0℃，进样量为20μl。DAD检测器的检测波长为250nm，检测时间为0-20分钟。The 8th peak of the primary active fraction separated by AKTA was further separated and purified by HPLC (Hitachi). The mobile phase is acetonitrile and ultrapure water. Use gradient elution, the specific parameters are as follows: 0-3 minutes, acetonitrile changes from 0% to 5.5%, ultrapure water from 100% to 94.5%; 3-50 minutes, acetonitrile changes from 5.5% to 7 %, the ultrapure water changes from 94.5% to 93%; 50-52 minutes, the acetonitrile changes from 7% to 100%, and the ultrapure water changes from 93% to 0%. The flow rate of the mobile phase was 0.8mL/min, the column temperature was 25.0°C, and the injection volume was 20μl. The detection wavelength of the DAD detector is 250 nm, and the detection time is 0-20 minutes.
冷冻干燥每一部分样品；Freeze-dry each part of the sample;
测细胞活性：将AC16消化后，铺于96孔板中，8000个/孔，每一组五个复孔。在5％CO 2饱和湿度37℃培养箱培养2小时，细胞贴壁。用培养基DMEM饥饿培养24小时后，替换成含10％FBS的DMEM、DMEM和含20％(体积比)不同馏分的培养基。培养24小时后，每孔加入10μl CCK-8试剂。孵育2小时后，于酶标仪在450nm检测吸收值。细胞活力(Cell viability)％＝(实验组 吸收值-空白孔吸收值)/(对照组吸收值-空白孔吸收值)×100％。 Test cell viability: After AC16 is digested, it is plated in a 96-well plate, 8000 cells/well, five replicate wells in each group. Incubate for 2 hours in a 5% CO 2 saturated humidity 37°C incubator, and the cells adhere to the wall. After starvation culture with medium DMEM for 24 hours, it was replaced with DMEM containing 10% FBS, DMEM and medium containing 20% (volume ratio) of different fractions. After culturing for 24 hours, add 10μl of CCK-8 reagent to each well. After incubating for 2 hours, the absorbance was measured at 450nm in a microplate reader. Cell viability%=(absorption value of experimental group-absorption value of blank hole)/(absorption value of control group-absorption value of blank hole)×100%.
3、实验结果3. Experimental results
1、新鲜羊水经UniSil 10-100 C18分离的色谱图如图4所示。细胞活力检测结果如图6所示。结果显示，图4中的P6、P7、P8峰在AC16细胞中具有主要生物活性。1. The chromatogram of fresh amniotic fluid separated by UniSil 10-100 C18 is shown in Figure 4. The cell viability test results are shown in Figure 6. The results show that the P6, P7, and P8 peaks in Figure 4 have major biological activities in AC16 cells.
2、P6、P7、P8峰HPLC结果如图5所示。和原始粗品羊水相比，结果表明UniSil 10-100 C18分离具有较高的分辨率，非常适合作为制备分离的第一步。如图7所示，细胞活力检测发现P8峰中含有的P8-2在AC16细胞中具有生物活性。以上结果说明较高纯度的P8-2是P8峰中以及羊水中能促进细胞增殖及的重要化合物之一。2. The HPLC results of P6, P7 and P8 peaks are shown in Figure 5. Compared with the original crude amniotic fluid, the results show that UniSil 10-100 C18 separation has a higher resolution, which is very suitable as the first step of preparation and separation. As shown in Figure 7, the cell viability test found that P8-2 contained in the P8 peak has biological activity in AC16 cells. The above results indicate that the higher purity P8-2 is one of the important compounds in the P8 peak and amniotic fluid that can promote cell proliferation.
