CN-122005614-A - Preparation method of sepsis therapeutic drug based on Syngnathus extract and drug

CN122005614ACN 122005614 ACN122005614 ACN 122005614ACN-122005614-A

Abstract

The invention discloses a preparation method of a sepsis therapeutic drug based on an Syngnathus extract and a drug, which specifically comprises the steps of raw material pretreatment, supercritical CO 2 extraction, column chromatography purification and preparation, wherein the high-efficiency extraction and purification of EPA and DHA in the Syngnathus are realized by optimizing supercritical CO 2 extraction parameters and column chromatography elution gradient, and the total content of EPA and DHA in the obtained active components is more than or equal to 80%. The medicine prepared by the invention can be prepared into oral preparations such as capsules, tablets and the like or injection, and has excellent stability and long effective period. Experiments prove that the medicine can regulate the 'pro-inflammatory-anti-inflammatory' balance by improving the content of SPM precursor substances (EPA and DHA) in sepsis model animals, obviously reduces the sepsis death rate and has high safety. The preparation method has stable process and strong controllability, and the prepared medicine has definite curative effect, provides a new medicine choice for sepsis treatment, and has higher clinical application value.

Inventors

Tu Yuexing
MENG JIANBIAO
GUAN WENQING
WANG QINGCHENG
XUE JING
SHEN QING

Assignees

浙江省立同德医院(浙江省精神卫生研究院)
杭州市临平区中西医结合医院
浙江工商大学

Dates

Publication Date: 20260512
Application Date: 20260206

Claims (10)