3、为了确定P8峰的疏水系数，用水分别配制P8峰、L-多巴和VB12标准化合物溶液，以安捷伦SB-Aq柱洗脱，具体洗脱条件如下：3. In order to determine the hydrophobic coefficient of P8 peak, prepare P8 peak, L-dopa and VB12 standard compound solutions with water respectively, and eluted with Agilent SB-Aq column. The specific elution conditions are as follows:
色谱柱：安捷伦SB-Aq，4.6 x 250mm，5micron；Chromatographic column: Agilent SB-Aq, 4.6 x 250mm, 5micron;
仪器：日立Primaide型高效液相色谱仪；Instrument: Hitachi Primaide type high performance liquid chromatograph;
参数：检测波长250nm，柱温25℃；Parameters: detection wavelength 250nm, column temperature 25℃;
流动相：乙腈，水；Mobile phase: acetonitrile, water;
样品处理：各样品用水溶解；Sample processing: each sample is dissolved in water;
洗脱：Elution:
时间(分钟)Time (minutes) 乙腈(％)Acetonitrile (%) 水(％)water(%)
00 00 100100
1111 00 100100
1717 55 9595
3030 1010 9090
4545 4545 5555
5050 100100 00
结果如图8所示。图8显示，P8的疏水性介于L-多巴与VB12之间。由 于L-多巴的辛醇/水分配系数Log P为0.05，VB12的辛醇/水分配系数Log P为1.897，因此，P8的辛醇/水分配系数Log P在0.05-1.897之间，更优选在0.1-1.897之间，更优选在0.5-1.897之间或在0.5-1.5之间。The result is shown in Figure 8. Figure 8 shows that the hydrophobicity of P8 is between L-dopa and VB12. Since the octanol/water partition coefficient Log P of L-dopa is 0.05, and the octanol/water partition coefficient Log P of VB12 is 1.897, the octanol/water partition coefficient Log P of P8 is between 0.05 and 1.897. It is preferably between 0.1 and 1.897, more preferably between 0.5 and 1.897 or between 0.5 and 1.5.
实施例3：鸡蛋中活性成分对修复心肌梗塞后心功能Example 3: Effect of active ingredients in eggs on repairing cardiac function after myocardial infarction
1、实验材料1. Experimental materials
(1)8周龄C57BL/6J成年雄性小鼠；(1) C57BL/6J adult male mice aged 8 weeks;
(2)小动物气麻装置；小动物呼吸机；小鼠气管插管和心梗模型无菌手术器械；(2) Small animal air anesthesia device; small animal ventilator; mouse tracheal intubation and myocardial infarction model sterile surgical instruments;
(3)尾静脉注射装置和注射器；(3) Tail vein injection device and syringe;
(4)
Figure PCTCN2020116079-appb-000001
高分辨率小动物超声成像***。 (4)
Figure PCTCN2020116079-appb-000001
High-resolution small animal ultrasound imaging system.
上述材料均从市售途经获得。The above-mentioned materials are all obtained from commercial sources.
2、实验方法2. Experimental method
(1)8周龄C57BL/6J小鼠在诱导箱中用5％的异氟烷诱导麻醉，医用胶带固定，拉直颈部，然后迅速经口腔用20g留置针气管插管接入呼吸机，进行机械通气，并切换气麻通道到呼吸机上，麻药量调为2.5％维持麻醉。呼吸机频率为115次/分钟，呼吸比1:1，潮气量为1.5毫升。将小鼠固定在手术台上，维持热板温度为37℃，将小鼠四肢与信号采集处理***连接，监测手术中心电图的变化。左胸部脱毛备皮，碘伏消毒并覆盖高压灭菌的纱布。左前外侧经第四肋间隙开胸，暴露心脏。打开心包，于左心耳下缘2mm处穿入，用7-0 prolene线经室间沟处结扎，经室间沟出结扎冠状动脉左前降支，会看到心尖变白，心电图ST段抬高。缝合肋间，肌肉和皮肤。消毒。关掉麻药，持续通气至小鼠苏醒。(1) The 8-week-old C57BL/6J mice were anesthetized with 5% isoflurane in the induction box, fixed with medical tape, straightened the neck, and then quickly connected to the ventilator with a 20g indwelling needle tracheal intubation through the oral cavity. Perform mechanical ventilation and switch the air anesthesia channel to the ventilator. The amount of anesthetic is adjusted to 2.5% to maintain anesthesia. The frequency of the ventilator is 115 breaths/minute, the breathing ratio is 1:1, and the tidal volume is 1.5 ml. Fix the mouse on the operating table, maintain the temperature of the hot plate at 37°C, connect the mouse limbs to the signal acquisition and processing system, and monitor the changes in the electrogram of the operating center. The left chest is depilated and skinned, disinfected with iodophor and covered with autoclaved gauze. Open the chest through the fourth intercostal space on the left anterolateral side, exposing the heart. Open the pericardium and penetrate 2mm from the lower edge of the left atrial appendage. Use 7-0 prolene thread to ligate through the interventricular sulcus, and ligate the left anterior descending coronary artery through the interventricular sulcus. The apex will turn white and the ST segment of the ECG will be elevated. . Suture the intercostal space, muscles and skin. disinfect. Turn off the anesthetic and continue to ventilate until the mouse wakes up.