1. A preparation method of a sepsis therapeutic drug based on an Syngnathus extract is characterized by comprising the following steps: A. Pretreating raw materials, namely crushing and sieving dry sea dragon medicinal materials to obtain sea dragon primary powder, soaking and stirring the sea dragon primary powder in ethanol solution with the mass of 8-12 times, filtering and washing under reduced pressure after soaking, mixing filtrate and washing liquid, concentrating under reduced pressure, and drying under vacuum to obtain sea dragon coarse powder; B. Supercritical CO 2 extraction, namely extracting the Syngnathus coarse powder by CO 2 , separating the extract by a separating kettle, collecting extract liquid, and concentrating the extract liquid under reduced pressure to obtain a Syngnathus coarse extract; C. Purifying by column chromatography, vacuum drying to constant weight to obtain Syngnathus active component; D. the preparation method comprises mixing the active components of Syngnathus with medicinal adjuvants, and making into final product.
2. The preparation method of the sepsis therapeutic drug based on the Syngnathus extract according to claim 1, wherein the raw material pretreatment in the step A is specifically: a1, taking a dried sea dragon medicinal material, crushing the medicinal material by a high-speed universal crusher, and then sieving the crushed medicinal material by a 40-60-target standard sieve, and collecting the sieved material as sea dragon primary powder; A2, adding 75-85% ethanol solution with the mass which is 8-12 times that of the sea dragon primary powder, placing the sea dragon primary powder into a constant-temperature soaking box at the temperature of 25-30 ℃ for soaking for 2-4 hours, and stirring every 30 minutes at the stirring speed of 50r/min; A3, filtering the soaked filter residue by adopting 200-mesh filter cloth under reduced pressure to remove insoluble impurities, and washing the filter residue with 4-6 times of ethanol solution with the same concentration for 2 times; A4, combining the filtrate with the washing solution, concentrating under reduced pressure until no alcohol smell exists, and then drying in vacuum to obtain the Syngnathus coarse powder, wherein the vacuum drying condition is 60 ℃ to minus 0.1MPa.
3. The preparation method of the sepsis therapeutic drug based on the Syngnathus extract according to claim 1, wherein the supercritical CO 2 extraction in the step B comprises the following specific procedures: B1, paving sea dragon coarse powder in a supercritical extraction kettle, wherein the paving thickness is 3-5cm, using 95-99% (v/v) absolute ethyl alcohol as entrainer, pumping the entrainer into the extraction kettle at a constant speed of 5-8% of the mass of the raw materials by a high-pressure pump, extracting at 30-40MPa and 45-55 ℃, continuously introducing CO 2 after purification at a flow of 20-30L/h, and extracting for 2.5-3.5h; After the extraction is finished, the extract enters a separation kettle along with CO 2 , wherein the pressure of the first separation kettle is 8-10MPa and the temperature is 40-45 ℃, and the pressure of the second separation kettle is 4-6MPa and the temperature is 30-35 ℃, so that the separation of CO 2 and extract is realized; And B3, collecting the extract in the separation kettle, placing the extract in a rotary evaporator, and concentrating under reduced pressure at 40-50 ℃ and minus 0.08-0.1 MPa until no alcohol smell exists, thus obtaining the crude extract of the sea dragon.
4. A method for preparing a therapeutic drug for sepsis based on an extract of Syngnathus according to claim 3, wherein: In the step B1, the extraction pressure is 35MPa, the extraction temperature is 50℃, CO 2 , the flow is 25L/h, the extraction time is 3h, and the using amount of the entrainer is 6% of the mass of the raw material.
5. The preparation method of the sepsis therapeutic drug based on the Syngnathus extract according to claim 1, wherein the column chromatography purification in the step C comprises the following specific contents: Dissolving crude Syngnathus extract with chromatographic pure methanol, fixing volume to 50-100mg/mL, filtering with 0.45 μm organic phase filter membrane, loading onto silica gel chromatographic column with volume of 5-8%, silica gel chromatographic column specification of phi 5cm×60cm, silica gel particle diameter of 100-200 mesh, activating silica gel at 110deg.C for 2 hr, and filling; C2, using petroleum ether-ethyl acetate mixed solution as an eluent, gradient eluting from 8:2 to 5:5 according to the volume ratio of petroleum ether to ethyl acetate, wherein the eluting flow rate is 2-3mL/min, and collecting one fraction every 50mL by adopting an automatic part collector; And C3, detecting the EPA and DHA content in each fraction by adopting a GC-MS method, combining the fractions with the EPA and DHA total content of more than or equal to 80%, and drying in a vacuum drying oven until the weight is constant to obtain the Syngnathus active component.
6. The method for preparing a therapeutic drug for sepsis based on Syngnathus extract according to claim 5, wherein the step C3 comprises the steps of detecting EPA and DHA content in each fraction by GC-MS method under the following conditions: Chromatographic column HP-5MS,30m×0.25mm×0.25 μm, sample inlet temperature 250 ℃, carrier gas of helium, flow rate of 1.0mL/min, split ratio of 10:1; Programmed heating, namely, the initial temperature is 80 ℃ and kept for 2min, and the temperature is 10 ℃ per min to 250 ℃ and kept for 10min; The temperature of the mass spectrum ion source is 230 ℃, the electron bombardment energy is 70eV, the scanning range is between 50 and 500 m/z, and the quantification is carried out by an external standard method.
7. The method for preparing a therapeutic drug for sepsis based on Syngnathus extract according to claim 5, wherein in step C2, the gradient elution of the eluent is specifically: 0-2h with petroleum ether: ethyl acetate=8:2, 2-4h with petroleum ether: ethyl acetate=7:3, 4-6h with petroleum ether: ethyl acetate=6:4, and 6-8h with petroleum ether: ethyl acetate=5:5.
8. A method for preparing a therapeutic agent for sepsis based on Syngnathus extract according to any one of claims 1-7, wherein the therapeutic agent is prepared by the above preparation method, and comprises oral preparation and injection.
9. The sepsis therapeutic drug based on the Syngnathus extract according to claim 8, wherein the oral preparation is a capsule or a tablet, and the mass ratio of the capsule is as follows: 30-50 parts of sea dragon active component, 20-30 parts of microcrystalline cellulose, 10-20 parts of lactose and 1-3 parts of magnesium stearate.
10. The sepsis therapeutic drug based on the Syngnathus extract according to claim 8, wherein the injection comprises the following components in parts by mass: 5-10 parts of sea dragon active components, 20-30 parts of polyethylene glycol 400, 10-15 parts of propylene glycol and 100 parts of water for injection; the preparation method comprises controlling pH to 6.5-7.5, filtering with 0.22 μm microporous membrane, packaging, and sterilizing at 121deg.C for 15 min.