(2)术后第二天，通过心脏超声测定心脏功能，根据左心射血分数，符合心梗指标的小鼠随机分为3组，每组8只。(2) On the second day after the operation, the cardiac function was measured by echocardiography. According to the left ventricular ejection fraction, the mice meeting the index of myocardial infarction were randomly divided into 3 groups, each with 8 mice.
(3)尾静脉注射对照5％葡萄糖、葡萄糖溶解的实施例2获得的P6和P8，每只每次100微升，隔天注射。(3) Inject the P6 and P8 obtained in Example 2 with control 5% glucose and glucose dissolved in the tail vein, 100 microliters each, and injected every other day.
(4)每周通过
Figure PCTCN2020116079-appb-000002
高分辨率小动物超声成像***检测心功能。观测4周，统计，同时计算心梗小鼠的生存率。 (4) Pass every week
Figure PCTCN2020116079-appb-000002
High-resolution small animal ultrasound imaging system detects heart function. Observe for 4 weeks, make statistics, and calculate the survival rate of mice with myocardial infarction.
3、实验结果3. Experimental results
(1)尾静脉注射对照5％对照葡萄糖、葡萄糖溶解的P6和P8小鼠存活率如图9所示，图中从上到下三根曲线依次对应P8、P6和对照。结果显示，P6、P8峰可以显著降低由于心肌梗塞导致的死亡。(1) The survival rate of P6 and P8 mice with 5% control glucose and glucose solubilized by tail vein injection is shown in Figure 9. The three curves from top to bottom in the figure correspond to P8, P6 and control in turn. The results show that P6 and P8 peaks can significantly reduce deaths due to myocardial infarction.
(2)如图10显示，P6、P8峰可以显著改善心肌梗塞小鼠左心室射血分数。(2) As shown in Figure 10, P6 and P8 peaks can significantly improve the left ventricular ejection fraction of mice with myocardial infarction.
(3)如图11显示，P6、P8峰可以显著改善心肌梗塞小鼠左心室短轴缩短率。(3) As shown in Figure 11, P6 and P8 peaks can significantly improve the short axis shortening rate of the left ventricle in mice with myocardial infarction.

Claims (10)

  1. 一种非人动物的羊水的提取物，其特征在于，所述提取物所含活性成分的辛醇/水分配系数Log P在0.05-1.897范围内，优选在0.3-1.5之间；优选地，所述提取物采用反相色谱分离得到。A non-human animal amniotic fluid extract, characterized in that the octanol/water partition coefficient Log P of the active ingredients contained in the extract is in the range of 0.05-1.897, preferably 0.3-1.5; preferably, The extract is separated by reverse phase chromatography.
  2. 如权利要求1所述的提取物，其特征在于，The extract of claim 1, wherein:
    所述羊水为来自禽蛋的羊水；优选地，所述禽蛋为胚龄为5-12天的鸡蛋，优选胚龄为6-11天的鸡蛋，更优选胚龄为7-9天的鸡蛋、更优选胚龄为7-8天的鸡蛋，或者为发育时期与所述胚龄的鸡蛋所处的发育时期相对应的鸡以外的其它禽类的蛋；优选地，所述禽蛋为家禽的蛋，如鸡蛋、鸭蛋、鸵鸟蛋和鹅蛋；或The amniotic fluid is amniotic fluid derived from poultry eggs; preferably, the poultry eggs are eggs with embryo age of 5-12 days, preferably eggs with embryo age of 6-11 days, more preferably eggs with embryo age of 7-9 days , More preferably, eggs of 7-8 days of embryonic age, or eggs of other avians other than chickens whose developmental period corresponds to the developmental period of the embryonic-aged eggs; preferably, the avian eggs are of poultry Eggs, such as eggs, duck eggs, ostrich eggs, and goose eggs; or
    所述羊水为非人哺乳动物羊水；优选地，所述羊水来自啮齿类动物；优选地，所述羊水来自胎龄为8-20天的啮齿类动物的胚胎，或来自发育时期与胎龄为8-20天的啮齿类动物的发育时期相对应的非人哺乳动物的胚胎。The amniotic fluid is non-human mammal amniotic fluid; preferably, the amniotic fluid is derived from rodents; preferably, the amniotic fluid is derived from embryos of rodents with a gestational age of 8-20 days, or from a developmental period and a gestational age of The embryos of non-human mammals corresponding to the developmental period of rodents of 8-20 days.
  3. 一种非人动物羊水提取物的制备方法，其特征在于，所述方法包括采用反相色谱从所述羊水中分离出辛醇/水分配系数Log P在0.05-1.897之间、优选在0.3-1.5之间的具有细胞增殖活性和/或心肌梗死后心功能恢复的馏分的步骤；优选地，所述方法包括实施两次反相色谱的步骤，其中，第二次反相色谱使用的反相色谱柱的分辨率高于第一次反相色谱使用的反相色谱柱的分辨率。A method for preparing a non-human animal amniotic fluid extract, characterized in that the method comprises using reversed-phase chromatography to separate the octanol/water partition coefficient Log P from the amniotic fluid in the range of 0.05-1.897, preferably 0.3- 1.5 between the steps of fractions with cell proliferation activity and/or recovery of cardiac function after myocardial infarction; preferably, the method includes the steps of performing reversed-phase chromatography twice, wherein the second reversed-phase chromatography uses reversed-phase The resolution of the column is higher than that of the reversed-phase column used in the first reversed-phase chromatography.
  4. 如权利要求3所述的方法，其特征在于，所述反相色谱所用的反相色谱柱的固定相以硅胶为载体，表面键合的非极性基团选自苯基、C8或C18烷基及其衍生物；使用有机溶剂和水作为流动相；优选地，所述有机溶剂选自甲醇、乙腈和乙醇。The method of claim 3, wherein the stationary phase of the reversed-phase chromatography column used in reversed-phase chromatography uses silica gel as a carrier, and the non-polar group bonded on the surface is selected from phenyl, C8 or C18 alkane. Base and its derivatives; organic solvent and water are used as mobile phase; preferably, the organic solvent is selected from methanol, acetonitrile and ethanol.