Description

Preparation method of sepsis therapeutic drug based on Syngnathus extract and drug Technical Field The invention relates to the technical field of medicine preparation, in particular to a preparation method of a medicine for treating sepsis based on an Syngnathus extract and a medicine. Background Sepsis is a life-threatening organ dysfunction caused by host reaction disorder caused by infection, is a common critical condition in severe medical science, and has the characteristics of high morbidity, high mortality, high treatment difficulty and the like. At present, sepsis treatment mainly comprises symptomatic treatment such as anti-infection, liquid resuscitation and organ function support, but lacks specific treatment drugs, and has limited clinical treatment effect. Proinflammatory Mediators (SPM) are lipid mediators which are produced by metabolism of polyunsaturated fatty acids (such as EPA and DHA) and have anti-inflammatory and proinflammatory effects, can restore the 'pro-inflammatory-anti-inflammatory' balance of an organism through the ways of regulating immune cell functions, inhibiting inflammatory factor release, promoting inflammatory cell apoptosis and the like, and have important potential in the treatment of sepsis. EPA and DHA are key precursor substances of SPM, and the in vivo content of the EPA and DHA directly influences the synthesis efficiency of SPM, so that the development of EPA and DHA-enriched medicaments is of great significance for the treatment of sepsis. The sea dragon is a traditional marine Chinese medicinal material, has the effects of tonifying kidney, strengthening yang, resolving hard mass, relieving swelling and the like, and modern pharmacological researches prove that the sea dragon contains rich EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), and the total content of the EPA and the DHA can reach 5-8% of dry weight. However, the application of the Syngnathus extract in sepsis treatment is not yet reported in domestic and foreign documents and patents at present, and the extraction method of EPA and DHA in the prior Syngnathus has a plurality of defects: For example, the extraction rate of the traditional solvent extraction method (such as n-hexane reflux extraction) is only 20-25%, and the total content of EPA and DHA in the obtained extract is less than 50%, and the risk of solvent residue exists (the n-hexane residue is easy to exceed the limit standard of Chinese pharmacopoeia (China)); Although the ultrasonic assisted extraction method can improve the extraction rate to 26-28%, the content of impurities (such as cholesterol and saturated fatty acid) in the extracting solution is higher, the subsequent purification is difficult, the purity is still difficult to break through 70%, and the medicinal requirement cannot be met. In addition, in the existing sepsis therapeutic drugs, no specific drug is supplemented for SPM precursors, and clinically common anti-inflammatory drugs (such as dexamethasone) can inhibit inflammatory response, but adverse reactions such as immunosuppression, gastrointestinal ulcer and the like are easy to cause after long-term use, so that the clinical application is limited. Therefore, there is a need to develop a preparation method of an extract of Syngnathus with high efficiency, stability and low residue, so as to obtain high purity EPA and DHA active components, and apply the EPA and DHA active components to sepsis treatment, and fill up the clinical blank. Disclosure of Invention The invention aims to provide a preparation method of a sepsis therapeutic drug based on an Syngnathus extract and the drug. The invention has the advantages of stable process, strong controllability, high purity of the prepared active components, high safety and good stability, and can efficiently extract and purify EPA and DHA in sea dragon. The technical scheme of the invention is as follows: a preparation method of a sepsis therapeutic drug based on Syngnathus extract comprises the following steps: A. Pretreating raw materials, namely crushing and sieving dry sea dragon medicinal materials to obtain sea dragon primary powder, soaking and stirring the sea dragon primary powder in ethanol solution with the mass of 8-12 times, filtering and washing under reduced pressure after soaking, mixing filtrate and washing liquid, concentrating under reduced pressure, and drying under vacuum to obtain sea dragon coarse powder; B. Supercritical CO 2 extraction, namely extracting the Syngnathus coarse powder by CO 2, separating the extract by a separating kettle, collecting extract liquid, and concentrating the extract liquid under reduced pressure to obtain a Syngnathus coarse extract; C. Purifying by column chromatography, vacuum drying to constant weight to obtain Syngnathus active component; D. the preparation method comprises mixing the active components of Syngnathus with medicinal adjuvants, and making into final product. In the preparation method o