  5. 如权利要求3所述的方法，其特征在于，所述方法包括两次反相色谱，其中，第一次反相色谱中，洗脱体积在51-731毫升之间的馏分，优选洗脱体积在250-504毫升之间的馏分，更优选洗脱体积在278-353毫升之间和/或在354-429毫升之间和/或在430-504毫升之间的馏分，或以其它相似色谱柱以同 比例所获相似馏分。用于第二次反相色谱；第二次反相色谱中，取洗脱时间在11-12.5分钟之间的馏分和13-14分钟之间的馏分，或以其它相似色谱柱以同比例所获相似馏分；The method according to claim 3, wherein the method comprises two reverse phase chromatography, wherein, in the first reverse phase chromatography, the elution volume of the fraction between 51-731 ml is preferably the elution volume The fraction between 250-504 ml, more preferably the fraction with an elution volume between 278-353 ml and/or between 354-429 ml and/or between 430-504 ml, or other similar chromatography The column obtains similar fractions in the same proportions. Used for the second reverse phase chromatography; in the second reverse phase chromatography, take the fraction with the elution time between 11-12.5 minutes and the fraction between 13-14 minutes, or use other similar columns with the same ratio Obtain similar fractions;
    优选地，第一次反相色谱的流动相中，有机溶剂的体积百分比梯度变化从5％到12％，水的体积百分比梯度变化从95％到88％；Preferably, in the mobile phase of the first reversed-phase chromatography, the volume percentage of the organic solvent varies from 5% to 12%, and the volume percentage of water varies from 95% to 88%;
    优选地，第二次反相色谱的流动相中，有机溶剂的体积百分比梯度变化从0％到7％，水的体积百分比梯度变化从100％到93％。Preferably, in the mobile phase of the second reverse phase chromatography, the volume percentage of the organic solvent varies from 0% to 7%, and the volume percentage of water varies from 100% to 93%.
  6. 一种从鸡蛋羊水中分离具有细胞增殖活性的成分的方法，其特征在于，所述方法包括：A method for separating components with cell proliferation activity from egg amniotic fluid, characterized in that the method comprises:
    (1)对鸡蛋羊水进行首次反相色谱分离，获取第6到第8峰的馏分；任选地，(1) Perform the first reverse phase chromatographic separation of the egg and amniotic fluid to obtain the fractions from the 6th to the 8th peak; optionally,
    (2)对第8峰的馏分进行第二次反相色谱分离，获取第2峰馏分；(2) Perform a second reverse phase chromatographic separation on the fraction of the 8th peak to obtain the fraction of the second peak;
    从而分离得到具有细胞增殖活性的成分；其中，步骤(1)和(2)使用的反相色谱柱均为C18反相色谱柱；Thereby, components with cell proliferation activity can be separated; wherein, the reversed-phase chromatographic columns used in steps (1) and (2) are all C18 reversed-phase chromatographic columns;
    优选地，两个步骤使用的流动相均为乙腈和纯水；其中，步骤(1)的反相色谱分离中，乙腈的体积百分比梯度变化为5到12％，水的体积百分比梯度变化为95％到88％；步骤(2)中，乙腈的体积百分比梯度变化为0％到7％，水的体积百分比梯度变化为100％到93％。Preferably, the mobile phases used in the two steps are both acetonitrile and pure water; wherein, in the reverse phase chromatographic separation of step (1), the volume percentage gradient of acetonitrile is 5 to 12%, and the volume percentage gradient of water is 95%. % To 88%; in step (2), the volume percentage gradient of acetonitrile varies from 0% to 7%, and the volume percentage gradient of water varies from 100% to 93%.
  7. 采用权利要求3-6中任一项所述的方法分离得到的来自非人动物羊水的活性成分。Active ingredients derived from non-human animal amniotic fluid obtained by the method of any one of claims 3-6.
  8. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-2任一项所述的提取物或权利要求7所述的活性成分，和任选的药学上可接受的载体。A pharmaceutical composition, characterized in that the pharmaceutical composition contains the extract according to any one of claims 1-2 or the active ingredient according to claim 7, and optionally a pharmaceutically acceptable carrier.
  9. 反相色谱柱在分离非人动物的羊水中具有细胞增殖活性的成分中的应用。Application of reversed-phase chromatography column in the separation of non-human animal amniotic fluid components with cell proliferation activity.
  10. 权利要求1-2中任一项所述的提取物或权利要求7所述的活性成分在制备促进细胞增殖和/或促进心肌梗死后心功能修复的产品中的应用。Use of the extract of any one of claims 1-2 or the active ingredient of claim 7 in the preparation of products that promote cell proliferation and/or promote cardiac function repair after myocardial infarction.
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